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Monitoring with Lichens - Monitoring Lichens

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NATO Science SeriesA Series presenting the results ofscientific meetings supported under the NATO ScienceProgramme.

The Series is published by lOS Press, Amsterdam ,and K1uwerAcademic Publishers in conjunctionwith the NATO Scientific Affairs Division

Sub-Series

I. Life and Behav iouralSciencesII. Mathematics, Physics and ChemistryIII. Computer and Systems ScienceIV. Earth and Environmental SciencesV. Science and Technology Policy

lOS PressKluwer Academic PublisherslOS PressKluwer Academ ic PublisherslOS Press

The NATO Science Series continues the series of books published formerly as the NATOAS I Series.

The NATO Science Programme offers support for collaboration in civil science between scientists ofcountries of the Euro-Atlantic Partnership Council.The types of scientific meeting generally supportedare "Advanced Study Institutes" and "Advanced Research Workshops", although other types of meetingare supported from time to time. The NATO Science Series collects together the results of these mee­tings. The meetings are co-organ ized bij scientists from NATO countries and scientists from NATO'sPartner countries - countries of the CIS and Central and Eastem Europe .

Advanced Study Institutesare high-level tutorial courses offering in-depth study of latest advances inafield.Advanced Research Workshops are expert meetings aimed at criticalassessment of a field, andidentificationof directions for future action.

As a consequence of the restructuring of the NATO Science Programme in 1999, the NATO ScienceSeries has been re-organised and there are currently fivesub-series as noted above .Please consult thefollowing web sites for information onprevious volumes published in the Series ,as well as details of ear­lier sub-series.

htlp:/Iwww.nato.inVsciencehnp:/Iwww.wkap.nlhtlp·/Iwww.jospress.nlhtlp://www.wtv-books.deLnato-pco.htm

I

-~­.'-V/I

Series IV: Earthand Environmental Sciences - Vol. 7

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Monitoring with Lichens -Monitoring Lichens

eclited by

Pier Luigi Nimis Department of Biology, The University, Trieste, Italy

Christoph Scheidegger WSL, Swiss Federal Institute for Forest, Snow and Landscape Research, Birmensdorf, Switzerland

and

Patricia A. Wolseley The Natural History Museum, Department of Botany, London, United Kingdom

Springer-Science+Business Media, B.v.

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Proceedings of the NATO Advanced Research Workshop on Lichen Monitoring Wales, United Kingdom 16-23 August 2000

A C.I.P. Catalogue record for this book is available from the Library of Congress.

ISBN 978-1-4020-0430-8 ISBN 978-94-010-0423-7 (eBook)

Printed on acid-free paper

AII Rights Reserved © 2002 Springer Science+Business Media Dordrecht Originally published by Kluwer Academic Publishers in 2002 Softcover reprint of the hardcover 1 st edition 2002 No part of the material protected by this copyright notice may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording or by any information storage and retrieval system, without written permission from the copyright owner.

DOI 10.1007/978-94-010-0423-7

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CONTENTS

1. MONITORING WITH LICHENS - MONITORING LICHENSAn IntroductionPier Luigi Nimis, ChristophScheideggerand PatriciaA. Wolseley

Section 1. Lichens asIndicatorsof Pollution

2. MONITORING LICHENS AS INDICATORS OF POLLUTION 7An IntroductionPier Luigi Nimis and Ole William Purvis

3. BIOINDICATION : CALIBRATED SCALES AND THEIR UTILITY 11David L. Hawksworth

4. BIOINDICATION :THE LA.P. APPROACH 21RandolphKricke and Stefano Loppi

5. BIOINDICA TION :THE COMMUNITY APPROACH 39Chantal van Haluwyn and Kok(c. M.) van Herk

6. ACCUMULATION OF INORGANIC CONTAMINANTS 65RobertoBargagliand IrinaMikhailova

7. LICHENS AS MONITORS OF RADIOELEMENTS 85Mark R .D. Seaward

8. BIOMARKERS OF POLLUTION-INDUCED OXIDATIVE 97STRESS AND MEMBRANE DAMAGE IN LICHENSDamienCuny, Maria Luisa Pignata, liseKrannerandRichardBeckett

9. KEY ISSUES IN DESIGNING BIOMONITORING PROGRAMMES IIIMonitoring scenarios, sampling strategies and quality assuranceMarco Ferretti and Walter Erhardt

Section 2.MonitoringLichen DiversityandEcosystemFunction

10. MONITORING LICHEN DIVERSITY 143AND ECOSYSTEM FUNCTIONAn IntroductionSusan Will-Wolfand ChristophScheidegger

11. METHODS FOR MONITORING BIODIVERSITY AND 147ECOSYSTEM FUNCTIONMonitoring scenarios, sampling strategies and data qualitySusan Will-Wolf, ChristophScheideggerand BruceMcCune

v

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12. MONITORING LICHENS FOR CONSERVATION : 163RED LISTS AND CONSERVATION ACTION PLANSChristophScheideggerandTrevorGoward

13. LICHEN MONITORING AND CLIMATE CHANGE 183GregoryInsarov andBurkhardSchroeter

14. MONITORING BIODIVERSITY AND ECOSYSTEM FUNCTION: 203FORESTSSusan Will-Wolf, Per-Anders Esseen andPeterNeitlich

15. MONITORING BIODIVERSITY AND ECOSYSTEM FUNCTION : 223GRASSLANDS, DESERTS, AND STEPPERogerRosentreterand David J.Eldridge

16. MONITORING LICHENS ON MONUMENTS 239AndreAptrootand Peter W.James

17. MONITORING MARITIME HABITATS 255AnthonyFletcherand Robin Crump

Section 3.Methodsfor MonitoringLichens

18. METHODS FOR MONITORING LICHENS 269An IntroductionPatriciaA. Wolseleyand David J. Hill

19. MAPPING LICHEN DIVERSITY 273AS AN INDICATOR OF ENVIRONMENTAL QUALITYJulietteAsta, WalterErhardt, Marco Ferretti,FrancescaFornasier,UlrichKirschbaum,Pier Luigi Nimis, Ole WilliamPurvis, Stergios Pirintsos,ChristophScheidegger,ChantalvanHaluwynandVolkmarWirth

20. IDENTIFYING DEVIATIONS FROM NA TURALITY OF LICHEN 281DIVERSITY FOR BIOINDICA TION PURPOSESStefano Loppi, Paolo Giordani,Giorgio Brunialti, DeborahIsocronoandRosannaPiervittori

21. EPIPHYTES ON WAYSIDE TREES AS AN INDICATOR 285OF EUTROPHICATION IN THE NETHERLANDSKok (C .M.) van Herk

22. USING LICHENS ON TWIGS TO ASSESS CHANGES 291IN AMBIENT ATMOSPHERIC CONDITIONSPatriciaA .Wolseley

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23. GUIDELINES FOR THE USE OF EPIPHYTIC LICHENS 295AS BIOMONITORS OF ATMOSPHERIC DEPOSITIONOF TRACE ELEMENTSRoberto Bargagli and Pier Luigi N imis

24. TRANSPLANTED LICHENS 301FOR BIOACCUMULA TION STUDIESIrinaM ikhailova

25. SAMPLE PREPARATION OF LICHENS 305FOR ELEMENTAL ANALYSISAna M ariaRusu

26. SULPHUR ISOTOPES IN LICHENS 311AS INDICATORS OF SOURCESBaruch Spiro, James Morrisson and OleWilliam Purvis

27. ESTIMATION OF CRITICAL LEVELS 317OF AIR POLLUTION (METALS) ON THE BASIS OFFIELD STUDY OF EPIPHYTIC LICHEN COMMUNITIESEugene Vorobeichik and Irina Mikhailova

28. MONITORING PHYSIOLOGICAL CHANGE IN LICHENS: 323TOTAL CHLOROPHYLL CONTENTAND CHLOROPHYLL DEGRADATIONKansri Boonpragob

29. CHLOROPHYLL FLUORESCENCE MEASUREMENTS 327IN THE FIELD :ASSESSMENT OF THE VITALITYOF LARGE NUMBERS OF LICHEN THALLIManfredJensen and Randolph Kricke

30. MEASURING BARK pH 333RandolphKricke

31. A PHOTOGRAPHIC QUADRAT RECORDING METHOD 337EMPLOYING IMAGE ANALYSIS OF LICHENSAS AN INDICATOR OF ENVIRONMENTAL CHANGEOle William Purvis,Lucy Erotokritou,PatriciaA .Wolseley,Ben Williamson,and Helen Read

32. SITE ASSESSMENT OF EPIPHYTIC HABITATS 343USING LICHEN INDICESFrancisRose and Sandy Coppins

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33. INDICATOR SPECIES - RESTRICTED TAXA APPROACH 349IN CONIFEROUS AND HARDWOOD FORESTSOF NORTHEASTERN AMERICAStevenB. Selva

34. MONITORING REGIONAL STATUS AND TRENDS 353IN FOREST HEALTH WITH LICHEN COMMUNITIES:THE UNITED STATES FOREST SERVICE APPROACHSusan Will-Wolf

35. BIODIVERSITY ASSESSMENT TOOLS - LICHENS 359ChristophScheidegger,Urs Groner,ChristineKellerand Silvia Stofer

36. USING LICHENS AND BRYOPHYTES TO EVALUATE 367THE EFFECTS OF SILVICUL TURAL PRACTICESIN TASMANIAN WET EUCALYPT FORESTGintaras Kantvilas and S. JeanJarman

37. USING CORTICOLOUS LICHENS OF TROPICAL 373FORESTS TO ASSESS ENVIRONMENTAL CHANGESPatriciaA .Wolseley

38. LICHENOMETRY 379DanielMcCarthy

39. TRANSPLANTING LICHEN FRAGMENTS 385FOR PROVENANCE-CLONE TESTSJean-ClaudeWalserandChristophScheidegger

40. ASSESSING CHANGES IN DENSITY AND CONDITION 391OF LICHENS FOR SPECIES RECOVERY PROGRAMMESPatriciaA. WolseleyandPeterW. James

41. MONITORING RED-LISTED LICHENS 395USING PERMANENT PLOTSAndre Aptrootand LaurensSparrius

42. A METHOD FOR DETECTING LARGE-SCALE 399ENVIRONMENTAL CHANGE WITH LICHENSGregoryInsarov

Appendix

List ofparticipantsat the NATO InternationalAdvanced 405ResearchWorkshopon LichenMonitoring(LIMON),OrieltonField Centre (West Wales, UK), 16-23rd August 2000

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PREFACE

The present volume originates from a NATO Advanced Research Workshop organisedby P.A. Wolseley (Administrative Director), and co-directed by G. Insarov and P.L.Nimis, which was held at Orielton Field Centre, West Wales,UK, over the period 16­220d August 2000. The workshop was attended by 63 participants and observers withrepresentatives from 2I different countries spanning temperate, tropical and sub-arcticregions in both northern and southern hemispheres. The rangeofexpertise was immenseand provided a valuable forum for debate.The workshop tackled two major areas wherelichen monitoring contributes to our understandingof environmental change, notablythose (a) caused by polJution, including the accumulationof substances and otherenvironmental disturbances and (b) the application of lichens as a yard-stick to assessthe impactofmanagement activities across a rangeof habitats. Each section includedpaper and poster presentations followed by a discusssion and formulationof specificactions. Mid-day field visits to topical monitoring sites to complement the sessionsfacilitated debate over the applicationof various methods. Considerable discussionfocussed on variation in methods and the challenges in achieving a single method overvast geographical and climatic regions where species and environmental conditions varywidely. Nevertheless, it was agreed to standardise wherever practical in order tofacilitate comparisons between regions, leading to greater acceptance and application ofthe methods by regional and national authorities and funding agencies. The limitationsofmethods designed to answer single questions were stressed, as welJ as the challengesin establishing monitoring where little or no environmental, or indeed lichenologicalinformation, yet exists.

As requested by NATO, we did not try to produce an "ordinary conferenceproceedings" volume, but rather a seriesof reviews followed by"recipes"of the widerangeofavailable methods.We have solicited additional articles from leading scientiststo produce a comprehensiveState-ofthe Art on "lichen monitoring". We areparticularly grateful to alJ authors for producing such high-quality papers within such ashort time period. Special thanks are due to our co-editorsof the three sections: OleWilliam Purvis, SusanWill-Wolf and David J. Hill, who together shared a great dealofthe editorial burden. Special thanks are also due to NATO, whose valued supportofthemeeting, under their Advanced Research Workshop Programme, has enabled our groupto re-define the aims and future direction of lichen monitoring. We hope that a clearervision has emerged, and that this will be visible in these pages.

Pier Luigi Nimis, Christoph Scheidegger and Patricia A.Wolseley

ix

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MONITORING WITH LICHENS - MONITORING LICHENS

An Introduction

P.L.NIMIS 1, C. SCHEIDEGGER 2 and P.A. WOLSELEy3

'Diparttmenta di Biologia, Universita di Trieste, via Giorgieri 10, 1­34127 Trieste, Italy ([email protected])2WSL, Swiss Federal Institutefor Forest, Snow and Landscape ResearchCH-8903 Birmensdorf, Switzerland ([email protected])3The Natural History Museum, Dept. ofBotany, Cromwell Road, LondonSW75BD, UK ([email protected])

Widespread changes in natural and managed environments in the last century have beenassociated with rapid developmentof technology with the capacity for massivedestruction of natural environments. This has been accompanied by large-scale naturaldisasters such as floods and droughts and by large-scale technical failures such asChemobyl, impacting greatly on human existence and welfare.It is the impact on socialconditions that has led to increasing interest in maintaining environmental quality andensuring that human activities do not threaten the ecosystem on which we depend. Thethreats to human health by water and air pollution led to early research on bioindicatorsin order to map and monitor the effects of pollution on selected organisms. However therangeof objectives to which biomonitoring is applied has grown steadily from waterquality and atmospheric pollution to heavy metal accumulation, climate change, and toenvironmental issues involving management of natural resources such as the effectsoffragmentation and habitat alteration, effectsof development on biodiversity as well asassessing conservation practices for rare or endangered species.

Lichens are among the most widely used biomonitors in the terrestrial environment.They are included in the fungal kingdom with an estimated 1.5 million species [2]ofwhich c. l/Slh are thought to be lichenised; that is, to have algal or cyanobacterialphotobionts that provide nutrients for the mycobiont. Fungi are frequentlyinconspicuous or have a strong seasonality so that they are often excluded frombiodiversity research. Many lichens are long-lived organisms with a high habitatspecificity so that they can be used to estimate species diversity and habitat potential atall timesofyear. Lichens are widespread in a rangeofhabitats from extreme conditionsof heat or cold, from deserts to tropical rain forests, from natural to managedenvironments. They may be found on all types of substrata such as trees, rocks andearth, as well as man-made substrata, allowing their use as biological monitors ofenvironmental conditions in urban and rural situations, as well as tropical and arcticareas. Most species are widespread, occur on more than one continent and typicalspecies numbers per hectare range from around ten to several hundred.

1P.L. Nimis, C.Scheideggerand P.A. Wolseley (eds.), Monitoringwith Lichens- MonitoringLichens. 1-4.© 2002 KluwerAcademicPublishers. Printedin the Netherlands.

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The literature on lichens as biomonitors is huge.This book attempts a synthesis, andto suggest areasoffuture developments.

The title of the book needs abriefexplanation. The term "monitoring" and morespecifically "biomonitoring" is being used for a varietyof studies, ranging fromanecdotal observations of a species to regularly repeated surveillance of complexbiological parameters, thus becoming an "omnibus-term" [3]. Expressions such as"monitoring air pollution with lichens" have become almost trivial. We wonderhowever whether they are correct. In order to avoid confusion, in this book we adoptsome definitions introduced by Hellawell [3] which are now generally accepted inecology and conservation biology [I].• Survey: An exercise in which a set of qualitative or quantitative observations are

made, usually by means of standardised procedures and within a restricted period oftime, but without any preconception of what the findings ought to be.

• Surveillance: An extended programme of surveys, undertaken in order to provide atime series, to ascertain the variability and/or rangeof states or values which mightbe encountered over time (but again without preconceptionsofwhat these might be).

• Monitoring: intermittent (regular or irregular) surveillance carried out in order toascertain the extentof compliance with a predetermined standard or the degree ofdeviation from an expected norm.However there are problems with these definitions in that a project with an

established method carried out for the first time would be a survey, and only whenrepeated would become surveillance or monitoring. But we consider that monitoring ismore a process than a result and that all projects where the method and sampling designprovide a basis for repetitive sampling should be termed monitoring, if the data can becompared with a predetermined standard. Bioindication studies and species inventoriesform the basis for much environmental assessment and where the documentation allowsa repetition of the study and a comparison with a norm it is considered to be monitoring.

As far as the term "indicator" is concerned, there are several, sometimescontradictory definitions. According to Hunsaker [4] an "indicator" is a characteristic oran entity that can be measured to estimate status and trendsofthe target environmentalresource. An "index" is a characteristic, usually expressed as a score, that describes thestatus of an indicator. For example, if lichens are the indicators, the lichen diversityscore can be the index. An index can be referred to as a "descriptor" [5]. The terms"indicator" and "index" obviously have a common origin. They both derive from theLatin "index" which is the second finger of the hand, that which we normally use topoint at something or somebody. With that finger, we can only point to one object at atime. Here lies the weakpoint of the term "indicator", one which has originated muchmisunderstanding. The biological response of organisms is complex and not a simplecause-effect. A single finger is often not enough for pointing to the complexityofbiological or environmental organisation. The demand for cheaper waysof estimatingbiological conditions or sustainabilityof management of the environment has led to aproliferation of bioindicators that have not been all rigorously tested and which have notbeen shown to have a specific applied value. The accuracyof these predictions mustdepend on the establishment of baseline standards or thresholds, so that the bioindicatorcan be used to monitor something.

Notoriously, lichens are sensitive organisms. Their response to environmental

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change may include biodiversity, morphology, physiology, accumulationofpollutants,etc. Such responses in the framework of"monitoringwith lichens" can be used as"indicators" for many complex factors, from climatic change to pollution.Bio"indication",however, is largely a matterofdata interpretation. On the other hand,"monitoringlichens" stresses the changeofthe floristic data dataper se, e.g.in Red Listprojects.

There are many cases in which monitoring the stateofan organism orofa groupoforganisms is a worthy aim initself.One case is that of biodiversity.As recently as1992the rapid escalation in lossofbiodiversity was addressed in Article 7 in the Conventionon Biological Diversity, where signatories for eachof the 168 countries are nowrequired to identify componentsof biological diversity important for long-termconservation and sustainable use of biodiversity. As shown in the second section of thisvolume, lichens are important in this context.

Another example has to do with air pollution. In monitoring for environmentalpollution one of the major arguments for using lichens is that the reactionoforganismsis a better surrogate of "environmental quality" than concentration measuresofa setofarbitrarily selected gaseous or particulate pollutants. This is a reasonable starting pointfor raising awareness on environmental quality. Lichens cannot"monitorpollution",but monitoring lichens can be important to evaluate its possible effects.

Identificationof the components of biodiversity still presents a problem in manyareasof the world, as well as in many taxonomic groups, where our knowledge isinadequate. Without basic data on environmental conditions or management historythere is no base-line information with which to test bioindicators. Whereas thedevelopment of biomonitoring has largely taken place in the developed world, there isan urgent need for it in other parts of the world in order to tackle a global problemofestablishing practical methodologies for biomonitoring using lichens.

The present volume originates from an Advanced Research Workshop proposed andsUPf0rted by NATO.Itwas held at Orielton Field Centre in West Wales,UK, from 16­23r August 2000, where it was attended by63 participants from21 countries, fromtemperate, tropical and near arctic conditions and including northern and southernhemispheres. This produced a great range of expertise, as well as highlighting someofthe problems in setting up monitoring where there is little existing environmental orlichenological information.We have tried to address someofthe problems in this book,by providing both a reviewofcurrent methodology and a practical guide.

This volume is divided into three sections:• Monitoring lichens as indicatorsofpollution.• Monitoring lichen diversity and ecosystem function.• Methods for monitoring lichens.

The first two sections are devoted to the presentationof the Stateofthe Art in therespective fields, while the third containsbriefpresentationsof methods which arecurrently used in lichen biomonitoring.We have tried to stress as much as possible theimportanceof sound methodological approaches. Sampling design, sampling strategy,and data quality evaluation were all too often neglected by biologists working in thefield of biomonitoring. Such issues are so important for the futureofthis discipline thattwo chapters were devoted to them in the first and second parts of the book.For the rest,overlappings and repetitions were difficult to avoid. We have tried to reduce them as

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much as possible, but we also accepted some degreeofoverlappingwhenever the sametopic was treated from different pointsofview.

The literature on lichens as biomonitors and the rangeofavailable methods are verylarge, and we cannot pretend to having been exhaustive. We do hopehoweverthat thisbook will provide an overviewof such an interesting andpromising field, and that itwill help to identify problems and pragmatic approaches, and to point the way to futureexcitingdevelopments.

Acknowledgements

The editors would like to acknowledge financial assistance from NATO for both the workshop and the bookproduction, the British Lichen Society and English Nature for financial assistance to the workshop, and theNatural History Museum for supporting this project. We would also like to acknowledge help from thefollowing people: Dr.Robin and Anne Crump at OrieltonField Study Centre who both hostedand contributedto the workshop; Frank Dobson for financial organisation; Stefano Martellos and Guido Incerti for editorialassistance, and John Wolf and Clifford Smith for their help in the preparationofthis volume.

References

I. Goldsmith,F.B.(1991)Monitoringfor Conservation and Ecology, Chapman and Hall, London.2. Hawksworth, D .L. (1991) The fungal dimension of biodiversity: magnitude, significance, and

conservation,Mycological Research 9S (6), 641-655.3. Hellawell, J.M. (1991) Development of a rationale for monitoring, in F.B. Goldsmith (ed.),Monitoring

for Conservation and Ecology, Chapman and Hall, London,pp. 1-14.4. Hunsaker, C.T. (1993) New concepts in environmental monitoring: The questionof indicators,The

Science ofThe Total Environment, Suppl., 77-95.5. Legendre,P.and Legendre, L. (1998)Numerical Ecology , Elsevier, Amsterdam.

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Section 1

MONITORING LICHENS

AS INDICATORS OF POLLUTION

editedby

Pier Luigi NIMIS and Ole William PURVIS

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MONITORING LICHENS AS INDICATORS OF POLLUTION

An Introduction

P.L.NIMIS1and O.W. PURVIS 2

'Dipartimento di Biologia, Universita di Trieste. via Giorgieri 10. 1­34127 Trieste. Italy ([email protected])2Department ofBotany. The Natural History Museum. Cromwell Road,London SW7 5BD, UK ([email protected])

Erasmus Darwin observed how lichens failed to grow near Copper Smelters at ParysMountain in Wales over 200 years ago. But it was not until sulphur dioxide, a productof fuel combustion, was identified as a major factor influencing lichen growth,distribution and health in the 1960's that the exponential growth world-wide in lichenbiomonitoring studies occurred with now well over 1500 papers published on thissubject, including several books (see [3]) and an on-going literature series published intheLichenologist. Today it is recognised that a wide rangeofother substances includingammonia, fluorine, eutrophication, alkaline dust, metals and radionuclides, chlorinatedhydrocarbons and'acid rain' may all be detected and monitored using lichens. Manycountries, particularly France, Germany, Italy, Switzerland, The Netherlands and US,are currently using lichens to monitor the effectsof gaseous and metal pollution usinglichens at both local and national levels, a trend set to continue. There are severalreasons why lichens have enjoyed such an extraordinary success in this field:• Lichens are ubiquitous and are currently increasing in many urban areas as a direct

consequence of decreasedS02leveis(see chapters 1-3, this volume).• They lack a protective outer cuticle and absorb both nutrients and pollutants over

muchoftheir outer surface from predominantly aerial sources.• Their symbiotic nature. The fungus is obligate; if either partner is damaged by

pollution this will result in a breakdown of the symbiosis, and ultimately to thedeath of the lichen.

• They are perennial organisms available for monitoring throughout the year.• Many lichen species accumulate high metal contents without exhibiting damage,

thereby permitting monitoring over wide areas.• Different methods exist providing opportunities for all ages and abilities.• Instruments are vulnerable to theft and vandalism.

In many biomonitoring studies lichens are considered to reflect "air pollution", "airquality" or "air purity" (e.g.as in the famous IndexofAtmospheric Purity (LA.P.)- seechapter 4, this volume). These terms, however, are not synonymous, and are inherently' fuzzy' concepts [I). Indeed the best approximation to a definition which one cantry toreconstruct sounds like:"air purity/quality is that thing which is assessed by indicators

7P.L. Nimis, C. Scheidegger and P.A. WolseJey (eds.), Monitoring with Lichens - Monitoring Lichens . 7-(O.© 2002 Kluwer Academic Publ ishers. Print ed in the Netherlan ds.

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of air purity/quality" [4]. In reality, biomonitoringtechniquesassess theeffects ofpollution and other environmentalchange on the bioticcomponentof ecosystems.However, as biologicaldata depend on several factorsotherthan pollution,biologistsoften find itdifficult to discriminate betweenthe effectsof pollutionand thoseofclimate,substrateecology, etc.In field monitoringit is very difficultto separatetheeffectsofmany intercorrelatedvariables. This isespeciallytrue forpollutionstudies, aspollutantconcentrationstend to becorrelatedwith the general levelof humanactivityand arethereforecorrelatedamong each other. This does not mean that theeffectsofindividualpollutantscannot be interpreted, but it does mean thatextremecare must betaken both indesigningappropriatestudies and in datainterpretation(seechapter9, thisvolume).

" Pollution" can be easily definedoperationallyin termsof concentrationslyingabove thresholdsfixed by law, which means that it must bemeasuredinstrumentally,and thatorganismsare not"cheaprecording gauges". The easyoperational-instrumentaldefinitionof "pollution" explains why this,contraryto the resultsof biomonitoringstudies,has entered into thelegislationofmost countries. Themonitoringofpollution,however,is difficult,becauseof:• the highnumberofpotentiallydangeroussubstances,• thedifficultyofestimatingtheir synergisticeffects,• the large spatial and temporalvariationofpollutionphenomena,• the high costsofrecording instruments, and hence,• the lowsamplingdensityofa purelyinstrumentalapproach.

Biomonitors, being widespread, permita higher sampling densitythan would bepracticalfor comparativelyexpensive physicochemicalmethods, which cancompensatefor the highvariabilityofbiologicaldata.It is importantto realise that lichens canneverdirectlyreplace technicalequipmentfor themeasurementof air pollution.However,they do enable rapid surveyof large areas and act as an alarm signalindicatingairpollution levels that can affect variousorganismsas well as identifyingareas whichshould bemonitoredby physicochemicalmeans.

The firstsectionof the present volume presents the Stateof the Art of the manydifferent methods by which lichens may be used to indicate airpollution asenvironmentalindicatorsin the broad sensesensu McGeoch [2].It summarisesthe hugebody of knowledgeon lichens as monitorsof the effectsof pollution,and at the sametime it points to futuredevelopments.This was not an easy task,consideringthe hugenumberof paperspublishedon this topic, and the wide arrayof differentmethods,conceptsandapproachesdevelopedover the last three decades.

The first threechaptersare devoted tobioindicationofpollutantsbasedon variousmethodsof assessing lichen diversity and theresponseof lichens topollutionfrom acommunityto species level.

The section opens with acontributionby Hawksworth (chapter3), tracing thedevelopmentof qualitative zonal bioindication scales from nineteenthcenturyobservationsto theirwidespreaduse throughoutGreat Britain from the 1970s. Thischapter is deliberatelycentred on the UKexperienceas methods and conceptsdevelopedthere were the starting point for averitableboom of lichen bioindicationworld-wide,whose developmentsare treated in the next two chapters.

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In chapter 4, Kricke and Loppi focus on the many 'IndexofAir Purity' ("LA .P.")formulas where a single numerical value is used to provide a quantitative indicationofair quality and their application in several countries.

In chapter 5, van Haluwyn and van Herk discuss qualitative ecological approachesin which species- and especially community-related informationis assessed to estimateair pollution, including the use of ecologicalindicator values of species, species groups,and communities.

The following chapters consider accumulation, down to the tissue level. Bargagliand Mikhailova (chapter 6) review the large bodyof literature dealing with lichens asaccumulatorsof trace metals, while in chapter 7 Seaward treats the useof lichens asaccumulatorsofradionuclides, from the early studies in the late 1950's when radioactivefallout from nuclear weapon tests became a cause of concern to national authorities, tothe post-Chernobyl period. As radionuclides are also trace metals, one might wonderwhy two chapters are needed here. The reason is that the histories of radioecology andof the analysisof non-radioctive metals in organisms have followed different, notalways parallel paths, partly becauseof the specialist natureof analytical facilities. Inour opinion- not limited to lichens - they would profit from converging into one and thesame research field.

Chapter 8, by Cunyet aI., takes us to an even lower level of biological organization:the impact of pollutants can be also measured by testing their effects on certainphysiological processes,now called "biomarkers".

Finally, in chapter 9 Ferretti and Erhardt consider issues like" design","sampling"and"quality", which areofuttermost relevance to the subjectofthis book. In the past,lichenologists have often underestimated the importance of an appropriate samplingdesign. Subjectivity is no longer acceptable, and standardization is fundamental for awide application of biomonitoring methods at the international level. While notintended to be an exhaustive review or a prescriptive manual to develop monitoringprogrammes, this chapter provides a useful framework for the complex processofsampling design and data analyses. Monitoring surveys need to be designed withspecific objectives in mind and should be testable according to rigorous statisticalprocedures. Lichenologists need only make a relatively small effort to present theirmethods in an internationally acceptable way, which means renouncing the plethora oflocal-personal methodological details, and concentrating on making the differentmethods operationally sound and applicable across very broad geographical scales.

Biomonitoring to assess the effects of pollution is certainly coming of age, andgovernments are starting to take notice [7]. An important step forward has been todevelop a unified method (see chapter 19, this volume) based on the German guidelines[6] and on the lichen mapping project organised by the Italian Environment Agency(ANPA). The influential Association of German Engineers intends to submit a slightlymodified version of these protocols to the European Committee for Standardisation inBrussels for adoption at a pan-European level [7].

The nature and impactofenvironmental pollutants is constantly changing. If lichendata are to be used to monitor or formulate regulatory decisions regarding air pollutionlevels, we need to know what levels are damaging to lichens and which gaseouspollutants (or other substances) are the primary or contributing cause of the observeddamage or distribution change [5]. Integrated monitoring programmes are clearly

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essential in conjunction with physicochemical measurements and are already beingcarried outin some countries,where lichenologists are carrying out studies investigatinglichen diversity as an indicator of atmospheric levelsof S02 and NOx in parallel withthe measurement of trace metals [7]. The field is ripe for fruitful collaboration betweena wide rangeof people from different backgrounds (ecologists, ecophysiologists,taxonomists, epidemiologists, analytical chemists, engineers, planners, etc.) tounderstand the specific effects of pollutants on lichens and on other components of theecosystem.

References

I. McCune, B. (2000) Lichencommunitiesas indicators of forest health,The Bryologist 103, 353-356.2. McGeoch, M.A . (1998) The selection, testing andapplicationof terrestrial insects asbioindicators,

Biological Review 73, 181-20I.3. Nash, T.H. III and Wirth, V . (eds.) (1988) Lichens, Bryophytes and Air Quality, B ibliotheca

Lichenologica30, Cramer, Berlin.4. Nimis, P.L., Lazzarin, G., Lazzarin, A ., and Skert, N . (2000) Biomonitoringof traceelementswith

lichens in Veneto (NE Italy),The Science ofthe Total Environment 255,97-111.5. Richardson, D .H.S. (1988) Understandingthe pollutionsensitivityof lichens,Botanical Journal of the

Linnean Society 96, 31-43.6. VOl (1995) Messung von Immissionswirkungen: Errnittlung undBeurteilungphytotoxischerW irkungen

von Immissionen mit Flechten -Flechtenkartierung zur Errnittlungdes Luftgutewertes(LGW), VDI­Richtlinie 3799.Blatt 1, Berlin.

7. Whitfield,1. (2001) Vital signs,Nature 411,989-990.

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BIOINDICATION: CALIBRATED SCALES AND THEIR UTILITY

D.L. HAWKSWORTH

Departamento de Biologia Vegetal IL Facultad de Farmacia,Universidad Complutense, Plaza de Ramon y Cajal, CiudadUniversitaria, £-28040 Madrid, Spain ([email protected])

1. Introduction

Awareness that lichen communities were affected by pollution arose at least by 1790,when Erasmus Darwin noted how they failed to grow near metal smelters on the islandofAnglesey in North Wales [7]. In about 1812 William Borrer observed that scarcelyany lichens could exist where the air was impure [50], presenting this in a mannersuggesting this was no new observation but something appreciated, but evidently rarelymentioned in print. Grindon [20] was more explicit in noting deteriorations in lichencommunities near Manchester due to tree-fellingand especially 'factorysmoke'.ItwasNylander [37], however, who first suggested that lichens could be used as a verysensitive hygrometer to actually measure the health of the air. The idea quickly spreadand there are numerous references to the sensitivity of lichens to air pollution or'smoke' in late nineteenth century European literature [22]. However, the firstpublication devoted entirely to the subject appears to be thatof Johnson [31] whoin1879 attributed lossesof lichens to 'smoke and fumes' from Tyneside and nearbycollieries.

This contribution traces the developmentof zonal systemsof bioindication fromsuch nineteenth century observations, discusses correlations with ambient pollutantlevels, and their wide-scale use by school children and students as well as researchersthroughout Great Britain from the 1970s. The utility of calibrated scales underameliorating conditions is assessed with particular reference to work in the UK wheredecreases in sulphur dioxide levels have been dramatic in many previously badlyaffected areas.

2.Uncalibratedzonal systems

Active fieldworkers soon recognized that all lichens did not respond in the same way ordisappear at the same time under pollution stress. Species on calcareous substrata [52]and nutrient-enriched bark [46, 36] in particular were documented as least affected. InSweden, Semander [46, 47] termed the area in cities with a depauperate lichenvegetation a 'struggle zone'('kampzon')and later [47] also recognized an inner' lichendesert' (Tavoken') with no lichens on trees, and an outer 'normal zone'('normalzon').

11P.L. Nimis, C. Scheideggerand P.A. Wolseley(eds.), Monitoring withLichens - Monitoring Lichens. 11-20.© 2002KluwerAcademic Publishers. Printed in the Netherlands.

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These zones were mapped in numerous cities in Europe between 1930 and 1970 [22,23]. In these, following the leadofVaarna[51], the struggle zone was generally beinginterpreted as the area with no foliose or fruticose lichens on trees, and the struggle zonewas subdivided into an inner struggle zone with stunted foliose lichens, and an outerstruggle zone in which stunted fruticose lichens started to appear; four zones wereconsequently often distinguished and mapped in this period.

Gradually the focus started to move to the behaviour of individual species ratherthan responsesof life-form categories, and lists ranked by their toleranceof urbanenviroments were drawn up [10]. Barkman [2] developed a 12-point IndexofPoleophoby to which species were assigned, and noted that the tolerancesof somespecies seemed to show some variation in different regions.

3. Calibratedzonalsystems

The early zonal systems were not correlated with particular levelsof pollutants.However, the tragedyof the 1952 London smog which led to numerous prematuredeaths had led the UK government to establish a national system of recording gaugesfor smoke and sulphur dioxide levels in rural as well as urban areas. By the late-1960ssome 1300 recording stations were operational. These provided physical measurementswith which species limits and zones might be correlated in this country and openedopportunities that were to make the UK a world leader in this aspect. The pioneer wasOliver 1. Gilbert who in 1965 [13] mapped the limitsof lichens on ash trees, asbestos­cement, and sandstone around Newcastle upon Tyne and also presented measurementsof pollutants from recording gauges. He was subsequently [15] able to recognize sixzones with different lichens and mosses on asbestos-cement roofs, acid stonework andtrees in the ranges of average annual sulphur dioxide levels of 30-170 figm-3 in lowlandBritain, primarily based on his studies around Newcastle-upon-Tyne; this system wasdescribed more fully in print two years later [16].

The examinationof historical lichen samples in both local and national museumsprovided important information regarding the historical impactofpollution. In WesternPark Museum in Sheffield, Hawksworth [21] found collectionsof pollution sensitivelichens made on moors near Sheffield between 1795 and 1807. Similarly Gilbert [14],found a small piece of bark in the Hancock Museum in Newcastle collected in 1812only 3 miles from the centre of Newcastle carrying eight sensitive lichen species. Onthis basis he established that over the past 150 years pollution had eliminated thisassemblage from over 25% of the county. Similar retrospective studies have beencarried out, and maps showing the changes in distributionofspecies in the British Isleswere soon being compiled [43].

Lichenology was a close-knit community in the UK in the late 1960s, but individuallichenologists tended to have in-depth knowledgeof relatively restricted areasof thecountry. Then in 1969, stimulated by the desire to learn moreof and map lichendistributions in England, Rose, Hawksworth and Coppins undertook a tour from theMidlands through the northofthe country and back, with Gilbertjoiningthem for partof the excursion [27]; 66 sites were studied in detail and many briefer observationsmade en route which included someof the most polluted and cleanest areasof the

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country. During the return car journey, and reflecting on the experiences from thisexcursion, an embryo 0-10 scale for lichen communities on trees was developed. Theissue was then whether these zones could be calibrated with actual measurementsofsulphur dioxide and smoke levels.

A study ofthe lichens and provisionalzones near recording gauges followed throughpersonal experience and writing to numerous colleagues to find what lichens occurrednear gauges where they lived. The results showed the best correlations to be with meanwinter levelsofsulphur dioxide and not at all with smoke, as Gilbert (13] had found tobe the case for lichens on asbestos-cement roofs. In 1970 the conclusions and scale werepublished inNature [24] with the different behaviourof trees with nutrient-enrichedbark allowed for and mapsofthe zones in England and Wales (Figure 1) as well as inmore detail for south-east England and in and around the cityof Leicester. Manycolleagues helped in checking detailsof zones in areas they knew, but the stricturesofthe journal meant muchofthe primary data was never published, although examplesofthe comparisons made between lichen zones and smoke and sulphur dioxide levels werepresented later [23].

Gilbert [17] designed a 0-6 zone simplified system for use by school children in anational survey, using species on stonework as well as trees and also one moss. User­friendly identification packs were developed and the results published inThe SundayTimes and a small book [33]. The results were similarto those published in 1970 [16]but with less detail and established for the first time that such scales could be developedand used by non-specialistsprovided appropriate explanatory material was available. Amore advanced student guide, with examplesof projects and notes on speciesidentification, was prepared by Hawksworth and Rose [26]; it was very popular andreprinted several times.

A different calibratedapproach was used by Seaward [44] using a single species,Lecanora muralis, and correlating the substrates on which it occurred with mean annualsulphur dioxide levels; that species only occurs on asbestos-cement tiles where S02levels are 200-240 ug rn", and does not colonize siliceous stonework until they arebelow 125 ug m".

The Hawksworth and Rose [24] system was quickly taken up not only in the UK butalso in adjacent partsofEurope where the same species and similar lichen communitiesoccurred, sometimes with fine-tuning to relate to local conditions (e.g. [8]). Its successand easeofuse led to the popular Al size wallchart illustrated by Claire Dalby with anaccompanying information booklet [30] published by what is now The Natural HistoryMuseum in London and British Petroleum Educational Services in 1981. It wassubsequently translated into eight different languages and published both as an A4laminated wallchart and as an annotated key toLichens and Air Pollution by theCompany ofBiologists and the Field Studies Studies Council [9]. Further, an updatedwide-ranging introductory overview with coloured illustrations was prepared byRichardson [40]. These products provided much needed identification aids for the non­specialist and were immensely important both for developing lichen monitoring andmaking it accessible to people of all ages and abilities. Subsequent community basedand formula-based systems developed, as well as those using transplanted lichens, arediscussed elsewherein this volume (see chapters 4 and 5), and so these are not treatedhere.

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6

:.....

Miles6

. 9+10

o!

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o KJlometres!

5

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Figure I. The Hawksworth and Rose Zones in England and Wales in 1970. with minor corrections (after[24]).

4. Speciestolerancesandpollutantlevels

The first author to link the toleranceofparticular lichens to actual sulphur dioxide levelsin the air was Skye in Sweden in 1958 [48], followed by Tallis [49] in northern Englandand Laundon [32] in London (Figure 2). These observations supplement the data oncorrelations embodied in calibrated zone scales but were again field-based. Laboratorystudies were however needed to establish beyond doubtcause-and-effect betweensulphur dioxide levels and lichen survival.

The firstexperimental studies were by Rao and LeBlanc [39] and Pearson and Skye[38] using sealedjars or flasks and gaseous sulphur dioxide. Hill [29], however,subjected species with different field tolerances to sulphite ions in solution and found

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good correlations with field sensitivities.These and other studies into the early 1970sare discussedby Baddeleyet all [1] and Richardson and Puckett [41]. As additional datafrom laboratory fumigations accumulated, some discrepancies appeared to emerge. Thetaskofcorrelating the two kindsofdata was tackled by Nash [34] who used Spearmancorrelations to compare experimental results with the Hawksworth and Rose [24] scale.He concluded that collectively the experimental data provides' strong evidence that thelatter'sscale does actually reflect sulfur dioxide sensitivity' even though the correlationwas not exact for all data sets.

o km 101···l e t.....1-L.J

Figure 2. Distribution ofXanthoriaparietinain London in relation to mean annual sulphur dioxide isolines (inpg m" ; after Laundon [32].Open circles =pre-. and solid circles =post-l950 records.

As laboratory studies are relatively short-term, the issue remains as to whether it ispeak values that are critical to lichen survival or seasonal (i.e. winter) or annual means.The effect of short-term peaks may depend on the physiological stateof the lichens;when not hydrated, high levels are likely to have little effect. The better correlationswith winter means found [12] may relate either to the lichens being more activephysiologically in that season, or to the somewhat elevated winter mean valuescompensating for peaks underdryconditions which have no effect.

Hawksworth and Rose [26] noted that the sensitivityof the same species might begreater in more oceanic than continental areas due to the greater lengthof time in theyear species were physiologically active in oceanic areas, but there is as yet little dataon this aspect. Heightened sensitivities from some species reported to occur aroundDublin in eastern Ireland [35] may be an artefact due to: (1) the area not havingequilibrated to falling sulphur dioxide levels (see below); and (2) the school childrenundertaking the study not being able to recognize and identify minute thalli.

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5.Applicationsunderamelioratingpollutantlevels

With hindsight, the calibrated scales developed in the UK at the endof the 1960s andearly 1970s benefited from relatively stable levelsof sulphur dioxide having beenmaintained for some years [19]. Early on caution was expressed as to how the scalesmight fare as bioindicatorsofactual pollutant levels in a situation with falling sulphurdioxide concentrations [26], something quite unknown at that time. By the late 1970s,however, levels of this pollutant had fallen to the extent that lichen reinvasion might beexpected to occur - and it did. Improvements, changes and encroachments towards citycentres were first noted around Leeds [28] andin London [42] as species started toextend their ranges into previously more polluted partsof cities. The situation inLondon became increasingly spectacular with species not seen since the 1730s-1780sreturning, and with a rapid re-invasion into the city taking place after the closureof apower station on the River Thames in 1983 [25] (Figure 3). In some cases there was adistinct movement of the zones, as around Liverpool and North Wales between 1973and 1986 [4], but the situation was not always so regular. While species did grow wheresulphur dioxide had fallen to levels which they would be predicted to tolerate from theirposition in calibrated zonal scales, the zones did not always reform in the same way.Forexample, where pollutant levels had fallen especially dramatically in London followingthe closure of a power station, groups of species characteristic of moderate sulphurdioxide pollution did not appear at all while those to be expected in cleaner air did; thisphenomenon where zones are missed out was termed' zone skipping' [25]. Gilbert [18]discussed the phenomenon further, and introduced the term'zonedawdlers'for lichensthat were slow to recolonize areas where they might now be expected (see chapter 4,this volume). Some lichens thriving at higher sulphur dioxide levels decrease as airquality improves; this has been documented forLecanora conizaeoides over 21 years insouth-east England [3].

By analyzing the enormous amountofdata accumulated through the British LichenSociety's Distribution Maps Scheme, Seaward [45] was able to show that many specieshad extended their ranges into formerly more polluted areas on a national scale since1992.Further, from data in the Scheme he was able to show howUsnea species that haddisappeared from an areaofca 68 000 km2 in the period 1800-1970had reestablished atmany sites within that area. A similar reexpansion has been mapped forParmeliacaperata [5]. However, in a particular lichen species, it cannot always be assumed thatthe full genotypic rangeof the lichen is actively recolonizing areas from which it hadbeen eliminated. In the case ofP. sulcata, ofthree genotypes recognized by the sizes ofITS amplification products in the UK, only one was found in areas where establishmentwas taking place [6]. The patchinessof recolonization, presumably largely due tochance introductionsofpropagules by birds, wind, or humans, means that attempts toapply calibrated lichen zones where air sulphur dioxide levels have fallen dramaticallywill tend to seriously over-estimate the current actual pollution levels.

Lichens which are particularly successful colonizers under ameliorating sulphurdioxide levels tend to be foliose and fruticose rather than crustose species, and are oneswhich often or only rarely reproduce asexually. In addition, those which prefer less acidor nutrient-enriched barks seem to be favoured [11].

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4 A Evernta prunastri

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Figure 3.Relationship between the sizes of the largest specimens offour lichen species fo und in north-westLondon in relation to the distance f rom the centre (Charing Cross). The arrows indicate disjun ctionsrepresenting periods ofexpansion in range. the most recent dated to J983on thallus size (after [25J).

6.Comparative utility

Experiencewith zonal scalesover the last 30 years hasshown themto be very effectivein reflecting sulphur dioxide pollution patterns, and wherecalibrated,mean annual ormean winter levels of that pollutant in areas where pollutantlevels have beenstable foratleast 5 years. They continueto haveutilityin such areas,and deserveto morewidelyapplied in less developed countries where physical measurementsof pollutants are

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absent or sparse. Under ameliorating conditions, however, recolonization is patchy, andwhere sulphur dioxide levels have fallen dramatically, zones are not reformed in thesame way;how long re-equilibration will take is unclear but some species are extremelyslow colonizers('dawdlers') while others spread more rapidly('skippers').

The calibration work undertaken does, however, hold for individual species in thezones. Under ameliorating conditions, therefore, the presenceof particular lichensprovides an immediate indication that mean sulphur dioxide levels are certainly lessthan the values at which the species is lost under increasing pollution levels. Forexample, the discoveryof sensitive parmelioid lichens in Cheshire immediatelysignalled improving conditions [6]. Indeed, in the recolonization process, those specieswhich do colonize are, on the whole, ones which would be expected to do so from thecalibrated zones to which they have been referred.

Just as this contribution was being finalized, a detailed study in Belgium comparingdifferent lichen bioindication methods in the same area with data collected from nearsulphur dioxide recording gauges was published [11]. The study concluded that not onlythe best correlations were those with mean winter levelsof that pollutant over a five­year period, but that studying selected lichens rather than all present was a satisfactoryapproach. I.e. the value of calibrated scales using a limited numberof species as hasbeen the practice in the UK was vindicated.

7. References

I. Baddeley, M .S., Ferry, B.W., and Finegan,E.1. (1973) Sulphur dioxide and respirationin lichens, inB .W. Ferry, M.S. Baddeley and D.L. Hawksworth(eds.), Air Pollution and Lichens. Athlone Press ofthe UniversityofLondon, London,pp. 299-313.

2. Barkman,J.J.(1958) Phytosociology and Ecology of Cryptogamic Epiphytes. Van Gorcum, Assen.3. Bates, J. W., Bell, J.N.B., and Massara, A .C. (2001) Loss of Lecanora conizaeoides and other

fluctuationsof epiphytes on oak in S. E. England over 21 years with declining S02 concentrations,Atmospheric Environment 35,2557-2568.

4. Cook, L.M., Rigby, K .D., and Seaward, M .R.D. (1990) Melanic moths and changes in epiphyticvegetation in north-west England and North Wales,Bioi. J. Linn. Soc. 39, 343-354.

5. Coppins, B.1., Hawksworth, D.L., and Rose, F. (2001) Lichens, in D.L. Hawksworth (ed.), TheChanging Wildlife ofGreat Britain and Ireland. Taylorand Francis, London, pp. 126-147.

6. Crespo,A., Bridge, P.D.,Hawksworth,D .L., Grube, M .,and Cubero, O.F. (1999) ComparisonofrRNAgenotype frequenciesofParmelia sulcata from long established and recolonizing sites following sulphurdioxideamelioration,Plant Systematics and Evolution 217,177-183.

7. Darwin, E.(1790) The Botanic Garden. a poem in two parts : Part I. Johnson, Litchfield.8. Deruelle, S. (1977) Influence de la pollution atmospheriquesur lavegetationlichenique des abres isoles

dans la region de Mantes (Yvelines),Revue Bryologique et Lichenologique 43, 137-158.9. Dobson, F.S. (1993) Lichens and Air Pollution, Richmond Publishing, Slough.10. Felfoldy, L. (1942) A variosi levego hatasa azepiphyton-zuzmovegetacioraDebrecenben,Acta

Geobotanica Hungarica 4, 332-349.II. Fox, B. W. (1999) The influenceof atmospheric pollution on the lichen floraof Cheshire, in E.F.

Greenwood (ed.),Ecology and Landscape Development: a history of the Mersey Basin. Liverpool,UniversityPress, Liverpool, pp. 185-193.

12. Geebelen, W. and Hoffmann, M. (2001) Evaluation of bioindicationmethods usingepiphytesbycorrelatingwithS02-pollutionparameters,Lichenologist 33, 249-260.

13. Gilbert, O.L. (1965) Lichens as indicatorsofair pollution in the Tyne Valley,in G.T. Goodman,RW .Edwards and J.M. Lambert (eds.),Ecology and the Industrial Society, Blackwell,Oxford, pp. 35-47.

14. Gilbert,O.L. (1968) Biological Indicators ofAir Pollution , PhD thesis, UniversityofNewcastle-upon­Tyne.

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15. Gilbert, O.L. (1968) Biological estimation of air pollution, in Commonwealth Mycological Institute(ed.),Plant Pathologist 's Pocketbook, Commonwealth Mycological Institute, Kew, pp.206-207.

16. Gilbert, O.L. (1970) A biologicalscale for the estimationofsulphur dioxide pollution,New Phytologist69,629-634.

17. Gilbert, O.L. (1974) An air pollution survey by school children,Environmental Pollution 6, 175-180.18. Gilbert, O.L. (1992) Lichen reinvasion with declining air pollution, in J.W. Bates, and A.M. Farmer

(eds.),Bryophytes and Lichens in a Changing Environment. Oxford Science Publications, Oxford, pp.159-177.

19. Gilbert, O.L. (2000)Lichens. Harper Collins, London.20. Grindon, L.H. (1859) The Manchester Flora. White, London.21. Hawksworth, D.L. (1967) Lichens collected by Jonathan Salt between 1795 and 1807 now in the

herbarium of Sheffield museum,Naturalist 1967, 47-50.22. Hawksworth, D.L. (1971) Lichens as litmus for air pollution: a historical review,International Journal

ofEnvironmental Studies 1,281-296.23. Hawksworth, D.L. (1973) Mapping studies, in B.W. Ferry, M.S.Baddeley and D.L. Hawksworth (eds.),

Air Pollution and Lichens, Athlone Press of the University of London,London,pp. 38-76.24. Hawksworth, D.L. and Rose, F. (1970) Qualitative scale for estimating sulphur dioxide air pollution in

England and Wales using epiphytic lichens,Nature 227, 145-148.25. Hawksworth, D.L. and McManus, P.M. (1989) Lichen recolonizationof London under conditions of

rapidly falling sulphur dioxide, and the concept of zone skipping,Botanical Journal of the LinneanSociety 109, 99-109.

26. Hawksworth,D.L. and Rose,F. (1974)Lichens as Pollution Monitors. Edward Arnold, London.27. Hawksworth, D.L., Rose, F., and Coppins, BJ. (1973) Changes in the lichen floraofEngland and Wales

attributable to pollutionof the air by sulphur dioxide, in B.W. Ferry, M.S. Baddeley and D.L.Hawksworth, D.L. (eds.),Air Pollution and Lichens. Athlone Pressof the Universityof London,London,pp.331-367.

28. Henderson-Sellers, A. and Seaward, M.R .D. (1979) Monitoring lichen reinvasionof amelioratingenvironments,Environmental Pollution 19,207-213.

29. Hill, DJ. (1971) Experimental study on the effect of sulphite on lichens with reference to atmosphericpollution,New Phytologist 70,831-836.

30. James, P.W.(1982)Lichens and Air Pollution. British Museum (Natural History), London.31. Johnson, W. (1879) Lichens and a polluted atmosphere,Hardwicke's Sci. Gossip 15, 217.32. Laundon, J.R.(1967) A study of the lichen floraofLondon,Lichenologist 3, 277-327.33. Mabey,R. (1974) The Pollution Handbook, Penguin Education, Harmondsworth.34. Nash, T.H. III (1988) Correlating fumigation studies with field effects,Bibliotheca Lichenologica 30,

201-216.35. Ni Lamhna, E.,Richardson, D.H.S., Dowding,P., and Ni Grainne, E. (1988) An Air Quality Survey of

the Greater Dublin Area carried out by Second Level Students, An Foras Forbartha, Dublin.36. Nienburg, W. (1919) Studien zur Biologie der Flechten. I. II . III.,Zeitschr . Bot. 11, 1-38.37. Nylander, W.(1866) Les lichens du Jardin de Luxembourg,Bull. Soc. Bot. France 13,364-372.38. Pearson, L. and Skye, E. (1965) Air pollution affects patterns of photosynthesis inParmelia sulcata , a

corticolous lichen,Science 148, 1600-1602.39. Rao, D.N.and LeBlanc, F. (1966) Effects of sulfur dioxide on the lichen algae, with special reference to

chlorophyll,The Bryologist 69, 69-75.40. Richardson, D.H.S. (1992)Pollution Monitoring with Lichens, Richmond Publishing, Slough.41. Richardson, D.H.S. and Puckett,K. (1973) Sulphur dioxide and photosynthesis in lichens, in B.W.

Ferry, M.S. Baddeley, and D.L. Hawksworth (eds.),Air Pollution and Lichens. Athlone Press of theUniversityofLondon, London, pp.283-298.

42. Rose,CL and Hawksworth,D .L. (1981) Lichen recolonization inLondon's cleaner air,Nature 289,289-292.

43. Rose, F., Hawksworth, D.L.,and Coppins,BJ. (1970) A Iichenological excursion through the northofEngland,Naturalist 1970, 49-55.

44. Seaward, M.R.D. (1976) Performance ofLecanora muralis in an urban environment, in D.H. Brown,D.L. Hawksworth and R.H. Bailey (eds.),Lichenology: Progress and Problems. Academic Press,London, pp.323-357.

45. Seaward, M .R.D. (1998) Time-space analyses of the British lichen flora, with particular reference to airquality surveys,Folia Cryptogamica Estonica 32, 85-96.

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46. Sernander,R. (1912) Studier ofvar lafvarnes biologiI. Nitrofila lafvar,Svensk Botanisk Tidskrift 6, 803­883.

47. Sernander,R. (1926)Stockholms Natur, Almqvist and Wiksell, Stockholm.48. Skye, E. (1958) Luftforenigfars inverkanpflbusk- och bladlavfloran Kring skifTeroljeverketi Nlirkes

Kvarntorp,Svensk Botanisk Tidskrift 52,133-190.49. Tallis, J.H.(1964) Lichens and atmospheric pollution,Adv. Sci. 21,250-252.50. Turner, D. and Borrer, W. (1839) Specimen of a Lichenographia Britannica, privately printed,

Yarmouth.51. Vaarna, V.V. (1934) Helsingin kaupungin puiden ja pensaidenjakalakasvisto, Ann. Bot. Soc zool.-bot.

Fenn. 'Vanamo ' 5 (6),1-32.52. Wheldon,J.A.and Wilson,A .(1907) The Flora ofWest Lancashire. privately printed, Eastbourne.

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BIOINDICATION: THE I.A.P.APPROACH

R. KRICKE 1and S. LOPPI2

lInstitut fir Botanik, Universitiit Essen , Universitdtsstrafle 5,D-45117,Essen, Germany (randolph [email protected])2Dipartimento di Scienze Ambientali, Universita di Siena , Via P. A.Mattioli 4,1-53100 Siena , Italy ([email protected])

1. Introduction

The symbiosis between algae and fungi enables lichens to colonisevarious apparentlyhostile places, like high mountains and deserts. However, the sensitivebalancebetweenthe symbiotic partners can be easily disturbed, as lichens aregenerallysensitivetoenvironmentalalterationsuch as changes in air humidity(forestry, urbanisation)and airpollution.

The correlationbetween lichen abundance and certain humanactivities wasrecognisedlong before their symbiotic nature. Erasmus Darwin, the grandfatherofCharles Darwin, illustrated in a poemof 1790 his observationson the effectsofa coppermine and smelting plant on the surroundingvegetation, includinglichens.

More scientific activity was spent on that topic in the secondhalfofthe19th century.Grindon [43],MacMillan [75] and Johnson [54] noted a vast decrease in lichen speciescomparedwith earlier reports due to"cuttingdown of old woods and the influxoffactorysmoke" [43].

In Europe,Nylander[88] and Arnold [2-7] were the first to publish scientific papersdescribingthe impactof large conurbations, such as Paris and Munich respectively,onthe lichen flora.Sernander[102] introduced the terms"lichen desert" and"strugglingzone" to illustrate theimpoverishmentoflichens in anurbanisedarea.

The conceptof lichen zones was used in many othersubsequentstudies, also incombinationwith phytosociological aspects (e.g. [10, 12, 14]). As a resultof thesestudies, it wasapparentthat sulphur dioxide (S02) was the main factor causing theobserveddecreasein lichen vegetation inindustrialisedand urban areas. Skye [103],Gilbert [37, 38] andHawksworthand Rose [46] were the first tocorrelatemean S02concentrationwith the lichen vegetation (see chapters 3 and 5, this volume).

Scales of S02 tolerance for lichen communities were establishedand widely usednot only in Britain, but also in other partsof Europe. Proposals for quantitativeestimatesof air pollutionwere alsopresentede.g. by Wilmanns andBibinger [121],again focusing on lichen communities.

21P.L. Nimis, C. Scheidegger and P.A. Wolseley (eds.), Monitoring with Lichens- MonitoringLichens. 21-37.© 2002Kluwer AcademicPublishers. Printed in the Netherlands.

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22

2.TheoriginalI.A.P.

At the end of the 1960s, amethodwas developedfor quantifyingenvironmentalconditionsusing lichens asbioindicators[23]. This Index of AtmosphericPurity (lAP)combinesthenumberofspeciesat the site withtheirsensitivitytowardsenvironmentalstressors,primarilyair pollution.The lAP is calculatedfor every site accordingto thefollowingequation(1):

lAP =...!!.-x(tQx !)IODIn

n =numberofspeciesQ =degreeoftoxiphobyf = frequency-abundanceofeach species

(1)

The degreeof toxiphobyQ is an empirical estimatederivedfrom differentmappingstudies in Europe [9, 14, 45] and must not be mistaken with the number ofaccompanyingspecies, also knownas Q,which will bediscussedlater.

A revisedversionof the lAP (equation2) was laterproposedby thesameauthors[65].

lAP =tCQx! )1 10

n = numberofspeciesQ = factorofaccompanyingspecies (see text)f = cover and frequency ofeach species

(2)

Here, the areaof cover and a modifiedfactorof sensitivitywere includedin themodel. Thissecondsensitivity index Q is definedby thenumberof speciesoccurringtogetherwith theindicatorspecies. Thus, stress-tolerantspecies have low Q values,whereas more sensitive species have higher Q values. Q can be computedby thefollowingequation(3):

m n

IISijQ = j=1 ;=1

mn = numberofspeciesm =numberofstations where the speciesofinterest is presentSij= equals 1 if speciesi is present at stationj (and species iis not the speciesofinterest)

(3)

Estimatesofdegreeofcoverare scaled into 5classes[65]:5) an abundantspecieswith a highdegreeofcoveron most trees,4) a frequentspecieswith a high degreeofcoveron some trees,3) an infrequentspecieswith moderatedegreeofcoveron some trees,2) a rarespeciesor onewitha low degreeofcover,and1) a very rarespecieswith a very lowdegreeofcover.

All lichensoccurringup to aheightof2 m, regardlessofexposure, were analysed.Using thismodel, every study site,consistingof 10-12 trees, isallocatedan lAP value.

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IndexofAlmospheri4:l'uril~' In Arv ida. P.Q.

I ll-5II (>-10III 11-20IV ~1 40

v ~ l -SO

VI ;' 110

»

'0'

23

Figure I . lAP-Zones in Arvida. Quebec (after [68]) .

This value permits local evaluation of air quality when correlated with apre-definedscaledevelopedfor the study area. The model, which included alsoepiphyticbryophy­tes, has proven to be simple to use, time- and cost-saving, and feasible, especially formapping the impactof main air pollutioncompoundssuch as sulphur dioxide (seeFigure 1) and fluorides [67-69]. Concentrationsof pollutants,mainly sulphur dioxideand fluorides, were also correlated to lichen damage in fumigationexperiments[66].

3.Similarand modifiedlAP-formulas

At about the same time, in Estonia the Indexof Poleotolerance(IP) was developed[107], a modelcomparableto the lAP, but using a"value ofpoleotolerance"insteadofthe"companion factor"Q. This model is based on empirical estimatesofenvironmentalsensitivity, as did the original lAP, derived from analysisof lichen synusiae. Therelative abundanceofeach lichen speciesof the total lichenvegetationon each tree isalso estimated. The IP can be computed by the following equation (4).

IP = i:.(aiXCi)I C.

n = numberof speciesai =species-specificfactorexpressingtolerancetopollutants(scalefrom I to 10)ci =degreeof cover foreach species(scale from I to 10)Ci =degreeof coverfor all speciesat the station

(4)

lAP and IP values proved to be related; both models can be used for air qualityassessment[76].

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24

In order to eliminate the influenceof different substrates on the lAP, Moore [78]introduced the ILA (Indexof Lichen Abundance, see equation 5) which uses thesensitivity factorQ following [65], and, additionally, a new"companion factor" Qs,which describes the average numberofcompanion species found on the same substratetype.

Qa xcILA = Qs xlO

n

Q.= factorofaccompanyingspecies regardlessofsubstrateQ.= factorofaccompanyingspecies occurring on same typeofsubstraten = numberofspeciesC = cover degree

(5)

Although statistical analysis showed that this modification was well suited to avoidthe interferenceof substrate-type, this method was not applied further. Also the IQ("Qualitatsindex")derived from the original lAP by Luhmannet al. [74] was used onlylocally and did not attract further interest.

Herben andLiska [49] examined the useof both the original lAP and a modifiedformula. The main focus was on the factorQ of accompanying species. Althoughcalculated and thus objective,Q is influenced by the data structure and by the methodoflichen analysis, and can lessen the validityof the lAP. Using Monte-Carlo simulation,where idealised lichen communities were correlated with several pollution loads, theimpactofseveral factors onQ and the lAP were analysed. To focus on the weightofQin the models, the original lAP [65] was used without the degreeofcover, so that lAP='LQ. With known stress simulations, correlations with the different lAP values wereundertaken, which showed thatQ and thus the validityof the indication system wasinfluenced by the following parameters: relative shareof sensitive species, shareofpoleophilic species, frequency distribution, sizeof the species pool, numberofexamined trees and their distribution within the study area. The authorsconcludedthatQ is subject to non-reproducible examiner subjectivity (e.g. selection and distributionofsampling sites), as is the case with most estimations.

4. Further developmentof thelAP

Kirschbaum [57] was the first to adapt the Canadian lAP and develop a quantitativemethod to describe the lichen vegetation in Germany. With the helpofa screening gridplaced on the trunkofsampling trees between 0.3 and 1.3 m above ground,standardisedestimatesof lichen coverage were made. At the same time, Kunze [63] introduced theuse of frequency values according to Raunkiaer [93], Ttixen [109] and De Vries [24],using a grid divided into 10 squares, also known as the"Flechtenleiter",Suggestionsconcerningthe useofcommon tree species were also made. Kunze [64] also showed astatistical dependence between lichen data and the effectsofcertain stressors, includingair pollution.

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25

A country-wide project aimingto map air quality at a 5x5Ian gridscale was carried out in TheNetherlands in the 1970s [25]. Atthe end of this study, a monitoringnetwork of ISO stations wasestablished, arranged along 8transects throughout the country,which started in 1977,became fullyoperational in1981, and continueduntil 1990 [110-111). Eachmonitoring station consistedofabout 10 free-standing waysidetrees, where cover estimates of alllichen species were made yearly.

5. The Swissproject

54 lichen species

lAP,= 'f,QxCI

lAP2= 'f,QxCxFI

lAP) = 'f,Q XCxF, V xS

lAPs ='f,QxCI

lAP6= 'f,CxFI VxS

lAP7= 'f,QXCI VxS

40 selected lichenspecies

lAPII= I.QxCI

lAPi2= 'f,Qx Cx F,lAP,) =IQxCxF

, VxS

n CxFlAPI6=I-­

I V xS

"QxClAPI7= L..,,--V xS

Figure 2. lAP-Formulas tested in the first phase of theSwiss project. (Q =factor ofaccompanying species ; C=%cover. scaled as follows: O.+.1.2.3.4.5; F =frequency­value (1-10); V = vitality (3 levels: very good. moderate.poorly developed) ; S = damage (3 levels: no. moderate.strong damage) (from [42]).

lAP20='f,QI

lAPI9= 'f,QxFI

lAPs = IF1

lAPIO= 'f,Q,

lAP9= 'f,QxFI

In Switzerland, a project wasstarted in the 1980s to develop anobjective and reproducibleindication model, sensitive towardsthe combined influenceof severalatmospheric pollutants [I, 50-51].Quantitative methods were chosen,like the lAP (sensu [65]) or theapproach of Kunze [63], sincethese allowed statistical testing ofboth lichen and environmental data.In the Swiss study, the lichens on500 trees in the vicinity of 13stations measuring air pollutants inthe Biel Region were analysed [50). Based on the original lAP formula, 9 furthercalculation models were developed, taking into account the following parameters: coverdegree, numberofcompanion species, frequency, vitality and damage. The lichen datawere processed in models using both the total numberofspecies, and a reduced datasetof 40 selected species. Thus, a totalof 20 different formulas were tested (Figure 2).Species reduction was introduced to eliminate species with no indicator properties, oreven negatively correlated with the lAP. The resultsof the first phase of the projectshowed that the highest correlation(R~0.98, p<0.05) between pollution data (meanannual valuesof 8 air contaminants) and the lichen vegetation was found when usingfrequency data within a sampling grid and the reduced species dataset(lAPIS method).Also IAP 20 showed a high correlation, but it was based on factor Q (numberofaccompanying species), with its associated drawbacks [see 49].

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26

2 3 5 6

802 S02 802 8°2 S02 S02

dust dust dust dust dust dustNO) N03 N03 NO) NO)Cd Cd Cd CdCu Cu CuPb PbCI

0,70

10,40

0,20

0,00 ------r------r------r------r-----,------,onumberof

eliminatedvariables 802

dustremainingvariables N03

CdenPbCIZn

0,75

~ 0,80

0,90

0,95

1,00

0,85

Figure 3.Behaviour ofR' due to gradualelimination of pollution variables (after [5IJ).

Even with only 4 stressors, the IAPl8model still showed high predictive values (R~0.93,p< 0.05, see Figure 3) thus proving the robustness of this approach [50].

In the second phase of the study, the validityof the IAPl8 was tested in otherregionsofSwitzerland, featuring a different species spectrum and reducing the numberofspeciesby merging difficult taxa and eliminating species of little or no bioindicativevalue [50]. Once again, IAP I8 showed high correlations with measured pollutionconcentrations. Other modelsthat werenot tested in the first phase of the projectwerealso highly significant. However, the factors used did not allow for sufficientreproducibilityand/or objectivity.

As a result of the Swiss project, the IAPI8 systemwas implemented as a nationalguideline for monitoring atmospheric pollution using lichens, replacing other indicationmodels, such as the"Lichen Index" [95-96],based on percentage cover.

6.Developmentof thelAP in Germany

Independently of the Swiss results, Rabe [91] developed a system called LuG!(Luftgute-Index) for the RWTOV (Rheinisch Westfalischer TechnischerUberwachungsverein),which was applied to describe the air pollution statusin the RuhrArea (Germany)(see Figure 4). This method employed an empirical species-specific

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27

sensitivityvalue (E), degreeofvitality(V) and degreeofcoverage(C) obtainedusing agrid (see equation 6).

(6)

n

ICxVxELuG! =-'1'-- _

Figure 4. Mapping ofair poilu/ion using the Luiil-method inDortmund (after [/20J).

millvery high stress (luGI 0,7)~ very high stress (l uGI 0.8)~ high stress (LuGI 0 ,9)_ high stress (luGl 1 ,0)

Eill rather high stress (l uG I 1,1·1,2) O,====i:::==:::E=:==3:==J1? km

nICI

Since lichen vegetation was extremely poor at that time, the degreeofcoverageandvitalityofLecanora conizaeoides was also used in bioindication.Although the methodwas used in anumberofstudies throughoutGermany,it could not be used as a standard,since theapplicationofempirical sensitivity and vitality values proved toocomplicated.

Moreover, the Swissresults showed theadvantageof using frequency valuesrather than estimatingper­centage cover.

For these reasons, theGerman VDI (Verein Deut­scher lngenieure)decided toadopt a reproducible andobjectivebioindicationme­thod to monitoratmosphericpollution using lichens.

Hence, in computingthe"Luftgutewert" (LGW) , theGerman guideline (VDIguideline3799, 1) [J16] isbased on the lAP18 formula.

The standardsofthis guideline are:• ascreeninggrid (50x20 em, divided into 10 equal squaresof lOxl0 em) is placed

1.5 m above ground on the most densely vegetated sideofthe tree trunk;• accordingto its size, the study area is divided intohomogeneousland units with a

sufficientnumberofsampling trees (e.g.6 trees for a grid lengthof I km);• based on theexperienceofthe Swiss study, the listofspecies to be used is limited

to 56 taxa, in which difficult species were merged;• Lecanoraconizaeoides is only used when it is the only species within thescreening

grid, since it grows well under conditionsof acid airpollution; an index is thencalculatedsolely on the degreeofcoverofL. conizaeoides;

• tree species are chosenaccordingto barkcharacteristics(e.g. roughness, acidity),and girth (Table 1);

• the LGW for each land unit iscalculatedas an averageof the LGWs ofthe singletrees; the standard deviation and the upper and lowerconfidencelimits are used totest the accuracyofthe estimationofthe real conditions;

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28

TABLE J. Tree classificationfor use within a project (girths in em in brackets) in the VDI system (J J6}) .

Trees with± sub-neutralbark

Acer platanoides (90 to 280)Fraxinus excels ior (90 to280)Juglan s regia (60 to280)Malus sp.(60 to 160)Populus sp.(80 to280)Ulmus carpinifolia (90 to280)

Treeswith± moderatelyacidbark

Acer pseudoplatanus (100 to280)Pyrus communis (80 to280)Robinia pseudacacia (90 to280)Tilia cordata (100 to280)Tiliaplatyphyllo s (100 to280)

Treeswith± acid bark

Alnus glutinosa (60 to 280)Betula pendu la (80 to 280)Prunus avium (60 to280)Prunus domestica (60 to 160)Quercus roburlpetraea (90 to280)

• the classificationof LGW values is based on the standard deviation across thewhole of the study area (which is influenced by the size and heterogeneityof thesampling area and the numberoftrees in each area);

• the LGW classes are interpreted on the basisof a predefinedscale of air quality(different for alpineregions).This VDI guideline was usedin several small and large-scalesurveys (e.g.[30,58­

61,87,117, 122], see also Figure 5), its strength being its high level ofstandardisation,enabling large-scale comparisons among studies. The German guideline wasimplemented also in France, in the regionofLyon, as a regional project formonitoringair pollution with the helpofschool children.

LGW -\'alue s air quality

0 ·7,2 • " cry poor

:> 7.2 -14,4 • very Iloor 10pour

:> 14,4·21,6 • poo,

:> 21,6 ·26,6 • poor 10 moderate

>28,8 ·36,0 • moderate

> 36,0 ·43,2 • J:ood

> 43,2·50,4 • J:ood 10 \'(' ry good

:> 50,4 " crygood

Figure 5. Mapping of air pollu tion in Bavaria using the VDI-guideline (aft er (J J7]).

7.DevelopmentofthelAP in Italy

Outside Switzerland and Germany, in Italy thelAPI S approach has been widely applied,starting from a pioneering study carried out in 1989 ([85], for a recent review see

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29

10050 60 70 80 90100-lichen biodiversity

60 L..- _

40

160

.£ 140~E120"­II)

~ 100oO!J§ 80...J

Figure 6.Relation between lichen depauperation (lOO-sum offrequencies) and lung cancer mortality in the Veneto region

(after (21J).

Piervittori [89]). The main modification adopted in Italy was a grid of fixed size (50x30em divided into10 units of IOxl5 ern), insteadof a grid adapted to fithalf thecircumferenceof each tree as originally used in Switzerland [84, 85]. This allowedtransformationof the lAP into alichen biodiversity index (LBI) [8].This method was tested against realpollution data at La Spezia(NWItaly), showing a good correlationwith S02 [84]. Later, themethodology was adopted in thewhole Veneto Region, in north­eastern Italy [86], where acorrelation emerged between lichenbiodiversity and lung cancer, as aresult of air pollution [21] (seeFigure 6). In Italy this methodologywas also implemented as a nationalguideline for monitoring the effectsof atmospheric pollution byphytotoxic gases (especially S02and NOx) using epiphytic lichens[83]. Suggestions on samplingstrategy, data processing and presentationof the results were also given, which aresimilar to thoseofthe VDI guideline.

In contrast to the German guideline, it is stressed that bioindication techniquesmeasure neither air pollution nor air quality, but rather they estimate the degreeofalteration from natural conditions by pollution-reactive componentsof ecosystems. Inother words, bioindication techniques evaluate the effects of pollution on the bioticcomponents of ecosystems. Biological data depend on ecological variables other thanair pollution, such as climate, substrate, light, dust, etc. and it is difficult to discriminatethe effectofair pollution from that of other environmental parameters on the frequencyand distributionof lichen species.For this reason, an operational definitionof"normalconditions", i.e.of baseline values, is fundamental in order to obtain proper scalesofenvironmental naturality/alterationfor the interpretationof lichen diversity data inbiomonitoring studies [72]. According to Piervittori [89] the numberof papers onbioindication with lichens published in Italy in the last decade exceeds 200.

8. Developmentofthe lAP in other countries

Several modifications to the original lAP formula have been suggested locally in manystudies world-wide,e.g. in Spain [22, 39], France [28], Slovenia [11-12] and Japan [44,80] but could not yet reach the status of a nation-wide guideline. The paperofAsta etal. (chapter 19, this volume) presents the first attempt to develop a unified guidelineincorporating several different methodological approaches based on IAPI8, with a viewfor wider application at a European level.

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30

9.Transplants

36

Besides in situ evaluation using native lichens, methods have also been developed usinglichen transplants. Lichens transplanted from unpolluted sites are exposed in an area tomonitor environmentalconditions, by assessing the degree of visible or measurabledamage to lichen thalli after a defined exposure period. A second VDI guideline (VDI3799, 2) [115] was proposed, which standardises methodsof collecting andtransplantingHypogymnia. Transplants are mainly used where where pollution has

impoverished the natural lichen vegetation to such an extentthat floristic evaluation is no longer worthwhile or possible.The basis for this bioindication method is to expose thalliofsensitive lichen species. Thalli are first removed from areaswith little environmental stress and transplanted into the studyarea (see Figure 7). A first descriptionof this technique wasgiven by Brodo [16], and modifications proposed by McCuneet al. [77].It should be noted that lichens must be allowed toadapt to the climatic conditions at the exposure site, so that theeffects of air pollution are not strongly blurred by thoseofclimate. Therefore, acclimatisationof several weeks to months

Figure 7.Boardfor lichenexposure (after [99]).

..

\

2.8

3.

-'-'~2.4

~o..J2.0 l-

1"tl

~ 1.2:;

08

1

°f~~~~~=;:~==;t;-~ ~ ~ k ~ ~ ~ ~[SO.] ppb

Figure 8.Relationship between the total chlorophyll content ofatransplanted lichen and the ambient S01concentration (after [70}) .

must be permitted [32].The transplant technique

has been used in a varietyofsurveys (e.g. [34, 52, 56, 70,100, 104-105, 118]). Speciesoften used in transplantexperiments includeHypo ­gymnia physodes in Europe[31, 34, 36, 48, 52, 70, 104,106, 119] and the generaRamalina and Usnea in SouthAmerica [19, 40-41, 71]. Asecond VDI guideline (VDI3799, 2) [115] was proposed,which standardises the use ofthalli of Hypogymnia phy­sodes for air quality moni­toringin polluted areas wherethe original lichen vegetation is strongly reduced.

As pollution is greatly declining in many areasof Europe and North America,transplant experiments are becoming less important. Even in highly industrialised orurban areas, lichen recolonization is well under way, which permits the floristicexaminationoflichencommunities [13, 35, 47, 55, 62, 73, 79, 90, 92, 101].

In some areas with high or increasing levelsofair pollution, lichen transplants arestill being used [17-19, 42]. In such cases, the vitalityofthe exposed individuals is the

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31

main aspect of evaluation.However, it is not easy to discriminate between responses tohuman-induced and natural stress. For example,parasitic fungi or grazing by arthropodscan cause visible damage, which could be misinterpreted as an effectofair pollution.

Reactionsof lichen specimens to environmental conditions are usually documentedphotographically (see chapter 31, this volume). Depending on the extent anddevelopmentof the visible reactionsof the specimen to the new exposure conditions(e.g.necrosis), conclusions can be drawn on the air pollution status. In addition, featuressuch as chlorophyll content (see Figure 8) or the degreeof plasmolysis can also beinvestigated.

10.Physiologicparameters

Investigations on the enzymatic activity [97], gas-exchange [20, 108] or chlorophyllflourescence [15, 26, 82], which indicate the vitalityof lichens, have also been carriedout. While these methods are already widely used in higher plants, there is a greatpotential for further research and development in the caseof lichens and othercryptogams.

The chlorophyll flourescence method provides a useful and objective criterium toassess vitality (see chapter 29, this volume).It has undergone much development overthe last few years, offering a handy and relatively inexpensive tool. With the aidofthechlorophyll flourescence values FvlFm, the qualityof the symbiotic status betweenalgal and fungal partner in the lichen can be examined. Vascular plants generally haveFvlFm valuesofapproximately 0.83, while lichens have values lying between 0.63-0.76[27]. Algal lichen partners react in an"all or nothing" way, with values either lyingbetween 0.63and 0.76or aroundO. In casesofFv/Fm= 0, the measured thallus area hasdied off. Aided by chlorophyll flourescence techniques, Jensen [53] showed that lichenswith necrotic parts carried out normal ratesof photosynthesis in those areas of thethallus that were not necrotic. The simple Fv/Fm value can support floristicbioindication methods and can provide objective assessmentsofthe reduction in vitalityobserved in exposed lichens during transplant experiments.

11.Discussion

As environmental conditions change and lichen vegetation responds to such changes,methods for evaluating environmental alteration using lichens need to be continuouslyadapted and improved to meet the demands of an ever-changing situation.However,"iflichen data are to be used to monitor or formulate regulatory decision regarding airpollution levels, we need to know what levels are damaging to lichens and whichgaseous pollutants are the primary or contributing causeof the observed damage ordistributionchange" [94].On the one hand, it is well-documented that epiphytic lichensrespond to atmospheric pollution, but it cannot be maintained that these organisms canbe used as indicators for general "air quality",i.e,a combined effectof a mixtureofpollutants [83, 113). A negative relationshipofmost species was observed only for802,

or the combination of802 and NOx which generally are strongly correlated, and thus

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32

biodiversity counts can only be used as a monitor for S02 [113]. In some situations,however,S02itself may be indicativeofthe general level of atmospheric pollution [21]." Epiphytic lichens may be useful monitors for pollutants other thanS02, but in that casespecies have to be separated with respect to their response to the pollutant in question.Methods for monitoring, for example NH3, could be devised by using such speciesweighting [114], but the greater sensitivityof most species toS02 will remain acomplicating factor" [113].

" In field monitoring it isvery difficult to separate the effectsof manyintercorrelated variables. This is especially true for pollution studies, as pollutantconcentrations tend to be correlated with the general levelof human activity and aretherefore correlated among each other...This makes it a complicated task to relateobserved biological changes to changes in the concentration of any single pollutant"[112]. However, the presence in a site of a given lichen vegetation allows, to a certainextent, the detection of deviations from "normal" or control sites (see chapter 20, thisvolume).

In the past, biomonitoring techniques focussed on major pollutants primarily relatedto industrial sources such as S02. In the future, such techniques will be useful indeveloping countries still experiencing high S02 concentrations, in order to complementand develop chemical measurements. However, for most countries of the northernhemisphere, it seems important to consider not only intense pollution phenomena, butalso rather diffuse and weak sources and different contaminants such as polycyclicaromatic hydrocarbons and also complex mixtures of several pollutants [33]. Lichenbioindication cannot evaluate environmental or ecological conditions on its own, andthe old-fashioned idea that lichens can replace physicochemical instruments should bedefinitely abandoned.

However, lichens can complement measurements, especially where there are nogauges, and can point to environmental problems in needof further instrumentalinvestigation. Furthermore, lichens alone cannot represent the full spectrum ofbiological effects of pollutants measured with conventional instruments.However, afterthe UNEP Earth Summitof1992,biodiversity has become a key issue in environmentalstudies and lichens should be regarded asof value on their own, threatened byatmospheric pollution ([29] and see section 2, this volume).

In conclusion, in a process that has evolved over the last 30 years, the useoffrequency as the main parameter for lichen bioindication models has proven to be themost useful, mainly as a consequence of the high "selective pressure"of the Swisstesting scheme in the 1980s. The valueof this method is reflected by its widestandardised application in other countries, such as Germany and Italy. However, due toquickly changing conditions, the present knowledge must continuously be criticallyreviewed [98], improved, and complemented by physiological techniques such aschlorophyll fluorescence, the use of biomarkers, etc.

12.Acknowledgements

RK i s grateful to G.B. Feige (Essen) for initiatinghim to the studyof lichens,and U. Kirschbaum (GieBen)whose opinion about bioindication was of greatvalue. SL would like to thank P.L. Nimis (Trieste) and K.Ammann (Bern) for introducing him to the lAP approach.

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13.References

1. Ammann, K ., Herzig, R., Liebendorfer, L., and Urech, M . (1987) Multivariatecorrelationofdepositiondata of 8 different air pollutants to lichen data in a small town inSwitzerland, in Advances inAerobiology, BirkhauserVerlag, Basel, 401-406.

2. Arnold, F. (1891) Zur Lichenenflora in Mtmchen,Ber. d. Bayer. Botan. Ges. 1, 1-147.3. Arnold, F. (1892) Zur Lichenenflora in Munchen,Ber. d. Bayer. Botan. Ges. 2, 1-76.4. Arnold, F.(1897) Zur Lichenenflora in Munchen,Ber. d. Bayer. Botan. Ges. 5, 1-45.5. Arnold, F. (1899) Zur Lichenenflora in Munchen,Ber. d. Bayer. Botan. Ges. 6, 1-82.6. Arnold, F. (1900) Zur Lichenenflora in Munchen,Ber. d. Bayer. Botan. Ges. 7.7. Arnold, F. (1900) Zur Lichenenflora in Milnchen,Ber. d. Bayer. Botan. Ges. 8.8. Badin, G. and Nimis, P.L. (1996) Biodiversityof epiphytic lichens and air quality in theprovinceof

Gorizia (NE Italy),Studia Geobotanica IS, 73-89.9. Barkman,J.J.(1958) Phytosociologyand ecologyofcryptogamic epiphytes, Van Gorcum, Assen.10. Barkman,J.1. (1963) Die epifyten-flora en -vegetaties von Midden Limburg (Belgie), Verhandl. der

KoninklijkeNeederl.Akad. van Wetenschappen, afd. Natuurk.• TweedeReeks 54,1-46.II. Batie, F. and Mayrhofer, H. (1995) Bioindicationofair pollution by epiphytic lichens in forest decline

studies in Slovenia,ProceedingsofBIOFOSP, Ljubljana 1995, 139-145.12. Batie, F. and Mayrhofer, H. (1996) Bioindicationof air pollution by epiphyticlichensin forest decline

studies in Slovenia,Phyton36, 85-90.13. Bellio, M.G. and Gasparo, D. (1995) Lichens as bioindicatorsof air quality: spatial and temporal

variation.La Spezia case study,Agricoltura Mediterranea, special volume, 224-232.14. Beschel, R.(1957-1958)Flechtenvereineder Stadte,Stadtflechtenund ihr Wachstum, Ber. Naturwiss»

Med. Ver. in Innsbruck 52, 1-158.IS . Branquinho, C.,Brown, D.H.,Maguas, C.,and Catarino, F. (1997) Lead (Pb) uptake and its effects on

membrane integrity and chlorophyll fluorescencein different lichen species,Environmental andExperimentalBotany 37,95-105.

16. Brodo, I. (1961) Transplantexperiments with corticolous lichens using a newtechnique,Ecology 42,838-841.

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87. Nobel, W .,Worm, R., Schritz,c.,Beron, J.,and Nitschke, C. (1999) Flechtenkartierungnach VDI 3799:MethodischeModifikationenund GrundlageflireineokologischeStadtplanung,Verh. Ges. Okologie 29:571-577.

88. Nylander,W .(1866) Les lichens du Jardin duLuxembourg,Bull. Soc. Bot. France 13, 364-372.89. Piervittori,R. (1999) Licheni comebioindicatoridella qualitadell'aria: statodell'artein Italia, in C.

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BIOINDICATION: THE COMMUNITY APPROACH

c.VAN HALUWYN 1 and C. M. VAN HERK 2

'Laboratoire de Botanique, Faculte des Sciences Pharmaceutiques etBiologiques, B.P. 83, F-59006 Lille cedex, France ([email protected]­lille2fr)2Lichenologisch Onderzoekbureau Nederland (LON,) Goudvink 47,Soest,3766 WK, Netherlands ([email protected])

1. Introduction

Two main methods for estimating air pollution with lichens were developed at the endofthe 1960s: the IndexofAtmospheric Purity (lAP) by De Sloover and LeBlanc [24] andLeBlanc and De Sloover [73], and the qualitative scale for estimating S02 pollution byHawksworth and Rose [56]. Since then, researchers have more or less modified these twobasic methods, taking into account regional knowledge on lichens and on patternsof airpollution. All methods currently in use can still be classified as"quantitative"and"qualitative",the latter often also indicated as the"communityapproach".

In the quantitative approach the complete lichen compositionofsample plots is reducedto a single value expressing air quality through a formula (e.g. lAP values). In the originaldefmition of the lAP, numerical values were assigned to species, expressing theirsensitivity to air pollution; the mean numberofcompanion species was often considered asa measureof sensitivity. In other methods, such as the German and the Italian guidelines[95, 157], species sensitivities are no longer used, and only the sumof frequenciesof(selected) species in a sampling gridof 10 units are taken into account (see chapter 4, thisvolume).

In the qualitative approach, on the contrary, auto- and/or synecological information onspecies, species groups, or communities is used to estimate air quality. The basicelementisthe species, each one having a rangeof tolerance to pollution which,if known, can beexpressed by ecological indicator values. These can consistof verbal expressions (e.g.sensitive, tolerant, etc.)or can be expressed as numbers on ordinal scales. Such informationcan be derived from field observations, from circumstantial evidence (e.g. bycomparingaspecies distribution with the distance from a pollution source), from correlations withmeasured pollution data, and from fumigation experiments in which species are exposed toknown levelsofpollution. Information on the conditionsofspecies (vitality, damage) maybe also used.

Speciescan begroupedaccordingto ecologicalsimilarities. Indicatorvaluescan beassignedto "groups of species" or to " communities". The term "community" is oftenapplied in a rather confusing way. Some authors used it also for species groups; others forcommunities in the senseof phytosociological syntaxa (e.g. associations).According to

39P.L. Nimis , C. Scheidegger and P.A. Wolseley (eds.), Monitoring with Lichens - Monitoring Lichens. 39-64.«:> 2002 Kluwer Academic Publish ers. Printed in the Netherlands .

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Will-Wolf [160] "community data consistofinformation on all partofthe lichen species inan area or subarea. Such information may include presence, abundance or density, cover,vigour, luxuriance or biomassof the species. Community data can be arranged at severallevelsof organization by habitat types or subsections, substrate, microhabitat conditions,etc". Groupsof species orcommunitiescan be used as indicators in the same way assingle species. Again, indicator values can be related tomeasurementsof singlepollutants,or can express a generalsensitivityto pollution. Exampleswill be given forthe most frequently used methods.

QUANTITATIVEMETHODS Sampling:

Delimitationof survey areaSampling unit

Selection oftreespeciesCommunityparameters(e.g,abundancescale)

Speciesrelatedinformation:Biodiversity

Cover,frequency, abundance,conditionetc.Matrix of sites x species

Matrix of sites xenvironmental infonnation

QUAUTATIVEMETHOD S

Calculation of a lingleindu value through amathematicalformula

Results may beinterpretedby :

A continuous predefinedscale

Mappingofindexvalues

Mapping of zones

Comparisonwitb pollutionmeasurements

Pbytolodologicaland/orItatisticaltreatment

An onlinal scale

Mapping of species,species groups orcommunities

Figure I. Main methodological steps of the quantitative andqualitative approaches topollutionmonitoringwith lichens.

Regardless of approach, the basic methodological steps are similar: selectionofsampling units,decisions about the numberoftrees in a sampling unit, the tree species to be

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used, the sampling area on the tree, and the parameters to quantify species' occurrences(Figure 1). Both methods generate biologicaldata,such as measurementsof biodiversityandofthe condition of communities.

The present chapter is devoted to the community approach. All procedures will beconsidered in which species-related information is important. Each levelof biologicalorganisation, from cell to community, isof interest in this context. To avoid overlap withother chapters in this volume, we concentrate on the useofecological indicator valuesofindividual species, species groups, and communities. As the number of published works isvery large, only a selection can be mentioned. We also suggest that apparently differentconcepts are closely related: individual species and communities can both provide usefulindicators, indeed one can use the indicator valuesof species for defming thoseofcommunities andvice-versa [97]. We conclude that the distinction between "quantitative"and "qualitative" methods is rather faint, since the ecological informationof individualspecies is also important in quantitative methods.

2. Indicator species

2.1 LICHEN SPECIES AS INDICATORS OF AIR POLLUTION

Since the mid-1950s, lichenologists realised that sensitivity to air pollution differs amongspecies.A wide rangeofdifferent sensitivities were established,and single species, as wellas combinationsof species, appeared to work well as bioindicators for mapping andmonitoring the effectsofair pollution.

The sensitivity of lichen species to pollutants may be estimated from several sources:(1) measurementsofair pollution may be compared with the lichens occurring at a site; (2)corroborative information may be used, usually derived from abiotic observations; (3)sensitivity may be estimated from the species composition at the investigated sites; or (4)from the general state (vitality) and the degreeof damage observed in the thalli; and (5)known sensitivity values may be used, derived from earlier studies.

A study of the impactof air pollution on lichens requires:(1) data on speciesdistribution; (2) auto-ecological information, e.g.indicator values for individual species; (3)an index expressing the species' sensitivity to air pollution; (4) the drawingofisoplethsona map. The results also depend on the selection and sizeof the sampling units and on thesampling procedures (see [128, 160],and chapter 9, this volume).Furthermore, every studyhas its own regional validity,dependent on the local flora, climate, etc.

A very large numberofstudies exists on air pollution and lichens, reviewed by Ferryetal. [38], Deruelle and Lallemant [29], Nash and Wirth [90], Richardson [110], Seaward[119], Van Haluwyn and Lerond [151], Piervittori [101], and the bibliographical listingsregularly published inThe Lichenologist by Hawksworth (1974-1979) and Henderson(1979 onwards).

Jones [67] was thefirstto compare the distributionofepiphytic lichens in England withknown levelsofair pollution, although he focussed on smoke rather than sulphur dioxide.Johnsen and Sechting [65] compared the distributionofepiphytic lichens and S02 isoplethsdetermined by 23 monitoring stations around Copenhagen (DK). They distinguished 5groups of indicator species whose distribution corresponded to known mean winter S02

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concentrations. The most famous qualitative scale, however, is thatof HawksworthandRose [56] for England and Wales (Tables 1-2),which was thefirstmajor attempt to linkspecies sensitivities to measured pollution data (see also chapter 3, this volume).

Zones 1 to 9 were equated to mean winter sulphur dioxide levels, zones 0 and 10 werequalifiedas "highly polluted" and "pure air", respectively. The scale was devised for use ondeciduous trees with either non-eutrophicated or eutrophicated bark. The resulting mapswere accurate within the rangeofS02 levels from"pure air" to 170 ug/rrr', level at whichall epiphytic lichens disappear. More recent studies showed that in some areas the airqualityzones established with this scale correlate with much lower S02 values (e.g.[91]).

The Hawksworth and Rose scale has been widely applied in Europe, sometimes, as inFrance, in amodifiedform [14, 26, 28,74].

TABLE 1.Qualitative scalefor the estimation ofair pollution in England and Walesusing lichens ofnon­eutrophicatedbark (ajier (56]). Nomenclature has not been updated.

ZoneS02

(J.lWmJ)

0 Eninhvtesabsent ?I Pleurococcus viridis s.l. nresent butconfinedto the base > 170

2P/eurococcus viridis s./. extends up the trunk:Lecanora conizaeoides presentbut Aboutconfinedto the base ISO

3Lecanora conizaeoides extends up the trunk; Lepraria incana becomes frequent on Aboutthe base 125Hypogymnia physodes and/orParmelia saxat ilis or P. sulcata appear on the bases

4 but do not extend up the trunks. Lecidea scalaris, Lecanora expallens and About 70Chaenoth eca ferruginea often nresentHypogymnia physodes or P. saxatilis extend up the trunk to 2.5 m or more; P.g/abratula, P. subrudecta, Parmeliopsis ambigua and Lecanora ch/arotera appear;

5 Calicium viride, Lepraria cande/ar is, Pertusar ia amara may occur; Ramalina About 60farinacea and Evernia prunastri if present largelyconfinedto the bases; P/atismatiaglauca may be present on horizontal branchesParmelia caperata present at least on the base; rich in speciesofPertusaria (e.g. P.

6 a/bescens, P. hymeneal and Parmelia (e.g.P. revo/uta - except in NE),P. tiliacea , About 50P. exasperatula (N-exposed); Graphis e/egans appearing; Pseudevern ia furfuraceaandAlectoria fuscescens present in unland areas.Parmelia caperata, P. revo/uta (except NE),P. tiliacea , P. exasperatu/a (N) extend

7 up the trunk; Usnea subfloridana, Pertusar ia hemisphaerica, Rinodina roboris (S) About 40andArthon ia imoolita (E) annearUsnea ceratina, Parmelia per/ata or P. reticu/ata (S and W) appear;Rinodina

8 roboris extends up the trunk(S) ; Normandina pu/chella and U. rubiginea (S) About 35usuallv presentLobaria pu/monaria, L. amp/issima, Pachyphia/e cornea, Dimere//a diluta or Usnea

9 florida present; if absent crustose flora welldevelopedwith often more than 25Under30specieson largerwell-lit trees

10L. amp/iss ima, L. scrobicu/ata, Sticta limbata , Pannaria ssp., Usnea articulata, U. purefilipendula or Te/oschistes flavicans presentto locallvabundant

Deruelle [27] showed that the results obtained in a region north-westofParis (France)were in perfect agreement with the data previously published for the UK. For furtherinformation see chapters 3 and 4 in this volume.

During the 1970s, several laboratory fumigation experiments were carried out todetermine the physiological basis for the drastic effectsofsulphur dioxide on lichens.Manystudies, however, failed to defme lethal thresholds. Only some species from acid bark,

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tested for their respiratory responses, appeared to correlate well with the scaleofHawksworth and Rose. For species from eutrophicated bark the correlation appearedto bepoor [5, 39], and actually, according to fumigation experiments by Tiirk and Wirth [138],damage by sulphur dioxide appears to be more severe at low pH values. Molina andVicente [86] found, with laboratory experiments, thatRamalina farinacea and Everniaprunastri could be useful indicatorsofacid rain.

Corroborative information to assess species sensitivity was applied before reliablemeasurementsofsulphur dioxide pollution were available. Before the 1950s, air pollutionstudies were mainly limited to casual observations on impoverished urban floras of westernEurope and Scandinavia. Sernander [122] recognized three zones for Stockholm (Sweden):desert, struggle and normal. The order in which lichen species appear at different distancesfrom town centres as a measure for toxitolerance was introduced by Haugsja [53] forNorway. He classified 28 species into 12 degreesof poleophoby. Different degreesofvitality were also considered. The author mapped both the absolute boundariesof thespecies, and the areas where they were developed normally.In the poleotolerance scaleestablished for Estonia by Trass [134, 135], the species were grouped into 10 classes (1-10)according to their sensitivity to environmental quality from "only natural landscapes andvegetation unaffected by culture" (zone 1) to "landscapes submitted to very stronginfluenceofculture" (zone 10).

TABLE 2. Communities ofeutrophicated bark and the zones in Table J to which they correspond (after (56]).Nomenclature has not been updated.

Zone0 Epiphytes absent1 Pleurococcus viridis s.l. extends up the trunk2 Lecanora conizaeoides abundant·L. exoallens occurs occasionallv on the bases3 Lecanora expallens andBuellia punctata abundant;B. canescens appears

4Buellia canescens common;Physcia adscendens andXanthoria parietina appear on the bases;Physciatribacia appears(Svexposed)Physconia grisea , P. farrea, Buellia alboatra, Physcia orbicularis. P. tenella. Ramalina farinacea,

5Haematomma coccineum var. porphyrium, Schismatomma decolorans , Xanthoria candelaria,Opegrapha varia and 0. vulgata appear;Buellia canescens and X parietina common;Parmeliaacetabulum appears (E)Pertusaria albescens, Physconia pulverulenta, Physciopsis adglutinata, Arthopyrenia alba. Caloplaca

6 luteoalba , Xanthoria polycarpa, andLecania cyrtella appear; Physconia grisea, Physcia orbicularis,Onegraoha varia and0. vuleata become abundant

7Physcia aipolia, Anaptychia ciliaris, Bacidia rubella. Ramalina fastigiata, Candelaria concolor andArthopvrenia biformis appear

8 Physcia aipolia abundant;Anaptychia ciliaris occurs with apothecia; Parmelia perlata, P. reticulatain SandW), Gvalecta flotowii, Ramalina obtusata, R. pollinaria, andDesmaziera evernioides appear

9Ramalina calicaris , R. fraxinea, R. subfarinacea, Physcia leptalea , Caloplaca aurantiaca, and C.cerina appear

10 As9

The worksof Barkman [7] and Skye [131] greatly stimulated future research.In theNetherlands, Barkman grouped 45 lichen species into 12 poleophoby classes. The indicatorvalue of each species was deduced from the distance they first appear from pollutantsources. Although not perfect, the indicator values of Barkman are remarkably similar tothose developed by Hawksworth and Rose in relation to S02 measurements.This highlightsthe significanceof S02 on epiphytic lichens during the 1950-1970s, even when using

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highly differentmethods and withoutpropermeasurements[146]. Suchqualitativescalesare of particular value in areas such as Eastern Europe [75, 112],China [133] and SouthAmerica [37] where directpollutionmeasurementsare scarce, while thesensitivitiesofindividualspeciesare unknown.The area aroundAnnabainAlgeria,for example, is undertheinfluenceof S02 andfluorideemissionsfrom a steel work and afertilizerplant. Thebiologicalimpact of thispollutionwas studiedby carryingout lichendistributionsurveys.It was possible to provide afairly rapidassessmentof air quality and to alert localauthoritiestowardsair pollutionproblems[152].Of particularinterestis the fact that somespecies which were known to bepollution-sensitivein NW Europe (e.g.Bacidia rubella,Caloplaca cerina) appearedas highlytoxitolerantin the Annabaarea.However,"it is clearthattolerancelimitscannotbereliablyemployedingeographicalareas other than those forwhichtheywereoriginallyworkedout" [54].

Speciescompositionat thecommunitylevelwasfirstused as a measureofpoleophobyof individualspeciesby LeBlancand De Sloover[73].Theseauthorsestimatedpoleophobyfor a given species by summing the number of other species present at theinvestigatedsites,and then takingthe averageof the sums for all sites where the specieswas present.Inother words, the mean number ofcompanionspecies was taken as an estimate ofpoleophoby (see chapter 4, thisvolume).Pollution-tolerantspecies occurring in species­poorcommunities,such asLecanora conizaeoides, usually score low values, contrary tospecies such asAnaptychia ciliaris, which is invariably present in species-richcommunities.The samesituationwasdescribedby otherauthors,e.g.by Jacobsen [64] inFlensburg (Germany) where these two species scored values of 6.54 and 20.67,respectively.In De Wit's scale [25],26epiphyticlichenswereclassifiedinto 8 groups.VanDobbenand Ter Braak [146]comparedthis scalewith that ofHawksworthand Rose [56],againfindinga goodcorrespondence. It is howeverquestionablewhetherthe same methodwould yield reliable results if applied today under theconsiderablylower S02 levelsprevailing in WesternEurope.The implicitassumptionmade is that S02 is the onlyecologicalfactor thatdetermineswhether a species is present or not. Indeed, a clearnegativerelationshipseems to exist between species diversity and high orincreasingconcentrationsof S02,obviouslyoverridingfactorssuch as climateandtopography(116].However,due to recentreductionsin S02 levels in manywesterncountries,thedistributionpatterns of lichens are no longercorrelatedwith S02concentrationsas in the recent past[120]. Some species seem to haveproblemswithrecolonisation,sincereproductivestagesof epiphytesare more sensitive to S02 than mature thalli [10].Recolonisationmay begovernedby otherfactorsthan S02; therecolonisationof London's oaks byepiphytesfrom1979 to1999 is poor incomparisonwith other tree species and with oaks inunpollutedregions.The highNOx levelsinirmerLondoncouldplayamajorrole in this context[II].

Vitality and damage caused bypollutantsmay be important todistinguishgroups ofspecies with differentsensitivity. Here we shall treat only symptoms ofmorphologicalalterationsandphysiologicalstress which are directlyobservableand measurablewithoutlaboratoryexperiments. This agreeswith the conceptof"bioindicator"sensu stricto i.e. "avisualtool producingstatementson thepollutionof a site withoutcarryingoutinstrumentalmeasurements" [42].The use ofbiomarkersasperformanceindicatorsof single species atmolecular-,cellular-andphysiologicallevels is dealtwith in chapter8, this volume.

Morphological alterationsof selected species can be used to assess the impact ofpollution.In a studyaroundNewcastleupon Tyne(UK), Gilbert[44] measuredvitalityand

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cover of Evernia prunastri (a S02 sensitive species) andLecanora conizaeoides (S02tolerant) along a transect from the city to the west. Deruelle and Lallemant [29J describedmorphological variations ofRamalina farinacea in relation to distance from Mantes(France). Morphological changesofterricolouslichens(Cladonia) were used as indicatorsof alteration in Siberian subarctic ecosystems [98]. According to Showman [125, 128],however, some care must be exercised in the use of injury as an indicatorof pollutioneffects.

Other factors, such as a severe winter, can cause injury to lichens [72]. Damage toepiphytic lichens caused by hail, wind and/or rain, was reported by Vonarburg [158] inSwitzerland. Schubert [113J used the reproductive strategyofLecanora conizaeoides as asymptom to estimate air quality, a factor previously incorporated in the scaleofBarkman[7] . L. conizaeoides indicates two different air quality zones depending on whether it isfertile or not. The same holds forAnaptychia ciliaris in the Hawksworth and Rose scale[56]. Schuster [114] used early development stages ofHypogymnia physodes, Cladoniaconiocraea and Usnea jilipendula as indicatorsof climatic conditions and environmentalpollution. Kauppi and Halonen [69] used the number, percentage cover and conditionoflichens to delimit five air quality zones. Lichen condition can also be estimated bymeasuring the longestspecimens ofUsnea hirta, Bryoria capillaris, B. fuscescens andHypogymnia physodes. Sometimes a population of a single species was used. Mikhailova[85] used the structure and density ofHypogymnia physodes populations, combined withdifferent experimental studies on the dispersalofsoralia, to assess the impactofa coppersmelter in the Middle Urals.

In the 1980s and 1990s, more data of S02 concentrations became available.In severalWestern European countries a dense networkof permanent measuring points wasestablished, not only forS02, but also for nitrogenoxides (NOx)and ozone(03) , and in afew countries for ammonia(NH3) and ammonium<NH4 ) ,the latter derived from data oncattle density [4]. Such data provided valuable opportunities to calibrate speciessensitivities with greater precision, assisted by multivariate analysis to study gradients(RDA ,CCA ,DCCA) .

In multivariate analysis, the so called species scores in an ordination diagram supply ameasureof species' sensitivity to air pollution, provided that eigenvalues are sufficientlyhigh and strong correlations exist between a certain axis and air pollution. Studies based onthese methods were first carried out in the Netherlands [23, 142-143, l53-154J andBelgium [61], where dense air quality networks are present. Biplotsof the responsesofepiphytic lichens were given in all mentioned studies; in some studies [23, 61, 143, 153]data on bark chemistry were also considered. RDA [142-143], CCA [23, 153-154] as wellas DCCA [61] proved to be useful tools. Van den Boomet at. [141] applied RDA asordination technique to assess the effect of air pollution on the epilithic lichensof 54megalithic monuments in the Netherlands (see chapter 16, this volume). Van Dobben andTer Braak [146] used multiple regression analysis of abundance or presence/absence valuesagainst environmental and pollution data (S02,N02, NH 3) to rank the sensitivityofepiphytic lichens to these pollutants, and scales were developed for c. 60 species in theNetherlands.In general, multi-co-linearitycan be a problem in such studies, e.g.that causedby a strong correlation between spatial patternsofS02andN02.

Over the last decade several attempts have been madeto fmally determine the auto­ecologyofseveral species. Ecological indicator values, expressing the ecological rangeof

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organisms with respect to several ecological factors are, at least in Europe, oneofthe mostfrequently used tools for summarising the complex bodyofknowledge on the ecologyofsingle organisms. For vascular plants, the best-known indicator values are those proposedby Landolt [71] for the Swiss flora, and by Ellenberg [35-36] for thatofCentral Europe.Indicator values are presently available for several other organisms, e.g. diatoms [140],bryophytes [34] and lichens.

W ith respect to lichens, the ecological behaviourof Central European and Italianlichens [70, 96, 163-164] was quantified by assigning indicator values regarding substrate,light, temperature,continentality, humidity, pH and nitrogen.The recent database on Italianlichens, published on the internet by Nimis [94], includes a large bodyof information foreach of the ca. 2,200 species in the Italian flora, including altitudinal and regionaldistribution, reproductive strategy, growth form, commonness/rarity, substrates, andecological indicator values for pH,air humidity, solar irradiation and eutrophication.

According to Nimis andMartellos[97], the useofindicatorvalues can becriticized,for two main reasons: 1) Theecologicalresponseof a species can vary indifferentportionsof its distributionalrange; indeed, theuncriticaluse ofindicatorvalues outsidethe area for which they weredevelopedcan lead to wrongconclusions: e.g. a lichen canbe strongly photophilousin thenorthernmostpartof its distributionalrange,much lessin its southernpart [103].2)The subjectivityofthese values; a fewexamplesshow thatindicatorvalues can be deduced from soundexperimentalwork (e.g. for lichens see [31,137, 167]);however, most of them, upon acloser scrutiny,reveal theirqualitativenatureofmere "expertjudgements"(12-13].

Despite these shortcomings,indicatorvalues are verypopular and widely used,especiallyin the fieldof applied ecosystemmanagement,since theypermitusers toproduce statementson the ecology of a site without carrying out instrumentalmeasurements;they have been also utilized to help interpretordinationdiagrams[22,100]. Indicatorvalues were also used forassigning toxitoleranceranges to differentepiphyticlichens. Wirth (163-164],and Kirschbaum and Wirth [70] used a 9-class ordinalscale for Central European lichens. Dietrich and Scheidegger[31] provided mean indicatorvalues for the Swiss Alps.Geiseret al. [43] determined the S02 sensitivity as a compilationof literature reports for all species known to occur in the Tongass National Forest (USA)based on a literature surveyof54 field studies in the UnitedStates, Europe and the formerSoviet Union. Of the 79 lichens listed, 17 species are both sensitive and widespread,making them useful indicatorsofair quality (see chapter 14,this volume).

Falling levelsof S02 over the last decades, especially in partsofWestern Europe andthe United States, has resulted in a re-invasionof many lichens species. Several studieshave used lichens as indicatorsofameliorating conditions where pollution-sensitive lichenswere monitored through repeated mapping, or with repeated visits at the same monitoringstations.For Ohio (USA) Showman [123-124, 126-127, 129] found thatParrnelia caperatawas a useful indicator to monitor the improvementof air quality. Henderson-Sellers andSeaward [57] and Seaward [1I7] documented air quality changes by mapping the re­invasionofLecanora muralis between 1969 and 1990 in the West Yorkshire conurbation(UK) (see Richardson [110] for additional data supplied by Seaward).

Another interesting exampleis the British mapping programof Usnea subfloridana, agood indicatorof falling S02 levels [IIO, 118]. Gilbert [51] presented a selectionofepiphytic species,which, from the literature and his own observations seem to be unusually

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fast or slow colonisersoflichendeserts in the UK (Table 3).

TABLE 3.Lists ofepiphytic lichens that are either «zone skippers) or «zone dawdlers» (after [51J).

Zoneskinners ZonedawdlersCandelaria concotor Calicium sp.Evernia prunastri Chaenotheca sao.Parme/ia caperata Chrvsothrix cande/arisP. perlata Diploicia canescensP. revo/uta Hvpocenomvce sca/arisP. subrudecta Lecanactis abietinaPhvscia aioolia Ooezraoha vuleataRamalinafarinacea Parmelia saxatilisUsnea subfloridana Parmeliopsis ambiguaXanthoria oolvcaroa Pertusaria amara

Rapid colonizers have been christened "zone skippers", slow colonizers "zonedawdlers"[55]. In general, lichens which are sensitive to increasing air pollution (manyfoliose and fruticose species) are often rapid re-colonizers when pollution is decreasing.Crustose species, on the contrary, are often slow colonizers.In Central Finland, in amapping study at 10 years interval, Poikolainenet of. [104] identified four groupsofspecies with different tolerance to air pollutants, and noted an increase in poleosensitivespecies due to decreasing air pollution.

For more than 30 years, sulphur dioxide has been consideredto be the main causeoflichen deserts. Fluoride, alkaline dust, acid rain and airborneeutrophicationhave also beenidentified as important factors.In some areas, oxidative pollutants such as ozone andperoxyacetylnitrate (PAN) are prominent. Nash and Sigal [88-89], and Ross[Ill] studiedthe sensitivityof lichens to oxidants in the Southern California mountains. Sigal and Nash[130] classified 32 species into 4 degreesof sensitivity (from very sensitive to tolerant).With the aidofmultivariate analysis, Gombert [52] proposed a sensitivity scale to 03 andN02 for epiphytic lichensofthe Grenoble area.In areas with F-emitting industries, fluoridedamage to lichens has been reported, and species groups with similar tolerance weredetected. The pioneering work by Mazel [80] and Martin and Jacquard [79] in Franceclassified lichens into different sensitivity classes. Gilbert [46] studied the effectsofairborne fluorides on lichensof wooden fence posts, acid rocks and trees at differentdistances from an aluminium smelter in Scotland, and distinguished three zones: a lichenand bryophyte desert, a transition zone, and a normal zone.

The effectsofeutrophication on lichens are well known; many studies report a recentincreaseof nitrophytic lichens. Barkman [7] summarized the most important sourcesfavouring the growthofnitrophytes: bark wounds, salt spray, dust, and dung from birds,cattle and dogs. Gilbert [48] studied the effectofalkaline dust on epiphytic lichens and barkpH along a gradient near cement works in Derbyshire (England), and found that severalnormally saxicolous species were growing on bark. Agricultural practices affecting lichenscan involve anything from the useof pesticides to inorganic fertilisers, toammoniavolatilised from animal waste, or pollution from farm vehicles [18]. The important roleofairborne ammonia from intensive cattle-breeding has been recognised only recently as asource of eutrophication. De Bakker and van Dobben [23] investigated speciescomposition, bark properties and estimatesof NH 3 along a transect, and used CCA asordination technique. Bark pH was found to be an important intermediate factor. Mean pH

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and standard deviation at which the lichens occurred was calculated for 25 species.Benfield [15] studied the transitionof lichen composition along a gradientof agriculturalactivity at Plymtree, Devon (England). Van Herk [154] investigated the responseofc. 80species to NH3by meansofmultiple regression analysis using pollution data(NH3,Nll,+,

NO x and S02)and tree girth in 900 monitoring stations (groupsofQuercus) in Frieslandand Drenthe (the Netherlands).Similar work was carried out in other areas.Hoffinann [61]studied the epiphytic lichen vegetation in Vlaanderen (Belgium) using DCCA as ordinationtechnique. Three auto-ecological propertiesof species distinguished by Wirth [163] wereentered as environmental indicators (moisture, acidity and nitrogen) and could be re­ordered according to the local behaviourof species. A scale for monitoring ammoniapollution in the Netherlands using lichens growing onQuercus was suggested by Van Herk[155], and 15 nitrophytic species were ordered into 6 classes.

The useof indicator species,ofcourse, is not limited to air pollution studies. Insarovand Insarova [62] suggested a5-c1assscale for lichens in the Negev desert (Israel) to assessthe effectsofglobal warming (see chapters 13 and 42, this volume). A notable example isthe useoflichensto monitor pollution by increasing tourist pressure in India [139].

2.2LICHEN SPECIES AS INDICATORS OF SOIL POLLUTION

The presenceofsome terricolous or saxicolous lichen species may be a diagnostic tool torecognize substrates contaminated by heavy metals. A review of all taxa observed onmetal-enriched environments was given by Purvis and Halls [106] and Cuny [21]. Pioneerwork was carried out in Britain by Gilbert in the 1970s and 1980s [49-50] as well as byAptroot and Van den Boom [3] and Lumbsch and Heibel [78]. So far, 291 taxa wererecorded. Purvis and Halls [106] listed species reported as specificofiron-rich and copper­rich rocks. Soils rich in lead or zinc may support a high diversityoflichens. Some speciesof Acarospora, Sarcosagium, Steinia and Vezdaea seem to be restricted to metal-richhabitats and may be useful indicators for the presenceofcertain metals. Recently, severaltaxa new to science were described from cadmium- and/or zinc-contaminated soils i.e.Micarea confusa [19] andPyrenocollema chlorococcum [3], or from old lead mines i.e.Coppinsia minutissima [78] (for further details see chapter 6, this volume).

3. Speciesgroupsas indicatorsofecologicalrequirements

There are many studies where indicator values are not assigned to single species orcommunities, but to groupsof species with similar ecological requirements. Diversemethods are used to assess and evaluate the indicator valuesof species groups. In moststudies on air pollution, at least some species inevitably receive the same indicator value asa resultofarranging them along ordinal scales. The species are often classified into groupssimply because their number is higher than the categoriesofthe ordinal scale.

According to Liska [76], mappingof ecologically similar species groups is moreinformative than thatofindividual species. "Groups ofspecies" may be especially useful inthe following cases: (1) different substrates are used to cover the survey area, (2) thespecies composition is used to study a problem with more than one dimension, (3) theecological behaviourofthe species cannot be easily expressed by an ordinal scale, and, (4)

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species groups are used as a substitute for communities.In some studies, insufficient trees are present in the survey area to map only epiphytes,

and additional observations may be made to fill gaps e.g.on wooden fence posts or acidrocks. The lichens occurring on these substrates, however, are different, and cannot beincluded into a single scale.In such cases, different scales can bedevelopedfor differentsubstrates, and these may be compared and fitted to each other usingcorroborativeinformation.This was done by Gilbert [46-47] in a study on the effectsoffluoride sourcesin England, Scotland and Scandinavia. He developed scales for acidophilous, saxicolous,lignicolous and corticolous species; terricolous species appeared less useful. A similarapproach was adopted by Gilbert [45] in a studyof lichen distribution around the cityofNewcastle upon Tyne (UK) in comparison with S02 levels measured by chemical gauges.He established a biological scale to estimateair pollution using 6 groupsoflichens, amongthem those colonizing acid stone works, bolesofdeciduous trees, old asbestos roofs, andcalcareous stone works. Each group was related to mean annual sulphur dioxide levels. Asix-zone scale for the estimationof mean S02 levels using speciesof human-madesubstrates (e.g.siliceous and calcareous walls, asbestos roofs) was established by Seaward[115] for the West Yorkshire conurbation (UK).

The study by Johnsen and Sechting [66] around Aalborg (Denmark) was oneofthe firstin which two independent influencesofair pollution on epiphytic lichens and mosses wererecognized and mapped. The authors compared the distributionof epiphytic species withmeasured S02 levels, dust deposition (mainly caused by cement plants), bark pH andsulphur content. They distinguishedtwo indicator groups.Species assigned to group 1 werevirtually restricted to the surroundingsof the cement factories, those assigned to group2had a wide distribution, but were, exceptLecanora conizaeoides, absent from the citycentre. Bark pH and S02 were found to be the most important ecological factors.

Jurging [68] used an arbitrarily determined weighted average derived from acombinationofexperimental and survey results to identify three groupsofepiphytic lichensfor dust fall (very resistant, resistant, and sensitive species), four groups for S02 pollution(from highly tolerant to highly sensitive) and three groups for a mixed pollution with S02and fluorides (from tolerant to highly sensitive).

The categorisationoflichensinto acidophytic, indifferent (neutrophytic) and nitrophyticwas applied by early authors, e.g. Sernander (121], Nienburg [92], and Rasanen (109].There is now general consensus that epiphytic species may be recognized as"nitrophytic"and "acidophytic"i.e,respectively mainlyXanthorion and Hypogymnietalia species (e.g.[163]). Nitrophytic species are usually associated with eutrophication, but in somecountries other factors such as dust may be important [77]. There ishoweverno agreementregarding the main factor(s) responsible for the occurrenceof nitrophytes: nitrogen ingeneral [7], onlyammonium [92], phosphorous [1], only a high pH [23, 33] or acombinationofhigh pH with at least some additional ammonium [156].Acidophytesareusually considered to be sensitiveto eutrophication.

Van Dobben [143] monitored the numberof acidophytic, indifferent and nitrophyticspecies at roadside stations in The Netherlands between 1977 and 1990, and found the firstgroup to be stable or slightly decreasing, while the latter was rapidly increasing. A similarstudy was carried out in the surroundingsof 'sHertogenbosch(The Netherlands)bycomparinghistorical data (1900:herbarium Wakker) with those from 1973 and 1988 [144].Van Herk [155] used nitrophytic and acidophytic species to mapammoniapollution in The

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Netherlands.NIW ("Nitrofiele Indicatie Waarde") and AIW ("Acidofiele IndicatieWaarde") were suggestedas new indicesto expressthe abundanceof these species groups,respectively, and were designed to be applied on tree species with primarily acid bark. Amap based on c. 5500 sampling sites was provided for about half the country, showing amarked effect of ammoniaonlichens.Changesof NIW and AIW between 1989and 1999are shown in Van Herk [156]; a comparisonof NIW and long-term airconcentrationmeasurementsofNH3 showed a strongcorrelation(R2 = 0.90,P <0.05). Hoffmann [61]used species groups (selectednitrophytes, acidophytes)to obtain a maximum correlationwith known levels of NH3 pollution (derived from cattle density, Vlaanderen, Belgium),and a IAP-like formulawas used to express the abundanceof the species; combinationsofthreeto sixacidophyticornitrophyticspeciesshowedgoodcorrelationswith pollutiondata.

Multivariate analysis enables to distinguish species groups with a similarecologicalbehaviour. Species groups may be identified by using their arrangement and mutualdistancein an ordinationdiagram(biplot).However,this usuallyprovidesonly informationabout a species optimum(centroid),while theecologicalamplitudemay be easily ignored.Cugny and Vincent [20] usedmultivariateanalyses to delimit 4 concentric zones aroundthe Toulouseconurbation(France),each with a specific lichenassemblage,and related to acertain degreeof urbanisationand traffic density. Gombert [52] carried outmultivariateanalyses in whichher ownobservationswere combinedwithecologicalindicatorvalues byWirth [163]. Six species groups weredistinguishedfor the Grenoble area (France), eachwitha differentresponseto light,humidity,pH andtoxitolerance.

Van Dobben and De Bakker [145] used RDA ordination toinvestigatetheecologicalbehaviour of some species which werepre-definedas nitrophytic,acidophytic,andindifferent in theNetherlands. In Van Herk [156], CCA sample scores were plottedtogether with the species'abundancevalues to assessdifferencesofecologicalamplitudewithinand amongthe groupsofnitrophytic,acidophyticand indifferentspecies.

Two-way indicatorspeciesanalysis(TWINSPAN) is another method regularlyappliedto classify species or sites into clusters. It produces a principal axis and dichotomiesin ahierarchicalclassification,with positive and negative indicator species identified at eachdivision[60].Pirintsoset at. [102]providedadendrogramshowingspeciesclusters,as wellas oneofclustersof sites for lichens,onPinusbrutianearThessaloniki(Greece).Wolseleyand Pryor [166] used TWINSPAN to identifyclusterswith 10 types of samples, for whichannual incrementsofQuercus petraeatwigswereused (see chapter22, this volume).Thesetwig-types could, on the basis of their lichencomposition,be linked tophytosociologicalassociations.In a second step,multivariateanalysis (PCO) was used to gain insights into(dis-)similaritiesamong phytosociologicaltypes andrelationshipswith environmentalfactors (acidification,eutrophication)and succession. According to Hoffmann [61],TWINSPAN can be considered as aone-dimensionalordinationtechnique,and mayunderestimatemultidimensionalrelationships. A data treatment using first TWINSPAN,and in a secondstep othermultivariatetechniquesmay thereforebepreferable,

4. The phytosociologicalapproach

So far, lichenologists have been little concerned with formally described lichencommunities(i.e. syntaxa in the sense of thephytosociologists)as indicatorsof air

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pollution. According to Nimis [93] communities derive from the overlapof species withmore or less similar ecological requirements in a given portionofecological space. Thus,they are perhaps better indicatorsof the conditionof the environment than individualspecies.An association may be defmed as "a plant communityof definite floristiccomposition" [40]. This defmition, however, applies also to higher phytosociologicalsyntaxa such as alliance, order, and class, in a similar way as species can be arranged intogenera, families, etc. The main advantageof the phytosociological approach is its holisticnature, as it takes the overall functional stateof a vegetation type into account [41]. Thecomplexityof biological organisation requires a high levelof standardisationof bothsampling strategies (e.g.the procedures to select sample plots, the sizeof the sample plot,etc) and data analysis. For instance, when selecting a wholetrunk as a sampling unit,associations from even different orders may be found within one releve, i.e. aChaenothecetum ferrugineae, belonging to theLeprarietalia on thedry sideofthe bole, aParmelietum revolutae belonging to theHypogymnietalia at breast height on the exposedside, and aXanthorietum candelariae belonging to thePhyscietalia adscendentis at thebase. On the other hand, too small a sampling unit easily results in releves with onlyfragmentsof an association. Sampling strategies and some aspectsofdata analysis in thephytosociological approach extend outside the scopeof this chapter, and are extensivelydiscussed by Barkman [7], and more generally by Braun-Blanquet [17]. Oneofthe reasonsofthe poor appreciationofthe phytosociological approach by lichenologists probably liesin its very poor methodological basis, where subjectivity plays a major role [93]; this is apity, because this approach, once the main faults are amended, is very powerful.

Since the mid 1950s, the phytosociological approach has been used with some successto study the effectsof air pollution. The famous workof Barkman [7] treated the mainepiphytic lichen communities of western Europe. Most communities were ecologicallycharacterized with terms such as "strongly acidophilous, strictly nitrophobous andtoxiphobous, photophytic, and ombrophilous, aerohygrophilous". Distribution mapsofmost associations occurring in The Netherlands were given, often showing areas withtypical stands, areas with fragments, and areas where the associations were missing.

The responseof lichen communities to air pollution was first studied by Beschel [16]who listed 42 species in 5 zones in the Austrian cities; among these, he recognised syntaxasuch asUsneion barbatae, Parmelion physodis, Xanthorion parietinae. The second workon this topic was that by Barkman [8] for the central partof the provinceof Limburg(Belgium). He distinguished 15 epiphytic vegetation types distributed along a gradientofspecies richness, the poorest ones being connected with towns, mines and factories. The airquality map was based primarily on phytosociological associations.The results showed thatepiphytic vegetation can be used as a good indicatorofair pollution.

Skye [132] identified five zones in the Stockholm region on the basisof speciescombination and distribution. These investigations were so detailed that this study stillprovides a model for data processing with multivariate analyses.

Van Haluwyn [147], and Van Haluwyn and Lerond [149-150] proposed aphytosociological approach to air quality monitoring with lichens based on the classicalproceduresof the French-Swiss School [17]. A statistical treatmentof c. 600 relevesdistributed in half the north of France revealed seven groups of epiphytic speciesdistributed along a hierarchical gradient of impoverishment, from associations throughalliances, orders and classes (Table 4).

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TABLE 4. Impoverishment oflichen vegetation in NorthernFrance (basedon 350 releves) as an effect ofpollution; romannumbersrefer tofivefrequency classes in groups ofreleves (after [149-150]).

Pleurococcus s.l. V V V III II III III III r III V II II II ILecanora conizaeoides V IV IV II V I II I ILepraria incana III II II IV I + r +Lecanora exoallens IV II IV I IV II III IIHypogymnia phvsodes V IV I V I II IIEvernia prun astri III IV III III III IIIPseudevernia furfur. II I rParmeliasulcata II IV V IV III IVDicranoweisia cirrh. I II II + + +Parmelia subrudecta III II III IVParmelia glabratula I III I IRamalinafarinacea I III IIParmelia subauri(era I II II IPhlvctis arzena II III + IIHypnum cupressif. I V +Pertusaria amara I I + +Parmelia caperata III IV II VParmelia revoluta V IIParmelia perlata I IIIParmelia reticulata +Buellia punctata III V IV III II V V III III IPhvscia tenella II II III III II V IV III IIIXanthoria oolvcaroa I II II I III II IPhyscia adscendens I II I + III III IIXanthoria parietina II III II I IV V IIIDiploicia eanescens I + I I II IICando xa nthostigma + II II I +Phvsconia grisea I + II I IV IPha eoph . orbicularis r I I III IIIXanthoria candelaria I + II + IRamalina [astigiata r + II II +Phvscia aioolia I + I IPhysconia distorta r I + + ILecidella elaeoehroma II I I IIParmelia acetabulum r II III IIIFrullania dilatata + + IIRamalina frax inea I IAnaptvchia ciliaris r IParmelia oastillitera rParmelia tiliacea +Parmelia soredians r I

There was enough evidence that the impoverishmentoflichen communitieswas relatedto air quality. Hence, an eco-diagnostic scaleofenvironmentalquality was proposed,usinga reduced datasetof 30 epiphytic species, chosen between the seven groupsofimpoverishment.In contrastto other studies, syntaxonomical knowledge is by no meansfundamental for the applicationofthis method,which was implemented in northern Franceas a regionalproject for monitoring air pollution by school children.

Wirth [162] provideda phytosociological scale for the estimationofacidic air pollutionin S Germany. Twenty associationswere classified into fourteen degreesof sensitivity:

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from 1 (low tolerance, e.g.Lobarietum pulmonariae) to 14 (high tolerance, e.g.Pleurococcetum vulgaris) (Table 5). Wolseley and Pryor [166] studied lichencommunitieson annual incrementsofQuercus petraea twigs in Wales(UK) , sampling linearwoodlandmargins, in which 10 twigs were randomly selected. The sampling units weredeterminedby each year's growth of twigs. The results showed a good correlation betweenenvironmental conditions and lichen communities.

The communityapproach is well suited for long-term monitoring, as shown by severalpapers which document changes in communities over time. Will-Wolf [159] showedthat"low-levelair pollution could affect native lichencommunities"three years after the startofa coal-fired generating station in Wisconsin (USA). Wirth [162] reconstructeda map ofepiphytic lichen communities, previously present in southern Germany, from historical dataand herbarium samples, and traced the impoverishmentof lichen vegetation due to airpollution. Some lichen associations disappeared, without being replaced by others, underthe influenceofacid rain and S02 (e.g. Gyalectetum ulmi, Opegraphetum rufescentis). Inother cases, a shift towards other associations was observed (e.g.Lobarietum pulmonariaetowards Pertusarietum hemisphaericae or moss communities,Parmelietum revolutaetowardsLecanoretum conizaeoidis). The replacementofa communityby anotherone canbe considered as a clear indicatorofenvironmental change. In Austrian forests submitted toalkaline deposition, the acidophilousPseudevernietum furfuraceae was replaced by thenitrophilousPhyscietum adscendentis [165].

TABLE 5.A phytosociologicalscalefor the estimation ofrelative (acidic) air pollution in southern Germany.Relative sensitivityscale (1: resistance low, 14: resistancehigh) (after Wirth [/62]).

1 Lobarietum pulmonariae subass.ofLobariaamplissima1 Nephrometum laevigati2 Gyalectum ulmi3 Usneetum florido-neglectae

3-4 Ramalinetumfastigiatae4 Parmelietum acetabuliwithAnaptychia ciliaris5 Usneetum filipendulae

5-6 Physcietum adscendentiswithPhysconiadistorta, Physciastellaris, P. aipolia6 Bacidia rubella- Aleurodiscusass.6 Leprarietumcande/aris7 Pertusarietum hemisphaericae8 Parmelietum caperatae8 Pyrenuletum nitidae9 Opegraphetum vermicelliferae

9-10 Porinetumaeneae10 Hypogymnia physodes- Parmeliasulcata community11 Chaenothecetum ferrugineae12 Buellietumpunctatae13 Lecanoretumconizaeoidis14 Pleurococcetumvulgaris

However, great care must be taken when reducing thecomplexnature and structureofacommunityto a simple community name. Syntaxa were generally supposedto maintaintheir compositionin time, but this assumption was challenged by Dirkse [32]: some

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characteristic species may become widespread and can loose their diagnostic value,whereas formerlycommon species may be restricted to special communities,becomingcharacteristic species. In fact, the conceptof "zone skippers/dawdlers"[55] showed thatlichen associations are not constant in time. Barkman [7, 9] consideredXanthoriapolycarpa as a faithful speciesoftheXanthorion parietinae on the basisofhis observationsin the Netherlands, while now this species is also extremely common in a wide rangeofother communities throughout the country. A precise definitionofwhatphytosociologistsmean by"syntaxon" is necessary, and complete releve lists should be provided in eachstudy.

Studies in which the complete lichencompositionis surveyed andmonitoredare veryimportant, since such data may become valuable in the future.Wolseley and Pryor [166]claimed that"phytosociological sampling provides a whole data set that wecanreinvestigate at a later date to detect environmental conditions which we may not havethoughtof'. There are many examples illustrating this statement. McCune [81] analysedthe"depression"oflichen communities along 03 and S02 gradients inIndianapolis(USA) .He observed that the longer the periodof exposure to air pollution, the stronger therelationships with lichen communities appeared. Purvis andBamber [105] studied theimpactofa gravel pit on the environmentofa nature reserve near London by monitoringParmelion communities onQuercus over a 5-year-interval.Considerablevariation inassemblage and composition was observed, especially a gradualreplacementofacidophilous species by nitrogen-tolerant lichens and the appearanceof fruticose lichens.Motejunaite [87] carried out a long-term monitoring project in Lithuania using a graphicalapproach for surveying yearly the epiphytic lichen communities, which allowed short-termchanges to be detected within one year. Lichen communities are included in the US ForestHealth Monitoring Program designed to monitor forest conditions since 1994. The mainobjectives are: to: (1) establish baseline studies; (2) detect and identify any change inclimate and air quality; and (3) provide substantial informationto the forest authorities.Theprogram is implemented in three phases. The first consists in data acquisition at sitesselected according to a systematic sampling design, plus special plots in urban andindustrial sites. The second phase elaborates a model for climateand air quality gradients.The last one applies the model to calculate gradient scores for additional plots. This long­term lichen monitoring program is currently ongoing in 32 states [43, 82, 84].Ofparticularinterest is the fact that the program is submitted to quality assuranceprocedures[83],whichare important for the future developmentofbiomonitoring(see chapter 9, this volume).

Attempts were frequently undertaken to correlate bioindication data with air pollutionmeasurements. Nowadays there is considerable debate as towhetherlichens can stillmonitor sulphur dioxide pollution. According to many authors[2,58-59, 117, 119, 150­151] it is clear that lichens respond increasingly to otheratmosphericpollutants. Seaward[119] produced the resultsofa multilinear regression analysis in which lichen diversity wastaken as a functionof air pollution and various climatic and edaphic factors, both for thelate 1960s and the mid 1980s (Table 6).

A significant decrease in the correlation between species diversity and sulphur dioxidelevels was found. Although the maximum tolerance levelsof separate species for S02 arestill valid, lichen communities are likely to remain fragmentary for a long time, even whenthe air is clean enough to allow fulldevelopmentofcommunitiestypicaloflow S02 levels« 30 ug/rrr'),

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TABLE 6. Correlation matrices produced in a multiple linear regression. in which species diversity is taken tobe a/un ction ofvarious fac tors operating in the late 1960s and in the mid-1980s in the West Yorkshire

conurbation (after [119J).

Lichen SulphurRainfal1

Popul.Altitude

North-D iversitv Dioxide density Westerl.

Late1960sLichen diversity 1.0000Sulphurdioxide level -0.463 1.000Rainfall 0.545 -0.391 1.000Populationdensity -0.276 0.451 0.057 1.000Al titude 0.601 -0.530 0.734 -0.114 1.000North-westerliness 0.482 -0.595 0.646 0.056 0.644 1.000Mid-1980sLi chendiversity 1.000Sulphurdioxide level -0.130 1.000Rainfall 0.496 0.060 1.000Population density -0.205 0.236 0.D15 1.000Altitude 0.539 -0.016 0.734 -0.088 1.000North-westerl iness 0.411 0.258 0.646 0.086 0.644 1.000

Examples areLobarion communities, or those typicalofbase rich bark(Ulmus) withspecies such asBacidia rubella and Gyalecta jlotowii, which are still on the decrease inlarge partsof Europe. In someParmelion communities of W Europe, on the other hand,recolonisation appears to be relatively fast, and species likeParmelia caperata, P. perlata,P. reticulata probably follow the zonesofHawksworth and Rose quite well [56].

In the opinionof thefirstauthorof this chapter [148], the indicator valueof lichensshould not be related to the effects of a single pollutant,but to the resultofantagonistic andsynergistic interactions among contaminants. The maps produced with theeco-diagnosticscale of environmental quality [149] in northern France agree well not only with thelocationof the main emitting sources and the prevailing winds, but also with differentenvironmental conditions (acidification,eutrophication, etc.), so that this scale seems toreflect the global qualityofthe environment.In the opinionofthe second author,however,one could wonder what could be the predictive valueofsuch a generalscale, consideringthatsome pollutants have antagonistic effects, and that the effectofother major pollutants(e.g. N02) is still obscure and only visible at very high concentrations. For a betterunderstandingofthe way in which lichen vegetation is affected by environmental pollution,we need to know what kind of pollution may be responsible for what kindofalterations inlichen communities.Therefore,there is a need for differentscales to be applied for differentpollutants (e.g.(155]for NH3) . Lichen communities are multidimensional entities, whichare affected by a wide range of (at least partly) independent primary factors. Multivariateanalysis has revealed that bark pH and toxic air pollutants are two major factors affectinglichens on freestanding trees in the Netherlands [156]. Air pollution by S02 seemed tocome through mainly by differences in toxitolerance,while effectsof NH 3 on speciescompositionare mainly the resultofa rise in bark pH.Obviously,all effectsofair pollutionon lichensdo notfitinto a one-dimensionalscale.To assess and quantify a deviation from anatural state is a very complex process, which probably involves integrationof the effectsof at least seven primary factors, i.e, light, temperature, continentality, humidity, pH,eutrophicationand toxic effectsof air pollutants. These are, in fact, the auto-ecologicalcharacters assigned to species by Wirth [163] and Nimis [94]. Every attempt tofit them

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into one dimension will be arbitrary.The three indicator concepts: individual species, species groups andcommunities,

are closely linked. The indicator valueof individual species can be used to determinethose of species groups. The reverse, estimatesof pollution sensitivity for singlespecies, can be derived from the analysisof lichen communities [136]. The indicatorvalue of a singlephytosociologicalreieve can be estimated by averaging the indicatorvalues of the species present in the releve. However, de Baere and Verheyen [22]maintained that there are clear differences between the indicator valueof species withdifferent cover/abundance.Nimis and Martellos [97] showed that indicator valuesassigned to single species permit the reconstructionof "virtualcommunities" similar toreal ones. One comes full circle: it is possible to use either single species orcommunities as indicators,the indicator valuesof species can also be used to definethose of communities. This has a potential interest for: (1) defining exactly what acommunityis, and (2) demonstratingthat communities must obey the rulesof nature,that they are not virtual concepts.

We conclude that the fundamental differences between "quantitative" and "qualitative"approaches are so small that this subdivision can be hardly maintained. "Quantitative" datacan be used in "qualitative" methods, and vice-versa. For instance, a phytosociologicalreleve includes both qualitative and quantitative information; the knowledgeof theindicator valueof a species is sometimes a prerequisite for optimising the efficiencyofsome lAP formulas, i.e. species without indicator properties are sometimes disregarded (seeGerman guideline [157]);mathematical-statisticalprocedures are increasingly applied todelimit species groups andvegetation units.On the other hand,qualitative information maybe also included in lAP formulas.In the early definitionofthe lAP [24], the poleophobyoflichen species was included. In the early Belgian studies, the indexofpoleophoby was thatproposed by Barkman [7]. Trass [134-136] introduced his own poleotolerance index in theformulaof a "Poleotoleranceof lichen synusiae". Insarov et al. [63] constructed a TrendDetection Index (TDI) to monitor climatic changes with epilithic lichens, defmed as thesumofcoveroflichen species with coefficients suitable for the detection of climatic trends.Rabe [108] developed a system called LuGI (Luftgute-Index) that was applied in the RuhrArea (Germany), which canbe calculated by a formula in which qualitative parameterssuch as an empirical species-specific sensitivity value (E) and a degreeofvitality(V) wereintroduced. An integrated methodin which both the"quantitative" and the"qualitative"approaches are used is probably the most appropriate. The auto-ecological informationprovided by individual species might allow the determination of eutrophication, geographicpatterns or climatic changes. On the other hand, a matrixof species and releves, asubstantial by-productof the quantitative methods, provides opportunities for multivariateanalyses, which may be carried out even without environmental data. The works ofDiamantopouloset al. [30], Pirintsoset al. [102] and Badin and Nimis [6] provide goodexamples. In the first two,the lAP methodofLelslanc and De Sioover was combined witha multivariate analysisofa site-by-speciesdata matrix (DCA and TWINSPAN).Badin andNimis [6] combined the BiodiversityIndex ofNimis [95] withmultivariateanalysesofboth a matrixofspecies and releves (60 species and 317 reieves) and a matrixofspeciesand stations. The lichen vegetation could be distributed between theParmelion and theXanthorion parietinae alliances, and a mixed vegetation characterised by speciesofboth Parmelion and Xanthorion . It is striking that very similar results were obtained

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with classicphytosociologicalmethods in northern France [149]. In these cases, theresults have provided,in addition toquantitativedata(lAP-index, or index of LichenDiversity),also "an ecologicalimage" ofthe survey area.

5. Communitiesas indicatorsof soil pollution

Some terricolous lichen communities may provide a diagnostic tool to tracemetalliferoussoils. Purvis and Halls [106] reviewed thesyntaxonomicdescriptionoflichen vegetationin metal-enrichedenvironmentsin relation to substratemineralogyand chemicalprocesses. Specific lichen communitiesfrom either iron (Fe) orcopper(Cu) rich substrates have been formallydescribed. Certain ironbearingrocks supporttwo main communitiesbelonging to theAcarosporion sinopicae, the Lecanoretumepanorae and theAcarosporetum sinopicae. The former is typical for vertical, sheltered,dry and overhanging rock surfaces whereas the latter occurs onexposed, sunny,horizontal surfaces [106-107, 161]. Recently, Purvis and Halls [106]describeda newalliance,Lecideion inopis, typical for coppermineralization.This syntaxon is found inopen, wellilluminatedsituations and ischaracterizedby Lecidea inops, Psilolechialeprosa and several Cu-rich ecotypes. In contrast withFe/Cu-containingrocks, nospecific lichen communityfrom either lead (Pb) or zinc (Zn)contaminatedsoils hasbeen formallydescribed. The Cladonietum rei, a newterricolousassociationdescribedby Paus [99], ischaracterizedby a typical assemblageof bryophytesand lichens(Cladonia rei, C. humilis, Peltigera polydactyla). This communityis indifferentto pH,occurs principallyin disturbed habitats, and is tolerant but notdependenton metal­pollutedsoils. Soil communitiesalong a heavy metalgradientweredescribedby Cuny[21] in northern France. Statistical analysesof both floristic and abiotic dataallowedattributionof a bioindicationvalue to several terricolouscommunities.Five speciesgroups could bedistinguishedaccordingto low- intermediate-and high trace metallevels (Cd, Pb, Zn) in the soil: two groups could beconsideredas metallophobe,twoother groupsincludedubiquitous species growing on both polluted and non-pollutedsites; the last group includedexclusivelyVezdaea leprosa and Sarcosagium campestrevar.macrosporum occurringonly on soils rich in lead and with a highCIN ratio.

6.Concludingremarks

Most ofthe approaches treated in this chapter are appropriate for estimating the effectsofS02 air pollution. While S02 levels are now low in many partsofEurope, this is not thecase in other partsof the world (e.g. China, India, Ukraine, Czech Republic, partsofIndonesia, Russia). The cheap methods treated here still have much value in these areas.Wherever S02 levels are considerably lower than in thepast,other factors seem to have anincreasing influence on lichen communities. Future lichen monitoring should take intoaccount the complex natureofall factors affecting lichen vegetation.

"Qualitative" methods have nearly the same requirements as "quantitative" ones. Inboth cases the main objective is that of providing standardised, reliable tools to assess theeffectsof air pollution, minimising variations caused by other factors (Figure I). The

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developmentof methodsin whichquantitativeandqualitativeapproachesare combinedis achallengefor the futureoflichenbiomonitoring.

7.References

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2. Ammann, K.,Herzig, R., Liebendoerfer,L., and Urech, M. (1987) Multivariate correlationofdepositiondata of 8 different airpollutantsto lichen data in a small town inSwitzerland, in Advances inAerobiology. BirkhauserVerlag, Basel, pp.401-406.

3. Aptroot, A . and Van den Boom, P.P.G. (1998) Pyrenocollema chlorococcum a new species with achlorococcoidphotobiontfrom zinc-contaminatedsoils and wood,Cryptogamie Bryologie Lichenologie19,193-196.

4. Asman, W.A.H. and VanJaarsveld,J.A. (1990) A variable resolution statistical transport model appliedfor ammonia and ammonium, Rijksinstituutvoor Volksgezondheiden Milieu, Bilthoven,RlVM report228471007.

5. Baddeley, M.S., Ferry, B.W., and Finegam,EJ. (1973) Sulphur dioxide andrespirationin lichens, inB.W . Ferry, M.S. Baddeley, and D.L.Hawksworth(eds.),Air Pollution and Lichens, The AthlonePressoftheUniversityofLondon,London, pp. 299-313.

6. Badin, G. and Nimis, P.L. (1996) Biodiversity of epiphyticlichens and air quality in theprovinceofGorizia (NE Italy),Studia Geobotanica 15,73-89.

7. Barkman,U . (1958) Phytosociology and Ecology ofCryptogamic Epiphytes, van Gorcum,Assen.8. Barkman, JJ. (1963) Die epifyten-floraen -vegetatiesvon M idden Limburg (Belgie), Verh. der

Koninklijke Nederl. Akad. van Wetenschappen, afd. Natuurk., Tweede Reeks 54,1-46.9. Barkman, U . (1975) Epifytengemeenschappen,in V . Westhoff and AJ. den Held (eds.),

Plantengemeenschappen in Nederland, Thieme& Cie, Zutphen,pp.272-286.10. Bates, J.W.,Bell, IN .B.,and Farmer, A.M.(1990) Epiphyterecolonizationofoaks along agradientofair

pollutionin south-EastEngland, 1979-1990,Environmental Pollution 68, 81-99.II. Bates,J.W.,Bell, J.N.B.,and Massara, A.C. (2000) Loss ofLecanora conizaeoides and otherfluctuations

of epiphytes on oak in S.E. England over 21 years withdeclining S02 concentrations,AtmosphericEnvironment 35, 2557-2568.

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13. Beinat, E., Nijkamp, P., and Rietveld, P. (1994) Value functions forenvironmentalpollutants: a newtechniquefor enhancingtheassessmentofexpertjudgements,Environmental Monitoring and Assessment30,9-23.

14. Belandria,G. and Asta, J. (1986) Les lichensbioindicateurs:la pollutionacide dans la region Iyonnaise,Pollution Atmospherique 109, 10-23.

15. Benfield, B. (1994) Impactofagriculture onepiphyticlichens at Plymtree, East Devon,Lichenologist 26,91-96.

16. Beschel, R. (1958) Flechtenvereineder Stadte, Stadtflechtenund ihr Wachstum, Bericht desNaturwissenschaftlich-Medizinischen Vereins Innsbruck 52, 1-158.

17. Braun-Blanquet,J. (1964) Pflanzensoziologie, Springer, Wien.18. Brown, D.H.(1999) Editorial,Lichenologist 31, I.19. Coppins, BJ. and Van den Boom, P.P.G. (1995) Micarea confusa a new species from zinc- and

cadmium-contaminatedsoils in Belgium and theNetherlands,Lichenologist 27,81-90.20. Cugny, P. and Vincent, J.P. (1996 ) Analyse faetorielle de ladistributionde la florelicheniqueen zone

urbaine. Mise en evidencede zones depollutiondans l'agglomerationtoulousaine, Bulletin de la Societed'Histoire Naturelle de Toulouse 132,41-47.

21. Cuny, D. (1999) Les impacts communautaires, physiologiques et cellulaires des elements tracesmetalliques sur la symbiose lichenique - mise en evidence de mecanismes de tolerance chez Diplosehistesmuseorum(Scop.)R. Sant., Thesis Univ. Lille 2, Lille.

22. De Baere, D. and Verheyen, R.F. (1987) Ecological indicatorvalues and theinterpretationofordinationdiagrams,Abstracta Botanica 11, 1-8.

23. De Bakker, AJ. and Van Dobben, H.F.(1988) Effecten van ammoniakemissie op epifytische korstmossen,een correlatiefonderzoek in de Peel, Rijksinstituutvoor Natuurbeheer,Leersum, RlN rapport 88/35, pp.1-48.

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ACCUMULATION OF INORGANIC CONTAMINANTS

R. BARGAGU I andI. MIKHAILOVA 2

'Department ofEnvironmental Biology, via P.A. Mattioli 4,1-53100Siena, Italy ([email protected])2Institute ofP/ant and Animal Ecology UD RAS, 8 Marta Str . 202,620144 Ekaterinburg, Russia ([email protected])

1. Introduction

Human activities release large amounts of exchangeable elements into the environmentand have become a major factor in altering biogeochemical cycles. This alteration isprogressively affecting the long-established steady equilibrium between theEarth'sprocesses and biological evolution [50]. Increasing body burdensof potentially toxicelements in organisms, even from remote regionsofthe Northern Hemisphere far fromsignificant sourcesof local pollution, have underlined the importanceof establishingreliable monitoring systems at different scales. A reliable appraisalof pollutantconcentrations in such an extremely variable compartment as the atmosphere needs astatistical approach based on a large numberof samples in both time and space (seechapter 9, this volume). The high costsof establishing and managing automaticmonitoring networks often limit the numberof sampling stations and/or the numberofpollutants considered. Thus, although very reliable, data from instrumental recordingmay be statistically weak and their integration with diffusion models cannot givereliable information about the deposition and impactof atmospheric pollutants onterrestrial ecosystems.

Biological accumulators are organisms that reflect the chemical contentof theirenvironment. Biological monitoring with accumulator organisms provides an essentialadjunct to instrumental recording.Hundreds of studies carried out over the last 30 yearsconfirm that lichens are among the most reliable accumulatorsof airborne inorganiccontaminants.The use of bioaccumulators is comparatively cheap, enabling coverageoflarge and remote areas, and provides current and retrospective information on theintegrated effectsofatmospheric pollutants and other environmental factors.

Lichens are ectohydric organisms which lack specialised structures for water andgas exchange and hence absorb gases and water with dissolved substances (includingmany pollutants) over much of their outer surface. The rich thallus branching of manylichen species and the large intercellular spaces within thalli facilitate trappingofparticulate pollutants.The chemical composition of lichens therefore largely reflects theavailability of elements in the environment. Lichens thus behave as long-livingcollectors of atmospheric pollutants, especially those associated with airbome particlessuch as trace metals, fluorine and radionuclides. In polar and alpine ecosystems where

65P.L. Nimis, C. Scheideggerand P.A. Wolseley (eds.),Monitoring withLichens- MonitoringLichens.'65-84.© 2002KluwerAcademic Publishers. Printed in the Netherlands.

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lichens constitute the bulk of the biomass, knowledgeof their role in environmentalbiogeochemistry is essential to establish the path of element cycling, the transferofpollutants to herbivores, and to predict possible effects of environmental and climatechanges. Ecophysiological studies of lichens subject to increased levels of potentiallytoxic elements can shed valuable new insights into the adaptationofliving organisms toenvironmental stress.

2.Sourcesof contaminantsin lichenthalli

Atmospheric deposition (wet and dry) is the main source of elements to lichen thalli.Wet deposition includes precipitation (rainfall, wash-out and snowfall) and occultprecipitation such as fog and dew. Under some conditions, occult precipitation may bean important sourceof water and element supply for lichens. Concentrationsofelements such as Pb are sometimes higher in fog than in rainwater [52]. Gaseous orparticulate elements in the atmosphere are also scavenged through dry deposition.Larger particles settle by gravitation (especially around cement factories, mining,smelting and metallurgical plants); fine particles (from about 0.01 to 100 urn indiameter) are removed continuously from air, accumulating on exposed surfaces. Theaerial inputof elements into ecosystems depends on element concentration andchemical form but is also affected by surface effects (e.g. interception) [95].Interception increases sharply on moist plant surfaces and cannot be simulated by inertor surrogate surfaces in automatic collectors. To evaluate the impactof atmosphericdeposition on terrestrial ecosystems and the transfer of potentially toxic elements toherbivores and ultimately to man it is therefore necessary to collect and analyse suitablespecies of lichens,mosses and vascular plants [6].

Owing to their slow growth rate, longevity, high surface-to-mass ratio and lackofprotective outer structures, the more resistant lichen species concentrate persistentatmospheric pollutants to readily detectable levels. For instance, an uptakeof sulphurdioxide by lichens 50 times higher than in vascular plants has been reported [94].Isotopic studies demonstrate a close relationship between the sulphur isotopecomposition in lichens and that in the atmosphere whereas soil was found to be the mainsourceofsulphur in pine needles [43]. Despite these and other results since the 1970s,which have failed to show significant transferof elements from bark to epiphyticlichens, the possible roleofthe substratum in lichen nutrition is still largely unknown.

The high specificityof most lichens for definite chemical typesof substratesuggests such interactions. The higher element content found in the lower thallussurface and rhizinae of terricolous lichens [38] was assumed to indicate elementexchange between the substrate and lichens. This possibility has also been postulatedfor epiphytic lichens [24, 81]. Brownet al. [21] showed that particulates trappedbetween the lichen thallus and bark may play a role in supplying elements to lichens,e.g. Parmelia sulcata growing directly on bark contained higher concentrationsof AI,Cr, Fe and Mg than did samples growing over epiphytic mosses.Moreover, levelsofthesame elements were much lower in adjacent surface bark uncolonised by lichens than indebris entrapped between the analysed thallus and the underlying bark. These resultsindicated that the presenceof the lichen leads to the accumulation of particulate

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elements beneath the thallus (i.e. lichens act as a filter for particles washed down thetree-trunkby stem flow). The role of rhizinae andof the lower surfaceof the lichenthalli in the uptakeofelements has yet to be fully investigated; however it seems likelythat a fraction of elements in water and/or particles in stem flow are directly accessibleto lichens.These elements may contaminate samples and cannot be ignored when usingepiphytic lichens for biomonitoring purposes. Barkman [10] found it more important toinvestigate the chemical compositionofthe water in contact with the bark than the barkitself. The chemical compositionof precipitation is changed after contact with treeleaves and bark and in turn the chemistry of these substrates is modified by precipitation(e.g. by altering acidity, adding or leaching elements). Atmospheric deposition, treeleaves and bark should therefore be considered as a system affecting the elementalcompositionofepiphytic lichens.

In general, the possible influenceof substrata on the elemental compositionoflichens depends on lichen species, bark properties, levels of environmental pollutionand the element in question. To minimise these effects inbiomonitoringsurveys it isnecessary to select species loosely attached to the substrate and to collect samples inareas of the tree relatively unaffected by stem flow. The lower surfaceof the thallusneeds careful cleaning (not washing) and whenever possible, the outermost partof thethallus, which has scanty or no connection with tree bark, should be selected forchemical analyses [3].

3.Elementaccumulation

Inorganic contaminants may occur in lichens as I) particles (adsorbed onto the thallussurface or within intercellular spaces), 2) ions bound to extra- or intracellular exchangesites, and 3) soluble intracellular ions.

3.1.PARTICULATE MATERIAL

Particulate material within lichen thalli includes unaltered particles ranging from subum to> 100 um trapped directly from the environment, and various metabolic productsincluding metal compounds and complexesof certain metals (especially Ca, Mg andCu) with organic acids produced by the mycobiont.

Lichens effectively trap airborne particulate matter (Figure I) both from naturalsources (e.g. soil, volcanic eruptions, sea salt aerosols, wild forest fires and biogenicvolatile non-methane hydrocarbons) and anthropogenic sources (mining, smelting andmetallurgical activities, combustion of fossil fuels, incinerationof refuse, cementproduction, etc.).

There is much direct and indirect evidence for particle trapping by lichen thalli.Indirectevidence includes an increase in ash and metal content in samples growing nearparticle emission sources [8, 13, 58]. The presenceofmetal-rich particles on the thallussurface and in intercellular spacesof the medulla has been demonstrated by SEM andelectron microprobes [33, 34, 73] (Figure 1). How particles reach these spaces is notclear. However, features of the thallus surface such as the absence or presenceof acortex, roughnessof epicortex, and sizeof pores could play a significant role in the

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amount and size of trapped particles. Comparing the chemical compositionofairborneparticulates and that ofParmelia caperata andP. rudecta collected around a coal-firedpower plant, Olmezet al. [63] found that the thalli did not collect all size particles withequal probability. Larger particles, probably deposited by impaction and/or drydeposition, were preferentially accumulated. While it is clear that trappingof large­sized particles allows the accumulationofextremely high concentrations of potentiallytoxic elements in lichen thalli, the significanceofPMIO's remains largely unquantified.Garty [33] suggested that the coefficientofvariation of lichen metal content in a givenlocality may be due to different sizes of metal particles. A low coefficientofvariationindicates a homogeneous spatial distributionof small particles whereas a highcoefficient indicates localized deposition of big-sized particles.

Figure J. Accumulation of aerial particulates from smelter emissions by the crustose lichen Acarosporasmaragdula.Zlatna, Romania. A) Pb-rich PMlOs on thallus surface (arrowed). B) Pb X-ray map ofsection ofA .smaragdulaapothecium (after [73J).

Higher concentrationsofelements are mostly found in older central partsof folioselichen thalli [8, 39]. This accumulation pattern could be at least partly due to theprogressive uptake of particles. This has important implications for biomonitoringbecause it means that thalli (or partsof thalli)ofthe same age must be usedif elementconcentrations are to be compared in different lichen samples [3].

Many large-scale surveys have found that most particles trapped by lichens aresimply soil and rock dust especially in so-called'clean' control and remote areas(Figure 2). This is readily demonstrated by the high, intercorrelated concentrationsofAI , Ti, Si and other lithophilic elements. Bargagli [5] showed that irrespectiveofmacrolichen species, average regional background concentrationsof various elementsquoted in the literature increased in proportion to AI, Ti or Fe content, from forest to

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more open environments such as farmland, upland prairiesand tundra. Thus in order toestimate concentrations of elements derived from anthropogenic sources in samplescollected in different environments, or in the same site but at different times, the effectof soil contamination must be minimized. This may be accomplished by normalisingraw concentrationsof elements in each lichen sample to residual ash content or toconcentrationsofsoil tracers such as AI, Ti, Si, Zr and Sc [5].

100000

10000 ECo

.9:1000 c

~100 e

CGlU

10 c0o

4 Cr

Figure 2. Average concentrations of elements in Parmelia sulcatathalli from background areas, differentlyaffected by soil contamination ofsamples (data from [37, 46. 8]for control areas I. 2.and 3-4.respectively).

Entrapped particles may remain unaltered within lichen thalli for long periods.However, even relatively insoluble compounds may over time be partly dissolved byorganic acids, or in polluted regions possibly by acid deposition, and may affectmetabolic processes (20). A wide rangeof particles may also be formed within or onlichens, particularly by crustose lichens growing on metal-rich rocks, where a widerangeofmetal-rich crystals occur, including relatively insoluble extracellular substancessuch as metal oxalates (e.g. Ca, Cu, Mn, Mg and Fe oxalates) and metal - lichen acidcomplexes (e.g. Cu-norsticticacid and Cu-psoromic acid [26, 72, 77]). Though lichensare known to alter rock chemistry, the quantitative significance of element uptake andrelease from the substrate by acid lichen compounds is unclear. Formationof copperoxalate was attributed to absorption throughrun-offrather than extraction from the rockitself[71].

3.2.EXTRACELLULAR BINDING OF CATIONS

Cations may bind to extracellular exchange sites in the cell wall and the outer surfaceofthe plasma membrane. The process is accompanied by a release of protons. Cation­binding is a rapid,passive physico-chemical process (18, 53] and may increase with celldeath, presumably by exposing exchange sites previously protected by the cellmembrane [75]. Few studies have attempted to identify the precise binding sites and

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cations are assumed to bind to a varietyof functional groups including hydroxyl,phosphate, amine and sulphydryl groups [18]. Both thephotobiontand mycobiontmaybe involved in cation binding; however, since the fungalpartnerconstitutes the bulkofthe thallus, most cations bind extracellularly to fungal cell walls [18]. Many free-livingfungi, especially filamentous species, are known to be effective biosorbentsof metalsand are attracting the attentionof biotechnologists[31]. The photobiontpresumablyplays a proportional role in the bindingofcations, because algal cells also have a highaccumulationcapacity [59].

Extracellularbinding is a reversible process and cations bound to exchange sites aredisplaced by cations with a higher binding affinity or higherconcentrationsbut loweraffinity. Metal ions are classified according to their ligand binding affinities andpolarizing power (charge/radius ratio) since such properties can determine biologicalactivity [55]. 'Class A' metals have a preference for O>N>S donors,'Class B' ,having apreference for S>N>O and borderline metals fall in between [55]:

• Class A metals: A1 3+, Ba2+, Ca2+, K+, Mg 2+, Na+andSr+

• Class B metals: Ag+, Cut, Hg2+and Pb2+

• Borderline: As2+, C02+, crt, Fe3+, Mn 2+, NiH, Sn2+, rt",V 3+ and Zn2+

Competitionexperiments have established a sequenceofion affinities for exchangesites:monovalentClass A< divalent Class A< borderline divalent < divalent Class B[53].The reversibilityofcation binding has important implications forbioaccumulationstudies because the contentof elements bound to exchange sites mainly reflects recentatmospheric deposition and may be readily exchanged by cations dissolved in rain or byshort-term exposure to pollutants. The predictablereplacementof one element byanother has been used to study movementsofmetals from cell wall to cell interior andthe effectsofdesiccation on intracellular metalconcentration[20].

The significanceof passive extracellularbinding in uptake and/or releaseofelements in lichens is indirectly shown by comparable ratios between theaccumulationfactors for Co, Sc and Zn in transplanted thalliof Parmelia sulcata and in "rag" (amuslin-likecloth impregnated with a resinous material) exposed at the same site [79).

3.3. INTRACELLULAR UPTAKE

In contrast toextracellularuptake, intracellular uptake is a slow and selective, energy­dependent, plasmamembrane-controlledprocess [18]. Despite the greater biologicalsignificanceof intracellular elements, few data are available on their uptake by lichensunder field conditions. Most results are from laboratory experiments and theirsignificance for field studies is unclear. Beckett and Brown [12] suppliedPeltigerathalli with Cd and found that intracellular uptake increased linearly over time,substantially dependent on temperature and stimulated by light. Because the Cd uptakewas competitivelyinhibited by other cations,particularlyMg, it was postulatedthat Cdutilised the carrier transporting Mg into the cell. These results were obtained using asequential elution procedure involving washing lichens with deionized water (to removeintercellularsoluble elements),displacementof exchangeableelements by a divalentcation and releaseofintracellular elements with total digestion in aconcentratedacid. A

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new step wassubsequentlyintroduced to release solubleintracellularelements andthoseexchangeablybound atintracellularsites [20].

It is important tounderstandthe relativeproportionsof extra- and intracellularelementsin lichens to evaluate the potential toxicityof accumulatedelementsunderfield conditions.

3.4. UPTAKE OF ANIONS AND NITROGENOUS COMPOUNDS

Few studies on anion uptake by lichens have been carried outcomparedwith those oncation uptake. Only phosphate[27], sulphite [28] and arsenate [57] uptake have beenstudied in any detail owing to thedifficulty in obtaining reliable measurements.Mechanisms of arsenate uptake by fungi,bacteriaand algae have been found to besimilar to thoseof phosphate[76]. The uptakeof these two anions by lichens is anactive process and hence their accumulation by dead thalli is negligible. The existenceof at least twoindependentsystems for arsenate uptake has beensuggested,whichfunction atdifferentarsenateconcentrations[76]. Studiesofanion uptake from complexsolutions have showncompetitionby sulfite andphosphateon arsenate uptake andenhancementby sulphate [57]. These results may haveecologicalimplications,becausethe environmentalbioavailabilityof phosphatesmay reduceaccumulationof toxicsulphite and arsenate ions. Alternatively, increased sulphate content in industrial areasmay favourphosphateuptake [76].

Both active and passive sulphite uptake has beensuggested for lichens withprevalentlypassive adsorption[28]. Extracellularanion exchangehas beenproposedwith proteins aspossiblecandidates for binding sites in fungal cell walls. Comparisonof uptake of cationic, neutral and anioniccomplexesof the uranyl ion byCladinarangiferina suggestedthat the cationic form is most readily taken up from solution,whereas the neutral and anionic forms are absorbed withintermediateand very lowuptake capacities [15]. The cationic form behaves like metal cations bindingextracellularly. Like arsenate, the uptakeof the anionic formofthe uranyl ion shows abiphasic pattern and occurs intracellularly. Solutionscontainingthe anionic formoftheuranyl ion have therefore been considered to be the most toxic for lichens [16].

Nitrogencompoundsas NH3 (orNH/ ions) and nitrates may bedepositedon lichensurfaces by wet and dry deposition. Lichens assimilate both formsofN from solution,but themechanismsand ratesoftransport are not wellunderstood.NH 3 is usually moreeasily absorbed than nitrate.Ammonium ions are initially bound to the cell wall andthentransferredto thecytoplasm[22].

4. Factorsaffectingaccumulationrate

4.1. INTERSPECIFIC VARIATION

Both particle trapping and ion uptake from solutions depend to a large extent onphysical aspects such as thallus type (fruticose, foliose or crustose) and othermorphological features such asbranching, wrinkling and roughness. Anatomicalfeatures such as the sizeofpores in the epicortex and densityofhyphae in the medulla

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may also influence the sizeoftrapped particles and their localisation and redistributionin the thallus. However, there have been few field studiesof the comparative uptakeability of different lichen species living in the same habitat. Differences in the contentof certain elements in species groups(Cladina vs. Thamnolia) were found in pristinelocations in Canada [54]. Addison and Puckett [1] reported significantly higher S-levelsin Evernia mesomorpha than in Hypogymnia physodes. Although morphologicalfeaturesof E. mesomorpha may enable more efficient trappingof gaseous andparticulate formsof S, differences in habitat (twigs and trunks) could alsoplayarole.Comparison of element uptake by morphologically similar speciesofParmelia did notreveal any clear trend [8]. In the genusCladina, small but statistically significantinterspecies differences in element concentrations have been reported in several studies[47, 64, 70]. These differences may reflect differences in thallus surface structure suchas the presence or absenceofan epicortex.

In addition to species morphology, the photobiont species appears toplayarole inaccumulation ability. In general, cyanolichens have higher concentrationsof manyelements than do lichens which contain green algae. The latter usually have morehomogeneous element spectra than cyanolichens [54]. Many cyanolichens have agelatinous thallus and their ability to reach higher levelsof saturation may facilitateenhanced deposition of aerosols.

4.2. INTRASPECIFIC VARIATION

The best way to avoid species-specific variations in element uptake and accumulation isto select the same foliose or fruticose species throughout the study area for a particularstudy. Important criteria to consider when selecting species are: (a) wide distributionthroughout the region; (b) relative resistance to gaseous pollutants, (c) capacity toaccumulate and retain metals (d) sufficient material should be available for replicateanalyses.

However, even selecting a single species does not necessarily guaranteereproducible results unless a rigorous sampling procedure is adopted. A recommendedmethod is given in chapter 23, this volume.

4.3. TEMPORAL VARIATION

The typical occurrence of higher element concentrations in older partsofthalli suggeststhat lichens retain elements efficiently and behave as time-integratorsof persistentatmospheric pollutants. However, retrospective studiesof element deposition by usingthis zonation pattern are premature [6] as an understandingofthe dynamicsof uptakeand leaching processes for each element in different-agedpartsofthe thallus is required.In reality, the thallus cannot be regarded as a medium where acquired elements remainunchanged and cannot be released. Although some data exists on ratesof elementtransformation and leaching, lichens are known to modify the chemistryofrainfall as itpasses over them. Pilegaard [66] found a significant negative correlation betweenrainfall and the contentof the several heavy metals in the thalli ofHypogymniaphysodes and Dicranoweisia cirrata. Puckett [68] explained a seasonal variation inmineral contentof lichens by dynamics of snow melting. The initial melt water is

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enriched with mineral substances and causes a sharp increase in element concentrationsin thalli, but subsequent melt water may leach out more elements.

In monitoring studies, it is important to know how quickly a lichen species reflectschanges in metal deposition patterns.Most studies dealing with temporal changes in theelement concentrationsof lichens rely on background values in herbarium material e.g.lead following introduction of lead-free petrol. However, pre-operational surveysofelement concentrations in lichens have become quite common in recent years, as theresultofa requirement for environmental impact statements prior to plant construction[69]. Temporal trends of element concentrations in lichens from industrial[32,40]andremote areas have been reported [14].

From transplant studies, we know that many lichen species respond to an increasein atmospheric pollution within a few months. With regard to falling environmentalpollution levels, several studies have reported a decrease in element concentrations inlichens [78, 89, 92]; however it is not clear whether this decrease reflected lossofpreviously accumulated elements or reduced content in tissues formed after thereductionofemissions.Deruelle [25] reported that Pb accumulated by lichen transplantsnear a road was lostin several months when the thalli were returned to the backgroundsite. According to Brown and Brown [20], this loss may be due to rainwater washingparticles from the thallus surface.

In general, field studiesof temporal changes in metal levels in lichens show thatvalues decrease after 1-2 years once emission ceases and the residence timeof manyelements in thalli is 2-5 years [92]. However, these estimates are only indicativebecause the residence time is affected by many internal and external factors, such asgrowth ratesof thalli, element speciation and location in cell fractions, as well asmeteorologicaland environmental conditions.

4.4. SPATIAL VARIATION

Spatial variations in the elemental compositionof lichens may reflect spatial changes inpollutant deposition (at different scales) as well as variations in the accumulationcapacityofthalli (for a given element load).These variations are mainly due to climaticand environmental conditions, which affect metabolism, growth rate and desiccationofthalli, depositionofelements and their availability. In background areas the compositionof lichens may change with altitude [4, 42] probably because the amount ofprecipitation and depositionof long-rangetransported elements such as Pb, Cd, and Znincreases with altitude.

Local topography and vegetation structure also significantly affect pollutantdeposition [82]. Single trees or trees in open woodland obviously obtain more particlesthan those in high dense stands.Takalaet al. [87] measured higher F concentrations inlichens from pines and birches than spruce and suggested that samples from sprucetrunks were sheltered from particulate F by twigs. Mechanical sheltering from pollutantswas also described by Perkinset al. [65] for epilithic lichens growing on different partsofawall.

Element contentofepiphytes may also depend on their height on the host tree (withhigher concentrations lower down [84, 87]) or on one sideoftree trunk [86]. In additionto the increasing availability of moisture and soil particles near the baseoftree trunks,

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these variations are due to the pattern and elemental compositionof stemflow. In fact,concentrationsof elements in precipitation increase as they move through the treecanopy, in the order crowndrip<throughfall<stemflow [2]. Tree inclination is alsoimportant for stemflow pattern. The upper partofa tree gets a large portion of rainwaterbut drying or desiccation is also faster. On strongly inclined trees with smooth bark,rainwater may collect in drops on the underside. Different microclimatic conditions ondifferent parts of the trunk (faster desiccation on the southern side, lower evaporationrates, sheltering by grass or epiphytic moss and long periodsof snow cover near thebase) may determine spatial variations in the elemental compositionoflichens.

Thus, standardized sampling procedures should include forest type, species ofphorophyte, degreeof tree inclination, position on trunk, bark morphology,microclimatic conditions and health of thalli. As snow, rainfall and drought affect theelemental compositionof thalli, the sampling period should be harmonised to makeresultsof time series studies or between different environments more reliable. Despitestandardized sampling procedures, even in homogeneous lichen populations, elementconcentrations may be quite variable ("biological variability"). As the frequencydistributionofmeasured values is close to normal [61], this variation is stochastic andthe representativenessofthe sampling can easily be improved by collecting a numberofindividual thalli in each sampling site [60].

5.Transplantstudies

Lichen transplants are used for monitoring air pollution especially in areas where nosuitable indigenous lichens exist. Relatively large foliose or fruticose lichens aregenerally used for trace element biomonitoring. Foliose species are preferable becausethey are generally more tolerant of gaseous pollutants. The general schemeof thisbiomonitoring approach is as follows: piecesofbark or twigs covered by healthy lichenthalli are transferred from unpolluted to polluted areas. Progressive accumulation ofelements is measured after definite exposure periods. Alternatively, lichens packedloosely in a fine nylon net ("lichen bags") were also exposedin several urban andindustrial environments (see [23, 36, 49] and chapter 24, this volume).

The main advantageof the transplant technique over collection and analysisofnaturally growing thalli is that this procedure can be standardized to a high extent.Standardization includes collecting transplanted material from the same typeofsubstrate with the same thallus size, and placing transplants in sites with similarmeteorological and environmental conditions (including position, orientation andexposure time).

A major problem of transplant surveys is establishing an appropriate exposureperiod. In heavily polluted environments, transplanted lichens may become saturatedwith elements and their surface structure and physiological performance may besignificantly altered. In addition to the accumulationof persistent atmosphericpollutants, many parameters can be measured for an indirect estimateofrelative levelsand biological effectsofatmospheric pollutants. These parameters include: chlorophyllcontent and degradation, ATP decrease, respiration and photosynthesis rate, increase inmembrane leakage, and peroxidation of membrane lipids [36]. Cell membrane damage

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in exposed lichens has been detected long before any indication of damage becomesapparent in the photobiont chlorophyll [35]. Since this damage may cause loss ofelements during rain, it is not surprising that concentrationsof some elements inexposed lichens may decrease.Thus, the exposure time should be established accordingto the purpose of the survey, climatic and environmental conditions of the study areaand the expected degree of stress for the selected species. In general, a nearly linearincrease in element bioaccumulation occurs in the firstfew weeks and most surveys arebased on one to three month exposure periods. However, under suitable environmentaland climatic conditions, longer periodsofexposure have also been recommended [85].

6.Lichen toleranceto inorganiccontaminants

Exposure of natural vegetation communities to increasing metal bioavailability initiatesselective processes among the species and only some will be able to adapt and survive.In cryptogams and higher plants, adaptation to metal pollution is quite rapid as it hasbeen observed at sites of recent (last 30 years) contamination. This tolerance is agenetically based phenomenon; probably, it is not metal-specific and in higher plantsshows a continuous gradation (from weak to strong) among ecotypes, physiotypes orraces [48]. In lichens metal tolerance has been demonstrated by field and laboratoryinvestigations. One example is the existence of natural undamaged populations nearemission sources where transplants showed signs of injury after several monthsofexposure [83]. Saxicolous lichens growing on metalliferous rocks have severalbiochemical mechanisms for metal detoxification, mainly consisting in thecomplexationof Cu, Zn, Fe, Mn, Mg and other cations by lichen substances anddepositionof the resulting compounds on the thallus surface and in the upper cortex[72]. Sarretet al. [77] studied the speciationofPb and Zn by powder X-ray diffraction(XRD) and extended X-ray absorption fine structure (EXAFS) spectroscopy and foundthat hyperaccumulation of metals inDiploschistes muscorum was achieved by theproduction of oxalate. Besides lichen acids and oxalate, other ligands such as citrate,malate and malonate, which have also been proposed as metal ligands for fungi [31],may also be involved in the immobilization and excretion of metals by lichens.However, not all lichen species growing in the same environment contain similarcompounds within their thalli. Therefore, this way of exclusion of potentially toxiccations is not a universal tolerance mechanism [18, 71].

Comparative laboratory investigations inPeltigera revealed reduced intracellularCd uptake by thalli from contaminated localities compared to those from backgroundsites, whereas the rate of extracellular bindingwas similar [12]. However, the reasonsfor reduced intracellular uptake are not well understood. Metal exclusion mechanismssuch as an altered transmembrane carrier system have been proposed, but were notconfirmed by following investigations [93]. Extracellular binding of large quantitiesofnative mineral ions has been hypothesized as a mechanism limiting inputof toxiccations to cell [93]. Decreased intracellular uptake was supposed as a reason for higherresistanceofphotosynthesis inPeltigera collected from contaminated sites than in thallicollected in unpolluted areas [19]. Resistance was assessed as the ratioofphotosynthetic rates after metal and after water treatment under laboratory conditions.

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Levels ofZn tolerance were proportional to the initial (field) Zncontentof the thalli.Resistanceofphotosynthesisin samples fromuncontaminatedlocalities was artificiallyraised by Znpre-treatmentone week before an experiment [19].

These results suggest that at least part of the metal resistance in lichens may bephenotypicallyacquired. Different species or communitiesof lichens may developdifferentdetoxificationmechanisms. However, in most speciesof foliose lichens usedin biomonitoringsurveys these mechanisms essentially consistof accumulationofpotentially toxic elements in the fungal cell wall and decreasedintracellularmetaluptake.

Goyal and Seaward [38] described some morphological and histological alterationsin Peltigera thalli from highly contaminated localities and interpreted them in termsofadaptations. An increase in the thicknessof the outer cortex and medulla, and higherdensity of rhizines may provide effective accumulationof metals in themycobiontpreventingdamage to the more sensitive photobiont. However, according to Beckett andBrown [11] these modifications could also be due to water regime, becausePeltigerathalli were collected from moss cover under background conditions and from naked soilat the mine site. Studies onHypogymnia physodes from urban sites [41] revealed theopposite, namely a decrease in cortex thickness and an increase in thicknessof thephotobiont layer.

7.Metal toxicityandtypicalconcentrationsof traceelementsin lichens

Toxic effectsofmetals on lichens under field conditions have been reported as suddenor gradual changes in lichen morphology [38],or as an increase in speciesabundanceorbiodiversity with increasing distance from emission sources [17, 29]. Along a metalpollution gradient, several hundred ppmofCu, Pb and Zn were reported as thresholdsfor the survivalofHypogymnia physodes and Parmelia squarrosa [74]. However, moststudies give rather ambiguous examplesof metal toxicity because they are based ontotal metalconcentrationsand disregard other environmental factors. Changes in lichenmorphology,biodiversityand element accumulation capacity are almost certainly alsoaffected by gaseous pollutants, especially S02, acidification and climatic factors. Theonly reportsof lichen death which can convincingly be ascribed to metal toxicity arethose in extreme environmental conditions such as beneath copper or barbed wire orgalvanised supports [6].

Field studies on metal toxicity require a detailed knowledgeof physico-chemicalforms and cellular locationof metals in the lichen thalli. Branquinho et al. [17] forinstance, studied physiological effectsof Cu-dust inRamalina fastigiata near a mineand they found that although the intracellular Cu uptake was muchsmaller(6%) thanthe extracellular, it explained the physiological changes and the survivalof the speciesin thesurroundingsofthe copper-mine.

In general, owing to the uptakeof particulate elements and theimmobilizationofsoluble forms in fungal cell walls, several speciesof epiphytic lichens are relativelytolerant to atmospheric pollutants and can be used asbiomonitors in urban andindustrialenvironments. As a rule, species having a wide ecological range such asHypogymnia physodes, Evernia prunastri , Pseudevernia furfuracea, Xanthoria

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parietina and several species of the genusParmelia s. lat. are among the mostfrequently used. In northern boreal forests and tundra, epilithic and epigeic lichensofthe generaCladonia and Cetraria have been extensively used and some speciesofUmbilicaria, Xanthoria and Usnea may even enable biomonitoringsurveys in remoteand extreme environmentsofthe Arctic and Antarctic [9,30).

The introductionofvery sensitive instruments for trace and ultratrace analysis andthe progressive standardizationof sample collectionand preparationprocedures make itpossible to establish contemporary background element concentrations in lichens fromdifferent regions (Table I). In general, samples of epigeic and epiphytic lichensrelatively unaffected by soil contamination have concentrations of elements in the rangeofTable I. Baseline concentrations of trace elements are necessary in order to assesspollution levels. As a rule, in most biomonitoring surveys baseline concentrationsofelements in the selected lichen species are estimated indirectly by analysing samplesfrom control areas. However, the values in Table I may be useful to establish whethervalues measured in abiomonitor species are clearly indicativeof environmentalpollution (e.g. being ordersof magnitude higher) or whether correction for soilcontaminationofsamples is necessary (see chapter23, this volume).

TABLE J. Background concentrations ofelements (pg e' dry wt.) in lichens (* = slightly contaminated by soilparticles) .

Italy [60] Hungary [87] Antarctica[9] Europe and European part ofEpiphytic Epigeic Epi1ithic America [14] Russia [89]*(Parmelia, (Cladonia (Umbilicaria Epiphytic/epilithic EpiphyticXanthoria ) convoluta) decussata ) (Hypogymnia (Hypogymnia

physodes) physodes)AI 350.0 185.0 180.0 363.0 638,0As 0.3 1.4 1,3Sa 3.3 2.5 23,0Cd 0.2 0.3 0.1 0.2 0,2Cr 1.2 0.4 0.5 1.7 2,2Cu 7.0 7.3 3.5 4.3 10,9Fe 290.0 197.0 200.0 480.0 664,0Hg 0.1 0.1 0,3Mn 20.0 9.4 9.0 35.0 149,0Ni 1.0 1.1 0.9 12.0 3,4Pb 4.0 7.4 0.3 28.0 8,6Ti 10.0 3.0 7.0V 0.6 1.1 0.7 1,5Zn 30.0 31.0 10.0 44.0 72,0

8.Di fferentiating sourcesof elements foundin lichens

As previously discussed, the elemental compositionof lichens depends not only onconcentrations of airborne pollutants, but also on climatic and environmentalconditions, which affect the metabolism and growth rate of lichens, and the depositionand availabilityof trace elements. The variability of results istherefore a probleminherent to biomonitoring studies and it is difficult to establish whether slightly elevatedlevels of one or more elements in lichens are due to intrinsic biological variability,natural sources of elements or local or remote human activities.Unfortunately, element

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concentrations in wet and dry deposition may only beof limited help ininterpreting theresults of biomonitoringsurveys. In fact, theaccumulationof airborne particles inlichens through interception, impaction or sedimentation differs from that in surrogatesurfaces in automatic samplers. Element pools in lichens and artificial collectors alsohave different temporal significance. Instrumental data often reflect element depositionrates over short or irregular time periods, whereas lichens aretime-integratorsofpersistent atmospheric deposition and the age (exposure time)of the thalli analysed isoften unknown. Besides, elements may be washed away by rain and snow and inpolluted environments saturationof extracellularbinding sites or physiologicalimpairment may prevent further uptakeofelements by lichens.

Isolated point emission sources are the easiest to identify by lichen monitoring, butin urban and industrial areas or in large-scale surveys there is a rangeof possiblesourcesof elements. In general, these sources are identified on the basisof elementdistribution patterns in the study area and by comparing the elementalcompositionoflichens with emissions from potential sources.

Enrichment Factors (EF) with respect to crustal composition[70] or to thecompositionofsurface soilsofthe study area (analyzed by the same procedures used forlichens [3]), have often been used to estimate anthropogenic contributions to theelementalcompositionoflichens. The general formulaofEF for elementx is:

EF = ([x]/[referenceelement])in lichens

([x]/[referenceelementj)in crust(1)

where AI, Ti, Sc and other lithophilic elements may be the reference element. As a rule,the more the EF values approximate unity, the more the elementofconcern derives itssource from soil particles, rather than from anthropogenic sources.

Besides determining the correlation coefficients between pairsof elementconcentrationsin the thalli, multivariate techniques such as factor analysis and principalcomponentanalysis have been applied to lichen monitoring data to identify sourcesofelements (e.g. [7,45,56,67,80]).

Isotope studies are another methodofdifferentiatingofelement sources in lichens.The impactof a point sourceof S has been detected over a distanceof severalkilometres [44] and relative inputsof natural andanthropogenicsources of Sdistinguished in lichens from regions with naturally high atmospheric Sconcentrations[91]. On the basisof Pb isotope composition,Monna et al. [51] recognized two mainanthropogenic sources and"natural"Pb contamination from active volcanoes in lichensfrom eastern Sicily.

Some sourcesof the most widespread inorganic contaminants aresummarizedinTable 2. The major source types have been classified in different ways and possiblesecondary associations between elements may change their profile under differentenvironmentalconditions. The impactofoil combustion andprocessingofoil products,for instance, are usually assumed to be reflected in lichen thalli by an associationbetween V and Ni concentrations. However, both elements may also beof crustalorigin. Associations between Br, Hg, Cd, Fe, Se and other elements are generallyconsideredto indicate emission from coal-fired plants, but coal fly ash has a chemicalcompositionquite similar to crustal materials and thebioaccumulationofelements such

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as Hg, Cd, Zn or Pb in lichens may also come from long-range atmospheric transport.Many "new" elements are increasingly entering our environment. Some lanthanoids

are componentsof alloys for magnetic materials and new high-temperaturesuperconductors. The installationofcatalytic converters in motor vehicles is expected toresult in emissionof elements such as Pt, Pd, Ru, Rh and Ce [50]. The possiblebiological effectsof increased environmental availabilityof these elements areunknown. Lichen biomonitoring will be a valuable adjunct to instrumental monitoringfor evaluating the spatio-ternporal deposition patterns of these pollutants providing anearly warning of their biological effects.

TABLE2.Somepotentially toxicelements and theiranthropogenic sources.

Al

As

Be

Cd

Cr

Hg

Pb

Pd, Pt,Rh,Ru

Se

TI

v

ToxicityAffects root growth and element uptake inplants, P metabolism in animals, implicatedin theAlzheimer'sdiseasePhytotoxic, believed to be carcinogenic,Black-foot diseaseHighly toxic to plants and animals,allergenic, some forms are carcinogenic,may cause ricketsInterferes withphotosynthesis, uptake andtransport of elements in plants.Carcinogenic, ltai-ltaidiseaseCr6

+ may cross cell membrane and is toxic toplants, animals and man, carcinogenicHighly toxic to plants, methylated Hg isbiomagnified in food chains, MinamatadiseaseCumulative poison, carcinogenic andteratogenic, may affect the intellectualdevelopmentofyoung childrenToxicity is not exactly known

Some plant species are accumulatorsof Seand can pose a danger to grazing animals,white muscle diseaseHinders seed germination, affectsphotosynthesis and transpiration.Tachycardiaand hypertensionPhytotoxic, in animals and man can be toxicwhen entering by wayofrespiratory system,carcinogenic

SourcesNon-ferrous metal-works, sheet metal, wiresand alloys

Mining and smelting, coal-firedpower plants,fertilizers, alloys, insecticide, fungicideElectrical insulators, aerospaceapplications,clock springs,X-ray windows, alloys.

Metallurgical plants, refuse incineration,mineral fertilizers, coal combustion,accumulatorsMetallurgy, production of paints, catalysts,tanneriesChlor-alkali plants, coal combustion, smelting,some typesofpesticides

Automobile exhaust (especially in the past),metallurgy, accumulators, cable coating, diecastingsAutomobile three-way catalytic converters

Electroplating, glass industry

Mineral processing, cement factories

Smelters, steelproduction,coal- andoil-burningplants

9.Conclusions

The ability for lichens to accumulate high levelsof elements far in excessof theirphysiological requirements closely correlated with atmospheric levels has led to theirwide-scale application as practical biomonitors of metal contamination. In large-scalesurveys and remote regions where few recording gauges are present, lichenbiomonitoring is oneof the only ways to determine the deposition patternsof traceelements (Figure 3). As a rule, this approach is based on the analytical determination of

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80

total concentrationsofelements in lichen thalli and is successfully applied throughoutthe world.

Clyster 1 10

9

8

7

~._-_ _--_ .

1~luster 2

I

b

10

9

8

7

. 3

2

o

Figure 3. Mapping element data in Veneto (NE Italy). showing spatial variation of element distributionaccording to two principal clusters ofmetals in a dendrogram. For each cluster. values were normalised andexpressed on a 0-/0 scale (aft er [62]).

Particle trapping and the passive complexationofcations to fungal cell walls seemto be the most frequent uptake mechanisms in epiphytic species most frequently used inbiomonitoring surveys.

Particles and elements bound to exchange sites may be lost (especially in desiccatedthalli) when climatic and/or other environmental conditions change. Environmentalfactors may also contribute to the variabilityof element concentrations within thallibecause they may affect lichen growth rate, element availability and the trappingofairborne soil particles. Thus, the selection and analysisofthalli (or portions of thalli)ofa similar age and according to strict sampling protocols is a prerequisite to theirsuccessful use as predictive quantitative biomonitors of inorganic contaminants.

As particles and soluble elements may also be derived from a variety of naturalsources, it is essential to distinguish man-made perturbations from intrinsic biologicalvariability and natural sources of inorganic contaminants.

Modern analytical systems for multielement determination at trace and ultratracelevels, in parallel with SEM/electron probe microanalysis and other techniques, providevaluable new opportunities to understand mechanisms of aerial particle uptake andpossible release under different environmental conditions.

Particulate characterisation, localisation, quantification, redistribution and/ortransformation(including element complexation by organic substances) may be studiedin small samples.An understandingof the effectsof different metal compounds, theirphysico-chemical forms and/or cellular location on lichen physiological processes willenhance the roleof lichens as sensitive biomonitors of the atmospheric depositionofinorganic contaminants. Itwill also provide an early warning system of their biologicaleffects in terrestrial ecosystems.

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88. Tuba, Z.,Csintalan, Z.,Nagy, Z.,Szente, K., and Takacs, Z. (1994) Samplingofterricolouslichen andmoss species for trace element analysis, with special reference to bioindication of air pollution, in B.Markert (ed.),EnvironmentalSamplingfor TraceAnalysis,VCH, Weinheim, pp.415-434.

89. Vestergaard N.K., Stephansen, V., and Rasmussen, 1. (1986) Airborne heavy metal pollution in theenvironmentofaDanish steel plant,Water, Air and Soil Pollution27, 1807-1814.

90. Vtorova, V.N. and Markert, B. (1995) Multi-element analysisofplants of forest ecosystems in EasternEurope,lzvestiya RAN, SeriyaBiologicheskaya 4, 447-454.

91. Wadleigh, M.A. and Blake, D.M. (1999) Tracing sources of atmospheric sulphur using epiphyticlichens,Environmental Pollution106,265-271.

92. Walther, D.A., Ramelov, GJ., Beck, J.N., Young, J.C., Callahan, J.D., and Marcon, M.F. (1990)Temporal changes in metal levels of the lichenParmotremapraesorediosumand Ramalina stenospora,southwest Louisiana,Water, Air and Soil Pollution53, 189-200.

93. Wells, J.M.,Brown, D.H.,and Beckett, R.P. (1995) Kinetic analysis of Cd uptake in Cd-tolerant andintolerant populations of the mossRhytidiadelphus squarrosus (Hedw.) Warnst. and the lichenPeltigeramembranacea (Ach.)Nyl.,New Phytologist129,477-486.

94. Winner, W.E.,Atkinson, CJ., and Nash, T.H. III (1988) Comparisonof S02 absorption capacitiesofmosses, lichens and vascular plants in diverse habitats, in T.H. III Nash and V. Wirth (eds.),Lichens,Bryophytesand Air Quality,Bibliotheca Lichenologica 30, J. Cramer, Berlin-Stuttgart, pp. 217-230.

95. Wittig, R. (1993) General aspects of biomonitoring heavy metals by plants, in B. Markert (ed.),Plantsas Biomonitors.lndicatorsfor HeavyMetals in the Terrestrial Environment, VCH, Weinheim, pp.3-27.

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LICHENS AS MONITORS OF RADIOELEMENTS

M. R. D. SEAWARD

Department ofEnvironmental Science, University ofBradford, BradfordBD7 1DP. UK ([email protected])

1. Introduction

Lichens are extremely efficient accumulatorsofchemical elements which are taken upfrom substrate solutions, deposited aerosols and rain; their thalli, particularly those withsoralia or isidia,provide effective surfaces for uptake. In some cases, lichens can beseverely affected by heavy metals, the latter's presence in all probability exacerbatingthe damaging effects of sulphur dioxide pollution [25, 26,44], and strong correlationsbetween pollution level, lichen distribution and chemical uptake are often observed. Towhat extent manyofthese elements are usefully employed in the lichen thallus is as yetunknown [76]; many lichens certainlyshow a resilience to heavy metal uptake, andhigh concentrations have been determined in species growing on metal-enriched soils[61,68].

Lichens have been shown to be highly effective monitorsof atmosphericcontamination from urban, industrial and automobile sources (see chapter 6, thisvolume). The slow metabolic activity and slow growth rateof lichens make themdepositories for, and monitors of, elements in prevailing environments. Before dataassembled from "polluted" sites can be appreciated, one must be awareof existingbackground levels; such baseline studies can reveal that there are often appreciably highand variable levelsof elemental accumulation in"control" material. These levels varyfrom habitat to habitat and from species to species. The proper useof lichens asindicators and samplersof ambient conditions is a valuable resource for theenvironmentalist [64, 65]. Lichen bioassays can be used as a technique for the periodicsampling of environmental elements, including retrospective field sampling by thecareful use of well-localised and dated herbarium material. Lawrey and Hale [39], forexample, have shown that the lead contentofone species sampled at 300 m from thesite of a road-bridge, constructed over the Potomac River, USA in 1965, to be 82 ppmin 1907, 128 ppm in 1938,343 ppm in 1958 and 1893 ppm in 1978. The higher leadcontent in 1958 is indicativeofan overall rise in atmospheric background levels, but thedramatic increase in 1978 is due to the new road traffic burden.

Lichens are also renowned for their ability to accumulateradionuc1ides;more than40 years ago,Gorham [24] reported that lichens in the Lake District,England containedthree times more radionuclides than higher plants. Even in remote areas, such as theArctic (e.g.[9, 74]) and the Antarctic (e.g. [5, 23, 57]), lichens efficiently accumulateand monitor fission products, particularlyl37es,atmospherically, and to a lesser effect

85P.L. Nimis, C. Scheidegger and P.A. Wolseley (eds.). Monitor ing with Lichens - Monitoring Lichens. 85-96.© 2002 Kluwer Academic Publishers . Printed in the Netherlands.

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stratospherically, derived from nuclear weapon testing and nuclear power plantmalfunction (cf. [1]). 137Cs and90Sr are the most harmfulof long-termcontaminants toman; they are readily absorbed by living material since they are chemically related tothephysiologicallyimportant K and Ca. They have received more attention in lichenstudies than any other radionuclides due to their role in thelichen>reindeer(caribou)>human food-chain. Not surprisingly, greater interest was shown in the useof lichens asbiomonitorsofradionuclides consequent upon the Chernobyl disaster (see below).

The efficient retentionof137Cs, with biological residence times of at least 5-8 years,is probably due to absorption by the mycobiont of a lichen in order to satisfy potassiumrequirements: an inverse relationship exists between137Cs and4°K activities, and a high137Cs uptake may reflect a K-poor substrate. The biological half-lifeof 137Cs inXanthoria parietina, for example, has been estimated to be 59 months [75], and, at adifferent location, 37 and 29 months forPseudevernia furfuracea and Cetrariaislandica respectively [28]. A dynamic model for radionuclide uptake by a lichen isprovided by Ellis and Smith [19]. Living lichens accumulate137Cs more effectively thandead lichens; Nifontova et al. [48], for example, have demonstrated that uptake byliving Umbilicaria is 28 times greater.

Differential accumulation by various species and ecological groupsof identicalterricolous species in different plant communities has also been shown. Gaare [21]found very large variations inCetraria nivalis within short distances (a metre or so) for134CS and I31Cs; increases occurred both with altitude and with exposure to wind (e.g.ridgetops), while differences betweenC. nivalis and Cladonia stellaris were related tosmall but important differences in their ecology rather than to specific variation inmorphology (i.e. poikilohydric efficiency), and therefore moisture content, at the timeof fall-out. Furthermore,Cetraria nivalis, being freeof snow at the timeof theChernobyl disaster, proved a better biomonitor.

2.Comparativestudies:Practicalitiesand protocols

Many problems face the researcher wishing to make temporal and spatial comparisonsof radionuclide levels. Such information as is available is fragmentary, measurementsprior to the Chernobyl disaster, for example, being almost non-existent for manyregions, particularly in Central and Eastern Europe. The problem is further exacerbatedby the wide variation in analytical methods and techniques employed, and theparticularradionuclides selected for investigation. Furthermore, the results obtained are expressedin a varietyofways, not only in termsofthe units employed but also the wet/dry stateof the samples examined, besides the need to compensate (a) for the decay rateoftheparticular radionuclide since fallout (or indeed calculated to the periodof the initialdisaster/shutdownat the nuclear reactor) and (b) for the increase in the lichen biomassover the monitoring period[49].In additionto all these factors, it is necessary to bear inmind that the lichens would have accumulated both natural and man-maderadioelements prior to the impact being measured and that these will have exaggeratedthis"background"level.

Standardization is also necessary in termsofthe choice of experimental material tobe analysed and the sites from which it is collected; these should as closely as possible

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exhibit uniform physical, chemical and biological properties, and each habitat should besubject to the narrowest possible amplitudeofenvironmental factors. The potential forinconsistency through the use of a numberofdifferent species/genera, each differing inits inherent capacity to accumulate the various radionuclides, and collected from a widerange of substrata and habitats experiencing various environmental conditions is all tooobvious from the literature. Furthermore, lichens are notoriously difficult to identify,and since species differ in their ecological behaviour and their physiological receptivity,taxonomic consistency is an essential prerequisite for any monitoring programme.It isalso desirable to have some knowledge of the physiological status of the material priorto the depositionofthe radionuclides (see e.g. [70]).

Topographical variations between one microhabitat and another can exert asignificant effect on a lichen's ability to accumulate airborne elements; seasonaldifferences, such as snow coverage, also play an important part, and changes in habitatover the monitoring period, such as the developmentof canopy over the site underinvestigation, cannot be ignored. Changes in pH brought about on the one hand byacidification and on the other by excessive useofagrochemicals can affect the mobilityof radioelements, eg137Cs. Although in most cases whole lichen thalli are studied,consideration should be given to the spatial distributionof radioelements within thesamples examined; for example, it has been shown that there is a vertical difference inaccumulation ability forCladonia (subgenusCladina) spp. which reflects the levelofmetabolic activity, but even the thallial parts deemed to be 'dead' are sinks forsignificant burdens [33, 43, 56].

In the lightofthe above potential for inconsistency, any protocol established shouldstandardize:(a) the choice of experimental material to be analysed;(b) the sites from which it is collected, each exhibiting as closely as possible uniform

physical, chemical and biological properties with a habitat subjected to thenarrowest possible amplitude of environmental factors;

(c) the methodofcollection and storage;(d) the analytical techniques employed; and(e) the numerical expressionofthe resulting data.

It should also be appreciated that to determine radioelement levels, particularlybackground and low levels, it is often necessary to use relatively large quantitiesofexperimental material; furthermore, if the programme is on-going, as for example in thecase of Poland (see below), present and future resampling at specific sites must notdeplete the resource. On both counts, this represents a major conservation problem forlichens due to their slow growth and unfavourable reaction to disturbance.

3.Biomonitoring

A wide range of taxonomically and ecologically different lichens have been employedto monitor radionuclides (a) in natural background areas, including uranium mines, (b)from nuclear weapon testing, and (c) from nuclear power stations. The most extensivelyused are terricolous speciesof Cladonia (subgenusCladina), mainly becauseof theirrole in thelichen>reindeer(caribou) > man food-chain: there is considerable concern

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regarding the ecological fate of radionuclides, particularly regarding the composition,growth and productivity of the lichen stands grazed by reindeer and caribou [18, 27, 31,32, 35, 42, 55, 76].

Terricolous lichens have also been widely used to monitor the extent and severityofradioactive contamination of the environment; for example, the highest137Cs levels(ca.2500 Bq kg") in terricolous lichens in Finnish Lapland were found during the mid­1960s; with the decrease in nuclear weapon testing, these had fallen to 74-460 Bq kg"by 1985, but after the Chernobyl disaster, levels up to 2100 Bq kg:' were recorded [56].Higher measurements, as one would expect, were recorded at that time in southernFinland. Background levels of 57-465 Bqkg" for 137Cs were recorded for similar lichenspecies in southwest Poland in 1979 [62]. A post-Chernobyl analysisof thetransuranium variation in Finnish terricolous lichens (unspecifiedCladonia spp.) showswide geographical variation in their deposition values [51], but a pattern emerges froma more detailed investigationof241pu in the southern halfofthe country [50].

Epiphytic lichens have also been employed to monitor radionuclides from nuclearweapon testing and from nuclear power stations; for example, Chibowskiet al. [15],who determined radioactive contamination of unspecified members of the Parmeliaceaefrom 1949 to 1996, have shown the relative impact of nuclear weapon testing and theChernobyl disaster, the137Cs levels peaking in the periods 1960-1963 and in 1986-1989respectively, and in 1978-1979 Nifontova and Kulikov [47] detected gradients inHypogymnia physodes from 650 Bq kg" in the vicinityofa power station in the Uralsreducing to 75 Bq kg" further away. Similarly, Pseudeverniafurfuracea has been usedfor monitoring, and limited comparative data from Austria for137Cs levels in thisspecies before and after the Chernobyl incident are available [16, 29,30].

According to Ecklet al. [17], epiphytic lichens tend to accumulate radionuclides toa lesser extent than do epigeic orepi1ithiclichens, there being a strong correlationbetween the content of the thallus and the substratum on which they grow, but not withtheir growth form. Sawidis [60] measured137Cs levels in the Thessaloniki areaofGreece after the Chernobyl disaster and showed that epiphytic lichens (1110-1706 Bqkg') had accumulated less than had terricolous mosses (5261-18848 Bq kg") and fungi(215-11418 Bq kg,l)whereas Papastefanouet al. [52] showed levelsof 1069-8436(­14560) Bq kg" in epiphytic lichens and 3963-5032 Bqkg" in terricolous lichens (cf.[17]),but only (271-)2064-4749 Bqkg" in terricolous mosses.

Saxicolous speciesof the generaUmbilicaria andLasallia have proved to be idealfor monitoring radionuclides derived from atmospheric sources [37, 38], since theydisplay morphological simplicity and can dominate habitats to such an extent thatreasonable biomasses are available for random sampling without depleting the stockavailable for future comparative monitoring; this is particularly important since lichensare extremely slow-growing (only one to a few millimetres per annum in temperatelatitudes). These lichens occur on saxicolous substrata which exhibit an acceptabledegreeofphysico-chemical uniformity. The thalli are relatively large, usually concave,and have minimal contact with the substratum, but uptake can vary from species tospecies. However, the abilityof a particular lichen to accumulate radionuclides mayreflect its ecological circumstances rather than its morphological, physiological andgenetic characteristics. Although Umbilicaria and Lasallia are apparently ideal

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biomonitors of radionuclides, unfortunately very limited data on them exist, as thesegenera have only occasionally been used for this purpose in European studies.

Earlier investigations into the useof these genera to monitor radioactivity insouthwest Poland[37,38]showed, for example, the effectiveness ofU. cylindrica andU. deusta as bioindicators of137Cs and that the radionuclide contentofthe thalliof allspecies examined increased with altitude. In surveys undertaken in 1978, 1979, 1986,1988 and 1990, material was collected from exposed rock surfaces, from the foothills(380 m) through to the higher altitudes of the Karkonosze mountains (1600 m) at 20sites, and numerous sub-sites, and used for a varietyofmonitoring programmes [66]. Intotal, seven species(U. cylindrica, U. deusta, U. hirsuta, U. murina, U. nylanderiana,U. polyphylla and Lasallia pustulata) were collected, each site supporting from one tofour species; sub-site collections were made to take accountofenvironmental variation,particularly in respectofaspect and exposure.

On 26 April 1986 the 4th unitofa 1000 MWe RBMK pressurized water reactor atChernobyl, 130km N of Kiev went out of control, releasing 3-5% of its radioactivestock into the atmosphere over a 10-day period. Radionuclides, reaching heights up to1500 m, were distributed throughout Europe according to weather patterns, anddeposited in various concentrations according to local precipitation. Radionuclidecompositionofthe fall-out had a characteristic"signature",egoa ratioof134CS : 137Cs. Innorthern Greece it was detected for the first time on 2 May, peaking during 5-6 Maydue to heavy rainfall coincident with the passage of a radioactive cloud, whereas insouthern France it reached a peak on I May, and the contentof airborne radioactivitydecreased by two ordersofmagnitude on 4 May after 10 mmofrainfall. Various"hotspots" throughout Europe were identified - some in press reports- but many wereinconsistent with weather patterns, high rainfall episodes, etc. One major route wascharted through Poland, Czechoslovakia, Austria, northern Italy, southern France andBritain; its general restriction to this pathway through Poland was confirmed by lichenmonitoring.

Radionuclide analyses of the post-Chernobyl collections (1986 and 1988)ofUmbilicaria and Lasallia were carried out using high resolution lithium-driftedGermanium detectors and a Nuclear Data 6620 Multichannel Analyzer system [67].Radioactivity data were normalised for decay to allow direct intercomparisonofspecific activity levels. In the lightof the Chernobyl incident, it was obviouslyappropriate to monitor changes in radioelement contentof lichens at locations stronglysuspected of having been subjected, in varying degrees,to deposition from a radioactivecloud which moved southwestwards over Poland during 28-29 April [71]. Work bySeaward et al. [67] demonstrated that this was indeed the case: the signatureofradionuclides accumulated byUmbilicaria, particularly the ratio134CS : 137Cs (0.54),was consistent with thatof the products accidentally released from Chernobyl, anddistribution patterns of deposition, as delineated from lichen monitoringof 137Cs,highlight pathways and "hot spots".

134CS and 137Cs levels in Umbilicaria and Lasallia were within the ranges <120­18263 and 471-36630 Bq kg" dry weight respectively; the137Cs measurements showedstartling increases (up to 165-fold) over those monitored seven years earlier using thesame species at the same sites, when a rangeofonly 18-226 Bq kg" had been detected[35].Lichens from land below 800 m showed less dramatic increases, the great majority

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of 137Cs measurementsbeing below 2000 Bq kg" and, as on the previous survey, theywere not correlated with altitude over this range. Above 1000 m, there was a highlysignificant correlation between137Cs and altitude ([63], Figure I).

30000

20000

•10000

I• • •• •

• •• • •• <0 '" • ••.. • • • •

I ••I , I , ,

400 800 1200 1600alt.trn) A

Figure J. Relationshipbetween JJ7Cs accumulation (Bq kg·') in Umbilicariaand Lasallia species (from[67j).

The variabilityofthe measurements no doubt reflecting"hot spots" generatedin thewake of the radioactive cloud's southwesterly movement; deposition across theKarkonosze mountain ridge rose dramatically from 2200 to 36630 Bqkg-lover adistanceofless than 9km ([63], Figure 2).

Some local variations in radionuclide accumulation inUmbilicaria are attributableto interspecific differencesof the lichens. Data on localmeteorologicalconditionsduring the period under consideration revealed that southwest Poland was subjected tounusually high levelsof rainfall in upland areas during the period 28-30 April 1986following five daysoflow rainfall, coupled with marked changes in wind direction andvelocity: 1.1 and 73.5 mmof rainfall fell on the highest ground (Sniezka) over theperiods 23-27 and 28-30 April respectively, and 9.8 and 45.1 mm respectivelyfell onthe nearby foothills (Jelenia Gora) to the north over the same periods; zero to lightWSW, Sand SE winds changed to strong to very strong NW and NNW winds on the28 April [62].

These data support the interpretations made by Smith and Clark [71]ofthe routeofoneparticularmajor radioactive cloud which passed over Poland.

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,..5000

CSSR

N

I-----2000-...,

\

5000,"fo 000

--'"\ ,-\ I20 000 20 090, ,," -....- - ....@.:36 000

Figure 2.Distribution olmes in southwest Poland in 1986 as delimited by accumulat ion in Umbi1icariaand Lasallia species (Bq kg·l) (from [67]).S = Sniezka (alt. 1603 m).

Other pathways for radioactive clouds emanating from Chernobyl were postulated,including some passing over Poland (e.g. [40]).In the lightof these interpretations andpress reportsof "hot spots", it was deemed necessary to investigate a remote uplandareaofsoutheast Poland (Bieszczady), 570Ian WSW ofChernobyl, and assumed to bevulnerable to high contamination. Collectionsof U. cylindrica made in August 1988had 137Cs levels (decay corrected to the date of the Chernobyl disaster)of881-4228 Bqkg". Although the characteristic ratio for134CS : I31Cs (0.57) clearly implicatedChernobyl as the source, surprisingly theI31Cs levels were considerably lower thanthose recorded from southwest Poland. These data should be compared with levelsdetermined for lichens obtained from several other European countries[7, 16, 20, 41].

Samples of Umbilicaria crustulosa collected from exposed vertical rock faces inCesme Peninsula, west of Izmir, in August 1988 [62] contained averagesof 610 and1329 Bq kg" (decay corrected to the dateofthe Chernobyl disaster) for134CS and 137Csrespectively (ratio 0.46). It would appear that these levelsof radionuclides in Turkishlichens were moderately low in comparison with levels recorded throughout much ofEurope. Turkey was undoubtedly the recipientofheavy contamination, as exemplifiedby high radionuclide levels in the 1987 tea harvest, which had to be destroyed;however, only a few widely spaced sites have so far been investigated and the"hotspots" went undetected by lichen monitoring.

Akcay and Ardisson [3] and Akcay and Kesercioglu [4] provide some useful dataon several radionuclides accumulated by three lichen species at two sites in Turkey; forexample, the authors determined137Cs levelsof2950 and 1035 Bq kg" inPseudevernia

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furfuracea collected from Izmir inSeptember1986 andSeptember1987 respectively,and 1159 Bq kg" for the latter date for material collected fromTrabzonon the BlackSea coast. Extrapolation of the measurements for all three species to the dateof theChernobyl disaster shows similar134CS : 137Cs ratios to those determined for Poland (seeabove). Additional information on the radioelementalcontentofseveraldifferentlichenspecies from these and other regionsofTurkey areprovidedby Akcay [2] and Sakaetal. [59].Unfortunately,it is difficult to interpret someofthe dataprovidedby all theseauthors due to taxonomic and numerical inconsistencies, but they should becomparedwith levels determined for lichens and other biota obtained from theMediterraneanregion (e.g. [6]).

In addition to the work in Poland, Austria, Turkey and Greece cited above, manyother partsof the world have been monitored with lichens for Chernobyl fallout,including Alaska [9], Canada [72, 74], Estonia [41], Finland [54], Greenland [73],Holland [69],Norway [13, 22], Romania [8), Russia [45], Sweden [58], but inevitablymostofthe work has concentrated on the area around the Chernobyl reactor [10-12, 14,46, 77] and in the Ukraine in general [36, 78].

It is clear from the Chernobyl work reported above that lichens are most effectivemonitorsof environmentalradioelements for time and spaceinterpretation. There isalso a wealthofevidence to show how lichens can be similarlyemployedtomonitor(a)both above and below ground testingofnuclearweapons/devices, (b) radioactivedebrisresulting from crashes to landof nuclear-poweredaircraft, satellites andspace-shipsand (c) accidental spillages fromnuclear-poweredsubmarines whichcontaminateshoreline biota. There is also a reasonable amountofdata on levelsofa very wide rangeof radioelementsderived from natural sources,particularlyin geothermal, volcanic andradioactive metal-rich areas. These sources andby-productsarising from miningoperations and from"normal" emissions from nuclear andlignite-burningpower­stations need to be monitored in order to establish creditablebackgroundlevels; in thisway, a clearer picture can be created in order to demonstrate the full effectsof anydisaster. Manyof these aspectsof radioelementmonitoring have beenadequatelycovered in the literature. However, it should be noted that such papers are rarely foundin mainstreamjournalslikely to be read by lichenologists; for example, many articlesemphasizethe techniques and methodology employed and therefore appear inspecialistjournals- indeed a chemically rather than abiologicallyoriented abstracting servicemay well prove more productive - and othercontributionsdealing withmonitoringcanbe found in almost any local, national or internationaljournal of environmental,ecological or biological interest.

For more general reviewsof radioelementuptake andaccumulationby lichens seeTuominen andJaakkola[76], Richardson [53] and Nimis [49] and for furtherpublishedsources see Jacquiot and Daillant [34] and theon-going literature searches by A.Henderson and T.L. Esslingerperiodically listed in The Lichenologist and TheBryologist respectively.

4.Concluding remarks

The above data summarise only a small fractionof environmentalmonitoringprogrammeswhich clearly demonstrate dramatic increases in radionuc1ide input to

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ecosystems via lichens, known to be highly efficient bioaccumulators. Interpretationofsuch data will only be feasible through a knowledgeof accumulation levels byparticular species at the same sites over a periodof time.It is strongly recommendedthat on-going biomonitoring programmes adopt protocols with standardized techniquesand rigorous methodology in order to credibly establish comparative temporal andspatial analyses.

5.Acknowledgement

I am most grateful to Professor PierLuigi Nimis for his comments on an earlier draft of this chapter.

6. References

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2. Akcay, H. (1995) Deposition of fission product radionuclidesin lichens and coniferousplants in Turkey,Journal ofRadio analytical and Nuclear Chemistry, Letters 200,147-158.

3. Akcay, H. and Ardisson, G.(1988) Radioactive pollution of Turkish biotas one year after the Chernobylaccident,Journal ofRadioanalytical and Nuclear Chemistry 128, 273-281.

4. Akcay, H. and Kesercioglu, T. (1990) A systematic study on the West Anatolia lichens related to theChernobyl fallout, Doga-Tr,Journal ofEngineering and Environmental Science 14,28-38.

5. Baeza, A., Miro, C., Paniagua, J.M., Navarro, E., Rodriguez, M.I .,and Sanchez, F. (1994) Natural andartificial radioactivity levels in Livingston Island (Antarctic regions),Bullet in of Env ironmentalContamination and Toxicology 52, 117-124.

6. Barci, G., Dalmasso, J., and Ardisson, G. (1988) Chernobyl fallout measurements in someMediterranean biotas, The Science ofthe Total Environment 70,373-387.

7. Bartok, K. and Mocsy, I. (1990) Studies upon lichen radioactivity, Revue Roumaine de Biologie, Bioi.Veget. 35,61-65.

8. Bartok, K. Mocsy,I., Bolyos, A .,and Dezso, Z. (1998) Studies onmCs content in lichens in mountainregions of Romania,Sauteria 9, 249-256.

9. Baskaran, M., Kelley, J.J., Naidu, A.S., and Holleman, D.F. (1991) Environmentalradiocesium insubarctic and arctic Alaska following Chernobyl,Arctic 44, 346-350.

10. Biazrov, L.G.(1993) Lichens as indicatorsofradioactive contamination,Journal ofRadioecology 1,15­20.

II. B iazrov,L.G. (1994) The radionuclides in lichen thalli in Chemobyl and east Urals areas after nuclearaccidents,Phyton 34, 85-94.

12. Biazrov, L.G., Desmet, G.,Janssens, A.,and Melin, J. (1994) Radionuclide content in lichenthallus inthe forest adjacent to the Chernobyl atomic power plant,The Science ofthe Total Environment 157, 25­28.

13. Bretten,S., Gaare, E.,Skogland, T.,and Steinnes, E. (1992) Investigationsofradiocaesium in the naturalterrestrial environment in Norway following the Chemobyl accident,Analyst 117, 501-503.

14. Chant, L.A.,Andrews, H.R.,Cornett, R.I.,Koslowsky, V., Milton, J.C.D., van den Berg, G.I., Verburg,T.G.,and Wolterbeek, RT . (1996) 1291 and 36Cl concentrations in lichens collected in 1990 from threeregions aroundChemobyl,Applied Radiation Isotopes 47,933-937.

15. Chibowski, S., Solecki, Y ., and Bystrek, J. (1998) The examination of gamma-emittercontaminationlevel of the lichens from eastern and south-eastern Poland, collected in the years 1949-1996,Journal ofRadioanalytical and Nuclear Chemistry 230,319-322.

16. Eckl, P., Hofmann, W.,and Tiirk, R. (1986) Uptakeofnatural and man-maderadionuclidesby lichensand mushrooms,Radiation and Environmental Biophysics 25, 43-54.

17. Eckl, P., Tiirk, R., and Hofmann, W. (1984) Natural and man-made radionuclideconcentrationsinlichens at several locations in Austria,Nordic Journal ofBotany 4,521-524.

18. Elkin, B.T., and Bethke, R.W. (1995) Environmental contaminants in caribouin the NorthwestTerritories,Canada,The Science ofthe Total Environment 160/161, 307-321.

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19. Ellis, K .M. and Smith, J.N. (1987) Dynamic model for radionuclide uptake in lichen, Journal ofEnvironmental Radioactivity 5, 185-208.

20. Feige,G.B.,Niemann, L., and Jahnke,S . (1990) Lichensand mosses - silentchronists of the Chernobylaccident,Biblioth eca Lichenologica 38, 63-77.

21. Gaare, E. (1987) The Chernoby1 accident: can lichens be used to characterize a radiocesiumcontaminated range?,Rangifer 7,46-50.

22. Gaare,E. (1990) Lichen content of radiocesium after theChernobyl accident in mountainsin southernNorway, in G. Desmet,P. Nassimbeni and M. Belli (eds.), Transfer of Radionuclides in Natural andSemi-natural Environments. Elsevier,London,492,50I .

23. Godoy, J.M., Schuch, L.A., Nordemann, DJ.R., Reis, V.R.G.,Ramalho,M .,Recio,J.C., Brito, R.R.A.,and Olech, M .A. (1998) I31Cs, 226.228Ra, 210Pb and 4()K concentrations in Antarctic soil, sedimentandselected moss and lichen samples,Journal ofEnvironmental Radioactivity 41, 33-45.

24. Gorham, E. (1959) A comparisonof lower and higher plants as accumulators of radioactivefall-out,Canadian Journal of Botany 37,327-329.

25. Goyal, R. and Seaward, M.R.D. (1982) Metal uptake in terricolous lichens. II. Effects on themorphology of Peltigera canina andPeltigera rufescens , New Phytologist 90, 73-84.

26. Goyal,R. and Seaward, M .R.D (1981) Metal uptake in terricolous lichens. I. Metal localization withinthe thallus,New Phytologist 89,631-645.

27. Hanson, W.e.(1967) Caesium-137 in Alaskan lichens, caribou, andeskimos,Health Physics 13,383­389.

28. Heinrich,G. and Remele,K. (1996) I31Cs, 9OSr, K " and Ca'" in lichens, mosses and vascular plantsofamountain area in Styria, Austria,Mitteilungen der Osterreichisch en Bodenkundl. Gesellschaft 53, 243­250.

29. Heinrich, G., Muller, H.J.,Oswald, K ., and Gries, A . (1989) Natural and artificial radionuclides inselected Styrian soils and plants before and after the reactor accidentin Chernobyl, Biochemie undPhysiologie der Pflanzen 185,55-67.

30. Heinrich, G., Muller HJ., Oswald, K ., and Wolkinger, F. (1989) Natiirliche und Tschernobyl­verursachte Radionuklide in einigenWasser-und Landpflanzen inSteiennark und Karnten,Phyt on 29,61-68.

31. Holleman,D .F.,Whire,R.G., and Allaye-Chan,A.C. (1990) M odellingof radiocesium transferin thelichen-reindeer/caribou-wolffoodchain,Rangifer, Special Issue3,39-42.

32. Holm, E.,and Persson, B.R.R.(1975) Fall-out plutonium in Swedishreindeer lichen,Health Physics 29,43-51

33. Holm, E. and Rioseco,J. (1987) '»fc in the sub-arctic food chain lichen-reindeer-man,Journal ofEnvironmental Radioactivity 5, 343-357.

34. Jacquiot, L. and Daillant, O.(1999) Bio-accumulation des radioelernentspar les lichens: revuebibliographique, Bulletin de /'Ob ervatoire Mycologique. 16,2-23.

35. Jones, B.-E.V., Eriksson, 0 ., and Nordkvist, M . (1989) Radiocesiumuptake in reindeer pasture, TheScience ofthe Total Environment 85, 207-212.

36. Kondratyuk,S.Y., Navrotska,I.L., Brun, G.O., Beznis, N.G., Gizbullina,V .K., Izotova,N .V., andLyugin, V.O. (1994) Study of radionuclide accumulation by lichens in the Ukraine, UkrayinskyiBotanischnyi Zhurnal51 , 46-52,(in Ukrainian).

37. Kwapulinski, J., Seaward, M .R.D., and Bylinska, E.A. (1985) I31Caesium content ofUmbilicariaspecies,with particular reference to altitude, The Science ofthe Total Environment 41,125-133.

38. Kwapulinski, J.,Seaward,M.R.D .,and Bylinska,E.A. (1985) Uptakeof226Radiumand228Radium by thelichen genusUmbilicaria, The Science ofthe Total Environment 41, 135-141.

39. Lawrey,J.D.and Hale, M.E. (1981) Retrospective studyofleadaccumulation in the northeastern UnitedStates,The Bryolog ist 84,449-456.

40. Mable,T. (1987)Sytuacja w Polsce w Zakresie Skazen Promieniotworczych po Awarii Radiologicznej wCzarnobylu, Chemiczne Zagrozenia Srodowiska w Polsce, Uniwersytet M arii Curie-Sklodowskiej,Lublin.

41. Martin,L.,Nifontova,M .,and Martin,J. (1991) Radionuclidesvariation in macrolichensin Estoniaafterthe Chernobyl accident,Proceedings ofthe Estonian Academy ofScience. Ecol. I ,42-51.

42. M attsson,LJ.S.(1972) Sodium-22 in the foodchain: lichen-reindeer-man,Health Physics 23,223-230.43. Mattsson, L.J.S. (1975) I31Cs in the reindeer lichen Cladonia alpestris: desposition,retention and

internal distribution,1961-1970,Health Physics 28,233-248.44. Nieboer, E., Ahmed,H.M., Puckett, K.J., and Richardson,D.H.S (1972) Heavy metalcontent of lichens

in relation to distance from a nickelsmelterin Sudbury,Ontario,Lichenologist 5,292-304.

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45. Nifontova, M.G. (1998) Concentrationsoflong-livedartificial radionuclides in the moss-lichen coverofterrestrial ecosystemsin the Ural-Siberianregion,Russian Journal ofEcology 29,196-200.

46. Nifontova, M.G. and Alexashenko, V.N. (1992) Content of90Sr and 134·Jl1CS in fungi, lichens, andmossesin the vicinityofthe Chernobyl nuclear power plant,Soviet Journal ofEcology 23, 152-155.

47. Nifontova, M.G. and Kulikov, N.V. (1981) 90Sr and Jl1Cs accumultion by some lower plants in thevicinityofBeloyarsk nuclear power station in the Urals,Ekologiya 12,94-97(in Russian).

48. Nifontova, M.G.,Lebedeva, A.V.,and Kulikov, N.V. (1979) Accumulation of90Sr and mCs in live anddead lichens,Ekolog iya 10,94-97(in Russian).

49. Nimis, P.L.(1996) Radiocesium in Plants of Forest Ecosystems,Studia Geobotanica 15,3-49.50. Paatero, J. and Jaakkola,T. (1994) Determinationof the241pU deposition in Finland after the Chernobyl

accident,Radiochim ica Acta 64, 139-144.51. Paatero, 1., Jaakkola, T., and Kulmala, S. (1998) Lichen (sp. Cladonia) as a deposition indicatorfor

transuranium elements investigated with the Chemobyl accident,Journal of EnvironmentalRadioactivity 38,223-247.

52. Papastefanou, C., Manolopoulou, M ., and Charalambous, S. (1988) Radiation measurements andradioecological aspectsof fallout from the Chernobyl reactor accident,Journal of EnvironmentalRadioactivity 7, 49-64.

53. Richardson, D.H.S.(1992) Pollution Monitoring with Lichens . Richmond Publishing,Slough.54. Rissanen,K . (1992) Lichens and plants obtained from permanent study plots in northern Finland as

bioindicators for radioactive fallout, in E.Tikkanen, M. Varmola and T. Katermaa (eds.),Symposium onthe State of the Environment and Environmental Monitoring in Northern Fennoscandia and the KolaPeninsula. University of Lapland Arctic Centre,Rovaniemi, pp.320-322.

55. Rissanen, K . and Rahola, T. (1989) Cs-137 concentration in reindeer andits fodder plants,The Scienceofthe Total Environment 85,199-206.

56. Rissanen, K. and Rahola, T. (1990) Radiocesium in lichens and reindeer after the Chernobyl accident,RangiferSpecial Issue 3,55-61.

57. Roos, P., Holm, E.,Persson, R.B.R., Aarkrog, A .,and Nielsen, S.P. (1994) Depositionof2lOPb, I37Cs,239+240pU,238pU, and 241Am in the Antarctic Peninsula area,Journal ofEnvironmental Radioactivity 24,235-251.

58. Roos, P.,Samuelson, C., and Mattsson, S. (1991) Jl1Cs in the lichenCladina stellaris before and afterthe Chemobyl accident, in L. Moberg (ed.), The Chernobyl Fallout in Sweden, Swedish RadiationProtection Institute, Stockholm,pp. 389-400.

59. Saka, AZ ., Cevik, U., Bacaksuz, E., Kopya, AI., and Tirasoglu, E. (1997) Levels of cesiumradionuclides in lichens and mosses from the province of Orduin the eastern Black Sea areaofTurkey,Journal ofRadioanalytical and Nuclear Chemistry 222, 87-92.

60. Sawidis, T. (1988) Uptake of radionuclides by plants after the Chernobyl accident,EnvironmentalPollution 50,317-324.

61. Seaward, M.R.D. (1973) Lichen ecologyof Scunthorpe heathlands.I. Mineral accumulation,Lichenologist 5,423-433.

62. Seaward, M.R.D. (1991) Biomonitoring radionuclides in eastern Europe, pre- andpost-Chernobyl,in Z.Ayvaz (ed.),Environmental Pollution and Control, Ege University, Izmir, pp 80-89.

63. Seaward, M.R.D. (1992) Lichens. Silent Witnesses of the Chernobyl Disaster. Inaugural lecture,UniversityofBradford, Bradford.

64. Seaward, M .RD. (1994) Measuring up to disaster: the necessity for valid baseline data, DisasterPrevention Management 3 (4), 17-26.

65. Seaward, M.RD. (1995) Use and abuse of heavy metal bioassays in environmental monitoring, TheScience ofthe Total Environment 176, 129-134.

66. Seaward, M.RD., Bylinska, E.A, and Goyal, R. (1981) Heavy metal content ofUmbilicaria speciesfrom the Sudety regionofS.W.Poland,Oikos 36, 107-113.

67. Seaward, M.R.D.,Heslop, J.A.,Green, D .,and Bylinska, E.A. (1988) Recent levels of radionuclides inlichens from southwest Poland with particular reference to 134CS and Jl1Cs, Journal of EnvironmentalRadioactivity 7, 123-129.

68. Shimwell, D.W. and Laurie, A .E. (1972) Lead and zinc contamination of vegetation in the southernPennines,Environmental Pollution 3,291-301.

69. Sioof, J.E. and Wolterbeek, B.T. (1992) Lichens as biomonitors for radiocaesium following theChernobyl accident,Journal ofEnvironmental Radioactivity 16, 229-242.

70. Smith, D.C.,and Molesworth, S. (1973) Lichen physiology. XIII . Effectsofrewettingdry lichens,NewPhytolog ist 72, 525-533.

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71. Smith, F.B.and Clark,MJ. (1986) Radionuclide deposition from the Chemobyl cloud,Nature (London)322, 690-691.

72. Smith, J.N. and Ellis, K.M. (1990) Time dependent transportof Chernobyl radioactivity betweenatmospheric and lichen phases in eastern Canada,Journal ofEnvironmental Radioactivity 11,151-168.

73. Strandberg, M. (1997) Distribution of mCs in a low arctic ecosystem in West Greenland,Arctic 50, 216­223.

74. Taylor, H.W., Svoboda, J., Henry, G.H.R., and Wein, R.W. (1988) Post-Chernobyl cesium-134 andcesium-137 levels at some localities in northern Canada,Arct ic 41,293-296.

75. Topcuoglu, S., Van Dawen, A.M., and Gungor, N. (1995) The natural depuration rateof mCsradionuclidesin a lichen and moss species,Journal ofEnvironmental Radioactivity 29, 157-162.

76. Tuominen, Y.and Jaakkola, T. (1973) Absorption and accumulation of mineral elements and radioactivenuclides, in V.Ahmadjian and M.E.Hale (eds.),The Lichens , Academic Press, New York,pp 185-223.

77. Van den Berg,GJ.,Tyssen, T.P.M.,Ammerlaan,MJJ.,Volkers,KJ., Woroniecka,V.D., de Bruin, M.,and Wolterbeek, H.T. (1992) Radiocesium lead in the lichen speciesParmelia sulcata sampled in threeregions around Chernobyl: assessment of concentrations in 1990,Journal of EnvironmentalRadioactivity 17, 115-127.

78. Wasser, S.P. (ed.) (1995)Accumulation ofRadionuclides by Cryptogamic Plants and Higher Fungi ofthe Ukraine. M .G.Kholodny Institute of Botany,Kiev (in Ukrainian).

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BIOMARKERS OF POLLUTION-INDUCED OXIDATIVE STRESS ANDMEMBRANE DAMAGE IN LICHENS

D.CUNY 1, M .L.PIGNATA 2

, I. KRANNER 3 andR. BECKETr

'Laboratoire de Botanique et de Cryptogamie, Faculte des SciencesPharmaceutiques et Biologiques. 3,rue du Professeur Laguesse, F-59006Lille Cedex, France (damien.cuny@wanadoofr)2Instituto Multidisciplinario de Biologia Vegetal (IMBIV, CONICET­UNC). Catedra de Quimica General. Facultad de Ciencias Exactas,Fisicas y Naturales. Universidad Nacional de Cordoba , Avda. VelezSarsfield 299,5000 Cordoba, Argentina ([email protected])3Institut fur Pflanzenphysiologie, Karl-Franzens Universitdt Graz,Schubertstrcfie 51, A-80lO Graz, Austria ([email protected])4School ofBotany and Zoology, University ofNatal, Private Bag X01,Pietermaritzburg, Scottsville 3209, Republic ofSouth Africa([email protected])

1. Introduction

Pollutants cause many kindsofdamage to organisms.Damage can occur at all levelsofbiological organization, from the components of individual cells to ecosystems (Figure1) [84]. Many studies have shown that plants and lichens can be used to assessenvironmental contamination. Traditionally, the rateof accumulationof contaminants,geographical distribution or morphological modifications have been studied in"indicator"species (see chapters 2-7, this volume). However, it is now realized that theimpact of pollutants can be measured more quickly by testing their effects on certainphysiological processesof these indicators. Suitable processes are now termed"biomarkers".An important consequenceofair pollution is the increased productionofreactive oxygen species (ROS) in organisms [71]. The aimof this chapter is to reviewthe conceptof biomarkers, outline mechanismsof free radical formation, damagingeffects of ROS, and protection mechanisms against these. The use of both thedeleterious effects and the defence mechanisms as actual and potential biomarkers inlichens is discussed.

2.Definitionsof biomarkers

The term biomarker is still not frequently used in the lichen literature, although theconcept has been employed for some time.

97P.L. Nimis, C. Scheideggerand P.A. Wolseley(eds.), Monitoring withLichens- MonitoringLichens. 97-110.© 2002Kluwer AcademicPublishers. Printed in the Netherlands.

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• The National Council Research (Washington DC) [63] defines the term as follows:"a biomarker is a xenobiotically-induced variation in cellular or biochemicalcomponents or processes, structures or functions that is measurable in a biologicalsystem or sample".

• According to Koemanet al. [49] a biomarker is the"modificationof a biologicalresponse which can be related to an exposure to, or toxic effectofan environmentalchemical or chemicals".

• Lagadicet al. [53] define a biomarker as "an observable and/or measurable changeat a molecular, biochemical, cellular, physiological or behavioural level, whichreveals the present or past exposure of an individual to a chemical pollutingsubstance".

• Van Gestel and Van Brummelen [80] define biomarkers as "any biologicalresponse to an environmental chemical at the below-individual level, measuredinside an organism or in its products indicating a departure from the normal status,that cannot be detected from the intact organism".

Figure 1 shows the relationshipbetween damaging effects of stress, the responseofan organism or community to stress, and its physiological status with respect to health.Damage caused by a stress to an organism occurs when its protection and repair systemshave been overcome.The terminal stage of this damage is the death of the individual orthe community, and the disruptionof the structure and functioningof the ecosystem.However, extreme physiological responses have little value as biomarkers, in the sameway as the classic dose-response curve flattens when dose is high [44]. According toErnst and Peterson [23] biomarkers should only be used when the extentofadaptationof a particular population to pollution is known. When using biomarkers in, forexample, transplant experiments, it is certainly important to carefully determine theirinitial values in uncontaminated material.

According to NASINRC [62] and Koemanet al. [49], three types of biomarkers canbe distinguished :• Biomarkers of exposure are responsesofan organism indicating that exposure to a

pollutant has occurred. They do not provide information whether the effects areharmful, beneficial or neutral to the organism. Examples include the inductionofmetalloproteins in plants exposed to trace elements [81] and the inductionofmono­oxygenase systems such as cytochrome P450 [60].

• Biomarkers of effects, in contrast to the previous category, show that the exposurehas affected the healthofthe organism. In most cases, harmful effects are studied,e.g.membrane damage[8,10,46,64].However, beneficial effects can also be usedas biomarkers, e.g. an increase in chlorophylls at polluted sites in transplantedUsnea amblyoclada [11].

• Subtle typesof biomarkers include increased toleranceof an organisms to apol1utant, or pol1ution-induced changes in the genetic composition orof anorganism [53].

Establishing suitable air pollution biomarkers in lichens involves the followingstages:• Studying the changes that take place in lichens growing in or transplanted to

ecosystems exposed to high levels of pollutants;

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• Demonstrating that under controlled conditions a given doseof a pollutant cancause a measurable response of a biomarker;

• Confirming that the biomarkers respond quantitatively to different levels of airpollutants and selecting biomarkers that could be employed in biomonitoringprograms by comparing the results with actual concentrations of pollutants obtainedfrom instrumental monitoring.

Stimulationof enzymeproduction

I HealthstatusI

IncurableDisease

CurableDisease

Stress

Health

ChangeofcommunitystructureDeathof the organismNecroticsymptoms

Chlorophylldestruction

Plasmolysisofcells

Membraneintegrity)

Electrolytesleakage)

Decreaseof ATP concentration)

Perturbationof respiration)

Variationofphotosynthesis)

Homeostasis Compensation Repairing IrreversibleDamages

Physiologicalcondition

Figure I. Range ofphysiological states ofan organism or community in response to stress modified afterDepledge et al. 1993 [I7]. Four states can be distinguished: first. an optimal healthy state maintained byhomeostasis mechanisms; second. a stressed state requiring compensatory mechanisms to re-establish thehealthy state; third. curable disease needing repair mechanisms; and fourth . incurable disease causingorganism death and change ofcommunity structure. According to the authors. the curve indicates the possiblerelationship between progress ion ofa healthy individual to a diseased state and ultimately to death.

Ideal biomarkers should be easy to measure, and produce distinctive symptoms thatare not confused with those caused by other environmental stresses.In practice, this ishard to achieve. In particular, the responses of biomarkers with a bell-shaped responseto stress are difficult to assess. Therefore, to successfully monitor environmentalpollution, very often more than one biomarker is required. Semi-quantitative andquantitative indices have been formulated that combine several biomarkers [57].Weighting of some biomarkers in these indices may be needed [61]. For example,

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Gonzalez and Pignata [35] and Levin and Pignata [55] proposed a Pollution Index (PI)in lichens based on the mathematicalcombinationofchlorophyll-adegradation, sulphuraccumulation and markersoflipid peroxidation(equation I).

PI = [(Phaeoph-aIChl-a) + (ST/SF) ] x [(MDA TIMDA F) + (HPCD TIHPCD F) ] (I)

Phaeoph-a, phaeophytina for each transplanted sample; ChI-a, chlorophylla; S,sulphur content, MDA, malondialdehyde concentration; HPCD, hydroperoxyconjugateddienes content. The subscript T refers to determinations made on transplanted samplesand F to those made on baseline material. This index has been demonstrated to beeffective for several lichen species in different biomonitoring programs and allows thedelimitationofzones with different levelsofatmospheric quality[II ,36-38].

When properly used, biomarkers can"forecast" impending harmful effects [80].Ideally, an environmental survey based on biomarkers can be used as a warning signalby early detectionofthe effects of pollutants, and by detecting pollution below the dosethat causes irreversible damage. Results from such surveys can be used to argue for amore intensive survey of the particular ecosystem.

3. Effectsof reactiveoxygen species onplantsandlichens

3.1. FORMA nON OF REACTIVE OXYGEN SPECIES

Free radicals are atoms or molecules with an unpaired electron. This unpaired electronis readily donated, and as a result, most free radicals are highly reactive. If notscavenged, they can cause the formationof further free radicals thus initiating freeradical chain reactions that finally can damage almost all biomolecules.Oxygen radicalsinclude singlet oxygene02), superoxide(02'-) , the hydroxyl radical("OH) , and togetherwith hydrogen peroxide (H20 2) are termed reactive oxygen species (ROS). ROSformation has been reported in chloroplasts, mitochondria, the endoplasmic reticulum,and the plasmamembrane [14,82].

In lichens, ROS formation has not been quantified directly following exposure to airpollution, but the damage they cause, and the defence mechanisms they induce, can beused as biomarkers. Interestingly, Minibayeva and Beckett [58] demonstrated thatdesiccation stress stimulates extracellularO£- and H20 2 production in lichens andbryophytes, suggesting that direct measurementsof ROS may in future provide a newsourceofbiomarkers.

Apart from the harmful effectsofROS, they can also play important roles in normalbiological processes, and pathogen defence. H20 2 for instance plays a role inlignification, and ROS possibly act as secondary messengers in parasitic infectionwhere they also have anti-pathogenic activity [4, 58].

Although ROS are produced during normal metabolism, most biotic and abioticstresses enhance their production.These include:• Parasitic infection [2,4];• Desiccation [52,76] and temperature stress [56];• Ageingofcells [I];• Xenobiotics like paraquat [41];

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• Air pollutants,particularly ozone,NOx,and S02.In lichens, fungi and plants, there is indirect evidenceof pollution-inducedROS

production e.g. for S02 [50], ozone [82], general atmospheric pollution [8, 19, 39, 65]and heavy metals [5, 30,32,47].

ROS are produced during many reactions (for comprehensive reviews see [1, 4, 21,42, 43, 52]). In higher plants, but not in lichens, ROS producing pathways are well­studied. The briefoverview that follows in 3.1. outlines someof the most importantROS producing pathways known from higher plants, and we encourage lichenologiststo study these pathways in lichens.

3.1.1.Singlet oxygenThe photosynthetic apparatus is an important site of10 2formation. Here, the trapping oflight by photosystems I and II causes the formationofexcited statesofthe chlorophyllmolecules. The high electronic excitation state resulting from the illumination of thechlorophyll molecule can transfer energy onto the 302 (triplet oxygen, ground stateoxygen) produced by photosynthesis, and raise it to a more reactive state, 102(equations2 and 3). Excess excitation or inhibition of photophosphorylation increases theproduction of'02, Singlet oxygen can react directly with polyunsaturated fatty acid sidechains to form lipid peroxides [42].

P + light

p* + 302

~ p* (excited pigment)

~ '02 + P

(2)

(3)

3.1.2. SUJleroxideThe superoxide anion is formed by the capture of an electron by oxygen or whenoxygen is exposed to ionising radiation. As much as 1-4% of the oxygen consumed bySaccharomyces cerevisiaecan generateO£- [28]. In biological systems, some enzymessuch as nitropropane dioxygenase, galactose oxidase, NAD(P)H oxidase, xanthineoxidase and some peroxidases catalyse oxidation reactions in which a single electron istransferred from the substrate onto oxygen to produceO£" (equation 4).

(4)

Autoxidation of some reduced compounds (e.g., flavins, pteridines, diphenols, andferredoxin; equation 5) can also transfer a single electron to oxygen to produce O2'- [4,41-43].

Ferredoxinreduced+ 302 ~ Ferredoxinoxidised+ O£' (5)

Compared to other oxygen radicals, O2'- is rather unreactive, and its cellularconcentration is very low«lO.I IM). It cannot react directly with membrane lipids tocause peroxidation,and cannot cross biological membranes.

Most O2'- formed in biochemical systems reacts withitself(non-enzymatically)toform H202 (equation 6).

(6)

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3.1.3. The hydroxyl radicalHydrogenperoxideformed by thepreviousor otherreactionsandO£- can reacttogetherto form the hydroxyl radical 'OH(equation7) in the iron-catalysedHaber-Weissreaction. The hydroxyl radical is the most reactive species known tochemistryhaving ahalf-lifeof Ins.

(7)

3.1.4. Hydrogen peroxideHydrogenperoxideis formed by manybiologicalreactions. For instance, O2'- is rapidlyconvertedto H202 by the enzymesuperoxidedismutase(SOD).

Most oxygen taken up by aerobic cells isreducedto waterby theadditionof fourelectronsto eachmolecule. This reaction(8) is catalysedby thecytochromeoxidasecomplexofthe innermitochondrialmembrane.

302 + 4e' + 4H+ ~ 2H20 (8)

Otheroxidasessuch asglycollateoxidase, urateoxidase,oxalateoxidaseand aminoacid oxidases,transfertwo electrons onto each oxygenmoleculeto form H202 [42,43]followingequation(9).

30 2 + 2e' + 2H+ ~ H20 2 (9)

3.2.DAMAGING EFFECTS OF ROS

ROS can cause considerabledamage to cells byattackingnucleic acids, lipids andproteins. Inparticular,' OH can attack anddamagealmost everymoleculefound inliving cells.It can, forexample, hydroxylatepurine andpyrimidinebases in DNA [42]producingsubstancessuch as8-hydroxy-2'-desoxyguanosine,thus enhancingmutationrates.

Oxidativedamage toproteinschanges theirconfiguration,mostly by oxidationofthe free thiolresidues of cysteine, thus producing thiyl radicals.These can formdisulphidebonds tootherthiyl radicals,causing intra- or inter-molecularcross-links.Some ofthesemodificationscan be used asbiomarkers[1].

ROS such as 102 and the'OH radical can alsoabstracthydrogen radicals frommembranouslipids thus initiatingperoxidation.For example,the perhydroxyradical(HOO') generatedby the protonationof O£- and ' OH can extractthe bis-allylichydrogenatom fromunsaturatedfatty acids (LH) forming lipid alkylradicals(L') thatare furtheroxidised by molecularoxygen to generate lipid peroxyradicals (LOO').LOO ' reacts with LH yielding LOOH and L·. A radical chain reaction is thuspropagated. Lipid peroxides decompose to produce aldehydes such as 4­hydroxynonenal(HNE) [24] and malondialdehyde(MDA) [79] and otherproductssuchas volatilehydrocarbonslike ethane andpentane. Thesealdehydesareconsideredto besecondarytoxic messengersthatdisseminateinitial free radical events. In thismanner,lipid peroxidesand theirdegradationproductscauseextensivedamage [24].

The consequencesof changes in lipid andproteinstructure,and theinactivationofmembrane-boundenzymes,are lossof membraneintegrity andselectivepermeability.Following membranedamage, leakageof electrolytesfrom cells can occur.Potassium

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leakage is frequently used as a measureofmembranedamage [31].Furthermore,ROScan causedysfunctionin photosynthesis.As mentionedearlier, ROS can be formed inthephotosyntheticapparatus, and their formation isenhancedunder stress [56].

3.3.DAMAGING EFFECTS OF ROS AS A SOURCE OF BIOMARKERS

3.3.1. Lipid peroxidation productsMany authors havesuccessfullyused MDA as a biomarkerto investigatethe effectsofenvironmentalpollution on lichens, yeast andvascular plants [8, 10, 13, 29, 47].Vel ikova et al. [83] showed an increase in MDA after an acid raintreatmentin beanplants.Prasadet al. [68] found an increase in MDAconcentrationafter zinctreatmentofBrassica juncea. In the lichenPunctelia subrudecta transplantedto a pollutedarea,Gonzalezand Pignata [35] observed an increase in MDAconcentrationproportionaltoits sulphur content. Cuny et al. [15] reported asignificant increase in MDAconcentrationsin Diploschistes muscorum exposed to heavy metals. Otherperoxidationproducts have also been used as biomarkers, e.g. hydroperoxyconjugateddienes(HPCD) [9, 55]. In Usnea amblyoclada transplantedto urban sites, high MDA, andHPCD concentrationsand high chlorophyll b/a ratios werecorrelatedwith highconcentrationsofparticulatematter, hydrocarbons,0 3 and H2S [10].

3.3.2. Membrane leakageIn lichens, membraneleakage hassuccessfullybeen used asbiomarkerof pollutionincludingS02 [25, 77] and heavy metals [3, 6, 7, 40, 66, 67, 78].Garty et al. [33] founda negativecorrelationbetweenmembraneintegrity and heavy metalconcentrationinRamalina duriaei .

3.3.3. Damage to photosynthesisAt the whole plant level,photosynthesiscan be used as abiomarkerof pollution. Forexample, Deruelle and Petit [18] found thatvehicularpollutioninhibitedphotosynthesisin lichens. Also, photosynthesiscan be used as abiomarkerof heavy metalpollution[30].

4. DefenceagainstROS

4.1. ROS SCAVENGING PATHWAYS

AntioxidantsscavengeROS, and some have already been used asbiomarkers.The mainfree radicalscavengersare reducedglutathione(j-glutamyl-cysteinyl-glycine, GSH) ,ascorbic acid (AA),a-tocopherol,and ~-carotene [21,41,52,75, 76].

4.1.1. GSHGSH reacts rapidly with'OH, '02and, together with AA is involved inremovalofH202

[27]. In addition to itsantioxidantfunction, GSH is involved in the formationofphytochelatins[(y-Glu-CyskGly] which, together withmetallothionins,are chelatorsofheavy metals [69]. In addition, GSH is a substrateof the enzymeglutathione-S-

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transferase (GST), which is involved indetoxificationof xenobiotics [54]. Anotherfunction ascribed toglutathioneis the maintenanceof protein thiol groups in thereduced state, and it is probably the main redox bufferofcells [34].

4.1.2. AscorbateAA also reacts rapidly withO£-,'OH and 10Z [42]. Apart from its role as anantioxidant,ascorbateplays a role in oxalate metabolism, probably acting as aprecursorofoxalate.GSH and AA are present in aqueouscompartments, their reducing power being used inradical and non-radical redox reactions.

4.1.3. a-Tocopherola-Tocopherol is the major radicalscavenger in biological lipid phases such asmembranes.It is a powerful scavengerof 10Z and of lipid peroxides. The tocopherylradical produced during antioxidant activity can be reduced back totocopherolbyascorbic acid. In plants, a-tocopherolappears to beconcentratedin photosystemII [52].

4.1.4. CarotenoidsCarotenoidssuch asp-caroteneact asantioxidantsin lipid phases byquenching10Zandmolecules with excited electrons produced byphoto-excitationor chemi-excitationreactions. They further react with peroxyl and alkoxyl radicals. In addition, thexanthophyll cycle pigments violaxanthin, antheraxanthinand zeaxanthindissipateexcess excitation energy thuspreventingformationof 10Z.

4.1.5. Other antioxidant compoundsOther compounds such as flavonoids, sugars, polyols, proline andpolyamines arebelieved to have antioxidantproperties (citations inSmimoff [75]). Many papersdemonstratethe antioxidantactivitiesof phenolic compounds in higher plants (forreview see [70]), but in lichens, very few studies have been carried out.

4.1.6. ROS scavenging enzymesEnzymes involved in scavenging cytotoxic oxygen species include SOD,ascorbateperoxidase (AP) and other peroxidases, mono- anddehydroascorbatereductases,glutathionereductase (GR) and catalase (for an overview see [22, 52]). All aerobicorganismscontainsuperoxidedismutases, metalloproteinscatalysingthedismutation ofOz'- to HzOz. Thus, O{ is removed rapidly and furtherconversioninto 'OH is prevented(10).

Oz'- + O{ + 2H+ ~ HzOz + 30Z

PeroxidasescatalyseHzOz-dependentoxidationofsubstrates (S) (equation 11).

SHz+ HzOz ~ S + 2HzO

(10)

(11)

Catalasesbreak down highconcentrationsof HzOz (equation12) very rapidly, butare much less effective thanperoxidases at removing HzOz present in lowconcentrationsbecauseoftheir low affinity (high Km) for this substrate.

2 HzOz ~ 2HzO+ 30 Z (12)

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An interplay between antioxidants and enzymes to scavenge ROS was firstsuggestedby Foyer and Halliwell[27]. They postulatedan ascorbate-glutathionecyclefor the scavenging of the H202 produced fromO{ by SOD . This cycle involvesreactionsofGSH , AA , GR , AP, mono- anddehydroascorbatereductases. Moreover, itmay also be linked toa-tocopherol[26].

4.2.ROS-SCA VENGING PATHWAYS USED AS BIOMARKERS

4.2.1. AscorbateCaviglia and Modenesi [12] observed an increase in AA inParmotrema reticulatumfollowing exposure toparaquat-inducedoxidative stress.Moreover, oxalatecrystalswere depositedextracellularlywhen this species wassubjectedto S02 pollutionorparaquat[59]. InDiploschistes muscorum exposed to high metalconcentration,Sarretetal. [72] observed storageofzinc as zinc oxalate dihydrate.

4.2.2. GSHSilbersteinet al. [74] evaluatedpossible protectionmechanismsinduced by airpollutionin Xanthoria parietina, a fairly pollutionresistantspecies, and inRamalina duriaei,which disappears rapidly in regions withpolluted air to investigate adaptationmechanismsthat allow airpollution-resistantlichens to survive inpollutedareas.Xanthoria parietina was shown to have multipleprotectivemechanisms,whichincluded an increase in GSH. In addition, Kong et al. [50] found a positivecorrelationbetweenGSH concentrationand S02 pollutionin lichens. Apart fromresponsesofGSHto environmentalpollution, in lichens desiccation stress inducespronouncedchanges inthe redox statusofthisantioxidant[51, 52].

4.2.3. ROS scavenging enzymesSchlee et al. [73] studied SOD activity inPseudevernia furfuracea, Cetraria islandica,Cladonia verticillaris and Hypogymnia physodes along an altitudinalprofile. SODactivity wassignificantlygreater inP. furfuracea, the majorproportionof the enzymebelongingto theCu/Zn-SOD isoform. These authorsconcludedthat low SOD activityin H. physodes is correlatedto stress sensitivity,especially to ozone, confirmingpreliminaryresults [19]. Moreover, Silbersteinet al. [74] found high SOD activity inpollutionresistant lichens. SODs respond to S02 fumigation, and theiractivitiesseem toincrease more rapidly than thoseofperoxidases[16,50].

Deltoro et al. [16] studied the following enzymesinvolved in defenceagainstoxidativestress caused by S02 inRamalina farinacea andEvernia prunastri: SOD , AP,catalase, peroxidase(Ee. 1.11.1.7) and GR. They observed an increaseofthe activitiesofthese enzymes inR. farinacea but not inE. prunastri, correlatingtheresistanceofR.farinacea to S02 fumigation with high activitiesofROS scavengingenzymes.

4.2.4. Lichen substancesHidalgo et al. [45] demonstratedantioxidantactivity in atranorin,divaricatinicacid,pannarin andl'-chloropannarinisolated fromProtousnea malacea and Placopsis sp.Silbersteinet al. [74] found increased parietinconcentrationsin Xanthoria parietinaexposed toenvironmentalpollution. Lichens containextraordinaryhigh amountsof

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various phenolic compounds [20, 48].It has already been demonstrated that thesesubstances have antibiotic, fungicidal and antiviral properties, can chelate metals, andinhibit spore germination, but their potential role as antioxidants is almost completelyunknown [52]. However, testing whether the concentrations of lichen products changein response to pollution would be a valuable contribution to establishing a new kind ofbiomarker.

5. Conclusions and outlook

In lichens, biomarkers that have already been used to study the effectsof pollutantsinclude lipid peroxidation products such as MDA, membrane leakage, photosynthesis,antioxidants and free radical-scavenging enzymes. As yet, antioxidants have receivedvery little attention and a more detailed investigation may reveal more sensitive waysofmonitoring pollution. Moreover, lichen substances, and direct measurementof R08 inlichens could become useful new sources of biomarkers.

The use of biomarkers requires skilled personnel and is expensive compared tobiomonitoring pollution with lichens using more traditional ways such as mappingspecies distribution or zone mapping. These techniques are cheaper compared to usingbiomarkers, but require considerable skill at lichen identification. Biomonitoring canreflect long term pollution patterns, while the major advantageof using biomarkers istheir ability to respond to pollution very rapidly and at a much earlier stage ofcontamination, i.e. long before visual symptoms and changes in community structureappear.

In most developing countries high priority is currently given to industrialdevelopment rather than to protectionof the environment, and controlof emissions isfrequently insufficient or does not exist at all. In these countries, biomonitoring, andparticularly the use of biomarkers, will rapidly indicate areas in which pollution is verybad. Information derived from such studies can be used to advise that governmentsurgently start instrumental monitoring in these areas.

In developed countries, pollution levels have generally decreased as a resultofenvironmental policies. However, while the concentrationsof some pollutants such as802 have declined, others have increased, e.g. ozone. Instead of contaminationpredominantly by802, the composition of pollutants has become more complex. Thisnew situation requires the development of new methods to provide authorities with an"early warning system". Biomarkers in lichens may form an integral partof these newmethods.

6.Acknowledgments

This work was partially supported by: the InternationalAtomic Energy Agency, Consejo de InvestigacionesCientificas y Tecnologicas de la provincia de Cordoba (CONICOR) and by Secretaria de Ciencia yTecnologiade la UniversidadNacional de Cordoba (SECyT) (Maria-Luisa Pignata); the Austrian ScienceFoundation (FWF), grant P12690-810,and from the Austrian Academyof Sciences (OAW), APART 428(Ilse Kranner); the University of Natal ResearchFund (Richard Beckett) and the Councilofthe Nord Pas deCalais Regionand FEDER (Damien Cuny).

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KEY ISSUES IN DESIGNING BIOMONITORING PROGRAMMES

Monitoring scenarios, sampling strategies and quality assurance

M. FERRETTI' and W. ERHARDT 2

'LINNJEA ambiente Sri, Via G.Sirtori 37,1-50137 Firenze, Italy([email protected])2UMEG, Zentrumfiir Umweltmessungen, Umwelterhebungen undGeriitesicherheit. Grossoberfeld 3, D-76135 Karlsruhe, Germany([email protected])

1. Introduction

" Design", "sampling" and "quality" are highly relevant to the subject of this book fordifferent reasons. First, although there is a wealth of experience in monitoring theeffectsof atmospheric pollution by plants (e.g. [35, 61, 62, 87]), it seems that theimportance of an appropriate sampling design is often underestimated [63, 112]. Indeedsubjectivity in sampling procedures was found to be the main source of data variabilityin bioindication studies based on lichens (e.g. [88]). Similarly, field sampling of plantsfor chemical analyses was proven to introduce errors exceeding 1000%, whereas all thesubsequent steps (e.g. storage, drying, homogenisation and chemical decomposition)may cause errors of up to 100-300% (e.g.[5,63, 112]).

The assessmentof the effects of human activities on natural resources is nowacknowledged as a key tool in environmental policy [10, 12, 13, 83, 96, 107]. Policyand decision makers usually need answers to questions like:"Is there any trend in airpollution effects on our (marine, coastal, freshwater, terrestrial)ecosystems?","Whereand when do air pollution effects on our ecosystems occur?", "At which rate areenvironment conditions changing?". To be effective, answers need to be valid at muchlarger geographical scales than the individual sites being monitored. The environment iscomplex and - since investigators cannot measure everything, every time, everywhere ­design and methodological issues become important to ensure robust estimationsoftemporal trends [74, 77], consistent large-scale screenings, and extrapolationsofresultsfrom sites to regions (e.g. [36,58,100, 107,Ill]).

Monitoring studies are defined as "systematic observationsofparameters related toa specific problem, designed to provide information on the characteristics of theproblem and their changes with time" [89], as"a processofdetecting whether changehas occurred, establishing its direction and measuring its extent" [30], or" .. tracking aparticular environmental entity through time, observing its condition, and the changeofits condition, in response to a well-defined stimulus" [99]. Although these definitionsdiffer, they all emphasise the time dimensionof (bio)monitoring, i.e. the difference

111P.L. Nimis, C. Scheidegger and P.A. Wolseley (eds.), MonitoringwithLichens- MonitoringLichens. 111 -139.© 2002 Kluwer AcademicPublishers. Printedin the Netherlands.

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between an assessment carried out at a given time (metaphorised by "aphotograph"byWittig [114] and a series of assessments over time - "a movie"). Thus, monitoringdesign should ensure validity and time consistencyof data and should enable randomfluctuations ("noise") to be distinguished from real directional trends("signals").Implicit to the above definitions is also the systematic and organised natureofmonitoring studies, that usually involve many steps, mostofwhich are subject to errors[112].There is thus a clear demand for documented design and quality assurance (QA).

Adequate design and QA are fundamental, especially when the operationalperspective changes from local studies to national and international programmes, wherethe comparability of results is particularly important (e.g.[8,37,56,66,75, 85,91,104,112, 113]). Methodological issues are also central to facilitate links and co-ordinationamong existing monitoring programmes [12, 75]. Statistical design [75, 76], anddifferences in definitions and in field methods [52] have been identified as the majorsourcesofinconsistency among monitoring programmes [80].

In this chapter we provide a general scheme for addressing the design of amonitoring programme (paragraph 2). We discuss the typesof error that should beconsidered at the design stage (paragraph 3), we identify critical aspectsof samplingdesign (paragraph 4) and we emphasise the importance of adequate QA (paragraph 5).Each of these issues would require a chapter byitself(e.g.[23]).Thus, this paper is notintended to be either an exhaustive review or a prescriptive manual to developmonitoring programmes. Rather, it provides a framework for going through the complexprocess of design. Fortunately, design issues tend to be similar among monitoringprogrammes, and can be expressed in terms of general questions such as: "where shouldI place my monitoring sites?", "How many sites do I need?", "What kindofindicators/indices do I need?" and so on. For this reason, reference is made to the richliterature on environmental monitoring rather than to the narrower issueof"bioindication/bioaccurnulation"with lichens. When needed, two categoriesofbiomonitoring studies will be considered: those using organisms livingin situ(autochtonous, often referred to as "passive bioindicators" [114] or as"detectors"[96]),and those using transplants ("active bioindicators" [114],"sentinels" or "bioassayorganisms" [96]). Some important issues related to data management, processing andevaluation are not addressed here, and are best covered elsewhere (e.g. [34, 55, 57, 97]).

2.Main issues inprogrammedesigning

The design process involves several steps, mostofwhich are reported in Table 1 andFigure 1. Clearly, they are all connected with each other, and the designer has toconsider them as a whole.

The design process requires certain aspects to be properly addressed, starting withrecognising generic issues, such as the existenceof a priori defined objectives. Theseinclude, for example, a legal mandate to comply with monitoring,(sensu Spellerberg[96 p.7]),the natureofthe problem to be examined, its determinants, their pathways andimpacts [26, 38, 44, 45, 63, 75, 79, 80, 99, 101]. Practical aspects like availableexpertise and resources are also relevant [115].

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HOW:measurementmethods

WHERE I WHEN:samplingdesign

r- ~.I General assessment/monitoringII area and theme ,-

-Conjecmre-Lega! mandate-Researchobjectives-Previousstudies-Intuition

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Figure 1. Relationships among the phases ofan assessment and monitoring programme (modified afterLegendre and Legendre [57}).

A peer reviewofthese issues will permit the operational identificationofobjectives,the kindofstudy needed, the indicator categories and reporting units, themeasurementquality objectives (MQOs) and data quality limits (DQLs), the acceptable errors fordifferent error types, the sampling strategy and tactic, and the reporting rules. All ofthese issues can be formally addressedif the whole programme is framed within a QAplan [94] (see paragraph 5). Stohlgrenet al. [101] identify and discuss several attributesthat long-term studies should fulfil for reliability and success. Manyof them fit theneedsofbiomonitoring(Table 2).

TABLE 2.Attributes ofsuccess for a long-term monitoring programme. Many are also relevant tobiomonitoring (after [101)) .

I Secure long-termfunding2 Developflexible goals3 Refine objectives4 Pay adequateattentionto informationmanagement5 Take anexperimentalapproach tosamplingdesign6 Obtainpeer-reviewand statistical reviewofresearchproposaland publications7 Avoid bias whenselectinglong-term locationofplots8 Insureadequatespatial replication9 Insureadequatetemporalreplication

10 Synthesizeretrospective, experimentaland related studiesII Blend theoretical and empirical models with the means to validate both12 Obtainperiodicresearch programevaluation13 Integrateandsynthesizewith larger andsmallerscale research,inventoryand monitoringprograms

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2.1. IDENTIFYING THE NATURE OF THE STUDY

The type of study is related to the scientific problem being examined. There aredifferent typesof environmental studies. Hurlbert [39] distinguished between"manipulative"and "mensurative"experiments, i.e. studies in which the investigatorcontrols circumstances, and those based on observation, respectively. Traditionally,biomonitoring falls in the second category, as the investigator has no chance to controlfactors such as the levels and dispersion of atmospheric pollutants.

A further distinction is that between mensurative studies on the effectsofa distinctperturbation, and those without evident perturbation.

According to Eberhardt and Thomas [21], the former is the caseof interventionanalysis (e.g. Before-After-Control-Impact,BACI), while the latter can be divided intofour main categoriesof field studies: analytical sampling, descriptive sampling,observational studies, and sampling for pattern. They "all depend on sampling, and maybe characterised by the way samples are distributed (allocated) overprospectivesampling units in the (target) population as a whole" ([21], see also paragraph 2.2).• Descriptive surveys are devoted "to obtain certain information about large groups"

[16], e.g. the numberof lichen species in a given area. With descriptive sampling,an efficient estimationof means and totals is possible, but differences amongsubgroups within a population remain unexplored.

• Analytical surveys enable comparison "among different subgroupsof thepopulation, in order to discover whether differences exist among them, and to formor verify hypotheses about the reasons for these differences" [16]. In this caseinferences from sampling over the entire populationofinterest are possible.

• Observational studies are narrower in scope. They aim to compare the effectsofe.g. a given stressor on groups of individuals subjected to different levels of thestressor. Observational studies are conducted on limited portions of the domainofinterests. They are similar to experiments, although an actual treatment is notfeasible.

• Sampling for patterns relies on geostatistics, and is mainly concerned withdescriptionofspatial patterns and map production. The final useofthe map shouldofcourse be well known.

2.2.IDENTIFYING THE OBJECTIVE(S)

2.2.1. How to avoid ambiguityThe importanceofan explicit and well defined objective, while mentioned several times(e.g. by Spellerberg [96 p. 183]), is worth stressing, as this is a major driverof thewhole design process. Examples of how different objectives may influence the designare given hereafter. Biodiversity loss and effects of air pollution are two importantconcerns in environmental sciences, and the diversityofepiphytic lichens can be (andhas been) used as an indicator in assessment and monitoringofboth (e.g.see chapter 4,this volume, and [54]). In this context, the aims of e.g.natural reserve managers maydiffer from those of public health authorities, which implies different designs (Table 3).Managers will be probably interested to learn the spatial distributionof lichen species,the most suitable environmental scenario for preserving diversity, the tree species

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hosting more lichens, and the conditions favouring highest diversity. In other words,they want to have as much of the ecological situation covered as possible. Healthauthorities, on the contrary, will be more interested in informationconcerninglichens asindicatorsof the effectsofair pollution.They will ask for data on a selectedspeciesofhost tree, with a limited number of confounding factors, and hence in as muchhomogeneous situations as possible. The consequencesof these two perspectives onsampling design are obvious (see Table 3).

TABLE3.Examples ofdifferent endpointsf or two differentpotential clients.

PotentialclientEnvironmental

AssessmentquestionAssessment Measurements

value endpoints endpoints

<model needed>

What is the spatialThe pattern of The mean/median

mean/medianS02 lichendiversityatHealthauthorities Air quality patternof air quality in

pollution over region sample location overregion x?

x region x

<no model needed>

What is theamount and The frequencyofResource

Biodiversitycompositionof threatened lichen

Same but samplemanagers threatened lichen speciesover the

speciesin regionx? region x

Once the natureof the study is identified, theunambiguousdefinition of theobjective(s) involves the explicitidentification of:

(i) the assessment question (and its related assessment andmeasurementendpoints,see e.g. Hunsaker [38]). In general, this question is placed by the"client" (resourcemanagers, policy and/or decision makers) and is based on the recognitionof genericenvironmental values to be protected (e.g., air quality,biodiversity). The assessmentendpointis more explicit (e.g.,S02pollution, lichen diversity, threatened species), andthemeasurementendpoint is a measurable ecological attribute related (directly or by amodel) to the chosen assessment endpoint. For example, S02 pollution can be indirectlymonitored by lichens, provided there exists a (conceptual, mathematical) model linkingS02 and lichens. On the other hand, lichen diversity can be directly monitored by thefrequency and abundanceoflichen species,and no model is needed (Table 3);

(ii) the target population. This is" the aggregateof units whosecharacteristicsdefine the desired scopeof inferenceofthe study" [75], or, in other words, the totalityof situations to which the conclusions are tobe applied. Recognitionof the targetpopulationis important as it drives the identificationof the sampled population (e.g."the aggregateof units from which a sample or subset is taken for inclusion in thestudy" [75]). The relationship between target and sampled population is importanttoassess the validityofinferences;

(iii) the geographical coverage (e.g.the area to be considered by the investigation).

2.2.2. Assessment or monitoring?It is also important to clearly define whether the objectiveof the study refers toassessment or to monitoring. Considering forest health, Innes [42] distinguishes amongthree kindsof surveys which can be adapted to most monitoring andassessmentprogrammes.

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First, surveys whose aim isto establish the condition within a region in a given year(for lichens as accumulatorsthe assessmentquestion would be:"What are theconcentrationsof metals in lichensof the regionx in the yeart?") withoutwishing todetermineany trend over time. This is a typicalassessmentprogramme.

Second, surveys aimedat detecting changes over time (the assessmentquestionwould be: "Did the concentrationsofmetals in lichens change in regionx?"). This is atypicalmonitoringprogramme.

Third, surveys aimedat identifying the cause of any detected spatial andtemporalpattern (the question would be: "What are the causesof the spatialand/ortemporalpatternsofmetalconcentrationsin lichens observed in regionx?") . Obviously, differentaims have an influence on sampling design and strategy.

2.2.3. Multiple objectives. changing priorities. conflicting opinionsWhile monitoringprogrammeswith a single objective (e.g. S02 pollution)arerelativelyeasy to manage, recent trends inenvironmentalmonitoring demand more flexiblesystems, able toaccommodatedifferent objectives. This is especiallytrue forlong-termstudies, whereobjectivesmay change as aconsequenceof changing environmentalpriorities[80],which makes it difficult to establish long-termobjectives(> 10 years). Inthese cases, it is oftenbetterto concentrate on short«5 years) ormediumterm aims «10 years). Results achieved in the short and medium term will help to identify the long­term aims [102]. In any case, as far as long-term studies areconcerned,themonitoringdesign should be"able toaccommodateperiodic frame update and samplerestructuringin order to address changes in thecompositionof the universeand changes in theperceptionof issues leading to new questions andconcerns"[77]. At best, the originalvision should besufficientlyflexible to permit the inclusionofemergingenvironmentalpriorities. In the caseof environmentalimpact assessment, or when the issue involvesconflictingopinions by different social groups (e.g. natureconservationand resourceexploitation),there may be different objectives for a single study. In these cases, it isimportantto prioritiseobjectives according to clearprocedures.For this purpose,anAnalyticalHierarchyProcess (AHP) has been suggested bySchmoldtet al. [90].

2.3.DOMAIN OF THE STUDY

The domain of a study is characterisedby ecologicalattributesand by spatial andtemporal scales.Ecologicalattributes canincorporatecertainenvironmentalconditions,such as speciesdistribution,and climatic, altitudinal orpollutiongradients.

Spatial and temporal variability are important in ecologicalmonitoring[3, 60, 92],being inherentpropertiesof natural resources [86], and should beconsideredcarefullywhendesigninga monitoringprogramme (e.g. [10, 19,45,69, 74,93,95]).

Spatial and temporal scales affect the overallsampling regime in termsof bothsampling density (or intensity: thenumberof sampling unitsin a given area) andsampling resolution (the numberof sampling occasions over a given time), andthereforedo affectmonitoringcosts. However, while theinfluenceofspace and time onmonitoringdesign is clear, the definitionofoptimal spatial and temporal scales is noteasy.Appropriatespatial and temporal scales can be selected either by the"client" (e.g.

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environmentalagencies that want to know about a given administrative area in a givenperiod),or by theinvestigator,or by both.

In any case, decisions depend on the natureofthe study (e.g. absence or presenceofa distinctperturbation,see paragraph 2.1), on theproblembeing examined(e.g. long­term faint trends or rapidphenomena),on the indicator being used, and on the availableresources.

The spatial scale is defineda priori when it coincides with administrativeorecological/geographicalboundaries. The time scale depends on thephenomenonunderinvestigationand on the choice of indicators. For example,monitoringthe effectsofairquality on the epiphytic lichens of France will require a long-term study(> 10 yrs)based onmonitoringsites representativeof the targetpopulation(the epiphytic lichensof France) with l-year as sampling resolution. On the other hand, monitoring thesummer developmentof ozone in the provinceof Florence (Italy) by meansofsymptomson sensitive plants wilI need ashort-termstudy«1 yr) based onmonitoringsitesrepresentativeofthe whole province area with al-weeksamplingresolution.

When a specificproblemarises, e.g to assess and monitor the effectsofa polIutionpoint source, or theeffectivenessof abatementstrategies, spatial andtemporalscalescan be much less clear. Scott Findlay and Zheng [92] maintain that "thedeterminationof characteristicscales is a statisticaldetermination".For example, they define thecharacteristicspatial scale for a stressor as" the sizeof the area over which the stressorlevels are statisticalIysignificantlyhigher thanbackgroundlevels".

Similarly, thecharacteristictime scale is defined as "a measureofhow faststressorlevels over the landscape respond to changes in sourceemissionrates". Unfortunately,the statisticaldeterminationofspatial and temporal scales was found to bedependentonthe influenceof sampling intensity and resolution. For example, the spatial scaleincreases with increasing sample size anddecreasingsampling resolution, while timescales increase with decreasingsample size and resolution [92]. This isbecausemoresampling units enhance the powerof statistical tests (see also Bennett andWetmore[7]). This means that thecharacteristicstressor scales can beexpanded/reducedas aresultofthe adopted sampling regime.

2.4.INDICATORS , INDICES, DESCRIPTORS

2.4.J. Identification ofindicators and indicesAn " indicator" is acharacteristicor an entity that can bemeasuredto estimate status andtrendsof the targetenvironmentalresource [38]. An"index" is a characteristic,usualIyexpressedas a score, that describes the statusof an indicator[26]. For example,iflichens are the indicators, the lichen diversity score can be the index.An index can bereferred to as a"descriptor"[57 p.27].

The developmentof indicators and indices is important inenvironmentalmonitoring[38]. The basis for a proper selectionof indicators is always therecognitionof theproblem, its determinants (actual or suspected) and theenvironmentalvalues onwhich effects are expected to occur [45] (Figure 2). For example, air polIution can bemonitoredby physicochemicalanalyses or bybioindicators: the formerprovidingquantitativeinformation on pollutantconcentrationsand on potentialexposurelevels,the latterproviding information about their effects.Biomonitoringis mainly concerned

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with response indicators, i.e.those that quantify the biological condition related to theassessment endpoint. Response indicators should demonstrate the following features[11,38,70]:• correlate with changes in processes or other unmeasured components such as the

stressorofconcern,• be appropriate for regional monitoring and apply to a broad rangeof resource

classes,• integrate effects over time,• be unambiguously and monotonically related to an endpoint, a relevant exposure or

habitat variable,or a stressor,• be quantified by synoptic monitoring (low natural variability),• be related to the overall structure and functionofecosystems,• be responsive to stressors of concern for management strategies,• have a low and standard measurement error,• have an historical data base or accessible data for developmentofa data base, and,• be cost effective.

FormulationandIndicator

Identification

IdentifystressorsFormulateassessmentquestionsDevelopconceptualmodelsSelect/researchindicators

ResearchIndicator

Evaluation

CharacterizevariancecomponentIdentify ncminalrsubnominalcriteriaPreapareexamplereportsDetermineoptimum sampling densitytomeetobjectivesSelectcoreindicators

Implementonregional/nationalscalePrepare annual statisticalsummariesPrepare periodicresourceassessmentContinueresearch indicatorevaluation

EvaluateperfoonanceIdentifyemerging assessmentissuesandcompareagainstcoreContinue research for newcosteffectiveindicators

Figure2.Processfor indicatordevelopment adoptedby USEPA EMAP.

In general, scientists are able to identify long listsof indicators, but these areseldom subject to formal, qualitative or quantitative review [1I]. The problem is how toorganize the existing knowledge for setting priorities. Priorities among indicators can beachieved in a scientific way by adequate decision-making process, like the Kepner andTregoe (K-T) analysis (e.g. [48], discussed also in[II]). Kepner and Tregoe [48]developed a method for reaching an unambiguous selection of the most suitableindicators for a given problem. The background of the process is that indicators/indicesshould satisfy desired characteristics, (called "wants") related to the main issueofconcern. When decisions should be made about candidate indicators, the system works

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as follows. First, a series of" wants" (W,....Wn) is listed by selected experts on the basisofexperience and existing literature (e.g. [11, 27,38,43]).Table 4 reports the "wants"according to Eichhornet al. [22]. Second,an advisory group is asked to give a score (S)to each "want". The expert scores are averaged for each "want"(SWI ...SWn) in order torank them according to relative importance. The suggested indicator/indices(1\....ln) arescored in relation to each "want"(SIIW\ ...SIIW n)' For example, if"want" number 2ofTable 1, is considered ("practicabilityin field work"), indicators/indiceswill be scoredaccording to their expected practicabilityin the field.Then, indicator/indices are rankedaccording to the score they receive for each individual "want". A weighted score iscalculated for each index by multiplying the "want" score by the index score andsumming for all "wants". This score will be used for the final rankingofthe indices.

TABLE 4. The "wants" and their area ofconcern adopted by Eichhorn et al.[22J for evaluating the value ofthe tree condition indices adopted by the EC and UNIECE Level Ilforest ecosystem monitoring (after [22J).

Want

Broad application

Practicability in field work

Status of methods

Reproducibilityand reliability

Connection

Correlationwith changes

Help in explain tree condition

Differential diagnosis

Early warning (anticipatory)

Area ofconcernThe expected range of ecological condition under which the index canbe properly usedThe expected practicability in field work,operational aspectsThe current status of methods, inclusive of existing references,definitions,etc.The expectedaptitude of the index to provide reliable andconsistentresultsThe level of connection with the other surveysThe expected relationship between theindex and the functioningoftheecosystemThe expected aptitude of the index to provide information about thecurrent status of the treesThe expected aptitudeof the index to provide distintive information,e.g.cause-effectThe expectedaptitudeof the index to provide early warning on futurechanges in tree condition

2.4.2. Integrated indices and data characteristicsBesides specific indicators (e.g. lichens) and indices (e.g. lichen diversity), integratedindices have some history in biomonitoring (see e.g. the biotic indices [96]). Morerecently, an attempt in combining concentrations of trace metals in lichens into acomposite index has been made by Nimiset al. [73].Problems related to the adoption ofsynthetic/integrated indices should be considered carefully [70]. For example, airpollution may cause different responses on different species, and in this case a syntheticindex may be useful, as it enables the simultaneous useofall the recorded symptoms,and the integration of data in an easy understandable way [27]. Relationships amongindices of tree crown condition have sometimes been used to derive integrated indicesoftree health: examples are the Sugar Maple Decline Index [67] or the early defoliation­discoloration combination to identify five tree damage classes in Europe. Combiningsymptoms in a composite index should consider factors such as population variability insymptoms expression and functional dependence between symptoms (the degree towhich the expression of one symptom constrains thatof another) [70]. Weightingofcomponent symptoms, permutation tests, transformation, normalisation andcombination approaches should also be considered carefully [70, 105, 57].

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Standardisationof componentsymptoms is neededto avoid that"variableswith thelargernumericvalues will overpowerthose with thesmaller" (70]. Weighting can benecessarywhen componentsymptoms have differentreliabilityor sensitivity.Equalweightis the rule when there is noclearbasis forweighting. In addition,the influenceof differentsample sizes should be taken intoaccount.Permutationallows testingwhetherdata fulfil theassumptionsofthestatisticalmethodthat is used (e.g.normalityin parametricstatistics). Transformation(linear and non-linear) is necessarywhenquantitativedescriptors of different nature are explored for relationship,or inmultidimensionalanalysis, when two variables are not linearly correlated. Thecombinationapproachmay beadditive, multiplicative,or intermediate[70]. Accordingto Legendreand Legendre[57 p. 32], "a variableis said to beadditiveif its valuecan beaddedwhile retainingthe samemeaningas theoriginal variable".When the additivepropertyis valid, theadditiveapproachis probablymore appropriatefor integratedindices.This is becausetheycorrelatewith theexpressionofsymptoms.

2.4.3. Measurement scalesData generatedby environmentalmonitoringcan beof variednature(biological,e.g.lichendiversity; chemical,e.g. metalconcentrationin lichens;and physical,e.g.size ofatmosphericparticles). They can bequalitativeor quantitative,and can begatheredaccordingto differentcontinuousand discontinuousmeasurementscales [57].Usually,discontinuous data generate from counting and can be computed according tofrequency, while valuesmeasuredaccordingto acontinuousscale can fall inwhateverposition.

The main typesof scales on which data can beexpressedare:nominal (e.g. sex,species,habitat type), ordinal(ranking hierarchicallythe data, like: rare,common,abundant),categorical(distancebetweencategoriesknown, withoutan absolutezero;ex: calendardates), andcontinuous,or rational(e.g.: length, metalconcentrations)[31].While themeasurementscale to be useddependson theproblemto beexaminedand onthe indicators/indices to be adopted, it should be clear that each scale has its ownmathematicalpropertieswith clearconsequenceswith regardto thepotentialfor dataprocessing[57].

2.5.REPORTING

Whateverthe objective,one should haveclearideas aboutthe kindof informationtheinvestigationwill provideand how the final data will bepresented.Accordingto Husch[40] andHusch et al. [41] (quotedby Cecconand Tabacchi[14)), the tables for the finalreportshould be designedbefore undertakingthe formaldesign of the survey. Forexample,the objectivemay be to assess S02pollutionestimatedby evaluatinglichendiversityover a given area andperiod(e.g. 5 years). The resultsshould include: (a)tables withstatisticaldescriptorsof lichen diversity at differentcalendardates andrelatederrors; (b) tables with anestimateofaveragesand relatederrors; (c) tableswiththe outcomesof statisticaltestsbetweensites/dates; (d) mapsof mean lichendiversityand residuals; (e) calibration curvesoflichendiversityvs. S02 concentration/doses,etc.The design is thereforeforced toensureenough replicationat site level to allow formeasurementof uncertainty,statisticaltests,adequatespatial andtemporalresolution,and should also accommodatecalibration sites. This procedure ensures a high

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consistency between survey design andits desired deliverables, is unambiguous aboutthe desired precision of theinvestigation, and forces the designerto identify potentialsources of uncertainty and to quantify the errors. Reporting rules are also important, astheir definition forces programme managers to consider what information should bedelivered,atwhat level of depth, and towhat target public.

3.Errors

Sampling and measurementerrors must be considered asan integral part of themonitoring activity from the initial design phase [112]. Table 5 reports detailedcharacteristicsof various errors for different steps of the investigations.Errors can beclassified into four major categories:sampling errors, assessment/measurement/classificationerrors, prediction errors caused by models, non-statisticalerrors. Theseerrors can occur in each part of the investigation[I 12], can be random or directional(according to the error type, Table 5), and slight or severe (according to theirmagnitude, Figure 3). In general, errors associated with sampling issues tend to behigher than the others. A distinctcategoryof uncertaintyis terminology, which oftenresultsin differentaspects receiving the same name [52].

TABLE 5.Possible errors, their quality and the associated risks to the various steps ofenvironmentalmonitoring. with regard to trace metals analysis (based on Wagner [1 J2]).

RI skofsertousProcedureand step M ain source and characteristicsof possible errors QWllltyohrre n errors

PlaIlDiDe,Definitionof thearea Spatial variability.heterogeneity Systematic+ Random HighSelectionof specimens Ecologicalor physiologicalvariability Systematic ModerateStratification Biological. physiological. spatialvariability Systematic ModerateSamplingmethod Representativity Systematicand/orRandom High (controllable)Numberofsamples Representativity Random HighSample mass Representativity Random LowTiming Temporal variability,trends Systematic or Random HighSamplingWea.hercondition Unreproducibledeposition,leaching.matrixeffect Systematic VeryhighPaclcaging Contaminatimor loss Systematic ControllableSampleconservation during sampling Losses duetometabolism,volatilisation, translocation Systematicor absolute Moderate

TransportatiOD Contamination,loss Systematicor absolute HighStorageShortterm Contamination,loss.metabolism,alteration of Systematicor High .Long term bindingform or weightbasis.speciation. solubility absolute veryhigbSample preparationCleaning. MUS hing Contamination,loss Systematic HighDrying Contamination,loss Systematic ModerateHomogenisotion Contamination,disregard of skeweddistribution Systematic HighSubsamp/ing Representativity Random Moderate

Samplepre-treatmentDigestion Contamination byreagents orcontainer, losses Systematic ControllableMatrixmodification Contaminationbyreagents Systematic Controllable

AnalysisInjection Inaccurateorbadly adjustedtools Random or systematic ModerateCalibroJion Physical,chemicalinterferences Randomor systematic ModerateDetection e.g,spectral interferences Systematic LowQuantification Baselineshift Random or systematic Very low

nata EvaluationAveraging Disregard datacharacteristics Systematic ModerateConfidenceinterval Disregard datacharacteristics Systematic ModerateTrenddetection Di"9!"' ddatacharacteristics Qualitalive High

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3.1.SAMPLING ERRORS

Sampling errors are generated by the natureof samplingitself- because a sample doesnot represent the complete population - and by the degreeofdata variability. In general,sampling errors can be controlled by augmenting the sample size and/or by renderingthe sampling design more effective [16]. Compared to other error sources, sampling canbe ofmajor importance (Figure 3). Examplesofthe importanceoferrors resulting fromsampling in biomonitoringstudies are reported by Markert [63], and Bargagli [5].Roella et al. [88] have shown how sampling density and the spatial allocationofsampling units are important driversofthe precisionofmapping in studies based on anestimateoflichendiversity on free-standing trees (see also [28]) (Figure 4).

Season ~~~~~••••••

Site~~~~~••••••••

Position on Crown ~~~~~~•••••

~Sample Treatment ....

Analytical Error ~Fr-r-,.....,....,....-r-.-.---r---r-r-r-T"""T-r-,.....,.--r-...,--,r-r-r-r-r-.

Position on Branch •••••••••••••••

o 20 40 60 80 100

% variationofconcentrations

Figure 3. Importance ofdifferent sources ofvariation ofCa and Ph concentration in holm oak (Quercus ilexL.) leaves (redrawn after Bargagli (5J).

3.2.ASSESSMENT ERRORS

Assessmenterrors incorporate measurement and classification errors. They can occurwhen the methodology is poorly standardised, when insufficient care is devoted to itsapplication, or when there are problems with e.g.instrument calibration.

3.3. PREDICTION ERRORS AND ERRORS IN MAPPING

Prediction errors can occur when the data are used in predictive models and/or togenerate maps. According to Kohlet al. [52],predictionerrors have different sources:(i) propagationofclassification errors in the model output; (ii) applicationofmodels ina rangeofdata"thathave been not covered in the constructionofmodels" [52 p. 365];(iii) erroneous models, either as resultsofa wrong build-up, orof increased numericalinstabilityof the model under extreme data ranges. Potential errors in mapping shouldbe addressed carefully, especially in relation to requirements placed bygeostatistics(thepopularisedname for theRegionalised Variables Theory [17,47,64,65]).

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SOUADPAC

SQUADPA E

SOUAml B

2! 21) t$ 10 •

~::";;_jO:" .~

SOUA [JlO, A

...16

Figure 4.Sampling sites (left ) and resulting lichen diversity maps (right) obtained fr om 5 field crews (A. B. C.D. E) operating indipendently in the same area with the same methodology (aft er Roella et al.[88J).

In particular, the widespread (and often blanket) applicationof commercialsoftware packages in reporting monitoring results can be a sourceoferrors. A typicalexample is the uncritical useofautomatic mapping programmes to illustrate patternsoflichen diversity or of metal concentrations in lichens. Such programmes can alwaysperform interpolations among neighbouring sampling sites: however, suchinterpolations make no sense when the sites are located e.g. in two different valleysseparated by high mountains. In other cases the kriging technique is used withoutparametrisation of the default variograms or interpolation is done with inadequatesampling intensity, e.g. when the distance among sampling stations is higher than theknown diffusion patterns of a single metal from an important point source.

3.4. NON-STATISTICAL ERRORS

Non-statistical errors are frequent, ubiquitous and can be very serious [52]. They usuallyoriginate from errors in measurement, sampling, and/or data processing. Examples aremistakes in data entry, programming errors and errors in defining the sample frame.

3.5.TERMINOLOGY AND DEFINITIONS

Different definitions for the same thing or similar definitions for different things can bea source of inconsistency among investigations. This is a problem when attempting to

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compare results orharmonised/standardisedmethodologies or to identify frameattributesofthe target population at large-scale. For example,Kohl et al. [52] report thedefinitionsof forests adopted by several European countries: minimum width variesbetween (and sometimes within) countries, ranging from 9 to 40 m, and minimum areafrom 0.01 to 2 ha. These differences create problems when attempting to comparestatistics or select common frame attributes. Somestandardisationofterminologywasattempted in the German guideline for lichenbiomonitoring[110], and a good exampleis the outputofthe IUFRO working group on forest terminology [20].

3.6. ERROR BUDGET

The total error can be quantified by determining the error budget to optimise theinterpretationof results. This procedure is especially well developed for forestinventories (e.g. [32]). The error budget provides a calculationof the total erroraffecting the survey estimates, which can be achieved by a mathematical model thataccounts for the various error sources. Kohl et al. [52] report the following formula byKish [49]:

MSE(y)

L:S;

4. Sampling design

meansquareerror

sum of all varianceterms(8,) frommultipleerror sources

squaredsumof the biases(B,)

(1)

Sampling design provides rules allowing monitoring sites(hereafterreferred to as"sampling units") to be properly selected and installed, and is always related to theobjectiveof the programme and to its domain. For example, sampling density can bevery differentif the investigation concerns an urban area, a region, or an entire country.Samplingdesign can be defined by the identificationof:• the survey form (how the survey develops through time),• the sampling strategy (the way in which sampling units are selected and allocated),• the sampling density (the numberofsampling units to be used), and• the sampling tactic (number and selectionofobservations in each sampling unit).

Before embarking into sampling it is important to know what the entity is and/or theattribute to be sampled, i.e. the population from which the sample should be selected(see paragraph 2.2). Although seemingly obvious, an explicit definitionof thepopulation to be sampled is rare in thebiomonitoringliterature; for example, it hasnever occurred in 125 studies concerning lichens carried out in Italy [2].

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4.1. SURVEY FORM

The survey form identifies the temporal dimensionof the investigation. Typically,monitoring is "tracking an environmentalentity through time" [99], which impliesrepeated observation of the same resource. This issue, almost neglected in lichenbiomonitoring, has received some attention in forest inventories. Forest surveys havemostly adopted three forms to estimate change: temporary form (independent repeatedsurveys in different sampling units), continuous form (in the same sampling units) andSampling with Partial Replacement (SPR) forms (some sampling units remain the same,some are replaced according to a designed process) [93]. Thus, different solutions arepossible, with the best choice depending on the natureof the study (e.g. use oforganismsin situ , or of transplants), and on the degreeof temporal autocorrelation ofthe data. Using the same sites has the major advantage that the varianceof changeestimates is reduced by the positive covariance between two subsequent samplings [93].However, this may change when the entity/attribute being monitored is subject tochanges through time which are independent from the phenomenon being evaluated. Forexample, the EU-UN/ECE forest monitoring programme [59] considers defoliation asan indicatorofair pollution effects on forests. Unfortunately, ageing of sample trees canbe an important factor in determining defoliation, and long-term observationson thesame sample trees can be biased by natural ageing, which can obscure the effects ofenvironmental stressors [103]. Therefore, while the permanent plotsof the continuoussurvey form can be of value for detecting environmental trends, much care is needed atthe interpretation stage and on how the observations are selected within each individualplot. This issue will be discussed further in paragraph 4.4.4.

4.2.SAMPLING STRATEGY

4.2.1. General remarks

Monitoring sites can be selected following either a design-based statistical approachwhich uses probabilistic methods, or by a model-based approach (e.g. [99]). These twoapproaches lead to different methods for making inferences, i.e. for reachingconclusions which are conceptually valid for the entire population from which thesample is taken [99]. In principle, the main advantage of a probabilistic sample is thatinferences are free of subjectivity, spatial patterns can be detected, and both design- andmodel-based data analysis can beperformed,

4.2.2. Locational methods

There is a huge amount of literature on sampling schemes (e.g. [16,18,33,46]),whichshows that, once again, sampling allocation depends on the aimof the study.Monitoring the effectof a point/linear emission source will probably need a typicalgradient study (sampling units located at increasing distance from the source, and/oralong the prevailing wind direction [1, 71]) and/or dense, emission-related samplinggrids [111]. On the other hand, investigations over an area where a distinct perturbationdoes not occur (e.g. large-scale screenings) will need a more regular distributionofsampling units. In general, sampling based on an objective (either random orsystematic) selection of sampling units is the most suited for providing unbiasedestimatesof a given attribute (e.g. lichen diversity) for a given area. In particular,

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systematic sampling (either aligned or unaligned, stratified or not) offers operationaladvantages. The selection of sampling units will be easier compared to a purelyrandomised procedure, and the spatial dependency of observations will be partiallycontrolled by regular spacing among units. This has further advantages for mappingpurposes [25, 108].

For monitoring purposes, strategies based on preferential/judgemental samplingshould be generally avoided (e.g.[78, 101]), especially when a new investigation has tobe carried out. This is because preferential sampling often results in the locationofsampling units along roads or tracks, or near easily accessible places which bydefinition are prone to biased environmental conditions (Figure 5). Such sites are theleast suited for providing data for mapping or for estimatesof statistical descriptorsofe.g. lichen diversity. Only when the aim is a comparison with preferentially chosen sitesalready investigated in the past, visiting the same sites can be permitted. Note, however,that this is a typical re-sampling rather than a completely new investigation.

Figure 5. Location of lichen biomonitoring sampling sites (solid dots) selected according to preferentialsampling in the pro vince ofPadova (Veneto Region. NE Italy) (after [72J). Note how sites tend to be locatednear settlements and motorways.

4.3. SAMPLING DENSITY

Sampling density (or intensity) controls much of the sampling error and -with aninverse relationship - the costs of the investigation. A very high sampling density may

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lead to low errors, but may be unsustainable economically. On the other hand, a verylow sampling density may provide cheap data, but their uncertainty is so high that theycannot be used [29, 50]. Steinnes [98] reports about the performanceof differentsampling densities for the moss surveys carried out in Norway in 1976 (43 samplingsites) and 1977 (490 sites). As expected, while the 1976 grid was still able to detectsome important pattern, the denser grid of 1977 was more effective both in termsofdetection range (Table 6) and for the detection of spatial patterns at a higher spatialresolution. However, these results should be evaluated also in termsofcosts, which arelikely to increase about ca. 10 times for the denser grid.

TABLE 6.Mean values and ranges 0/element concentrations in Hylocomium splendensobta ined by the 1976(43 sampling sites) and 1977 (490 sampling sites) Moss Surveys in Norway. Values in mgkg-I (after [98J).

Element Mean Range1976 1977 1976 1977

Na 322 346 83-1900 40-6580AI 890 945 80-5860 120-8970CI 171 188 40-410 30-810Sc 0.16 0.18 0.03-1.07 0.02-2.23V 3.7 3.6 0.3-10.1 <0.5-64Cr 2.1 2.8 0.3-8.3 0.2-128Mn 235 310 30-720 22-1240Fe 663 687 130-3700 110-7350Co 0.41 0.32 0.09·1.57 0.03-3.73Cu 6.6 7.5 2.6-28.8 1.3-82

Zn 40 41 11·80 12·241As 0.57 0.52 0.13-2.33 0.05-3.54Se 0.39 0.47 0.05-1.09 <0.10-2.84Sr 7.4 6.5 1.6-18.7 1.3-34.7Rb 9.1 11.3 1.1-26 1.2-52Mo 0.13 <0.2 0.05-0.44 <0.2-1.8Ag 0.12 0.10 0.02-0.59 <0.05·1.38Cd 0.29 0.30 0.07-0.86 <0.01-1.5

Sb 0.28 0.30 0.05-0.94 0.03-1.96Cs 0.29 0.19 0.04-1.21 <0.01-2.06La 0.49 0.72 0.08-1.51 0.07-15.2Sm 0.10 0.07 0.01-0.27 0.006-0.89Pb 37 26 6-148 1-181Th 0.10 0.12 0.01-0.37 0.01-2.55

Classically, the appropriate samplingdensity can be unambiguosly definedaccording to the following formula[108]:

n~(ta e:)' (2)

where:n= numberofsampling units,t= Student t at defined probabilitylevelp(for large sample sizesandp= 0.05,t=1.96),CV=coefficientofvariation,E= relative error (% of mean).

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This formula enables the sampling density to be determined regardlessof the sizeofgeographical areas.

Different formulas were reported by Spellerberg [96 pp.79-80]. However, the useofsuch equations impliesa priori knowledge about the expected variabilityoflichen data,which is in part dependent on local environmental conditions.

Whenever possible, it is recommended to optimise sampling density by apreliminary survey (e.g.[Ill]). Otherwise, information from other studies in similarenvironmental conditions may be used. In this case, it is better to adopt a conservativeapproach, i.e. a larger coefficient of variation into equation (2),if this is economicallyfeasible.

An example of different grid densities leading to different errors according toequation (2) is shown in Figure 6. Note how after a certain threshold the reductionoferrors becomes very costly in termsofsampling efforts.

35,-------------------------,

30

25

~ 20

~~ 15 n: 864 (18 .7 km grid)

J ~10 / / n: 1351 (14 .9 km grid)

5 ", - -/~n:2401(11 .2kmgrid)

20000160001200080004000o~~====================±JoNo of sampling sites

Figure 6. Relative error computed according to equation (2) in relation to the number of sites (assumedCV=75%; t=I .96; p=O.05). The dashed circle identifies sampling numbers aI/owing relative errors of3.4 e5% respectively . The figures within the graph indicate actual sampling numbers and the required grid sizesf or Italy (after {28} . modified).

Considering lichen biomonitoring, Table 7 suggests sampling densities for differentgeographical scales and types of study, which can be adopted when information on thevariabilityof lichen data is not available, and when there is no chance for developing aformal sampling design study.

TABLE 7. Possible grid densities for different geographical scales and types ofstudy. Data are in km.

Geographicalscale<5 >5-100 >100-1000 >1000km2 km2 km2 km2

Distinctperturbationoccurring O.25xO.25 O.5xO.5/6x6 Ixl/12x12 unusual

Before-After O.25xO.25 O.5xO.5/6x6 Ixl/12x12 unusual

No distinctperturbationoccurring O.25xO.25 /O.5xO.5 0.5xO.5/ 6x6 3x3/12x12 >9x9

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4.4. SAMPLING Txcnc

Sampling tactic concems the size, internalorganization,hierarchy, number andselectionofobservationswithin the sampling units.

4.4.1. SizeDependingon the natureof themonitoringprogramme, the sizeof the samplingunitscan be expressedin termsofboth surface andnumberofobservations.In general, thesize of the plot should be able to capture the within-sitevariability. Conceptually, anadequate plot size can be defined as that"whose furtherenlargementproducesaninsufficientgain in information for the added cost" [53]. Datavariability,of course,depends on the local conditions,on the indicator and on the adopted index. In addition,the sizeof the sampling unit must bebalancedwith the grid density: for example, withunits of0.25xO.25 km, 0.25km is also the maximum densityof thegeographicalgrid.The adequatenumberofobservationsper sampling unit depends on the sizeofthe latter(when fixed-area plots are used) or ona priori selected numbers, in which case someindication can come from equation (2).

4.4.2. Shape, organisation and hierarchyIn general, very little attention has been paid in Europe to the designofbiomonitoringsites. In most cases, the methods only indicate e.g. thenumberof trees to be used formonitoringlichen diversity, or thenumberof tobacco plants tomonitorthe effectsofozone (e.g. [82, 109]). Much less(if anything) is said about how and on what surfaceindividual observationsshould be taken, and/or howtransplantsshould be installedwithin the sampling unit, although some attempts are being made indesigning anational lichen diversity network for Italy [28]. Pros and consof differentplot typesshould considerdifferentaspects, such as the benefitof reducingborderline(uncertain)situations, the easeof field work, the temporal and spatialdependencyof observations(see Hurlbert[39]), and the potential for area-relatedstatistics. Typical samplingunitshapes refer to points, lines, or surfaces which can be combined indifferentways [9].Circularshapes offer the lowestperimeter/surfaceratio, which minimisesthe chanceofmeetinguncertainsituations like trees standing exactly on theperimeterof the unit. Inaddition, a circle is much easier to identify, being defined by two data only, centre andradius.

Samplingunits can be organised as: (i) an individual sampling site with no furthersubdivision, or (ii) sampling sites (Primary Sampling Units- PSUs) within whichSecondarySampling Units (SSUs) are installed. In the latter case, thedescriptor(e.g.lichen diversity index)of the PSU can be the (weighted) average among SSUs. Ahierarchicalsystem of PSUs and SSUs can help to distribute theobservationsoverspace, to ensure adequate replication, to improveestimationandprecision,and to permittesting within and between PSUs [39]. SSUs can be distributedin clustersthroughoutthe PSU surface, which improves accuracy [39] and may also mitigate practicalconstraintstypicalofselecting observations within the sampling site (Figure 7).

4.4.3. Spatial dependencyThe selection anddistributionof theobservationsover the areaof the sampling units

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helps also in controlling the statistical propertiesof the data. Ideally, indicators shouldbe randomly distributedwithin a sampling unit, in order to ensure independencyofobservations. However, the chance to capture the variability of the sampling unitincreases when the observations (e.g. sample trees) are evenly distributed; this helpsalso to avoid the spatial dependence of the observations. However, ensuringrandomisation and an even distribution of observations at the same time can be difficult.In many cases, even the simple location of randomly selected observations over thesampling unit can be very time consuming. For these reason, secondary sampling unitsare reasonable alternatives (Figure 7).

1 km) ( ) (

O~)

o 0o • • 0

In. 0 • ~-0.:••-. 00o 0

0 o 0c

1 km) ( ) (

O(!O 0O. • 0

n • • le_o.:••

·0 00o 0

0 o 0c

A. Sampl ing the trees closest to the centre B. Sampling the trees closest to the centre, C.Sampling thetrees into subplots .(regardless the quadrats) 3 trees per quadrat 3 trees per quadrat

Figure 7. Effects of different sampling tactics on the spatial distribution of I2 hypothetical sample individuals(trees, solid circles) selected from the same populat ion (solid plus open circles) at a given sampling unit oflxl km. A: the lxl sampling units is considered with no further distinction. In this case the shape is aquadrat, and the e.g. 12 individuals closest to the centre are sampled. regardless of their spatial locationwithin the quadrats; B: the lx l sampling unis is considered as PSU and divided into 4 quadrats funct ioningas SSUs. Then. 3 individuals per SSU (the closest to the centre ofPSU) are selected; C: In this case. the Ixlquadrat is the PSU. Circular SSUs are selected fr om the centre ofeach subquadrat (radius equal to 250 m,e.g. 14 of I PSU side lenght} and 3 individuals per SSU are selected. taking into account their distance fromthe centre ofSSU (thin line) (after Asta etal..chapter 19. this volume [4)} .

4.4.4. Temporal dependencyAlthough this issue is not addressed at all in other biomonitoring works, those usingautochthonous indicators (such as epiphytic lichens) seem to prefer a sampling based onthe "nearest trees" concept, e.g. the suitable trees closest to the geographical co­ordinates of the sampling unit. When such trees arc monitored through time, thisselection procedure can create problems for trend analysis. If a fixed number of treesper sampling unit is selected, and these trees are the same through time, the followingpoints should be considered: (i) the valuesof the attribute can be biased by ageing, or(ii) by succession dynamics, (iii) trees may die or be harvested (thus disappearing fromthe sampling unit), (iv) new trees may grow in [51]. Only fixed area plots provide asample which is strictly identical over time, since data can be always referred to surfacearea. At the best, all trees within the sampling unit should be considered. While thisseems very demanding in termsof work load, candidate trees must generally fulfilseveral requirements in terms of age,diameter, inclination,vicinity to other trees and soon, which means that not all trees are suitable for sampling.

4.4.5. Potential for area-related statisticsAnother problem concerns the extent to which estimates based on sampling units with

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constant numberof nearest trees can be interpreted in termsof area-related attributes.Preto [84] states that"only sample plots with constant area or with variable samplingprobability provide unbiased estimates of most trees and standparameters."If notadequatelysupported by a clear design, statements like "45% of the study area has alichen diversity between 5 and 10" are problematic, especially when used to makeassertions about the proportionofa given area subjected to differentSOz concentrations(e.g.[72]).

4.4.6. Observational units for transplantsTransplantsare frequently used inbiomonitoring: lichen and moss bags, as well ashigher plants can be introduced in a study area for monitoring purposes. When usingtransplants, it is always possible to have the exact numberofobservationsrequired bycosts and precision. A difficulty may be that of bringing bioindicators and the necessaryfacilities to the sampling site under complex terrain condition. In addition,maintenanceand watering may be needed (e.g. for higher plants), although this problem can besolved by adequately designed infrastructures (e.g. water containers with timers).Depending on the typeoffacility, the above issues can be a serious constraint which hasoften resulted in biased site selection, because easy accessible sites are often preferred.

4.4.7. Observational units for bioindicators in situAutochtonous bioindicators (tree, lichens, herbs) do not require infrastructures andmaintenance, but may be subject to other formsof restriction, such as preciserequirements in termsof species, age, dimension and location. This often impliesmeasurementsand observations to check whether these requirements are met. Thus,although field crews are not asked to carry infrastructures and to visit the site regularly,they may have considerable problems in finding individuals that meet therequirements,and considerable time may be necessary to carry out the field procedure.

5.QualityAssurance

Quality Assurance (QA) "is an organised groupofactivities defining the way in whichtasks are to be performed to ensure an expressed levelofquality" [68]. This means thatthe data should be the resultofa process in which all steps are carefully and correctlyaddressed, from design to data collection, processing and reporting [43]. QA is wellacknowledgedas an important tool in biomonitoring [24, 81, 85, 106, 112, 113]. Themain benefitof a QA programme is the improvementof consistency, reliability andcost-effectivenessof the programme through time. For example, long-term monitoringprogrammes may last for decades, and the time factor can have a strong impact on theimplementationofthe work by the personnel involved [94]. A QA procedure is criticalfrom the early stagesofmonitoring programmes, and a QA plan is fundamental, sinceitforces programme managers to identify and evaluate the majorityof factors involved inthe programme [94] (Table 8). In addition, the assessmentof data quality permits amathematical managementof uncertainty, which can result in a more appropriatepresentation and useof the data [26]. Four main activities areconsideredin a QAprogramme [15] (Table 9):

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• Quality Management (QM). It concerns the proper designof the project and itsdocumentation and it has the major benefit of ensuring that activities are performedin a proper way.

• Quality Assurance (QA). It concerns the first steps of data quality evaluation,including the use and documentation of Standard Operating Procedures (SOPs). Ithas the benefit of providing consistent methods with known and verified dataquality.

• Quality Control (QC). It mostly concerns the training, calibration and controlphases. Its major benefit is to ensure that data are appropriately collected andquality assurance is carried out.

• Quality Evaluation (QE). It mainly concerns the statistical evaluation of dataquality. It allows precision and accuracyof determinations to be evaluated,providing a basis to evaluate data comparability.

TABLE 8.Typical contentsofa QualityAssurance Plan(QAP) (after[94]).

Section

TitlepageApproval page

TableofcontentsIntroduction

Data qualityobjectives

Program organizationandresponsibilitiesData collection

Dataevaluation

Content

Identifies the planDocuments the awareness and acceptanceofthe QAPby management and theindividuals responsible for itsimplementationProvidesthe report structureBriefpresentationofthe monitoring program's purpose,scope,descriptionoffield work. Serve as quickreference to the program and to prevent having to go toanother document for basic information on the programitself.Defines and documents the work objectives and thequalityofdata needed to meet thoseobjectives.Identifies the major participants in the monitoringprogram and their roles andresponsibilities.Focus on the Quality Control (QC) data collection andhow it relates to the actual field/lab data collection.Discuss QC sample types, siteselection, frequency andsampling procedure.Documents how QC data will beevaluatedand used toensure the technical aspectsofthe monitoring areappropriate and complete.

In particular, Quality Control is aimed at ensuring the comparabilityof the resultsfrom different sites and the reproducibility of data collected by different observers.Failure to harmonise methodologies may make temporal and spatial comparisonmeaningless. This is a key issue for investigations aiming to generate representativeresults at large-scale (national, international) and in the long-term. Methods are not theonly source of inconsistency: changes in personnel, external conditions, measurementlocations, spatial coverage, and measurement frequency [6] can be important as well.Use of inter-comparisons, maintenanceof methodological meta-data bases and dataquality assessment are imperative for long-term monitoring programmes. In case of

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chemical analyses, samples should be stored for possible future analysis when newtechniques will replace the current ones.

TABLE 9. QA activities, areas ofconcern and related benefits (after (15. 43]).

Activity andareasof concernQuality ManagementProper designPlanningModelsDefinitionofdata quality requirementsRelevant samplesError structurePeer evaluation and reviewDocumentationImplementationofQA programmeQuality AssuranceData qualityobjectivesUse ofstandardUse ofstandardoperatingprocedures (SOPs)Verificationand validationDocumentationQuality ControlEvaluation samplesTrainingand useofSOPsPrecision determinationsCalibrationControl chartingQuality EvaluationUse ofstandardsReplicationBlanksInspections and audits

6.Conclusions

Benefit

Considers if the right questions are being askedAllows comparabilityclassificationsIdentifies criticalmeasurementsvariablesConsiders datausers' needsEvaluates numbers and representativenessPartitions sampling andmeasurementerrorJudges correctnessRecord design processAchieves data gualityrequirements

A ids method selectionAllows quality control and evaluationProvides consistent useofmethod with known data qualityDocuments sample integrity and dataconsistencyProvidesevidenceofactivities and quality

Basis for statistical controlPromotes statistical controlDefines random variation and allows accuracyassessmentReduces or eliminates biasDocuments statistical control

Allows precision and accuracy determinationsProvides ongoing evaluationMon itorscontaminationProvides objective evaluationand basis forcomparability

Biomonitoring is increasingly recognised as a useful tool to collect information aboutstatus and trendsof the effects of air pollution and environmental changes on naturalresources. While this is important for biologists, a full scientific acceptance ofbiomonitoring beyond the biologists' community will be reached only when allmethodological questions have been clarified and standardised.

To date, with few exceptions, the issues related to design and quality assuranceprocedures are probably the least explored. This is unfortunate for a varietyofreasons.Design is a fundamental partofmonitoring programmes.Although data fromparticularsites can be important, policy and decision-makers usually require information at largergeographical scales, which means that a statistical approach is needed for extrapolationfrom a site to a region. In addition, adequate designs can ease the integrationofdifferentprogrammes into a more comprehensive one.

Collecting environmental data is an important activity, but data are useless or evendangerous if they do not meet expressed levelsofquality. In the past, most monitoringprogrammes emphasised the results, while much less care was given in reporting figuresabout the extent to which data were considered valid, comparable and credible. This

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was, from a historical perspective,fully understandable, but that time is over. More andmore environmental agencies ask and force environmental scientists to provide theirdata with estimatesof uncertainties, intercalibration tests, and validation. Proper QAprocedures and adequate design will beof great help in biomonitoring, improving thestrength of its methodological basis and facilitating environmental biologists toovercome the traditional problems of acceptance and credibilityoftheir studies.

7. Acknowledgements

The authors are grateful to three anonymous referees for their helpful and stimulating comments. We are alsoin debt with P.L. N imis (Universityof Trieste,Italy) for the thorough revisionofthe manuscript. Additionalthanks to G. Bnmialti and P. Giordani (University of Genova, Italy) and to A. Cozzi (LINNJEA Amb iente,Firenze, Italy) for providing important references.

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1976 Norwegiansurvey, in B. Markert (ed.),Plants as Biomonitors, YCH, Weinheim, pp. 381-394.99. Stevens, D. (1994) Implementationof a national monitoring program,Journal of Environmental

Management 42, 1-29.100. Stoddard, J.L., Driscoll, C.T.,Kahl,J.S.,and Kellog,J.P.(1998) Can site-specifictrends beextrapolated

to a region? An acidificationexample for the Northeast,Ecological Applications 2 (2), 288-299.101. Stohlgren, TJ., Binkley, D., Veblen,T.T., and Baker, W.L. (1995) Attributesof reliable long term

landscape scale studies: malpracticeinsurance for landscape ecologists, Environmental Monitoring andAssessment 36, 1-25.

102. Stout, B.B. (1993) The Good, the Bad and the Uglyof monitoringprograms: defining questions andestablishing objectives,Environmental Monitoring and Assessment 26, 91-98.

103. Strand, G-H. (1995) Estimation of the difference in crown vigour for 2280 coniferous trees in Norwayfrom 1989 to 1994, adjusted for the effectsofageing,Environmental Monitoring and Assessment 36, 61­74.

104. Summers, J.K. and Tonnessen,K.E . (1998) Linking monitoring and effect research: EMAP's intensivesites network program,Environmental Monitoring and Assessment 51,369-380.

105. Swamee, P.K. and Tyagi, A. (1999) Formationofan air pollution index, Journal of the Air and WasteManagement Association 49,88-91.

106. Tallent-Halsell,N .G. (1994) Forest Health Monitoring 1994. Field Methods Guide, EPN6201R-94/027,U.S. EnvironmentalProtection Agency, Washington, D.C.

107. Urquhart, N.S., Paulsen, S.G.,and Larsen, D.P. (1998) Monitoring forpolicy-relevantregional trendsover time,Ecological Applications 8 (2),246-257.

108. Van Meirvenne, M . (1991) Characterizat ion of soil spatial variation using geostatistics,RijksuniversiteitGent, Fakultet van deLandbouwwetenschappen, Laboratorium voor AgrarischeBodenkunde,PhD Thesis, 45 pp.

109. YDI (1995) Mapping of Lichens for Assessmentof Air Quality, VDI 3799-1. VDI HandbuchReinhaltung der Luji, pp. 1- 24.

110. YDI (1999) Biological measuring techniques for thedeterminationand evaluationof effectsof airpollutants on plants.- Fundamentals and aims.VDI3957-1 (1999) VDI Handbuch Reinhaltung der Luji ,pp.I-27.

III . Wagner, G. (1993) Large-scale screeningofheavy metals burdens in higher plants, in B. Markert(ed.),Plants as Biomonitors, YCH, Weinheim, pp,425-434.

112. Wagner, G. (1995) Basic approaches and methods for quality assurance and quality control in samplecollection and storage for environmental monitoring,The Science ofthe Total Environment 176,63-71.

113. Wagner, G., Mohr, M.-E., Sprengart, J., Desaules, A., Muntau, H., Theocharopoulos,S., andQuevauviller,P. (200 I) Objectives, concept and designof the CEEM soil project, The Science of theTotal Environment 264, 3-15.

114. Wittig, R. (1993) General aspectsofbiomonitoringheavy metals by plants, inB. Markert(ed.),Plantsas Biomonitors, YCH, Weinheim, pp. 3-27.

115. Wolterbeek, H.Th. and Bode, P. (1995) Strategies in sampling and sample handling in the contextoflarge scalebiomonitoringsurveysoftrace elements air pollution,The Science ofthe Total Environment176,33-43.

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Section2

MONITORING LICHEN DIVERSITY

AND ECOSYSTEM FUNCTION

editedby

ChristophSCHEIDEGGER and SusanWILL-WOLF

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MONITORING LICHEN DIVERSITY AND ECOSYSTEM FUNCTION

An Introduction

S. WILL-WOLF' and C. SCHEIDEGGER 2

'Department ofBotany, University ofWisconsin, 430 Lincoln Drive,Madison, WI53706-1381, USA (swwo/[email protected])2WSL, Swiss Federal Research Institute, CH-8903 Birmensdorf.Switzerland ([email protected])

Concern about maintaining the biodiversity of lichen species and communities has beenan issue with lichenologists for many years. Beginning over 100 years ago with effectsof air pollution (see Section 1, this volume), and expanding in the last 50 years toinclude effectsof land management and fragmentationof natural habitats, concernabout the lossof lichen biodiversity in connection with human modificationof naturalenvironments has led to many studies designed to assess patterns and monitor trendsoflichen biodiversity and community composition world-wide (reviews by [1, 7]). Morerecently, recognition of the regional, continent-wide, and even global scaleofthreats tothe normal functionof lichens in ecosystems [10] has fostered studies and monitoringefforts designed to assess the large-scale impactof multiple threats to the structure,composition, and function of lichen communities (e.g. [5]). As modem conservationconcepts have expanded to include traditionally neglected groups of organisms, it hasbecome more appreciated that understanding and maintaining the biodiversityof taxaother than vascular plants and vertebrate animals must be addressed explicitly [2-4, 6].Lichens demand specific conservation strategies [7] and approaches to monitoring [9].The desire to compare and evaluate the most current approaches to monitoring lichenbiodiversity and ecosystem function was the motivation for the chapters in Section 2ofthis volume.

The chapters in this section cover a diverse arrayofapproaches to monitoring lichenbiodiversity and ecosystem function, from focussing on one or a few species tomonitoring the biodiversity of lichens across whole regions. One thing these chaptershave in common is that all address monitoringof lichen abundance, diversity, and/orcommunity composition. Response to air pollution is only oneofmany possible factorsconsidered to affect lichens, in contrast to chapters in Section 1ofthis volume.

In chapter 11 Will-Wolf, Scheidegger, and McCune review methods for monitoringlichen biodiversity and ecosystem function with lichens. This chapter is designed tocomplement the chapter by Ferretti and Erhardt (chapter 9) on key issues in designofbiomonitoringprograms, which should be read first.

Chapters 12 and 13 focus on a particular monitoring goal that can be addressed inmany different situations. In chapter 12, Scheidegger and Goward review methods for

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monitoring lichens for conservation, emphasising rare species, the developmentofRedLists, and conservation action plans. In chapter 13, Insarov and Schroeter reviewapproaches to using lichen communities to monitor climate change, evaluatingmonitoring approaches for their ability to factor out other causesofvariation.

Chapters 14-17 address monitoring lichens in a particular habitat, reviewingmethodology for answering monitoring questions common to those habitats. Chapters14 and 15 address methods for investigating lichen community response to multiplecauses in widespread habitats, and review protocols to address research andmanagement questions at multiple scales world-wide. They cover both detailed studydesigns intended to address local biodiversity issues and rapid survey monitoringprotocols using subsets of lichen communities as monitorsof large-scale ecosystemfunction and biodiversity. Chapters 16 and 17 address monitoring lichens in restrictedhabitats,and concentrate their reviews on studiesdone mostly in Europe.

In chapter 14, Will-Wolf, Esseen, and Neitlich review methods for monitoringforest lichens, focusing on monitoring effectsof forest management and habitatfragmentation on lichenbiodiversity, as well as large-scale programs using lichens tomonitor forest ecosystem function and changes from multiple causes.

In chapter 15, Rosentreter and Eldridge review methods for monitoring lichens ingrasslands, deserts, and steppes; they focus primarily on effects of grazing on thesesystems. They review methods for monitoring biodiversity, but emphasise methods forusing lichens to monitor ecosystem function.

In chapter 16, Aptroot and James review efforts to monitor lichens on humanmonuments, surveying efforts worldwide, but concentrating on monitoring projects inEurope.

In chapter 17, Fletcher and Crump review monitoringof lichens in maritimehabitats, concentrating on Great Britain because resultsofmany studies in other areasare not publicly available.

Methods for monitoring saxicolous lichensare emphasised in chapters 13, 16, and17, and are included in chapter 12. Epiphytic lichens are emphasised in chapter 14 andare included in chapter 12. Terricolous lichens are emphasised in chapter 15, and areincluded in chapter 12. Lichens of tundra are referred to in chapters 13 and 15, but alsosee Toemmerviket al. [8] for monitoringoftundra lichens.

All chapters in this section stress that the designofa monitoring study needs to becarefully considered to ensure that sampling protocols are appropriate to meet thedesired goals. Careful documentation and data archiving are essential for the successfulcontinuationoflong-termmonitoring projects.

References

I. Bates, J.W. and Fanner, A .M. (eds.) (1992) Bryophytes and Lichens in a Changing Environment.Clarendon Press, Oxford.

2. Galloway, DJ. (1992) Biodiversity: a lichenological perspective,Biodiversity and Conservation 1,312­323.

3. Hawksworth, D.L. and Kalin-Arroyo, M.T. (1995) Magnitudeand Distribution of Biodiversity, in V.H.Heywood and R.T. Watson (eds.), Global Biodiversity Assessment. Cambridge University Press,Cambridge,pp. 107-192.

4. Jonsson, B.G. and Jonsell, M. (1999) Exploring potential biodiversity indicators in boreal forests,Biodiversity and Conservation 8, 1417-1433.

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5. McCune, B., Dey, J., Peck, J., Heiman,K., and Will-Wolf, S. (1997) Regional gradients in lichencommunities of the southeast United States,The Bryologist 100 (2),145-158.

6. Pharo, EJ., Beattie,AJ., and Pressey, R.L. (2000) Effectivenessof using vascular plants to selectreserves for bryophytes and lichens,Biological Conservation 96, 371-378.

7. Scheidegger, C., Wolseley, P.A., and Thor, G. (1995) Conservation biology of lichenised fungi,Mitteilungen der Eidgenbssischen Forschungsanstalt jUr Wald, Schnee und Landschafl70, 1-173.

8. Toemmervik, H., Johansen, RE .,and Pedersen, J.P. (1992) Use ofmultitemporal Landsat image data formapping the effects of air pollution on vegetation in the Kirkenes-Pechenga area in the period 1973­1988,NORUT report 2024/1-92,Tromsa,pp.32.

9. Will-Wolf, S.,Hawksworth, D .L.,McCune, B.,Sipman,HJ.M.,and Rosentreter, R. (in press) Assessingthe biodiversityof lichenized fungi, in G.M. Mueller, G.F.Bills, and M.S. Foster (eds.), Measuring andmonitoring biological diversity: standard methods for fungi. Smithsonian Institution,Washington,DC.

10. Wolseley, P.A. (1995) A global perspective on the status of lichens and their conservation,Mitteilungender Eidgenossischen Forschungsanstalt fiir WaldoSchnee und Landschafl70, 11-27.

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METHODS FOR MONITORING BIODIVERSITY AND ECOSYSTEMFUNCTION

Monitoring scenarios, sampling strategies and data quality

S.WILL-WOLF I, C. SCHEIDEGGER 2 andB. McCUNE 3

IDepartment ofBotany, University ofWisconsin, 430 Lincoln Drive,Madison. WI, 53706-1381. USA ([email protected])2WSL Swiss Federal Research Institute. CH-8903 Birmensdorf,Switzerland ([email protected])3Dept. ofBotany & Plant Pathology. Oregon State University, CordleyHal/2082, Corvallis , OR, 97331-2902, USA ([email protected])

1. Introduction

In this chapter we review strategies for monitoring lichenbiodiversity and formonitoring ecosystem function with lichens. Concern about threats to lichenbiodiversityworldwide has led to increased interest in monitoring the statusof lichensand assessing threats (e.g.reviews [1,32]).The word"health" referring to the degreeofmaintenanceofnormal, natural ecosystem function, as in " forest health" or" ecosystemhealth", is widely used and accepted in some partsof the world (e.g. [17]), but not inothers, so we use the phrase"ecosystemfunction" for the concept in thischapter. Formonitoringof ecosystem function, we focus on methods that can be applied on broadscales and/or to detect responses to multiple causes other thanpoint-sourcepollution.This chapter is intended to complement the detailed discussion by Ferretti and Erhardt(chapter 9, this volume)of issues in the designof monitoringprograms to assesspollution responseof lichens. Most of that discussion is relevant to our focus, and ourdiscussion builds on that chapter.

We define monitoring as studies designed to assess trendsof change over time aswell as variation in status across an area for lichencommunitiesor indicatorsofecosystem function. We consider any study which is designed to be repeated over timeto assess trends a monitoring study, even before repeat sampling has been completed.We emphasise quantitative methods for monitoring lichens.

The definitionof "community" used in this chapter is very broad, referring to agroup of lichen species growing together, but allowing broad interpretationof the sizeof a community. Our usage thus includes both very fine-scale definitionsof acommunity(for instance the lichen communityofvertical facesofgranite tombstones ina local area) and much broader definitions (for instance the lichencommunityof oakforests in eastern North America).We do this to emphasise that similar questions about

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the relationshipof lichen community composition to environmental and managementvariables can be addressed at different spatial scales.

2.Monitoringgoals, resources,andmethodology

Two different monitoring goals are addressed in the second sectionof this volume,monitoring biodiversity of lichens and monitoring ecosystem function. A major goalofbiodiversity inventory and monitoring is to assess the statusofand monitor changeofthe entire lichen community as completely as possible. Monitoring ecosystem functionwith lichens, on the other hand, uses lichens as indicatorsof the conditionof anecosystem or of change in one or more ecosystem functions. This is often done mostefficiently with a selected subset of the lichen community.

2.1.MONITORING BIODIVERSITY OF LICHEN COMMUNITIES

One common goal of monitoring lichen biodiversity is to track the statusofrare species.Another goal of biodiversity studies is to explicitly relate responseofthe entire lichencommunity to responseof more easily monitored subsetsof lichens. In this way,investigators may calibrate the subsets as indicatorsof biodiversity and ecosystemfunction at a few sites, when only the subsets are used in more widespread monitoringprogrammes. Such studies provide the large-scale context in which unique sites andhotspots of lichen biodiversity are embedded.

Those studies that monitor intensively the biodiversity of entire lichen communitiesare typically conducted at unique or relict sites. Because of the small geographic scale,conclusions are less applicable to a broader context. Nevertheless, monitoring thesespecial sites is important.

2.2.MONITORING INDICATORS OF ECOSYSTEM FUNCTION

One of the most important research agendas for lichens currently is to assess themagnitudeof impactsof human ecosystem management on lichen communities andspecies. Examples are response to forest and rangeland management practices, andmonitoring the effectof climate change (an exampleof inadvertent global effects ofhuman activities) on lichens. Studies have in common a design to assess variation fromone or a few management factors, and control for or average out variation from othersources. So they have aspectsof sample design in common with community-basedmethods for monitoring pollution response (see chapters 4 and 5, this volume). Animportant characteristicofhuman management practices as causal variables is that theyare generally widely distributed across regions, and are somewhat equivalent in scaleand patternof geographic distributionto "non-point source" pollution as discussed inchapters in Section 1. Examples include effectsof forest management practices onforage lichens used by caribou in British Columbia [24, 28]; impactsof grazing inrangelands on biotic soil crusts (see chapter 15, this volume); and effectsof forestmanagement practices on nitrogen-fixing epiphytes associated with old-growth forest(see chapter 14, this volume).

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These studies usually focus on a subset of the lichen community as an indicatorofecosystem quality, for example epiphytic macrolichens in forests or soil lichens insteppe. Monitoring with easily sampled subsetsof lichen communities, because it ismore economical than complete inventories, is usually selected in programmes designedfor widespread application and frequent monitoring.

2.3. LARGE-SCALE MONITORING OF ECOSYSTEM FUNCTION

Large-scale monitoring of ecosystem function requires the following components: asimple field method that can be used by many different investigators (oftennonspecialists), a repeatable field method for collecting meaningful data, collaborationamong many scientists and government agencies, compelling links between theorganisms and societal values (the public must value either the organisms themselves orecosystem conditions that they indicate),and timely, clear, interesting products (reports,scientific papers, web sites, etc.)

Indicatorsof function should point to ecosystems where a problem exists or isemerging. They should also tell us when and where ecosystem function is improving.Lichen communities have been demonstrated to be useful indicators for the conditionofmany different ecosystems [17].

2.4. SUBSETS OF LICHEN SPECIES FOR MONITORING

The two most commonly selected kindsofsubsetsof lichen species for monitoring arehabitat subsets and lichen guilds or morphological groups. Commonly used habitatsubsets include epiphytic lichens on tree trunks or lichens on vertical rock faces.

The term"guild" is often used in ecology to refer to a group of organisms whosefunctional roles in the community are similar; for lichens guilds are usually definedbased on morphological similarity and presumed functional similarity, as withcyanolichens. When macrolichens are sufficiently common, they are frequentlymonitored as an indicator of response of all lichens. Dividing species into guildspostfacto to aid in data interpretation and predictionis more common than recording data inthe field by guilds or groups. Field recordingof data only by guilds or groups is notappropriate for monitoringofbiodiversity, but has been found to be a valuable indicatorof ecosystem function in some programmes. Such approaches have been recommendedfor rapid survey monitoring of ground lichens in arid lands (see chapter 15, thisvolume), forage lichens for mammals (e.g. [28]), andof lichen biomass in forests (seechapter 14, this volume).

The way in which response of a subset of lichen species or habitats to variablesofinterest relates to responseofall species in all habitatsis a question extremely relevantto monitoring studies. Intensive small-scale studies to calibrate the response of subsetsto all species will enhance understandingof the generality and predictivenessof thetrends indicated by response of subsets. Subsets of lichens shown to be particularlysensitive to the variablesof interest in the monitoring programme are good subjects formonitoringofecosystem function. For example, epiphytic cyanolichens in forests havebeen found worldwide to be moderately sensitive to air pollution, and also sensitive to

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forestmanagementpractices and habitat fragmentation (reviewed inchapter14, thisvolume).

2.5.PERSONNEL

In many partsof the world there are not enough trained lichen experts to do all themonitoring that is needed, nor is there enough money to pay expert personnel to do themonitoring [2, 17] so monitoring protocols which can be performed bynonspecialistsare the only choice for such areas. Becauseof time and budget constraints, and thepressing need for answers, we must compromise between our desire to use skilledlichenologists as field personnel and practicalitiesofaccomplishingthe task.

For macrolichens surveyed with time limits (2 hrs for a 0.4 ha forest plot), trainednonspecialists were able to find 65-90%of the macrolichen species found by alichenologist [18]. However, in a recent testof a field sampling protocol (without timelimits) that included crustose lichens, a nonspecialist recorded less than one thirdofthespecies a lichenologist was able to find [27]. The obvious conclusion from these studiesis that nonspecialists should collect field data only for macrolichens, and perhaps for afew very easilydistinguishedcrustose species. The useof nonspecialists should bebased upon strict training and quality control procedures. Where the supply and supportfor lichen specialists as field personnel exists, as in central and western Europe (seechapter 35, this volume), crustose lichens can be included in sampling procedures forwidespreadmonitoring programs.

Monitoring of special sites is more easily done by individualinvestigators,whilelarge-scale monitoring usually needs to be done by collaboration between investigators.Monitoring of whole-communitybiodiversity and monitoring for rare species (seechapter 12, this volume) must be conducted with lichen specialists who can distinguishrare species.

3. Designofsamplingprotocols

Many issues relating to designofsample protocols for monitoring lichens are covered inFerretti and Erhardt (chapter 9, this volume) andWill-Wolf et al. [32]. Issues discussedhere are thoseof particular importance to monitoring biodiversity, or ecosystemfunction at large scales.

3.1. HABITAT CHARACTERISTICS AND ENVIRONMENTAL VARIABLESIMPORTANT TO LICHENS

3.1.1. Macrohabitat variablesGeographic distributionof lichen species varies with mostof the same large-scaleenvironmental variables to which other biota respond. Climate and its interaction withlandform explain muchof the variation in lichen speciescompositionwith elevation,topography, and oceanic versus continental climate. Shade, moisture, and availabilityofwoody substrates make forested versus non-forest habitat important. Habitatfragmentation and other human land use variables, such as urbanisation, intensityof

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agricultural or pastoral use, and forestry management, are increasingly important aspredictors of lichen species distribution.

3.1.2. Habitat structure and microhabitat variablesMost lichens have some degree of substrate specificity, so lichen communitycomposition varies with such substrate variables as soil type, rock type, and tree barkpH. Lichen species composition also varies with within-site differences in light regimeand moisture status, so slope and aspect of rock face, soil shading by macrovegetation,position in forest layers, gaps, or edges, tree age, and dead wood are examplesofmicrohabitat variablesofimportance to lichens.

Although most monitoring projects have focused on epiphytic lichensof the trunklayer, other major lichen habitats should be included in a rangeof project types.Intensificationof human land-use often parallels the destructionof the lichenvegetation, sometimes even by the removalofsubstrate such as rock outcrops or cairns.Additionally, saxicolous lichens growing on anthropogenic substrates such as mortarmay significantly contribute to the biodiversity in rural areas [27]. The advantageofrestricting a survey to epiphytic lichens is the considerable cost reduction (typically byabout 60%). However, data quality is equally high for all substrates and epiphyticlichens only [26].

3.2. ESTIMATING ABUNDANCE

Abundance may be estimated or measured in a varietyofways to address questionsof'how many' or'how much' for lichens (e.g.frequency, cover, and biomass for speciesor larger groupings). For monitoring studies, abundance estimates should be at leastsemiquantitative, and should be demonstrated to be repeatable over time.

Two common quantitative approaches to assessing abundance for lichens arepresence in multiple units such as microplots or trees (frequency) and estimatesofcover. Frequency in sample unitsof fixed size has a long history of use in ecologicaland lichenological studies, but because frequency is dependent upon sizeofsample unit,comparison between studies with different sizesofsample unit is difficult. Therefore, iffrequency is the desired measure of abundance in a monitoring program, allinvestigators should use the same plot size or sizes. In a project which aims at detectingchanges in lichen diversity related to land-use intensity, a frequency- based abundanceestimation is being used in a European project on biodiversity assessment tools (seechapter 35, this volume). Cover estimates have an equally long historyofuse, and areindependent of the plot size used (e.g. [9, 13,19,28,30]).However,cover estimates canvary widely between field personnel, so standardisation, training, and qualityassessment are essential to keep variation in cover estimates low enough to berepeatable for monitoring.For some conifer forests, litterfall can be a relatively reliableestimatorofcanopy lichen standing crop biomass (see chapter 14, this volume).

3.3. WITHIN-SITE SAMPLE DESIGN

Within-site sample design can range from visual estimationof abundanceof lichenswithin one large plot to more complex subsampling of microplots within a macroplot.Subsampling is usually based on stratification by microhabitat factors known to be

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related to variation in species composition. The size of the sample unit can be balancedby the number of replicates to obtain meaningful data for a wide range of sample unitsizes. However the kindsofdata and their accuracy, and the time and costof the totalsample effort involved will vary considerably.

McCune and Lesica [20] found tradeoffs between species capture and accuracyofcover estimates for 3 different within-site sample designs for inventory of macrolichencommunities in forest plots. On average, whole-plot surveys captured a higherproportionof species than did multiple microplots, while giving less accurate coverestimates for species. The reverse was true for microplots, with lower species captureand much better cover estimates for common species. Belt transects fell between theother two methods. Time for surveys generally was the reverse of plot size - microplotsurveys took the longest, followed by belt transects, with whole-plot surveys the fastest.Scheideggeret al. [27] found a similar pattern in a baseline survey of the intensivelymanaged Swiss plateau. About 20% of the10m2 plots were without lichens (average 9species/plot), whereas lichens were found on all of the 500 m2 plots (average 28species/plot).

For complete inventory of lichen communities, McCune and Lesica [20]recommended a combined strategy: ocular surveyof an entire large plot to maximisespecies capture, plus survey of many small microplots to increase accuracyofabundance estimates for common species.

3.3.1 . Within-plot sample designWithin-plot sample design involves choices of what sizeof macroplot to use andwhether and how finely to stratify (subdivide) the sample design with microplots tobalance time and effort with the desire to quantify the importanceof differentmicrohabitats.While all important microhabitats should be at least inspected in a wholeplot survey, the needto quantify different microhabitats varies with the kind ofcommunity and the purpose of the study. Macroplots represent one kindof generalhabitat, such as forest type or rock type, and typically include several different lichenmicrohabitats. Microplots represent one specific microhabitat, such as tree trunk andaspect,or horizontal or vertical rock face on a particular rock type.

For many monitoring studies, macroplots are permanently marked for resurvey.Permanent photopoints are a useful addition to sampled plots. Permanent microplotshave been a common choice for monitoring saxicolous lichen communities, but theyhave been also used on tree trunks. Problems with permanent trunk plots includechanges in size and bark characteristics over time, and tree death. Trunk microplotsrelocated in a random or other unbiased manner for resurveys are a better choice thanpermanent plots for long-term monitoring studies.

3.3.2. Plot size and number ofreplicatesWe discuss macroplots and microplots separately. Some sample designs include onlyone or the other typeofplot,but many sample designs include microplots nested withinmacroplots. Braun-Blanquet releves for lichens generally use plots in size rangescorresponding to what are described here as microplots(e.g.[6]).

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Macroplots. Macroplots for lichen surveystypicallyrange from about 0.03ha to about 1ha. Choice of size is often constrained by study designs that simultaneouslyaccommodate macrovegetation studies. Macroplots can be nested, for instance with asmall macroplot for sampling small trees nested within a larger macroplot for samplinglarge trees [5, 26].

Number of independent replicate macroplots in studies cited in biodiversity chaptersvaries from 1 to over 100. With the exception of large regional monitoring programs,the numberofreplicates is generally inversely related to the sizeofthe macroplot used.Studies with fewer than 3 replicates were generally biodiversity inventoriesofunique orrelict lichen communities where more sites were unavailable. Repeat monitoring inthese cases is not as statistically robust, nor are conclusions as reliably applicable to alarge populationof similar lichen communities, but much can still be learned frommonitoring lichens in unique situations.

The investigator needs to define what is being represented and what degreeofprecision is needed for repeatability before the numberofmacroplots can be specified(see chapter 9, this volume). For example, in the USA Forest Health Monitoringprogram sampling protocol,all macroplots in an entire geographic region are the samplepopulation [19].

Microplots. Microplot size, shape, and number of replicates are tailored to the substrateand characteristics of the macroplot being represented. For tree trunks, soil and rockplots, microplotsof 0.01-1.5 m2 and 5-60 replicates, and for tree branches, lengthsof0.2-1 m and 25-100 replicates have been used in the studies cited in other chaptersofthis section.McCune and Lesica [20] found that microplot sample number needed to behigher for ground than for other forest layers to achieve equivalent species capture(Figure 1). Species-areacurves have been constructed to aid in determining adequatesample numberof microplots for other northwestern USA conifer forests [8] and forsaxicolous lichensin eastern USA [14, 15]. Generally, number of replicates variesinversely with plot size. If the microplot is not sufficiently larger than the largestindividual encountered, skewed relationships between plot species richness and otherfactors may be deduced [22].

3.4.SAMPLE SITE LOCATION

Effective monitoringof lichen biodiversity or monitoring lichen communities asindicatorsof ecosystem function often requires information on macrovegetation, siteenvironmental characteristics, and other variablesof interest such as climate and airquality. Co-locating lichen study plots with macrovegetation plots in collaboration withother researchers is an efficient and economic way to acquire such data. Manyof themonitoring studies cited in chapters in the second sectionof this volume used thisstrategy. Sample design for the lichen study then is organised around existing sampledesign. Often existing national forest monitoring programmes are taken as a basis, andthe monitoringofepiphytic lichens is integrated into this existing network [19, 25].

There are three major ways to relate environmental variables and other potentialcausal factors to lichen biodiversity or functional response in monitoring studies: 1.Distribute sample sites along an important continuous gradient and relate numbers

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representing aspects of lichen biodiversity and function to values or indicesof thegradient variable; 2.Define categoriesofthe variables of interest and allocate replicatedsample sites to areas within the categories as defined; or, 3. Distribute sample sites atrandom or evenly across the area to be monitored anda posteriori either correlate lichensite data with site values for continuous variables or group sites within categoriesdefined byvariables for data analysis. One can also allocate sample sites in these waysfor the purpose of averaging across variables notofprime interest.

Figure 1.Species-area curves for bryophytes and lichens combined for three strata (branch epiphytes. trunkepiphytes. ground) of a forest in Montana. USA. One old-growth (solid line) and one second-growth stand(broken line) are shown for branch epiphytes and trunk epiphytes. Three old-growth stands and three second­growth stands are shown for the ground layer. Reproduced by perm ission from McCune and Lesica f20} .

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3.5.SAMPLE STRATIFICATION

Because of the strong influenceof both macro andmicrohabitatvariables on lichencommunities, there is no particular geographic scale above about 0.001 m2 at whichlichen communities can be considered"homogeneous".Stratification can be used tocontrol the allocationof sampling effort among different spatial or environmentalsubunits of the data, while retaining the ability to calculate unbiasedwhole-sampleestimatesof communityparameters, such as species richness, or relative abundanceoflichen guilds. For example,a study area may be stratified by randomlysamplingwithingrid cells (e.g. [19]) or other spatial divisions. Sampling may also be allocated to giveequal effort by environmental factors (Figure 2, [6]) or by land use categories.

Investigators should design survey protocols so monitoring can answer the largest­scale questions appropriately. Intensificationof within-plot sample design to increaseprecisionof within-plotestimatesof lichen community variables should not draw somuch of the sample effort thatbetween-plotsample size (numberof independentreplicate macroplots) is reduced below the number needed to adequately address thelarge-scale monitoring questions within the resourcesofthe study.

Hierarchical or nested designof sampling strategy allows efficient allocationofsample effort so adequate sample size is obtained to answer monitoring questions atseveral scales. A nested plot design is being implemented in a project whose aims are tocompare lichen biodiversity within and among land-use gradients across Europe (seechapter 35, this volume). Given the limited resources and time, it is beyond the scopeofthe project to collect complete species lists. The focus will be to gather quantitativefloristic data that are robust againstvarious typesof sampling errors and which have astrong relation to the sample plot size. At eachof6 independentlocations in a country,16 macroplots will be located in a 1Ian square grid. At each macroplot,12 lichenmicroplots (releves) will be placed in eachofthree habitats, boulders, tree trunks, andsoil. One microplot will cover a surfaceof 50x40 em. Within plots, there will be asample size of 12 plots/macroplot to describe the lichencomposition of eachmicrohabitat, and a sample sizeof 36 plots/macroplotto summarise the lichencompositionofan entire macroplot. There will be a sample sizeof 16 macroplots/lIangrid to monitor variation in lichen composition and relation to localmacrohabitatvariables. And there will be a sample sizeof 6 grids/country to describe lichencompositionand relationship to regional macrohabitat variables.

Response of lichen communities has been shown to differ at different scales. Forexample, Dettki and Esseen [4] found higher species richness and abundance in naturalas compared to managed forest stands (314rrr'). In contrast, they found higherabundance but no difference in species richness in natural versus managed forestlandscapes(2500 ha). Another striking example is the differential expressionof lichencommunities on hardwoods vs. conifers at different spatial scales in thesouth-easternUSA . Lichen communities everywhere in the world are strikingly different onhardwoods (angiosperms) vs. conifers. While this isofobvious importance at the scaleof individual trees,it was not correlated with regional gradients in epiphytic lichencommunitiesin the south-eastern U.S. (Figure 3), primarily because both conifers andhardwoods are well distributed throughout the region [19]. Thus, stratifying orjust

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recording hardwood vs. conifer in the data is crucial when comparing individual stemsbut can be irrelevant at large spatial scales.

100

90

80

70

'E 60Q)

l: 50Q)

a.40

30

20

10

0Non-forest ForestCruslose Cruslose

Non-forest ForestFoliose Foliose

• Coline-submontaneDLower montanefZIUpper montaneImSubalplne

Non-forest ForestFrulicose Frulicose

Figure 2. Mean percentages of total species per plot of crustose, foliose. and fruticose lichens along analtitudinal gradient in the Pre-Alps of Switzerland. Non-forest and forest strata are plotted separately.Reproduced by permissionfrom Dietrich and Scheidegger [6].

Spatial autocorrelation describes a situation in which sample plots close together aremore similar to each other than to plots in similar habitats but further away. Some forestlichen species have been shown to have relatively small-scale dispersal limitationsacross distancesof 100 m or more [26] (see also chapter 14, this volume), but otherfactors leading to spatial autocorrelation have rarely been investigated for lichens. Thepossibility of spatial autocorrelation at many scales is an almost universal design issue[10] in studies involving sampling of natural vegetation (as opposed to experimentalstudies). Failure to acknowledge the effect of spatial autocorrelation is oneofthe mostcommon practices leading to pseudoreplication [10].

3.6.MINIMUM-EFFORT WHOLE-PLOT SAMPLE DESIGN AS CONTEXT

Incorporationof standardised low-budget macroplot sampling is an extremely usefulfeatureoflarge-scalemonitoring studies (e.g. all Europe or North America, or northernhemisphere boreal forest). Region-wide studies are not only useful for answering large­scale questions but also for providing context for more intensive studies. Widestandardisationof a macroplot protocol enhances comparability between intensivestudies (often using different sample protocols) at different locations, and betweenintensive local studies and the background context ("normal condition")ofthe region inwhich they are embedded. The US Forest Service Forest Health Monitoring Programsample protocol [19] is currently being used for such a purpose in the USA, and the all-

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Europe biodiversity assessment project (see chapter 35, this volume) is intended toserve this purposein Europe.

Overlay : Physcia millegrana

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Figure 3.Scores fo r fo rest plots on lichen community gradients in south-eastern USA. Axis 1 corresponds to aregional climatic gradient (cool mountain climates to right. warm coastal climates to left) . Axis 2 correspondsto an air quality gradient (plots with lower air quality toward the bottom of the graph). A) Non-metricmultidimensional scaling (NMS) ordination diagram with radiating environmental vectors from the centroidofpoints. The length ofthe line is proportional to the r' ofthe indicated variable with the axes; the directionindicates the direction of increasing values in the graph. B) Overlay of Physcia millegrana,a pollution­tolerant species. Size of the symbol is proport ional to abundance, with the smallest symbol meaning absence.C) Overlay of mean annual temperature as a fun ction of score on Axis 1. Reproduced by perm ission fro mMcCune et al.[19].

4. Analysis of data

4.1.ECOLOGICAL DATA AND STATISTICAL ANALYSIS

Distribution ofindividual lichen species response along gradientsof environmentalvariation, like thatofmost plants, should be expected to be modal (humped) rather thanmonotonic except along short gradients [7, 11]. Even whenindividual species responseis monotonic,relative contribution to community composition is often modal (Figure 4).Investigatorsshould choose analysis procedures with this in mind [30] or alternatively,construct lichen composition indices or other composite variables that varymonotonically before using standard parametric statistics

Quantitative comparison of variables and indices related to lichen communitycompositionwithenvironmental variables, macrovegetationvariables,or other variables

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of interest can be structured in two general ways. Lichen community variables for a setof independent replicate plots can be compared with values of other variables usingcorrelation or regression techniques. For hierarchically structured (stratified) sampledesigns, summary values (such as averages) for lichen communities from plots in two tomany categories can be compared with summary values for other variables from thesame plots and/or categories using ANOVA or similar techniques. The natureofstratification of many ecological data sets (for example trees within plots within foresttype or landscape unit, or microplots within forest layer within macroplot, etc.) oftenrequires statistical analysis to be designed similar to blocked and/or nested analysisofvariance rather than full-factorial ANOVA, even when non-parametric tests are used.Treating blocked or nested data as fully independent replicates is an exampleofpseudoreplication [10]; this must be avoided in order to generate valid probabilityvalues for statistical tests.

4.2.USE OF LICHEN SUBSETS

Different groupsof lichens often respond differently to environmental or managementvariablesofinterest. Partitioning dataa posteriori for analysis by lichen guild can oftenhighlight groupsof species particularly responsive to variation in selected factorsofinterest. The different response of cyanolichens vs. alectorioid lichens vs. green-algalfoliose lichens to variation in forest age in USA Pacific Northwest is one example [16].In another example, Dietrich and Scheidegger [6] showed that the proportionofepiphytic species in three morphological guilds differed among altitudinal belts, andfurther that the pattern was different for two habitat subsets (Figure 2).

4.3.MULTIVARIATE DATA ANALYSIS

Investigators should always consider multiple factors relating to observed patternsofdifferences in species composition and other community variables. Multivariate dataanalysis techniques are useful for investigating patternsof variation in communitycomposition and exploring the relationship of composition to multiple potentialexplanatory variables [3, 7, 11]. Exploratory techniques such as ordination andclassification aid in sorting out factors relating to variation in community composition[12, 21, 23]. They are particularly useful both to investigate the relationshipofcommunity composition to known variables of importance and to detect the occurrenceof variation not related to external variables already part of the analysis. Multivariateanalyses can also aid in selecting appropriate scaling for comparisonof environmentalvariables and indices of lichen response. McCuneet al. (Figure 3, [19]) generatedregional climate and air-quality gradients on which to track responseof lichencommunities using non-metric multidimensional scaling (NMS) ordination. Directgradient analysis techniques such as canonical correspondence analysis (CCA) andredundancy analysis (RDA) are appropriate for studies in which the external factorsofmajor concern have been previously identified [29].

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Time or Geography Constant

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Figure 4. Theoretical response curves comparing success of individual lichen species (top) with relativeimportance of species in communities (bottom) along an air-pollution gradient (change over time ordifference across a geographic area). Even when a species' success changes monotonically along thegradient. relative contribution ofthat species to a lichen community may be modal. Responses ofspecies andcommunities along other environmental gradients often generate similar patterns. Relative contribution ofaspecies to communities along an environmental gradient is sensitive both to differential responses ofotherspecies and to competitive interactions among species. Adapted by permissionfrom Will-Wolf f30] .

5.Dataquality:evaluation,documentation,and archiving

5.1.EVALUA nON OF DATA QUALITY

Ferretti and Erhardt (chapter 9, this volume) point out that evaluationofdata quality hastoo often not been incorporated into design of lichen monitoring programmes. Theyreview several aspectsof data quality evaluation related to collection and analysisofsample data. It is critical to assure that errors associated with monitoring protocols arelow enough that changes can be detected, or detected soon enough to address goals andtrigger action as a resultofmonitoring.Assessment and controloffield sampling errorsare always important; they are especially critical when nonspecialists are involved insampling procedures. McCune et al. [18] determined the minimumperformancestandards for nonspecialists to achieve desired measurement quality objectives for time­limited macroplot surveys in a nation-wideforest monitoring program in the USA; thesestandards are incorporated into training programs and certification standards for fieldcrew, and into data quality assessment programs. Scheideggeret al. [27] evaluated thespecies captureof specialists and a nonspecialist for sampling microplots (releves).Specialists overlapped by 85% in species captureof foliose, fruticose, and crustoselichens, while a nonspecialist recorded only 30% of the crustose species a lichenologistwas able to find (without time limits).

In large-scale and long-termmonitoring projects, it is likely that many differentlichenologists will participate in laboratory identification of collected field samples.

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Agreement on standards for species identification becomes essential for validcomparison of data across identification specialists and over time. Taxonomic advancesneed to be incorporated into identification standards, but compensation for such shiftsneeds to be made when data are compared (e.g. [31D.Failure to address either of theseissues of species identification may seriously compromise any quantitative comparisonsor assessment of trends being monitored.

5.2. DOCUMENTA nON AND ARCHIVING

AlI sample protocol, data summary, and data analysis procedures need to bedocumented in sufficient detail to be duplicated by other professionals fromdocumentation alone [32] (see also chapter 9, this volume). Documentation should bestored in secure archives,preferably in more than one place. Electronic data files shouldbe accompanied by metadata files that define alI data fields including units on numbers,and formulas for calculating alI summary indices. Lichen species data files should beaccompanied by metadata files documenting lichen species identification criteria,species concepts for those taxa in transition, and a history of any changes in delimitationof taxa. Voucher colIections should be made by all specialists responsible foridentification of taxa.

Hard copy and electronic versions of raw data and data summaries should bearchived in a platform-independent standard format (ASCII is a commonly acceptedstandard format for electronic data) in multiple locations. Electronic technology can beexpected to continue to change at a fast pace in the foreseeable future, so platform­independence should be a major criterion in choiceofelectronic formats for archivingdata.

When reporting detailed results of data analysis, investigators should include name,source, and version number of software used. Popular reportsoffindings and executivesummaries should include at least one or two references to more detailed scientificreports.

6.Conclusions

Three general issues are critical to design of effective monitoring studies.1) The goalsofthe study need to be formalIy stated and carefulIy considered to ensure

that sampling protocol, including managementof error and natural variation, isappropriate to meet the desired goals of the monitoring project.

2) Sample protocol must be consistent with both human and financial resourcesestimated to be available over the entire expected duration of the monitoringprogramme.

3) Repeated data quality evaluation, careful documentation and data archiving areessential for the successful continuation of long-term monitoring projects. Ferrettiand Erhardt (chapter 9, this volume) consider these issues in detail.Use combined macroplots and microplots for studiesof lichen community

biodiversity. For monitoring of lichen communities as indicatorsof biodiversity orecosystem function, use subsets of lichens and macroplot sampling primarily. Always

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considerstratificationto effectively allocate sampling effort and/or represent spatial orenvironmental variation.

7. Acknowledgments

Will-Wolf acknowledgesfunding support from NATO to attend the LIMON workshop in August 2000, andfunding support from the USDA Forest Service during theproductionof the manuscript. We thank ananonymousreviewerand the editorsofthis volume for helpfulcritiqueofthe content andorganisationofthechapter. John Wolf contributedmany editorial suggestions thatimproved the readabilityof the text. KandisElliot adapted the figures.

8. References

I. Bates, J.W. and Farmer,A.M. (eds.) (1992) Bryophytes and Lichens in a Changing Environment,Clarendon Press,Oxford.

2. Brown, M.1., Jarman, S.1., and Kantvilas, G. (1994) Conservation and reservationofnon-vascularplantsin Tasmania, with special reference to lichens,Biodiversity and Conservation 3, 263-278.

3. Clarke,K.R . (1993) Non-parametricmultivariateanalysesofchanges incommunitystructure,AustralianJournal ofEcology 18,117-143.

4. Dettki, H. and Esseen, P.-A. (1998) Epiphytic macrolichensin managed and natural forest landscapes: acomparison at two spatial scales,Ecography 21,613-624.

5. Dietrich,M., Stofer, S., Scheidegger, C., Frei, M., Groner, U., Keller, C., Roth, I., andSteinmeier,C.(2000) Data samplingofrare and common species forcompilinga Red Listofepiphytic lichens,Forest.Snow and Lands cape Research 75, 369-380.

6. Dietrich, M. and Scheidegger, C. (1997) Frequency, diversity and ecologicalstrategiesof epiphyticlichens in the Swiss central plateau and the pre-Alps,Lichenologist29, 237-258 .

7. Gauch, H.G.Jr. (1982) Multivariate Analysis in Community Ecology , CambridgeUniversity Press.8. Geiser, L.H., Derr, C.C., and Dillman, K.L. (1994)Air quality monitoring on the Tongass National

Forest . Methods and baselines using lichens, United StatesDepartmentof Agriculture,Forest Service,Alaska Region,Report RIO-TB-46.

9. Greig-Smith,P. (1983) Quantitative Plant Ecology . Studies in Ecology V. 9. University of CaliforniaPress,Berkeley and Los Angeles.

10. Hurlbert, S.H. (1984) Pseudoreplicationand design of ecological field experiments,EcologicalMonographs 54,187-21I.

II. Jongman, R.H.G., Ter Braak, C.1.F.,and Van Tongeren, O.F.R.(1995) Data Analysis in Community andLandscape Ecology, CambridgeUniversity Press.

12. KantviIas, G. and Minchin, P.R. (1989) An analysisofepiphytic lichencommunitiesin Tasmaniancooltemperate rainforest,Vegetatio 94,99- I 12.

13. Kuusinen,M . and Siitonen,J. (1998) Epiphytic lichen diversity in old-growthand managedPicea abiesstands in southern Finland,Journal ofVegetation Science 9, 283-292.

14. Lawrey, 1.D. (1991) Thespecies-areacurve as an indexofdisturbancein saxicolouslichencommunities,The Bryologist94, 377-382 .

15. Lawrey, J.D. (1992) Natural andrandomly-assembledlichencommunitiescompared using the species­area curve,The Bryologist95, 137-141.

16. McCune,B. (1993) Gradientsin epiphyte biomass in threePseudotsuga-Tsuga forestsofdifferent ages inwestern Oregon and Washington,The Bryologist 96, 405-4 II .

17. McCune, B. (2000) Lichencommunitiesas indicatorsofforesthealth,The Bryoiogistl03, 353-356 .18. McCune, B., Dey, J., Peck, 1., Cassell, D., Heiman, K., Will-Wolf, S., and Neitlich, P. (1997)

Repeatabilityofcommunitydata: species richness versus gradient scores in large-scalelichen studies,TheBryologist 100, 40-46.

19. McCune, B., Dey, J., Peck, 1., Heiman, K . and Will-Wolf, S. (1997) Regional gradients in lichencommunitiesofthe Southeast United States,The BryologistlOO, 145-158.

20. McCune , B. and Lesica, P. (1992) Thetrade-offbetween species capture and quantitativeaccuracy inecological inventoryoflichens and bryophytes in forests in Montana,The Bryologist95, 296-304 .

21. Oksanen, J. (1988) Impact of habitat, substrate and microsite classes on the epiphyte vegetation:interpretation using exploratory and canonical correspondenceanalysis,Annales Botanici Fennic i 25, 59­71.

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22. Oksanen,J. (1996) Is the humped relationship between species richness and biomass an artefact due toplot size?,Journal ofEcology 84,293-295.

23. Pharo,EJ. and Vitt, D.H. (2000) Local variation in bryophyte andmacro-lichencoverand diversity inmontane forestsofWestern Canada,The Bryologist 103,455-466.

24. Rominger, E.M., Allen-Johnson, L.,and Oldemeyer, J.L. (1994) Arboreal lichen in uncut and partially cutsubalpine fir stands in woodland caribou habitat, northern Idaho andsoutheasternBritish Columbia,Forest Ecology and Management 70, 195-202.

25. Roth, I., Scheidegger,C., and Lussi, S. (1997) Rote Liste der Flechten: aufBaumen leben 700 Arten ­wieviele sind bedroht?,BUWAL-Bu//etin 4/97,35-38.

26. Scheidegger, C.,Frey, B .,and Walser, J.-C. (1998) Reintroductionand augmentationof populationsoftheendangeredLobaria pulmonaria : methods and concepts, in S. Kondratyuk and B. J. Coppins (eds.),Lobarion lichens as indicators ofthe primeval forests of/he eastern Carpathians. Kiev, pp. 33-52.

27. Scheidegger, C., Stofer, S., Frei, M., Groner, U., Dietrich, M., Roth, I., and Keller, C. (1999)Biodiversitiitsmonitoring Schweiz: Operationalisieren von Z9 Flechten, WSL, Birmensdorf,pp. 12.

28. Stevenson, S.K. and Enns, K.A. (1993)Quantifying arboreal lichens for habitat management: a review ofmethods, British Columbia MinistryofForestry, Victoria, B.C.,IWIFR-42.

29. Van Dobben, H.F. and Ter Braak,CJ.F. (1998) Effectsof atmospheric NHl on epiphytic lichens in theNetherlands: the pitfallsofbiological monitoring,Atmospheric Environment 32, 551-557.

30. Will-Wolf, S.(\988)Quantitativeapproaches to air quality studies, in T.H. III Nash and V. Wirth (eds.),Lichens. Bryophytes and Air Quality, BibliothecaLichenologica30, J. Cramer,Berlin-Stuttgart,pp. 109­140.

31. Will-Wolf, S. (1998) Lichens of Badlands National Park, South Dakota, USA, in M.G. Glenn, R.C.Harris, R. Dirig, and M.S.Cole (eds.), Lichenographia Thomsoniana: North American Lichenology inHonor ofJohn W. Thomson. Mycotaxon Ltd., Ithaca, N.Y., pp. 323-336.

32. Will-Wolf, S.,Hawksworth, D.L.,McCune, B.,Sipman,HJ.M., and Rosentreter, R. (in press)Assessingthebiodiversityof Iichenized fungi, in G.M. Mueller, G.F. Bills and M.S. Foster (eds.),Measuring andMonitoring Biological Diversity: Standard Methods for Fungi. Smithsonian Institution Press,Washington,DC .

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MONITORING LICHENS FOR CONSERVATION: RED LISTS ANDCONSERVATION ACTION PLANS

C. SCHEIDEGGER 1 and T. GOWARD 2

I WSL, Swiss Federal Research Institute, CH-8903 Binnensdorf,Switzerland ([email protected])2Herbarium, Department ofBotany, University ofBritish Columbia,Vancouver, B.C, V6T 2BI, Canada. Mailing Address: Edgewood Blue,Box 131, Clearwater, B.C., VOE INa, Canada ([email protected])

1.Specificproblemsfor theconservationof lichens

1.1.THE MUTUALISTIC WAY OF LIFE

Lichen-formingfungi are mutualistic symbiotic organisms. The mycobiont coexistswith one or more algal or cyanobacterial photobionts.Conservationbiology of lichensdeals, therefore, with more than one organism, although it is the fungal partner, ormycobiont, which is generally the target for conservation. It is also themycobiontwhich determines the systematic positionoflichens.

The indeterminate patternofgrowthofmulticellular fungi has enabled lichen fungito adopt an astonishing arrayof growth forms, and, as poikilohydric organisms, tooccupy microniches not available to most other life forms. Many species have evolved arequirementfor substrates (e.g.sheltered tree bark, large old logs,dry decaying wood)that are themselves by-productsofadvanced succession in more dominant ecosystems.Such species are often sensitive to various formsof anthropogenicdisturbance,including intensive agricultural and forestry management.

In keeping with their need to secure nutrients mostly from their immediateenvironments, e.g. atmospheric dust, rainwash, lichens are highly efficientaccumulatorsofenvironmentalimpurities. Many species are, therefore, highly sensitive to air, water,and soil pollution. Some species have suffered catastrophic decline inindustrialisedregions as a resultofthese pollutants. With the introductionofindustrialsmokestacksinthe 1970s, these declines have become established over large geographic areas [77].

1.2. WIDE DISTRIBUTION - DISJUNCT AREAS

The incidenceof circumpolardistributions is much higher in lichens (and othercryptogams) than in most other macroscopic organisms, including plants, mammals, and

163P.L. Nimis, C.Scheidegger and P.A. Wolseley (eds.), Monitoring withLichens- MonitoringLichens. 163-181.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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birds. At boreal latitudes, nearly 60-70% ofall macrolichens occur more or less aroundthe world insuitablehabitats[1]. Unfortunately,widely distributedspecies tend toreceive a low priorityfor conservation, even in portions of their range where they aretrulyendangered.This tendencycan be highlydetrimentaltoconservationoforganisms,such as lichens, in which speciesconceptsare not yet fully settled. Certainly at leastsome taxa currentlybelievedto be circumpolarwill eventuallybe shown tocontaintwoor even several moregeographically limitedentities.

9

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Figure 1. Thallus development in Lobaria pulmonaria: a: lsidioid soredia are released fro m an adult thallusand auach on a suitable habitat. b: Development into stratified. spathuliform juvenile thalli within 1 year. c:After 4 years, isidioid soredia have developed into thalli only about 2 mm broad. d: After about 5 years, theearly stages of lobe differentiation can be observed. e: At about 8 years, laminal growth by secondarymeristems leads to the f ormation offoveae and ridges.f After about 14years. soralia develop at the thallusmargins and on the laminal ridges, while upwards-growing lobes become canaliculate, and downward­growing lobes are spoon-shaped. g: Meristematic growth zones of lobes growing upwards often divide intothree daughter meristems (see *) . h: First apothecia are f ormed on ridges of lobes growing downwards. i:Marginal soralia inactivate meristematic growth zones (arrow) ofold lobes. These stop growth but continueto produce diaspores. k: Parts of the thallus can get lost and old lobes often fo rm marginal and laminalregenerative structures (arrowhead). Figure after [54].

1.3.LONG GENERATION TIME

Lichens are poikilohydric organisms and in many habitats theyremain in ananabiotic state for much of each year. Further, thephotobiontbiomass is mostly verylow and netproductionis accordingly low, at least ascomparedwith thatof higherplants.This low productivity oftencorrelateswith slow growth rates and, in some cases,

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exceptionally long life spans. Although only limited data are available on longevity inlichens, the extrapolated maximum ageofsaxicolous species in hot and cold deserts isgreater than 500 and several thousand years, respectively [7, 35). Though the lifespansof most epiphytic lichens are probably much less, some individual thalli are certainlyable to persist for a century or more, an interval commensurate with the"lifespans"ofthe trunks and branches on which they grow. Longevity is likely to be importantespecially for species in which reproduction is initiated only after a prolonged juvenilephase (Figure I); it is also certainly crucial for the maintenanceofrare species in whichan inability to recolonise efficiently is limiting (see below) (Figure I).

104. ECOLOGICAL CONTINUITY

The tendencyof many rare lichens to have long generation times, coupled withinefficient mechanisms of dispersal, makes them dependent on habitats with a lowintensity and frequencyof disturbance [48, 49). Favoured habitats include rockoutcrops, talus slopes, and primeval forests with a low incidence of fire and other stand­replacing events. Yet extensively managed habitats can also sometimes support suchspecies dependent on ecological continuity [75]. Examples include pasture woodlandswith a low tree harvesting intensity, and xeric terricolous habitatswith low levels ofcompetition from vascular plants as well as from the animals that graze them.

1.5.SURVIVAL OF SMALL POPULA nONS THAT ARE NOT REALLY VIABLE

Fragmentationof forested landscapes can entrain dramatic declines in many forest­dwelling lichens. Widespread and common species have become discontinuous,especially during the past century, the ranges of many formerly widespread andcommon species have become discontinuous, with the resulting populations becomingincreasingly reduced in size. This tendency can create a high potential for geneticbottlenecks- an observation especially applicable to epiphytic species associated withold-growth forests. Although forest fragmentation has in many regions been occurringfor a century or more, this interval in fact represents only a few generationsofepiphyticlichens associated with old-growth forests. Conditions permitting the originalestablishmentof such species may no longer exist. In Europe, both the medievallandscape and its characteristic epiphytic lichen cover are now largely relictual; even themost stringent efforts to preserve the remaining old stands are unlikely to prevent thelossofyet more old-growth-associated lichens.

1.6.CLONAL POPULATIONS OF SOREDIATE/ISIDIATE SPECIES

Vegetative dispersal is the sole means of propagation for many epiphytic and terricolousspecies.Clonal populations, therefore, are probably rather frequent in lichens. Europeanlichenologists have noted a gradual reduction in the productionofapothecia during thepast two centuries,at least in some regions [11, 50]. Although lichen population biologyis poorly understood at a molecular level, studies on1. pulmonaria indicate that fertilepopulations are genetically more diverse than sterile populations. At the same time,however, high genetic diversity is not necessarily prerequisite to the developmentof

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luxuriantpopulations[78]. Indeed, small anddecliningpopulations ofL. pulmonariamay actuallysupporta ratherhigh genetic diversity.

2. Monitoringfor rareandendangeredspecies

Current efforts toconserve rare andendangeredorganisms generally involve datagenerationfrom oneoftwo verydifferentapproaches.The firstofthese can bereferredto as the habitat orecosystemapproach,while thesecondis appropriatelytermedthespecies-basedapproach[26]. Theecosystemapproachwill not bediscussedhere (for asummary, see [26]).

Species-basedconservationinvolves, as a first step, the carefulmonitoring ofindividualtaxa. Theobjectivehere is to identify those specieswarrantinghigh priorityfor conservation.Once theendangermentstatusof such species has beenthoroughlydocumented,conservationand recovery plans can bedeveloped[26]. In recenttimes,and under theguidanceofIUCN, this approachhas led both to thedevelopmentofRedLists, i.e. lists of quantifiablyrare andendangeredspecies,and to thepreparationofstatus reports. In both cases, nationalproceduresoften follow the IUCNguidelineswithspecific adaptationsto regional or national levels andrequirements[19, 21]. Thefollowing chapterprovides brief discussions of both thesesapproaches,the firstofwhich iswidelyused e.g, in Europe, while thesecondhas beenappliedmost notablybyCanada. We recognisethat other,somewhatintermediateapproachesexist, but lackofspaceprohibitstheirinclusionhere (for asummary,see [43]).

3. Red Lists

Early attemptsto categorisespecies-at-riskaccording to the severityof the threatsfacing them date from the 1960s (fordiscussion, see [26]), though they bear littleresemblanceto currentpractices. The general aimofa modemRed List is toprovideanexplicit,objectiveframeworkfor theclassificationof speciesaccordingto their risk ofextinction[30]. MorespecificallyRed Lists are intended to:• providea systemthat can beappliedconsistentlyby differentpeople;• improveobjectivityby providingclearguidanceon howdifferentfactorsaffecting

risk ofextinctionshould be evaluated;• providea system that will facilitatecomparisonsacrosswidelydifferenttaxa;• give people using listsof threatenedspecies a betterunderstandingof how

individual species were classified.A wide rangeofdata is required, mostof it quantitative,to achieve thedesiredlevel

ofobjectivity. The informationneeded for a Red List isspecifiedin thecriteria,and thedegreeofextinctionrisk is given in the threatcategories. In the caseof inconspicuousorganisms particularly (including most crustose lichens), theavailable data areinadequatefor thedevelopmentofRed Lists, andspecificmonitoring projectsmust bedesignedto collecttherequiredinformation.

3.1.THE THREAT CATEGORIES

Candidatespeciescan be assigned to anyof three"threat"categories,i.e. criticallyendangered(CR), endangered(EN) and vulnerable(VU) . All of these categoriesare

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recognised as conferring "threatened" status upon the species in question. Species thathave disappeared worldwide are considered extinct (EX). Species surviving in botanicalgardens but not in the wild are considered extinct in the wild (EW), while species nolonger occurring in a country or region are considered regionally extinct (RE) [19, 21].A near threatened category (NT) is reserved for species that do not qualify for athreatened category at the present time but are expected to do so in the near future.Taxawhich neither face serious decline nor have a restricted areaofoccurrence are classifiedas least concern (LC). Species for which additional data are needed in order todetermine an appropriate Red List status are called data-deficient (DD). Finally, speciesnot considered during a RedList project are referred to as not evaluated (NE) [31]. Alltaxa listed as critically endangered qualify also for vulnerable and endangered, and alllisted as endangered qualify also for vulnerable.

3.2.THE CRITERIA

The IUCN has adopted five criteria intended to reflect varying formsof risk faced bydeclining species.Criterion A is related to past and future reductions in population size,while criterion B estimates the risk arising from both continuing decline and a smallareaofoccurrence or occupancy.In criteria C and D, the focus is on small populationswhich additionally suffer ongoing decline or in which all individuals are in onesubpopulation. Finally, criterion E considers the riskof future extinction. A morecomprehensive discussion can be found in the official text published by IUCN [31].

3.3.THE PARAMETERS

Red List criteria are determined according to a varietyof parameters. These arediscussed below.Definitions of the parameters as used in IUCN [31] are given in italics,followed by explanations on how each parameter can be used in a lichenologicalmonitoring project.

3.3.1. Population and population size (criteria Band C)In the Red List literature a population is measured as the number ofmature individualsofthe taxon.

This measure, unfortunately, is hardly applicable to lichens, in which many speciesare inconspicuous, and are likely to be found only by chance. Even in careful surveysoflichen diversity the proportion of the total flora reported is often determined by theamount of time spent examining plots. Obviously, the number of "counted" orextrapolated individuals is mostly a rough estimate, intended primarily to provide alower limit of population size. If repeatabilityofa monitoring program is desired, it isoften useful to neglect thalli smaller than a given minimum length or diameter. Forexample, when monitoring for biodiversity on 100 m2 permanent plots thalli smallerthan 5 mm in diameter were excluded [56].

For endangered macrolichens, very detailed counts of individuals have often beenreported. In Newfoundland, a threatened populationof Erioderma pedicel/atum wasinvestigated both by amateurs and by foresters. Here the resulting population sizeofabout 3000 individuals was used to defend a high conservation priority for this species,

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including mitigation measures in future management plans [41, 51].Nevertheless, in thecase of many crustose species, as well as some macrolichens (e.g. mat-forming andpendulous fruticose lichens), it is often impossible to delimit a separate individual. Tosome extent, this problem can be solved by adopting the conceptof the "functionalindividual" (Hallingback,pers. comm.), especially for epiphytic lichens, which dependfor their survival on the continued existence of the host tree. Because the single mostimportant causeofdeath for epiphytic lichens is the deathoftheir phorophyte host, allconspecific thalli inhabiting that phorophyte can for practical purposes be considered afunctional individual. This approach makes population studies of epiphytic lichensrelatively easy because mortality of the lichen functional individual can then be relatedto the mortalityofthe phorophyte [55].

Another approach to estimating the population size in lichens is to measure aspecies' abundance or biomass; for example, the summed lengthofthalli has been usedas a measure of population size forUsnea longissima [16]. Of course, a preciseestimation of population size is important for rare species only; and when time­consuming censuses are performed for highly localised species, this focus is very usefulin the preparationofRed Lists. For more common species, however, extrapolation fromrepresentative surveys is usually sufficient for a rough estimateof regional populationsize. For example, the Swiss national population ofHypogymnia physodes has recentlybeen estimated to consist of at leastI.608xl09 individuals[53].

3.3.2. Number and size ofsubpopulations (criteria A, B, C and D)Subpopulations are defined as geographically or otherwise distinct groups in thepopulations between which there is little genetic exchange.

The size of a subpopulation seems to us rather subjective; it depends on practicalaspectsof the area being considered. For example, the British Red List is based on anational gridwork consisting of 100km2 squares [10] while in the Swiss counterpart,subpopulations were considered to occupy a range of about 400km2 [53]. However,because many lichen species are rather poor dispersers and because even rather smallareas can therefore be assumed to support independent populations, it may be useful toadopt subpopulation areas somewhat smaller than this.

3.3.3. Mature individuals (criteria A, B, C and D)Reproducing units within a clone should be counted as individuals, except where suchunits are unable to survive alone (e.g. single pseudopodetia in Stereocaulon).

Problems related to the concepts of individuals are discussed under theparameter"population".

3.3.4. Generation time (criteria A, C and E)Generation time is the average age of parents of the current cohort (i.e. new-bornindividuals in the population).

In lichens, generation time can therefore be defined as the average age at which athallus has produced diaspores (i.e. whether ascospores, isidia, soredia, or thallusfragments) which led to the establishment of a succeeding generation. For conveniencea period of three generations is used in the assessmentof Red List status. Generationtime can of course differ considerably among lichen species. Foliicolous species, for

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example, are usually short-lived. Competitive epiphytic species live considerably longerand usually die only upon the death of their phorophytes (see above). This observationholds true not only for the functionalindividual but also for individual thalli, which canpersist for many decades, notwithstanding reported high ratesof biomass turnover.However, depending on climatic conditions and the maximum age attainable by aphorophyte in different parts of its range, generation time can vary considerably evenwithin a given lichen species. The estimated generation time forL. pulmonaria inCentral Europe is probably several decades because significant diaspore productioncommences only at about 35 yearsofage, afterwards continuing for many decades [55].For the purpose of Red List status, at least a century would be required forL.pulmonaria to attain its third generation. This corresponds well with the upper timelimits usually considered in Red List designations,which is set at 100 years [31].

3.3.5. Reduction (criterion A)A reduction is a decline in the number ofmature individuals ofat least the percentagestated under the criterion over the time period specified. although the decline need notbe continuing.

In regions with a long tradition of lichen floristics and well-curated lichencollections, historic decline is one of the most powerful criteria for developing RedLists. This parameter has been used in a semiquantitative way in most traditional RedLists. However, with the modem, more quantitative definition of this criterion, itbecomes difficult to compare past with present, because the methods used in earlysurveys usually differed considerably from those employed today. Only recently, forexample, has it become possible to survey an entire country or region over a shortperiod of time [72]. Modem surveys also tend to be much more intensive than theirearlier counterparts. For example,analysisofa selectionofwell-known macrolichens inSwitzerland has shown that many species thought to have undergone reduction in thepast actually now occur at a greater number of sites than had formerly beendocumented. Anticipating this result Scheideggeret al. [53] included in their studyseveral species for which no evidence of past reduction could be deduced. The ratio ofhistoric to modem collections was then used to correct for different levelsof samplingintensity between past and the present surveys. The same authors also found thatsurveys of subpopulations gave better results than did analysesof the collection sites[52]. Difficulties such as these are significant only in regions in which samplingintensity is not uniform.Elsewhere, in regions with a more complete knowledge of pastdistribution it is possible to accurately estimate reduction by simply revisitingpreviously known sites [66].

3.3.6. Continuing declineA continuing decline is a recent, current or projected future decline. which is liable tocontinue unless remedial measures are taken.

The quantitative estimation of a continuing decline requires an intensive monitoringof the target species. If these direct measures are not available, then an indirectestimation via the decline of the habitat area or habitat quality is possible.However, it isoften difficult to find data for specific lichen habitats.

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3.3.7. ExtremejluctuationExtreme jluctuations can be said to occur in a number of taxa when population size ordistribution area varies widely, rapidly and frequently, typically with a variationgreater than one order ofmagnitude.

Extreme fluctuations are likely to occur in rare ruderal species. Although thisparameter seems not to play an important role in epiphytic lichens, it may be moresignificant in the life cycles of some foliicolous or terricolous taxa with both shortgeneration time and high turnover rates.

3.3.8. Severely fragmentedThe phrase "severely fragmented " refers to the situation in which increased extinctionrisk to the taxon results from the fact that most ofits individuals are found in small andrelatively isolated subpopulations.

This is a very important parameter in lichens. Many regional Red Lists and florasreport taxa that occur on single trees [10] or on a few boulders [3].

3.3.9. Extent ofoccurrence (criteria A and B)Extent of occurrence is defined as the area contained within the shortest continuousimaginary boundary drawn to encompass all known, inferred or projected sites ofpresent occurrence ofa taxon.

Because most lichen species are rather widely distributed, at least relative to themaximum allowable areaofcoverage for Red List status, only a few endemic species(see [18]) are likely to qualify for a listing under this parameter. When preparing aregional Red Lists, however, extentof occurrence can be important to document,especially in the case of subpopulations at their ecological limit.

3.3.10. Area ofoccupancy (criteria A, B and D)Area of occupancy is defined as the area within its "extent of occurrence, which isoccupied by the taxon ",

According to IUCN protocol, this parameter can be estimated by summing the areasofgrid cells in which the target species occurs. Mapping units for national surveys canvary significantly from region to region, e.g. whether a 10'x6' [minutes] grid [37, 68,74], a IOxlOkm grid [10,17,27]or a 20x20km grid [15]. All of these grid units,however, are larger than the 50km2 area required for a CR listing under criterionB. Afiner grid system is therefore required to determine the areaof occupancy with asuitable degreeofresolution.

In a recent national survey of epiphytic lichens of Switzerland, Dietrich [15]determined areaof occupancy from a random sample where one observationrepresented 50krrr'. This level of resolution, however, is practical only when the areabeing surveyed is comparatively small. Even in a country the size of Switzerland, 826observation plots were needed. A much larger number of plots would be required inmost other countries, though the actual number might be kept to a minimum throughhabitat stratification. For example, habitat types with depauperate lichen floras areunlikely to support rare and endangered lichens, and need not be surveyed.

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3.3.11. Location (criteria B and D)The term "location " defines a geographically distinct area in which a singlethreatening event can rapidly affect all individuals ofa species .

The actual sizeofa location varies according to the nature of the threat(s). In regionssubject to industrial air pollution, the sizeof a location can be in the rangeof severalhundredkm",The geographic extentofa location also depends on forest and landscapemanagement practices. In regions with predominantly small-scale management, areasonable size for a location is 1 ha [53].

3.3.12. Quantitative analysis (criterion A, E)A quantitative analysis is defined here as any form of analysis which estimates theextinction probability ofa taxon based on known life history. habitat requirements, andany specified management options.

Along with expected declines due to habitat loss, a high degree of fragmentation intosmall populationsis a typical response especially of epiphytic lichens. The latterresponse will lead to even greater future declines. However, the estimation of thisdecline is often hampered by lack of data, which are needed for a population viabilityanalysis (PVA). Although it is unlikely that life cycle data can be collected for morethan a handfulof species, PVA can be carried out for some flagship speciesof highconservation relevance. Further, such a PVA could allow lichenologists to estimate thefuture declineofecologically similar species, such as competitive late-stage speciesoftheLobarion communities.

3.4.DATA COLLECTION AND SAMPLING DESIGNS

Data collection for a Red List project involves careful monitoring, i.e. 'intermittentsurveillance carried out in order to ascertain the extent of compliance with apredetermined standard or the degreeofdeviation from an expected norm' [28]. For anygiven species, the norm can be defined in termsof population size, geographicdelimitation or degree of fragmentation. When undertaking a Red List project, it iscrucial that the following sequential set of criteria be addressed from the outset:purpose,method, analysis, interpretation, and fulfilment [73].

3.4.1. Purpose: what is the aim ofthe Red List project?Red List projects aim at checking the statusofall lichen species or of a subsetofthem,e.g. the macrolichens. Follow-up, by contrast, is usually undertaken in order to detectsignificant changes in species status since the first Red List. Here the goal is todetermine to what extent conservation measures (or other factors) have arrested earlierdeclines. This often involves revisiting historical sitesof rare species and searching forhitherto overlooked or new sites.

In order to avoid loss of time and energy, the specific objectiveof a particularsurveillance programme must be decided in advance, i.e., whether it is intended toprovide baseline data for: (1) future Red List assessments, (2) mapping projects, and/or(3) biodiversity studies.Survey frequency and data reliability should also be specified atthe outset.It is important to ensure that a high degreeof reliability can be attained, as

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between-crewerrors are relatively frequent in monitoring programs that focus onspecies richness [40].

3.4.2. Method: How can the parameters/or Red List criteria be measured?The (cost) efficiencyofany large-scale monitoring programme is decisively influencedby its sampling design [58]. Attempts to monitor lichens for Red Lists must cope withthe problemof how to provide representative information on rare and endangeredspecies. The presenceofrare species can often not be predicted with precision andit isusually through the field knowledgeofan experienced Iichenologist that rare species arefound. Although this" sixth" sense for rare species can be learned, itneverthelessremains highly subjective and depends on a viablelichenological tradition.Unfortunately, field knowledge is now generally in decline in many countries, evenwhere outstanding traditions in lichen floristics have formerly existed [37].

The vast majorityoflichen species now considered to be endangered were formerlymuch more abundant than they are today (see [10, 53, 63, 76]). This trend seems likelyto continue into the foreseeable future, at least for some species groups, e.g.cyanolichens and calicioids.It is, therefore, important to apply Red List criteria not onlyto rare species but also to species that, thoughwidespreadand common today, maybecome the threatened speciesoftomorrow. To be ofgreatest value in the longtenn,asampling design should thereforeinclude assessmentsof all species,especiallyasregards frequency occurrence. It should also take into account therequirementofstatistical analysis for random sampling. Too often the importanceof random samplingis underestimated, with the result that many population estimates are nearly worthless[61].

From the wide varietyof sampling methods available [12], the most useful for thepurposeofmonitoring biodiversity is the stratified random sample approach, in which aregion is subdivided into strata, and each stratum is then sampled randomly [73]. In onerecent Red List project involving epiphytic lichens, for example, the study area(Switzerland) was stratified into 5 geographical strata, 2 vegetation formations and 6altitudinal strata [14]. Biodiversitysurveys must often remain very incomplete, as thereis time to visit only a small fractionof the total study area.Between-replicatesamplevariation is most easily minimised when very small sampling units (e.g. 500 m2

) aremonitored. Using this approach common and widespread species are well represented,though localised species (e.g. species restricted to rare habitats or invariably scarcewhen present at all [46]) could easily be missed. Nevertheless a recent surveyof 826small (500nr') permanent plots, a fractionof rxio" ofthe study area, yielded more than60% oftotal regional epiphytic lichen richness [15].

If only a few specific habitats aredisproportionatelyrich in rare species, then astratification for these habitats could contribute to an even more effective samplingdesign for rare taxa. However, habitatspotentiallyoccupied by rare lichens typicallycover a very broad geographic range. Collecting data specifically for rare species istherefore more efficiently accomplished by examining larger mapping units. In thiscase, lichen-rich habitats are more likely to be visited and monitored in the contextofsystematic traverses and intelligent random meanderings [59]. This method is veryeffective especially in areas where target species havepreviouslybeen reported andwhere more detailed surveys are intended to add information onpopulationsize and

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delimitation. What is more, experience has shown that the"intelligentrandom meander"approach is more highly regarded by amateur lichenologists (who often carry out themajorityoflichen monitoring) than the more standard "small plot" approach.

3.4.3. Analysis: how are the data handled?Akcakaya et al. [2] have recently developed the software programme RAMAS Red List(http://www.ramas.com/). which assists in the calculationof Red List status. Even so,each project will have its specific interpretation of parameters, which will requirecustomised methods for data analysis. In all cases the general characteristicsofmonitoring data such as trend, cycle and noise [73] have to be distinguished.While it istrue that cyclic trends in lichen populations have not yet been reported, noise and trendstill remain to be separated - not an easy task in the caseofthese long-lived organisms.

3.4.4 . Interpretation: what might the data mean?IUCN protocol has established firm guidelines for interpreting field data in the contextof Red List assessments (see [31]). Though it is beyond the scopeof this paper todiscuss these interpretations in detail, we can observe that the ecological andevolutionary significanceofRed List status is supposed to be universal for all groups oforganisms. For example, an 80% reduction in beluga whales should theoretically havebiological implications and evolutionary consequences equivalent to those that wouldattend an 80% reduction inLobaria pulmonaria. Unfortunately this assumption, thoughprecautionary, is difficult to apply meaningfully, for it seems to require that speciesconcepts should be uniform and equivalent for all taxa on earth, which is so far not thecase.

3.4.5 . Fulfilment: when will the aim have been achieved?The goal of conservation politics related to Red Lists can be roughly summarised asfollows: Sustainable management of natural resources and conservation measures forthreatened organisms should both make Red Lists shorter and shift the red listed speciesdownwards to lower threat categories. The goals of monitoring for Red Lists willtherefore be achieved as soon as no species qualify for the Red List criteria.

3.5. STRENGTHS AND LIMITS OF RED LISTS

Over the past 15 years national Red Lists have been published for many countries andnatural regions; for a review, see [63], [4, 5, 6, 8, 9, 10, 20, 29, 32, 33, 34, 36, 38, 42,44,45,47,53,57,60,62,64,65,67,69, 70, 71]. At the same time, a preliminary globalRed List has been compiled [62]. These Red Lists constitute a most valuable sourceofinformation. Nevertheless, Red List protocol has undergone major changes during thisperiod. The earliest Red Lists were often prepared by one or two regional authoritieswho were able to apply threat categories rather consistently across their local flora. Thisapproach, however, made it difficult to align Red List designations across politicalboundaries. A major advantageof the new IUCN Red Lists protocol is its greateremphasis on quantitative estimatesofregional or global risk.Rarity and vulnerabilityofpopulations at the national level are thus now intended to reflect the probabilityofregional extinction. This emphasis builds on the strengthof earlier Red Lists by

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ensuring greater interregional compatibility, and thus making possible a more broadlybased assessment of at-risk status.

4.The statussurvey:A case study

4.1.COSEWIC: A CANADIAN APPROACH TO AT-RISK DESIGNATION

By world standards, Canada is a sparsely populated country in which naturalecosystems remain largely intact. Until recently, the impact of human activity in mostregionsof the country has been negligible, and conservation efforts have accordinglytended to receive low priority. Nevertheless, no fewer than 12 speciesof mammals,birds, fish, and molluscs formerly endemic to Canada are known to have gone extinctwithin the past century, while another 15 species have lately disappeared within theCanadian portionsoftheir ranges [13].

Responsibility for rare species conservation in Canada is shared between two levelsof government. Provincial and territorial governments have sponsored monitoringprogrammes at a regional level, mostly under the auspicesofConservation Data Centres(CDCS) and Natural Heritage Information Centres (NHICS), while the federalgovernment has taken the lead in promoting monitoring at the national level. To date,however, only the latter initiative can be said to be truly underway, at least as concernslichens; the CDSS and NHICS have as yet developed no authoritative provincial lichentracking lists. In the following account, we therefore focus on initiatives conducted atthe national level only.

Canada's enormous size and sparse research capacity has prompted the Canadiangovernment to adopt a nationally co-ordinated process for the official designationofendangerment status. Since 1978, the designation of species-at-risk in Canada has beenundertaken by the "Committee on the Status of Endangered Wildlife in Canada", widelyknown as COSEWIC . COSEWIC operates at arms' length from government, though itis funded by the Canadian Wildlife Service. Its purpose is to determine the statusofwild species (including subspecies, varieties, and nationally significant populations)suspectedof being at risk in Canada. The committee is comprisedof representativesfrom all provincial and territorial wildlife agencies, four federal agencies, three nationalnon-government organisations, and the chairs/co-chairs of various specialist groups.The committee meets annually to consider status reports on candidate species.

In keeping with its name, COSEWIC was originally intended to assign at-risk statusto "wildlife" species only, i.e.,fish, birds, mammals, reptiles, and amphibians, thoughvascular plants were informally understood to be appropriate for status designation. In1994 however, the COSEWIC mandate was expanded to include lichens and mosses,while in 1995 it was expanded again to encompass lepidopterans and molluscs. Thus,though lichens are now included under its aegis,many other organisms are not.

4.1.1. Status reportsThe credibilityof the COSEWIC designation process rests on the availabilityofaccurate and detailed information on the status of candidate species.This information isassembled and made available through the preparationof lengthy status reports. Theformatof COSEWIC status reports has been standardised to ensure a high level of

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documentationfor all candidate species.An impressionof the levelof detailrequiredfor thepreparationofthese reports can begarneredfrom Table 1.

TABLE I . Standardised format ofa COSEWIC status report .

ABSTRACT

FIGURES

SECTION I : SPECIES INFORMATIONI . Classification and nomenclature2. Description

Local field charactersIllustrations

3. Biologicaland economic significance4. Distribution

Locality citationsExtant populations recently verifiedExtirpated populationsHistorical populations of unknown statusPotential sites for investigationErroneous reportsStatus and location of cultivated materialBiogeographic and phylogenetic history

5. General environment and habitatcharacteristics

ClimateAir andlor water quality requirementsPhysiographic and topographiccharacteristicsEdaphic factorsDependence on dynamic factorsBiological characteristics

6. Population biologyDemographyPhenologyReproductive biology

7. Population ecology

8. Land ownership and managementresponsibility

General natureofresponsibility9. Management practices and experience

Habitat managementPerformance under changed conditionsCultivation

10. Evidence of threats to survivalHabitat destruction or modificationOverutilisationof speciesDisease or predationOther natural or manmade factors

II. Present legal or other formal status

SECTION II : ASSESSMENT OF STATUS12. General assessment13. Status recommendation14. Recommendedcritical habitatIS . Conservationrecommendations

SECTION III : INFORMATION SOURCES16. References citedin report17. Other pertinent publications18. Collections consulted19. Fieldwork20. Acknowledgementsand knowledgeable

individuals21. Other information sources22. Summary of materials on file

SECTION IV : AUTHORSHIP23. Initial authorship of status report24. Maintenance of status report

Responsibilityfor commissioningstatus reports falls to thesubcommitteechairs/co­chairs. For lichens, it is thevascularplantsubcommitteeco-chairwho initiates a statusreport. This is a lengthy process, and involves: 1) developinga priority listofcandidatespecies (see below); 2) securing the necessary funding; 3)contactingand contractingknowledgeableindividuals to prepare the reports; and 4) guiding the reports through tocompletion. Oncecompleted,the reports aresubjectedto peer review by a paneloflichen specialists within thevascular plants subcommittee.The reports are thensubmittedto COSEWIC for deliberationat its annualdesignationmeetings.Only then isofficial at-risk statusdetermined. COSEWIC status reports forvascularplants, mosses,and lichens are based both on existinginformationand on field-work toestablishthevalidityofearlier literature reports. In the past, COSEWICdesignationswere meant toreflect the statusof candidatespecies solely within Canada's borders, i.e, withoutreference to their status in the United States or otherjurisdictions.More recently,

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however, an attempt has been made to consider the "rescue effect", i.e. the likelihoodthat species, though potentially endangered in Canada, will eventually re-establish fromone or more populations outside the country. Species judged to be subject to the rescueeffect are now accorded a lower status designation than would apply given strictadherence to the designationcriteria.

4.1.2. The threat categoriesIn the past, COSEWIC has assigned candidate species to one of seven categories, i.e.,not-at-risk, vulnerable, threatened, endangered, extirpated, extinct, and data-deficient.More recently, however, COSEWIC risk categories have been modified to reflectadvances in conservation biology, and to more closely align with the IUCN categories(see above). Canada's current risk categories can be defined as follows:• Extinct (X): a species (i.e., including subspecies, variety, or geographically distinct

population) that no longer exists.• Extirpated (XT): a species no longer occurring in the wild in Canada, but extant

elsewhere.• Endangered (E): A species facing imminent extirpation or extinction.• Threatened (T): A species likely to become endangered if limiting factors are not

reversed.• Special concern (SC): A speciesjudgedto be particularly sensitive to human activity

or natural events.• Not at risk (NAR): A species that has been evaluated and found to be not at risk.• Data deficient (DD): A species for which there is insufficient scientific information

to support status designation.

4.1.3. At-risk designationCOSEWIC endangerment status is assigned on scientific merit alone; political, socialand fiscal implications are not considered. By the same token, the assignmentofat-riskstatus by COSEWIC carries no legal import; its primary function is merely to drawpublic and government attention to specific instancesof impending extirpation orextinction. This approach of course presupposes that the responsiblejurisdictionscan bedepended upon to act within their mandates to offset these trends. Unfortunately, thishas not always been the case.

The assigning of at-risk categories has traditionally been consensus-driven and, inconsequence, relatively non-quantitative. This practice has raised concerns regardingpossible inconsistencies in the designationof at-risk status across different taxonomicgroups. These concerns have lately become more vocal, following recent attempts tosecure at-risk legislation in a federal endangered species act. In response, COSEWIChas now adopted quantitative designation guidelines modelled on the IUCN Red Listcriteria (see above). This move toward a more defensible protocol has extended even tothe processofprioritisingcandidate species for COSEWIC assessment.

4.1.4. Status oflichen conservation in CanadaInitiatives to conserve Canada's lichen flora are at an early stage. Indeed, the firsttentative steps toward lichen conservation in this country can be said to have begun onlya decade ago, in the early 1990s, when the British Columbia Ministryof Environment

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commissioneda seriesofstudies on the statusofepiphytic lichens associated with old­growth rainforests. Species included in these studies areHeterodermia sitchensis,Hypogymnia heterophylla, Nephroma occultum, and Pseudocyphellaria rainierensis[22,23,24].The resulting reports adopted a COSEWIC format (Table 1),and were later"donated"to COSEWIC for status designation.Unfortunatelyonly oneofthese species,Heterodermia sitchensis, was eventually accorded at-risk status as"endangered"; theother species are currently listed as "special concern".

More recently, Wolfgang Maass completed a COSEWIC status report onEriodermapedicellatum [39], a globally rare species restricted in North America to maritimeCanada [51].

Another step toward lichen conservation occurred with thecommissioning,byCOSEWIC, ofa report on the rare lichensofCanada. In this report, Gowardet al. [25]called attention to 112 lichen species considered to be at risk in Canada. Most of theseare macrolichens. At least 30 species are believed to be in serious decline, while sixspecies appear to be extirpated in Canada. These areAlectoria fallacina, Heterodermiahypoleuca, Leptogium azureum, L. byssinum, L. dactylinum, and L. rivulare - mostofwhich, however, are still present in adjacent portionsofthe United States.

4.2.STRENGTHS AND WEAKNESSES OF THE COSEWIC APPROACH

The COSEWIC approach has three basic strengths: I) its high levelofscientific rigour;2) itstransparency; and 3) its consistencyofapproach. Scientific rigour ensures that at­risk status is accorded only to species really in dangerofextirpation orextinction- withobvious benefits to the expenditureofpublic funds on recovery programs.Transparencyinvites input from sources outside government and industry, thus tending to promote anacceptable degreeofobjectivity.And finally, consistencyofapproach allows organismsfrom a wide arrayof taxonomic groups to receive equal attention by Canada'scommitteeon endangered species - subjectof course to adequate levelsof knowledgeamong taxonomic specialists.

It is also possible to point to at least three weaknesses with the COSEWIC approach.First, COSEWIC embraces only the most charismatic taxonomic groups, allof themmacroscopic. As a result, only a small percentageofCanada's total species are currentlyeligible for at-risk designation. Second, the processofdeterminingspecies status is veryslow and involved, with only 513 designations having been completed in more than twodecades. This is testament both to the rigourof the COSEWIC process, and moreparticularlyto the shoestring budgets on which thiscommitteehas operated since itsinception. Finally, as already noted, COSEWIC status designation has no legal import;it recommends but does not oblige.

What does the future hold for Canada's rare and endangered species? Actually thereis reason for cautious optimism. Political pundits predict that the long-proposed federalspecies-at-riskact will come into effect within the next few years. Should this happen,COSEWIC would receive legal status; and its designations, once ratified by parliament,would result in themandatorypreparationofrecovery plans for all species designated asthreatened or endangered.

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5. Conclusions

Recent methodological developments in the preparationofRed Lists and status surveyshave strengthened the reliabilityof both these approaches to at-risk designation.Ongoing efforts to harmonise the use ofat-risk categories will provide a basis for evengreater consistency between them. Notwithstanding these positive trends, however,there is not too much reason for optimism regarding the futureof lichen conservation.While the powerofRed Lists and status surveys to assist in conservation managementhas increased, their levelof acceptance by politicians and conservation authoritiescontinues to lag behind. The practical utility of these tools appears to depend less ontheir scientific merit than on the cost-effectiveness of the recovery plans that flow fromthem. What is more, in the harsh bureaucratic reality of conservation practice, theaestheticsof the target organisms are often at least as important as their ecologicalsignificance. Unfortunately, lichens are seldom accorded a high ranking forconservation: a stateof affairs certainly in part related to their small size, generalinconspicuousness, and lack of big brown eyes. Even lichens that have been welldocumented as critically endangered often fail to receive an adequate level ofconservation priority. This unsatisfactory situation is likely to change only when lichenshave acquired a higher public profile. Clearly, these organisms have yet to achieve theirfull potential political import. We urge our colleagues to join us in sharing - withpoliticians, conservation authorities, the media, and the broader public - our collectivesense of the importanceof lichens, not only as biological chinking and surpassingmodels of symbiosis,but also as monitors of ecosystem functioning.

6.Acknowledgements

For constructivecommentson the manuscript, we thank Susan Will-Wolf, Per-Anders Esseen,Erich Haberand Ruben Boles.John Wolf also contributed many editorial suggestions.

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40. McCune, 8., Dey, J., Peck, J., Cassell, D., Heiman, K., Will-Wolf, S., and Neitlich, P. (1997)Repeatabilityofcommunitydata: species richness versus gradient scores inlarge-scalelichen studies,TheBryologist 100 (1), 40-46.

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conservation, Wiley, Chichester, pp. 205-217.47. Randlane, T. (1998) Red List of Estonian macrolichens [Eestisuursamblikepunane nimekiri],Folia

Cryptogamica Estonica 32,75-79.48. Rose, F. (1976) Lichenological indicatorsof age and environmentalcontinuityin woodlands, in D.H.

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50. Scheidegger, C. (1995) Early developmentoftransplanted isidioid sorediaofLobaria pulmonaria in anendangeredpopulation,Lichenologist 27, 361-374.

51. Scheidegger, C. (1998)Erioderma pedicel/atum: a critically endangered lichen species,Species :Newsletter ofthe Species Survival Commission IUCN 1998 (June), 68-69.

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53. Scheidegger,C., Dietrich, M.,Frei, M.,Groner, U.,Keller,C., Roth, 1.,Stofer, S.,and Clerc, P. (2001)EpiphytischeFlechten der Schweiz, in C.Scheidegger, P. Clerc, S. Lussi and F.Cordillot (eds.), RoteListe der baum- und erdbewohnenden Flechten der Schweiz, BUWAL, WSL, Bern (in press) .

54. Scheidegger,C., Flachsmann, S., Zoller, S., and Frey, B. (1997) Naturschutzbiologiebei Flechten:Konzepte und Projekte,Kleine Senckenbergische Reihe 27, 167-175.

55. Scheidegger,C., Frey, B.,and Walser, J.-C. (1998)Reintroductionand augmentationof populationsoftheendangeredLobaria pulmonaria: methods and concepts, in S. Kondratyukand BJ. Coppins (eds.),Lobarion lichens as indicators ofthe primeval forests ofthe eastern Carpathians , Phytosociocentre,Kiev,pp.33-52.

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57. Scholler,H.,Cezanne, R.,and Eichler, M.(1996) Rote Liste der Flechten (Lichenes) Hessens, HessischesMinisterium des Innern undflirLandwirtschaft, Forsten undNaturschutz,Wiesbaden.

58. Scott, C.T. and Kohl, M. (1993) A method forcomparingsampling designalternativesfor extensiveinventories,Mitteilungen der Eidgeniissischen Forschungsanstalt fUr Waldo Schnee und Landschaft 68, 1­62.

59. Selva, S. (1998) Searching for Caliciales in the AdirondacksofNew York, in M.G. Glenn, R.C. Harris,R . Dirig and M.S. Cole (eds.), Lichenographia Thomsoniana : North American Lichenology in Honor ofJohn W. Thomson, Mycotaxon Ltd.,Ithaca, New York, pp.337-344.

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60. Shibuichi, H. (1998) Red data list of lichens in Saitama Prefecture, Lichen - News Bulletin of theLichenological Society ofJapan 11(2), 25-26.

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63. Thor, G. (1995) Red Lists - aspectsof their compilation and use in lichen conservation, inC.Scheidegger, P.A. Wolseley and G.Thor (eds.),Conservation Biology ofLichenised Fungi. Mitteilungender EidgenossischenForschungsanstalt fur Wald, Schnee und Landschaft, Birmensdorf, Switzerland, pp.29-39.

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Norway -Sommerfeltia 23, 1-258.68. Tiirk, R.(1990) Lichen mappingin Austria,Stuttgarter Beitriige zur Naturkunde, Ser. A 456, 67-72.69. Tiirk, R. (1996)Rote Liste der Flechten Salzburgs , Naturschutz Beitrage, 18, Amt der Salzburger

Landesregierung, Salzburg.70. Tiirk, R., Breuss, 0 ., and Ublagger, J. (1998) Die Flechten im Bundesland Niederosterreich,

Wissenschaftliche Mitteilungen aus dem Niederosterreichischen Landesmuseum 11, 1-315.71. Tiirk,R. and Hafellner, J. (1999) Flechten. Rote Liste gefahrdeter Flechten (Lichenes)Osterreichs. 2.

Fassung, in H. Niklfeld (ed.),Rote Listen gefiihrdeter Pflanzen Osterreichs , Austria Medien Service,Graz, pp. 187-228.

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LICHEN MONITORING AND CLIMATE CHANGE

G. INSAROy l andB. SCHROETER 2

lInstitute ofGlobal Climate and Ecology, 20b Glebovskaya Str., Moscow107258, Russia ([email protected])2Botanisches Institut , Universitiit Kiel, Olshausenstr. 40, D-24098Kiel, Germany ([email protected])

1.Global climatechangeand its effects on biota:ProjectionsofanIntergovernmentalPanel on Climate Change

I .I .GLOBAL CLIMATIC CHANGES

Anthropogenic increase in the concentrationofgreenhouse gases is leading to warmingof the Earth's surface and other climatic changes. Current projections issued by theIntergovernmental Panel on Climate Change (lPCC) are based on a numberof globalclimate models and scenarios described in the IPCC Second Assessment Report (SAR)volume [22].We summarise briefly resulting projectionsofclimate change as follows.

Global mean surface air temperature will increase relative to 1990 by 1°C to 3.5°Cby 2100. Average sea level will rise 15-95 cm from its present level by 2100.Widespread reduction in the diurnal rangeof temperature is projected. A generalwarming will lead to an increase in the occurrence of extremely hot days and a decreasein the occurrenceofextremely cold days. Increase in precipitation intensity and extremerainfall eventsare anticipated. Severity of droughts and floods will increase in someplaces and decrease in others. In high latitudes precipitationand soil moisture willincrease in winter. In the Arctic and Antarctic,a maximum surface warming is expectedin winter, but little surface warming is expected in summer.It is worth noting thatregional projections are less reliable than global ones.

Winter snowline in the Northern Hemisphere may move northward by 5-I0°latitude, and the snow season may be shortened by one month or more, depending onsnow depths. Snowline in mountains in the temperate zone will move upward about140-I70 m for every 1°C increase in temperature. Ice shelves and, to a greater extent,mountain glaciers will experience retreat and collapse. As a result, bare ground will beexposed. It is expected that 30 to 50%of glacier mass will disappear by 2100. Theextentofpermafrost zones is likely to decrease; it is projected that the total permafrostarea in the Northern Hemisphere will shrink by 16%. As a resultofpermafrost meltingand hydrology regime change in high mountains, steep slopes will become moreunstable, and landslides and avalanches will increase in intensity and frequency. Allthese changes influence lichens directly, or indirectly through changes in their habitats

183P.L. Nimis, C. Scheidegger and P.A. Wolseley (eds.). Monitoring withLichens - MonitoringLichens, 183-201.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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and/or changesIII species interactions within lichen communities and with otherorganisms.

1.2. SPECIES DISTRIBUTION, BlOME AND FLORISTIC ZONE SHIFT

Anticipated changes in terrestrial ecosystems are summarised in another IPCC-SARvolume [71]; projections for each of ten regions are described in yet another IPCCreport [72]. Lichens themselves are mentioned very few times in connection withanticipated changes in tundra. Below we describe briefly some projected changesimportant for lichens.

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Mean Annual Precipitation

Figure 1. Distribution ofthe world's major biomes correlated with mean annual temperature and meanannual precipitation (reproduced from [71]. with permission).

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In the temperate zone, a 1°C change in mean annual temperature corresponds to ashift in isothermsofabout 140Ian in latitude, or 170 m in altitude. As resultof globalwarming between 0.1°C and 0.35°C per decade, species are expected to move 14-50Ianper decade towards the poles (in latitude), or 17-60 m per decade upwards in elevation.This shift will be more pronounced at junctionsoffloristic regions and at other locationswhere climatic gradients are more sharp.

Assuming that CO2 (the main greenhouse gas) concentration will be doubled (2xCO2 condition) [71], substantial partsof biomes will experience climatic conditionsdifferent from those under which they currently exist (Figure 1), and as result largeforested areas will change from current to new vegetation types. Global models projectthat averaged over all zones, 33%ofcurrently forested areas could be affected by thesechanges. The greatest changes will occur in high latitudes, about 65% in the borealzone, and the least in the tropics, about 7.5% in tropical rain forests. Net loss of forestarea could be as much as 17% in the boreal zone despite itsencroachmentinto currenttundra, 14% in tropical rain forests, and 9% in tropical dry forests. Temperate forestcould increase by 4.5% [71]. Tundra could lose one-third to two-thirdsof its currentarea [72].

1.3.BIODIVERSITY CHANGE

Climate change can affect biodiversity directly through physiological responseofspecies, or indirectly by altering relationships between species. Biodiversity will beadversely affected when habitatdegradation combined with fragmentation takes place;significant and irreversible species loss could be a resultof such combination. Climatechange in combination with other factors like land use and deforestation may locallydecrease species diversity. Opportunistic and highly mobile species will become moreabundant; they may replace slower growing species and species requiring more stableconditions [23, 71].

Species may become permanently extinct when local extinction cannot be reversedby re-immigration from surrounding areas. Coastal zones suffering from sea levelrising, and summitsof low mountains currently covered by alpine tundra which canbecome forested as result of an upward shift of tree line, are such areas. Due todisappearance of alpine glaciers, species requiring cold environment for survival maybecome locally extirpated. On the other hand, areas liberated from snow and ice willbecome available for colonisation by plants and lichens.

2.Effectsof climatechangeson ecophysiologicalprocessesin lichens

Climatic factors influence lichen growth and ecology through major physiologicalprocesses. Rain, fog, dew and vapour pressure control thallus water content [45].Experimental surveys prove the importance of thallus water saturation to a numberofmetabolic processes: gas exchange, nitrogen fixation, translocation of photosynthates fromalgae to fungus and their transformationwithin the fungus [42]. Maximal summer, minimalwinter, and average annual temperatures affect lichens' net photosynthetic rate.Therefore,temperature influences lichen biomass. Changes in environmental temperature imply

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changes in thallus temperature that can generate physiological modifications. Thesemodifications could result in the appearance of new ecotypes [42] and for shifts in aspecies' range later on.

There are many reports in the literature on the influence of climatic factors on lichenecophysiology (e.g. [28,40]). In this section we focus on lichensofAntarctica, for tworeasons: (1) the rate of projected climate change in the polar regions is greater than formany other regions of the world [71]; (2) lichens playa major role in Antarcticterrestrial ecosystems [16].

Becauseoftheir poikilohydrous nature, the physiological propertiesoflichens differsubstantially from thoseof higher plants. Most lichen species are highly resistanttoenvironmental stresses such as high or very low temperatures and strong light includingUV in the desiccated, inactive state, but many lichen species are also able to withstandextreme environmental conditions in the hydrated state. As a resultof this strategy,lichens grow more slowly than higher plants [15].

Effects of climate change on the primary productivityof lichens are bestinvestigated in areas where data reveal a long-term trendin climate change and wherelichens form a major partof the vegetation. This is especially the case in the ice-freeecosystemsof the Antarctic continent and the adjacent islands. Here, lichens togetherwith mosses dominate the terrestrial ecosystems while only two native flowering plantsare present. In the maritime Antarctic (i.e. the west coastof the Antarctic Peninsulanorth of 67°S) a substantial increase in air temperatures over the past 50 years has beendetected: summer air temperatures have increased by> 1°C at Signy Island [66] whilewinter temperatures have increased by 5°C in the same period [67]. This warming hasled to a substantial southward spread of the two native flowering plants. Monitoringofselected populations over a periodof 27 years has revealed a significant increase innumbersofindividuals and populations at sites in the maritime Antarctic [66].

No comparable data are available for lichens mainly due to their slow growth andlow reproductive capacity. However, long-term data sets on the microclimaticconditions and photosynthetic activityofoneofthe most prominent fruticose lichens inthe maritime Antarctic,Usnea aurantiaco-atra, reveal that lichens do not necessarilyprofit from an increase in temperature [63]. Even though the physiological performanceof all investigated lichen species in the Antarctic is temperature-limited[16, 41] anddata by Schroeteret al. [62,63]reveal that this is true almost throughout the entire year,an increaseofthe thallus temperature in the active stateofabout 0.5-1°C may lead to areductionof annual carbon gain by 90%. This reduction is mainly due to increasedrespiratory carbon losses due to higher temperatures [63]. Especially in winter when theinput of solar energy is drastically reduced to almost zero, increasing temperatures leadto a dramatic increase of the respiratory carbon loss.During spring, summer and autumnwhen light levels are above the light compensation pointof photosynthesis duringdaytime, an increase in temperature is frequently correlated with a decreasein relativeair humidity, and this results in faster desiccation ratesofthe poikilohydrous organismsas long as no rain or snow is available as a source for rehydration. If snowfall or rainfalloccurs in summer, the lichen thallus is often hydrated and becomes active during night­time with substantial respiration loss and dries out quickly during daytime when aphotosynthetic carbon gain would be possible [60].

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Even though more data on different species and from different ecological conditionsare necessary to draw a full picture, one can conclude that lichens will not profit froman increase in ambient air temperatures in the same way as flowering plants as long asany increase in temperature is not paralleled by sufficient water availability.In the longrun,dry lichen heath formations such as theUsnea-Himantormia-Iichen heath may bereplaced by grasslands dominated by the nativeDeschampsia antarctica and otherintroduced, highly competitivegrass species.

3. Monitoringeffects ofclimatechangeon lichenbiodiversity

3.1. INTRODUCTION

Lichen communities demonstrate strong correlation with climatic factors. Changes inlichen biomass, cover, frequency, species diversity and community indices followchange of such climatic gradients as temperature, humidity, UV, etc. alongenvironmental gradients. When lichens remain on the same substrate, climate affectsindividual lichen species and interactions among species like competition andfacilitation. If substrate is non-living, community change is a compositionofthese twoeffectsofclimate on lichens. If substrate is living, resulting effect includes both effectsabove and indirect effects of climate change affecting substrate species andcommunities. The latter case includes tree species composition change in all typesofforest biomes, which influences epiphytic lichens, and taiga/tundra boundary shift,which influences both epiphytic lichens in taiga and epigeal lichens in tundra. Theupward shiftofsnowline and the melting of glaciers in mountains provide new habitatsthat likely will be colonised by lichens. In these cases, climate change due toanthropogenic causes seems to be the most realistic and parsimonious explanation forfuture lichen community change and shifts in rangesoflichen species.

Two types of studies of effectsof climate change on lichen biodiversity arediscussed in this section. Large-scale monitoring systems are partsofcomplex systemsusually aimedto study the combined effectofair pollution and climate change on forestecosystems. Monitoring along small-scale gradients like altitude or distance fromseashore aimes to ascertain the relation between climatic factors and lichen biodiversity,broadly defined as species diversity and community composition.

3.2.LARGE-SCALE MONITORING

McCune et al. [51] established a large-scale (over 200 sampling plots) gradient studyofepiphytic macrolichen communities across several states in the southeast United States.Macrolichen abundance was estimated according to a four-point scale by observinglichens on all woody plants and fallen branches found in the 0.38 ha plots excluding the0.5 m basal partoftrees and shrubs. A strong lichen community gradient was found tocorrespond to macroclimatic gradients from the coast to the Appalachian Mountains andfrom the south to the north. Both climate gradients are linked to temperature, beingcooler in the north and in the mountains.There is substantial species turnover along theclimatic gradient, and an increase in lichen species diversity with elevation and latitude.

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It is suggested that possible climate warming may cause northward and upward shiftsoflichen communities in the region. These initial observations will serve as a baselineagainst which future change in conditionof lichen communities can be documentedafter repeated samplingofpermanent plots (see chapterII , this volume).

Monitoring of epiphytic lichens has been conducted for ten years at 14 sites acrossSweden [3). Lichen cover on tree trunksofPinus sylvestris and Betula pubescens hasbeen estimated by a line-intercept method. Lichen community indices followed south­north and east-west gradients as well as growing season duration. However, because ofnatural variabilityof lichen communities it is difficult to predict their change on thebasis of predicted changeofa single factor.

Lichen monitoring systems can detect the combined effectsof background airpollution and climate change.It is very difficult to distinguish one effect from anotheron the basisofthe predicted change of a single factor becauseofthe natural variabilityof lichen communities, though large-scale monitoring programs have been designed todojustthat. Another reason of this difficulty is additional"noise" due to climate changeeffects on trees,in addition to direct effects on lichens and their interaction. This noiseessentially masks the signalof climate change effect on lichen communities. Large­scale lichen monitoringof forest ecosystems comprises an integral part of complexmonitoringof economically important natural resources. It will be expedient to useexisting lichen monitoring systems and other lichen studies using replicated sampleplots established for different aims. Previously collected data should be incorporatedinto future projects. Among such systems are the large-scale gradient studiesofepiphytic macrolichen communities in the United States, the national epiphytic lichensmonitoring system in Sweden (both mentioned above), and the long-term and large­scale ecological observation system established in Switzerland [7, 8). Other candidatestudies are the Finnish country-wide observationsof lichens on conifers [44], epiphyticlichen monitoring in the Pacific Northwest, USA [13], and background monitoring ofepiphytic lichens in national reserves in Russia and adjacent countries [26, 34).

Map information on lichen distribution in areas far from emission sources, includingthat available electronically as Geographical Information Systems data [13, 19, 54, 65)also can be used in climate change monitoring with lichens. On the other hand, datacollected in monitoring surveys for climate change can be used for lichen biodiversitystudies, conservation and other aims.

3.3.SMALL-SCALE STUDIES ALONG CLIMATIC GRADIENTS

3.3.1. Altitudinal gradient studiesEpiphytic lichens ofPinus nigra [55] and Fagus sylvatica [56) stands on MountOlympos, Greece were recorded (using an importance value combining cover andfrequency) along an altitude gradient up to 1510 m above sea level.It was shown thataltitude is the most important factor determining the spatial heterogeneityof thoseepiphytic lichens. The altitude of 1200 m is considered as an ecotone for lichens on bothphorophytes because differences are detected in lichen community structure below andabove this limit. The same importance value was used in a studyofepiphytic lichens onQuercus pubescens along an altitude gradient of 0 to 900 m in Tuscany, central Italy

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[47]. Great differences in community structure were found along the gradient, and thealtitudeof500 m was identified as an ecotone.

Biomass of fiuticose lichens growing on branches and trunks ofAbies balsamea inthe mainlandofGaspePeninsula, Quebec, decreased with increasing altitude from 720to 1068 m [1].

Zobel [76] investigated the indicator valueof 19 lichen species on the trunksofPinus sibirica, Picea obovata andAbies sibirica around a pulp and paper factory in theKhamar-Daban Mountains, east Siberia. Lichen presence/absencehas been estimatedfor more than 1000 trees sampled along an altitude gradientof450-1500 m. Only twospecies,Parmelia caperata andP. olivacea , were found to exhibit a similar response topollution at all altitudes. For the remaining species influenceof air pollution differedsignificantly at different altitudes.

Magomedova [48] studied the change of epigeal and epilithic lichen communitiesalong an altitude gradient in the Northern Ural Mountains. It was demonstrated that inmountain tundra, lichen species biodiversity,lichen cover and number of species withingenera are rather stable at altitudes of 1000 to 1400 m; above 1400 m biodiversity andnumber of species within genera decrease while lichen cover increase. The typeofdominant lichen community also changes along the altitude gradient.

Holien [20] found that species diversityof crustoseCaliciales on trunksof Piceaabies was significantly higher at higher than at lower altitudes, over a rangeof 110-430m in central Norway.

In the western partofGissar ridge, central Asia, the coverof lichens on trunksofJuniperus seravshanica and J. semiglobosa increases with an altitude increase from1800 to 2800 m whereas lichen species diversity decreases [35].

3.3.2. Coast-mainland gradient studiesA sharp decline in lichen species biodiversity occurs from the northern maritime (200species) to the McMurdo dry valleys in the continental zoneofAntarctica (6 species)[16].

Hansen [18] found that the numberof corticolous lichen species increases withdistance from the outer coast in south-west Greenland, whereas terricolous andsaxicolous lichen species demonstrate the opposite tendency, but not as clearly as forcorticolous lichens.

Decrease in epiphytic lichen cover on trunksofPinus sylvestris with distance fromthe sea coast in the Kola peninsula, Russia was shown by Insarov and Pchiolkin [30].

3.4.OTHER SPATIAL AND TEMPORAL STUDIES

The examplesof gradient studies presented above do not cover all numerous casestudies and types of climatic gradients. For instance, numberof species and abundanceoflichens increase significantly in south-north direction in Iraq from Baghdad to Mosul;rainfall increases in this direction as well [64]. Both biomass and thallus lengthofAlectoria sarmentosa on branches ofPicea abies in northwestern Sweden increasedwith increasing distance (5-100 m) from the forest edge [9]. Lichen species diversity ontrunk basesofPicea abies in middle boreal Finland increased with increasing distance(0-100 m) from south-facing forest edges [43]. Fryday [12] showed a strong relationship

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between snow-bed lichen communities and climate in the Scottish Highlands. Theeffectsof global warming will almost certainly change the distribution of snow-bedsand associated lichen communities [12].

Evidence of climate change influences on lichen community structure andbiodiversity is not restricted to their changes along climatic gradients. Temporalchanges in single habitats have been documented as well. Follmann[II] compared thestate of lichens in coastal central and northern Chile for two periods, 1960-1965 and1989-1991, and reported that the mean cover-abundance index declined for almost allofabout 300"frequent"and "numerous" species. 14 lichen species could not be foundduring the second survey; lichen species diversity decreased by about 45% in fourcharacteristic formations, and some lichen associations important on the landscapepartially disappeared or experienced significant impoverishment. These changes havebeen attributed to "a slow aggravation of the xeric climate in the northern Chile sincethe past century" and to extraordinary episodic tropical rainfalls during the last decadesin the Atacama Desert caused byEl Nino events [72, pp. 195-196]. Soaking and watershock due to the 1982/1983El Nino event were fatal for many lichens on the GalapagosIslands ([74], cited after [11D.

Some studies presented in this paragraph examine the combined effectof climaticand other factors on lichens. Large-scale lichen monitoring is a partof nation-wideprograms, namely the Forest Health Monitoring Program in United States, and theNational Swedish Environment Monitoring Programme. Both are comprehensiveprograms dealing with influenceof air pollution and other variables on forestecosystems. A study in the Khamar-Daban Mountains was undertaken mainly for airpollution indication with lichens, and altitude was a factor masking influenceof airpollution on lichen communities.Other studies can be characterised as lichen ecologicalstudies along climatic gradients.

In the next paragraph we describe a special lichen survey aimed to detect climatechange.To our knowledge, this is the only study of this kind.

4.Detectingclimatechangewith lichens:long-termmonitoring

The main objectives of the study are: (1) to defme how a change in lichen biodiversitycaused by an alteration of climate can be detected, (2) to provide baselines against whichthese changes can be detected and measured. Monitoring studiesof lichen communities todetect climate change include [33]:• developing an effective samplingprocedure for lichen measurement,and field surveyof

lichensusing this methodology;• assessing lichen sensitivityto climatic stress;• constructing an Integrated Index of lichen community state having the highest

resolutionas it relates to climatechange detection.A survey has been performed to detect effects of climate change in the Ramon

Nature Reserve in the Central Negev Highlands, Israel (Figure 2). The reserve is locatedat the junction of twobiogeographicaland floristic zones, Irano-Turanian (steppevegetation) and Saharo-Arabian(truedesert) [69].Detailed descriptionsofthe reserve andthe region can be found in [6, 27, 33, 77]. For our survey it is essential that there is a

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strong altitudinal temperature gradient, namely 1°C change in yearly meantemperaturecorrespondsto an altitudinal shiftof55 m. This shift is nearly three times as sharp asthat for the temperate zone. Though aprecipitationaltitudinal gradient also exists, it isnot so sharp as the temperature one. Vegetation is sparse and patchy in distribution.Numerous stones and rocky outcrops in the reserve are covered exclusively by lichensattached to the rocks.

100km- Skm

Figure 2.Sampling plots ofthe epilithic lichen gradient study in the Central Negev Highlands (reproducedfrom [33J).

Unfortunately, there are still no generally accepted methodologies to develop regionalforecastsofclimate change. Moreover, existing predictionsof the changes in climate areinsufficiently detailed [22]. The only justified and widely used forecast is a2.soC increasein global yearly mean temperature by the end of the next century [22]. This introducessubstantial uncertainties in forecasting the ecological consequences of anticipated globalclimate change.

Lichen sampling has been undertaken on several sampling plots, i.e. flat horizontalcalcareous rocks or big stones. All rocks wereofthe same stratum,i.e. rock type, surfacecharacteristics(roughness, slope, exposure), and shading conditions. Small or unstablerocks, as well as rocks with evident anthropogenic damage were not used. Whileselecting plots, noa priori information on lichen abundance at a plot was taken intoaccount. Lichens were measured on rocks with a rulerof 10 em length madeofflexibleplastic. When measuring epilithic lichens, the ruler was placed onto a rock arbitrarilyand directed northward.The beginning and endofthe intersectionofeach lichen thalluswith the ruler (transect) was recorded. Several transects were chosen within a plot,separated by distancesofup to several meters, but all placed on the surfaceofthe samerock.

Two surveys have been conducted. The first was an auxiliary study aimed to assesslichen species sensitivity to global warming. The twelve plotsestablishedin courseof

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the first one have been located along an altitudinal gradientof530-1100 m (Figure 2). Asample designof ten transects per plot has been adopted. Twenty-two lichen specieshave been recorded: their cover, frequency, and intraplot and interplot variancesoflichen cover have been estimated. Under the assumption that(I) anticipatedchanges inclimate at a certain altitude are equivalent to those observed whilechangingelevationand (2) lichen cover depends linearly upon altitude, the slopes, intercepts and standarderrorsoflinear regression against altitude for coverofeach species have been estimatedas well. The slope values are used as species sensitivity to temperature change.

Trend Detection Index (TDI) is a weighted sum of lichen species cover with weightcoefficients chosen so as to ensure maximum ability to detect global climate trends,

(1)where:

K - numberofspecies,ai- coverof'r-thspecies, i = 1.2•...•K.w,- weightcoefficientsto be chosen, i = 1.2•...•K .

We assume that changes in lichen species cover are proportional to a change intemperature for each species with its own coefficientcharacterisedby lichen speciessensitivity. Because the regional forecastsofclimatechange for the Negev desert are notsufficiently detailed yet, we adopted the most justified global forecast, i.e.2.5°C increase inglobal yearly mean temperature by the year 2100 [22] while planning the constructionofthe lichen monitoring system. This is the first step to a comprehensive study that shouldinclude regional projections for both temperature and precipitation change, and projectionsofchange in diurnal variability of moisture available to lichens [46].

Weight coefficientsw,have been estimated to maximise the signal/noise ratio. Herethe signal is the expected change in TDI due to climate change; noise is thecharacteristicof spatial variability and sampling errorof species cover masking thesignal. Weight coefficientsw, appeared as functionsof lichen speciessensitivityandtheir SD estimates, intra- and interplot variabilityof lichen cover, and the climatechange value to be detected (see details in [33]).

While planninga system to monitor climate change with lichens, one should first,choose a site located at a certain altitude for periodicmeasurements. Then, three majorparameters have to be determined: a minimal change in a stateof the environment(climate change)LJF to be detected, number of plots M within a site (reserve), andnumberoftransectsm within a plot at which lichens are measured. The valueofLJF is2SC; it is set by the IPCC forecast. Assuming that 1°C corresponds to a 55 m shiftalong an altitudinal gradient, a 2.5°C temperature change valuecorrespondsto a 140 maltitudinal shift. EstimatesofM andm for given weight coefficients have been selectedto maximise the trend detection capacityof the system, and to take into accountlogistical issues.Itwas shown that for the Ramon Nature Reserve the optimal samplingscheme is 2-5 transects per plot scheme, and numberofplots should be 40-50.

A second, basic survey has been undertaken along the 900 m isohypse. It is aimed toprovide information on current lichen community state. This information should beaggregatedtogetherwith lichen species sensitivity data by TDI. TDIassessmentis thevalue against which future changes in lichen community influenced by global warmingcan be detected and measured. In this survey 42 plots with four transects per each plot

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have beenestablishedand 27 species have been recorded [33]. TDI value and its SDhave been calculated [29]. It was found that a minimal changeof L1FO.8°C isdetectable. Such resolution appears sufficient in viewof global warmingof 2SCpredicted by the IPCC for the endof the 21st century. As a result, an early warningsystem for local ecological consequencesofglobal warming is proposed.

5.Methodologyto designlong-termlichenmonitoringprojects

More direct and subtle influencesof climate change on lichens can be manifested inareas where substrate structure remains the same. Lichen physiology, growth, biomass,communitystructure and distribution can all change in space and time in accordancewith changeof climatic factors. Background air pollution isanothercause of suchchanges. Natural variabilityof lichen communities and sampling noise mask theevidenceof climate change lichens can provide. This is why it is difficult to attributecausalityof observed lichen community structure and distribution to a single factor.Usually we can see a change, but we are not able to argue that it is definitely caused bytemperature increase, or by background air pollution level change, or by a combinationof the factors, i.e. by global change.An observed response to change in climaticparameters is notproofthat the causeof the change is uniquely linked to the impact.Izrael et al. [38] suggested a procedure to estimate the probability that the observedlong-term changes in ecosystem state occurred by natural reasons. To makerelationships between long-term climate and background air pollution trends and lichenindividual and community change more clear, lichen long-termmonitoringis needed.Monitoring includes observation, assessment and projectionof changes in ecosystemsand their elements caused by anthropogenic influences [26, 37]. The longer the periodofobservation, the more accurate are conclusions about amplitudeofchanges [38, 68].

The methodologyof epilithic lichen monitoring for detecting climate changedescribed in paragraph 4 is a modificationof themethodologyof long-term epiphyticlichen monitoring for detecting background air pollution (see [26, 32, 34, 39] andchapter 13, this volume). Its main characteristics are:• line-interceptmethodoflichen measurement;• selectionofmonitoring sites in protected areas far from emission sources;• selectionof sampling plots and transects, or model trees withouta priori

information on lichenpresence/abundanceat a plot andtransects/modeltrees;• sampling within as narrow ecological stratum as possible to reduce influenceof

other than target factors;• optimisationofsample design for routine observations;• constructionof Trend Detection Index in the form given byequation(I); TDI

resolution capacity is reinforced by applying lichen species sensitivity toanthropogenic factors to be detected.The line-interceptmethod provides a more accurate estimationof lichen cover for

fixed sampling timeif compared with the releve methods [25]. It is approved for theIntegratedMonitoring Programmeof the InternationalCooperativeProgrammeunderthe umbrellaofthe UN ECE Convention on Long-RangeTransboundaryAir Pollution[70].

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Another way to reduce the influenceof anthropogenic factors other than climaticvariables, is to select survey areas free from such human influence as forestry,agriculture, roads, etc.,and far from air pollution emission sources. Protected areasofnational and regional level often can be used. Sample plots within a survey area shouldbelong to as narrow strata as possible to reduce the impactof natural variability in thelichen community. This means that substrate, altitude, slope and aspect should be thesame for all sample sites. To exclude influence of tree age on lichen monitoring resultsfor epiphytic lichens sampled on tree trunks, tree age and position of sampling unit ontree trunk should be the same as well.

The need to fix sample tree age during long-term lichen monitoring means thatsample trees at the same site should not be permanent for sequenced observations. Thiscomes into conflict with forestry studies where permanent sample plots are establishedfor stand growth study, and reduces the possibilityof combined use of permanentsample plots for forest inventory and lichen monitoring. Difficulties in sample treeselection for long-term epiphytic lichen monitoring in relatively xerophytic formations,such as in the Mediterranean region can arise because the number of trees per samplingplot can be small and not all age classes be represented. Monitoring of epilithic lichenscan be an alternative solution in some regions [33]. Non-living substrate also reducesthe variabilityof lichen communities, which should be considered as a noise in courseof long-term lichen monitoring of climate change.

Indices related to formula (I) are widely used in lichenology. When wi=l and allother coefficients are zero, formula (1) gives the cover of the speciesi. When wi=1 forall species, TDI yields the overall species cover. Whenw, is the sensitivity to airpollution for the lichen speciesi belonging to a certain "poleotolerance class" TDI iscalled the Index of Poleotolerance. Whenw,is the mean numberofother lichen speciesencountered in association with the speciesi within the studied area, TDI is a form ofthe Index of Atmospheric Purity (lAP, see chapter 9, this volume).The proposed TrendDetection Index of lichen community state can be used to quantify response to anyexogenous factor.It provides higher detection capacity than other indices that are linearcombinationsoflichenspecies cover [33, 39].

The useofbiological indicators in monitoring systems requires detailed justification.To propose a given parameter of a species for monitoring, one should investigate themagnitude of its response to potential change in the environment and its naturalvariability. The resultsof such an investigation allow one to estimate the resolutionofthe proposed variable, i.e. its ability to detect a response to a particular environmentalfactor under consideration. The investigation has to be done prior to starting routinemonitoring while doing an auxiliary survey. In case all species' parameters areseparately not effectively enough in detecting trends, the useof the integrated indicesmight be helpful [32]. However, the procedureofcombining individual indices into anintegrated one could and should be a subject for optimisation. This is demonstrated byFigures 3 and 4. The overall cover(wi=W2='''=WK=I), K=22, number of recordedspecies, showed no relation to altitude in a gradient study in the Negev desert (Figure4), while the sum of species cover with the optimal coefficients given by formula(I)revealed a trend (Figure 3). Thus, the proposed index (TDI) appears efficacious indetecting the climate-driven changesin lichen cover.

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There are different ways to estimate lichen sensitivity to climate change. In addition tomeasurements along climatic gradients described above, species range analysis andresultsof laboratory experiments on the influenceofclimatic factors on lichens can beused [28].

y=0.3x - 207.7

r=o.77F1•10=33.57p<O.001

1050950

••

••

• •••

650 750 850

Elevation,ma.s.1.550

160

120

800....><isI- 40

0

-40450

Figure 3. Trend Detection Index trend with altitude in Central Negev Highlands.

160

120

<f..: 80Q)>00

E 40Q)>0

0

-40450

y= O.06x+ 6.59

Figure 4. Overall lichen cover trend with altitude in Central Negev Highlands.

This methodology can be modified to detect change caused by a combinationoffactors, such as temperature change combinedwith precipitation change. To do this, weshould use generally accepted global or regional projectionson precipitation changethat could be available in the near future. Background air pollution also should beincluded in the combinationoffactors for which changes can be detected.

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6. Globalperspectives:wheretomonitorlichens

In polar regions and high mountains, areas that may be liberated from snow and ice as aresult of anticipated global warming are areas for potential lichen colonisation.Experience gained in the course of lichenometric studies can be a starting point forlichen monitoring in such areas (see e.g. [49, 50, 61], chapter 38, this volume, andadditional references in these articles). In Antarctica, an international research program,Regional Sensitivity to Climate Change in Antarctic Terrestrial Ecosystems [57] hasbeen established. This program provides a co-ordinated investigationof the effectsofglobal climate change on natural ecosystems. An altitudinal and latitudinal gradient,Antarctic Environmental Gradient (AEG), is being used to examine consequencesoffuture climate change on terrestrial and limnetic ecosystems. Lichens and mosses arethe key functional groups of primary producers in the terrestrial ecosystemsofAntarctica and therefore are the focusof the investigations. The AEG is used at thecommunity and ecosystem levels to analyse the factors that control species richness andfunctional group diversity, and how changes in these will affect ecosystems structureand functioning. At the level of single species, the physiological response to abioticvariables under changing climatic conditions as well as phenotypic plasticity andgenotypic variationand their interaction along the AEG are key questions.

Lichens of coastal zones and of low mountain summits may become locally orpermanently extinct from these habitats becauseof flooding of coastal zones andforestation of summits.Lichen monitoring in rocky habitats in the coastal zone [4, 5, 10,59] could be the basis for future surveysofflooding. For monitoringofhigher plants onmountain summits, the Global Observation Research Initiative in Alpine Environments[14] is under development. Its approach is focused on summits of different altitude,from the lower limit of the alpine belt up to the limitsof vascular plants. It can beslightly modified to accommodate monitoringoflichens.

Ecotones frequently coincide with sharp climatic gradients. Therefore ecotones aresensitive to climate change because many species have their distributional marginsthere. Climate change will cause lichen habitats to change in these areas, and it willaffect lichens directly as well. Forest-tundra ecotone, forest-steppe ecotone, savanna­steppe ecotone and steppe-desert ecotone all are important areas for lichen monitoringto be included in climate change studies. Vegetation monitoring experience in suchareas should be taken into account, see Holten and Carey [21] for instance. Boundariesof phytogeographical and floristic regions often coincide with isotherms or isohytes [6]that is whyjunctionsof floristic regions are also frontier areas where climatic gradientsare sharp.Ifwe adopt Takhtadjyan's [69] system,we see that the Circumboreal region hasthree points ofjunctionwith two other regions: with Eastern Asiatic and Irano-Turanianregions in the Sokhondo Range in Chita region, Siberia, Russia,with the Irano-Turanianand Mediterranean regions in Anatolia, Turkey, and with the North American Atlanticand Rocky Mountain regions in the Canadian Rockies, Alberta, Canada. Lichensofthelast two areas are studied rather well, but to our best knowledge no monitoring surveyshave taken place there yet. An auxiliary lichen monitoring survey in the SokhondoNature Reserve was undertaken in 1986 [31]. Lichen monitoring in the Central Negevdesert, at thejunction of the Irano-Turanian, Saharo-Arabian, and Mediterraneanregionswas established in 1996-1997 [33], as discussed above.

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Lichen monitoring in the context of background air pollution or climate changestudies aims at detecting local consequencesof global effects. The resultsof lichenmonitoring studies in one place can be generalised to other ones with care. The scale ofcorrect generalisation should not exceed a floristic province [69].

While planning long-term lichen monitoring projects for ecotones and boundariesofphytogeographical and floristic regions, it is worth taking into account that livingsubstrates are also subject to climate change effects. A tree species can replace another,or steppe can appear in a previously forested area. As a result, lichen communities candisappear from the area. Climate change effects on a single tree species might affect theresponseof lichen communities monitored on this tree in the area. In all cases, lichencommunities on non-living substrates give a clearer response to climate change (signal)than lichen communities on living substrates, because in the last case the variabilityofsuch substrates constitutes an additional noise masking the signal.

In conclusion, it is again stressed that geographical areas where projected climaticchanges are the fastest and where lichen monitoring studies should be primarilyconducted include:• Polar regions;• Mountains;• Coastal zones, especially thoseofsmall islands;• Biome boundaries,junctionsof floristic regions.

To detect climate change, lichen sampling on non-living substratesis preferable tosampling on living ones.

7.Concludingnotes

Climate change can influence lichen communities. It can also change the role lichensplay in high latitude ecosystems that include lichen and bryophyte, or lichen, bryophyteand vascular plant groups [52, 53, 58]. Lichen monitoring atjunctionsofphysiographicprovinces and along existing climatic gradients in tundra can reveal trends in speciescomposition, biomass, carbon and nutrient pools [17].

Lichens play an important role in carbon exchange between atmosphere andterrestrial biota through photosynthesis and respiration, and they contribute to globalcarbon sequestration. Furthermore, they influence ozone content in the loweratmosphere through the release of volatile organic compounds (chemical precursorsofozone) [75]. These processes and properties are subjectsof interest to partiesof theUnited Nations Frame Convention on Climate Change (see [73] and references there),and are subjects for prospective lichenological researches.

The scienceofclimate change is progressing. More detailed projections for regionaland seasonal climate changes are becoming available (see e.g. [24]). A reviewofbiological consequences of global warming has appeared [23]. More accuratepredictions for change of terrestrial ecosystems influenced by climate change can beexpected, such as the quantitative assessment of potential shifts for 80 individual treespecies in the eastern United States [36] and resultsof the large-scale internationalexperiment on temperature manipulation to examine variability in species responseacross climatic and geographic gradientsoftundra ecosystems [2].This progress should

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be taken intoaccountin creatingandmodifyingsystems tomonitorlichens under globalclimatechange.

8. Acknowledgements

This chapter has benefitedfrom thesharedinsights of Dr. Irina Insarova. Thanks are due to Prof.SergueiSemeno vand Dr.TrevorGoward for valuable advices. The commentsfrom twoanonymousreviewersgreatlyimproved the manuscript.

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2. Arft, A.M., Walker, M.D., Turner, P.L., Gurevitch, J., Alatalo, J.M., Molau, V., Nordenhall, V.,Stenstrom, A .,Stenstrom,M ., Bret-Harte, M.S.,Dale, M.,Diemer, M.,Gugerli,F., Henry, G.H.R.,Jones,M.H .,Hollister, R .D.,Walker, LJ.,Webber,PJ.,J6nsd6ttir, r.s.,Laine, K.,Levesque, E.,Marion, G.M.,Melgaard,P.,Raszhivin,V., Starr, G.,Totland,0 .,Welker, J.M ., and Wookey, P.A.(1999)Responsesoftundra plants toexperimentalwarming: meta-analysisof the internationaltundraexperiment, EcologicalMonographs 69(4), 491-511.

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haufigstenBaumarten: Ein methodischerAnsatz zur Beurteilung von Umweltveranderungenim Wald undim Freiland,Botanica Helvetica 106,85-102.

8. Dietrich, M. and Scheidegger, C. (1996) The importanceof sorediatecrustoselichens in the epiphyticlichenfloraofthe Swiss Plateau and thePre-Alps,Lichenologist 28 (3), 245-256.

9. Esseen, P.-A. and Renhorn, K .-E. (1998) Edge effects on an epiphytic lichen in fragmentedforests,Conservation Biology 12,1307-1317.

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17. Hahn, S.C., Oberbauer, S.F., Gebauer, R., Grulke, N .E., Lange, O.L., and Tenhunen, J.D. (1996)Vegetationstructureand abovegroundcarbon and nutrient pools in the Imnavait Creek watershed,in J.F.Reynolds and J.D.Tenhunen(eds.),Ecological Studies 120, Springer-Verlag, Berlin-Heidelberg ,pp. 109­128.

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20. Holien, H. (1996), Influence of site and stand factors on the distribution of crustose lichensof theCaliciales in a suboceanic spruceforest areain central Norway,Lichenologist 28 (4), 315-330.

21. Holten,1.1.and Carey,P.O.(1992)Responses ofclimate change on natural terrestrial ecosystems in Norway,Norsk InstituttforNaturforskning,Trondheim,59 pp.

22. Houghton, J.T., Meira Filho, L.G.,Callander, B.A., Harris, N.,Kattenberg, A., and Maskell, K. (eds.)(1996) Climate change 1995. The science ofclimate change, University Press, Cambridge.

23. Hudges, L. (2000) Biological consequences of global warming: is the signal already apparent? Trends inEcology and Evolut ion 15 (2),56-61.

24. Hulme, M .,Mitchell, J.,Ingram, W.,Lowe, J.,Johns, T., New, M.,and Viner, D. (1999) Climate changescenarios for global impacts studies,Global Environmental Change 9, S3· S19.

25. Insarov,G. (1982) Epiphytic lichen sampling on tree trunks, inProblems ofEcological Monitoring andEcosystem Modelling, Gidrometeoizdat Publisher, Leningrad, 5, 25-33 (in Russian, summary in English).

26. Insarov, G.E., Filippova, L.M., and Semenov, S.M. (1986) The Methods of Assessment of EpiphyticLichen Flora Statein Relation to Background Natural EnvironmentPollution, in Y.A. Izrael (ed.),Research on Environmental Pollution and its Effects on the Biosphere. Proc. Third Meeting of theInternational Working Group on UNESCO MAB Project No14,29March-30 April 1985, Yalta, USSR,Gidrometeoizdat Publishers, Leningrad,pp. 123-131.

27. Insarov, G. and Insarova, I. (1995) The lichensofcalcareous rocks in the Central Negev, Israel, IsraelJournal ofPlant Sciences 43:53-62.

28. Insarov,G.and Insarova,I. (1996)Assessmentoflichen sensitivityto climatechange,Israel Journal ofPlantSciences 44,309-334.

29. Insarov, G. and Insarova,I. (in press) Long-term monitoringof lichen communities response to climatechange and diversityoflichensin the Central Negev Highlands, Israel,Bibliotheca Lichenologica.

30. Insarov, G.E.and Pchiolkin,A .V. (1985)Quantitative characteristics ofthe epiphytic lichen flora state inthe Kandalaksha nature reserve, All-Union Institute for Gidrological and Meteorological Information •World Data Center and Nature Environment and Climate Monitoring Laboratory, Moscow (in Russian).

31. Insarov, G.E.and Pchiolkin,A .V. (1989)Quantitative characteristics ofthe epiphytic lichen flora state inthe Sokhondo nature reserve, Nature Environment and Climate Monitoring Laboratory, Moscow (inRussian).

32. Insarov, G. and Semenov, S. (1993) Evaluation of epiphytic lichen monitoring data, in J. Cerny (ed.),Symposium on Ecosystems Behaviour: Evaluation of Integrated Monitoring in Small Catchments , CzechGeological Survey Publisher,Prague, pp. 136-137.

33. Insarov, G., S. Semenov and Insarova, I. (1999) A system to monitor climate change with epilithiclichens,Environmental Monitoring and Assessment S5 (2), 279-298.

34. Insarov, G.E. (in press) Monitoring of epiphytic lichens exposed to background air pollution:Conservation implications,Forest Snow and Landscape Research.

35. Insarova, 1.0. and Pchiolkin, A.V. (1988) Comparison of epiphytic lichen flora characteristics in threeCentral Asia nature reserves, in Problems of Ecological Monitoring and Ecosystem Modelling,GidrometeoizdatPublisher, Leningrad,11,97-104(in Russian, summary in English).

36. Iverson, L.R. and Prasad, A.M. (1998) Predicting abundance of 80 tree species following climate changein the Eastern United States,Ecological Monographs 68 (4),465-485.

37. Izrael, Y.A.,Filippova, L.M.,Semevsky, F.N.,Semenov, S.M.,and Insarov, G.E. (1978) On principlesofecological monitoring of environment under background air pollution,Proceedings ofthe USSR AcademyofScience 241, 253-255 (in Russian).

38. Izrael, Y.A., Filippova L.M., Insarov G.E., Semevsky F.N., and Sernenov S.M. (1985) BackgroundMonitoring and Analysis of the Reasons of Global Changesin Biota State, in Problems of EcologicalMonitoring and Ecosystem Modelling, Gidrometeoizdat Publishers, Leningrad, 7, 9-26 (in Russian,summary in English).

39. Izrael, Y.A.,Filippova, L.M., Insarov, G.E.,Semevsky, F.N.,and S.M. Semenov (1986) Methodologicalaspects of implementing terrestrial biota background monitoring, in Problems ofEcological Monitoringand Ecosystem Modelling 9, 7-21, Gidrometeoizdat Publisher, Leningrad (in Russian, summary inEnglish).

40. Kappen, L. (1988) Ecophysiological relationships in different climatic regions, in M. Galun (00.), CRCHandbook ofLiehenology. Vol. II, CRC Press,Boca Raton,Florida,pp.37-99.

41. Kappen, L. (1993) Lichens in the Antarctic region, in E.1.Friedmann (ed.),Antarctic Microb iology,Wiley-Liss, New York,433-490.

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42. Kershaw,K.A.(1985)Physiological ecologyoflichens.CambridgeUniversityPress, Cambridge,279 pp.43. Kivisto, L. and Kuusinen, M. (2000) Edge effects on the epiphytic lichen flora ofPicea abies in middle

boreal Finland,Lichenologist 32 (4),37-398.44. Kuusinen, M.,Mikkola, K., and Jukola-Sulonen,E.-L. (1990) Epiphytic lichens on conifers in the 1960's

to 1980's in Finland, in P. Kauppi. P. Anttila, and K. Kenttiimies (eds.), Acidification in Finland.Springer-Verlag, Berlin, pp.397-420.

45. Lange,O.L. (1988) Ecophysiologyof photosynthesis: Performanceof poikilohydric lichens and homoiohydricMediterraneansclerophylls,Journal ofEcology 76, 915-937.

46. Lange, O.L., Schulze, E.D., and Koch, W. (1970) Experimentell-okologische Untersuchungen anFlechten der Negev-Waste. II . COl-Gaswechselund Wasserhaushalt vonRamalina maciformis (Del.)Bory am naliirlichen Standort wahrend der sommerlichen Trockenperiode,Flora 159, 38-62.

47. Loppi, S., Pirintsos,SA , and De Dominicis, V. (1997) Analysis of the distribution of epiphytic lichenson Quercus pubescens along an altitudinal gradient in a Mediterraneanarea (Tuscany, central Italy),Israel Journal ofPlant Sciences 45,53-58.

48. Magomedova, M.A. (1986) The altitudinal distribution of lichens along the mountain "Kosvinskykamen", in P.L. Gorchakovsky (ed.),Flora and vegetation ofprotected areas. The Ural Scientific Centreofthe USSR AcademyofScience, Sverdlovsk, pp.103-118 (in Russian).

49. McCarthy, D.P. (1997) Habitat selection and ecology ofXanthoria elegans (Link.) Th.Fr. in glacierforefields: implications for lichenometry,Journal ofBiogeography 24, 363-373.

50. McCarthy, D.P. (1999) A biologicalbasis for lichenometry? Journal ofBiogeography 26,379-386.51. McCune, 8., Dey, J., Peck, J., Heiman, K., and Will-Wolf, S. (1997) Regional gradients in lichen

communities of the southeast United States,The Bryologist 100 (2),145-158.52. Melick, D.R. and Seppelt, R.D. (1997) Vegetation patterns in relation to climatic and endogenous

changes in Wilkes Land, continental Antarctica,Journal ofEcology 85 (1),43-56.53. Nash III, T.H.and Olafsen, A.G.(1995) Climate change and theecophysiologicalresponse of Arctic lichens,

Lichenologist27 (6),559-565.54. Nimis P.L. (2000) Checklist ofthe Lichens ofItaly 2.0, Universityof Trieste, Dept. of Biology, IN2 .0/2,

http://dbiodbs.univ.triestc.it/55. Pirintsos, S.A., Diamantopoulos,1., and Stamou, G.P. (1993) Analysis of the vertical distributionof

epiphytic lichens onPinus nigra (Mount Olympos, Greece) along an altitudinal gradient,Vegetatio 109(1),63-70.

56. Pirintsos,S.A., Diamantopoulos,1.,and Stamou, G.P. (1995) Analysis of the distributionof epiphyticlichens within homogeneousFagus sylvatica stands along an altitudinal gradient (Mount Olympos,Greece),Vegetatio 116 (I),33-40.

57. RiSCC (2000) Regional Sensitivity to Climate Change in Antarctic Terrestrial Ecosystems. AnInternational Research Program on Antarctic and Peri-Antarctic Terrestrial and Limnetic Organismsand Ecosystems , http://www.up.ac.za/academic/zoology/scar/risccspwgb.html

58. Robinson, A.L., Vitt, D.H.,and Timoney, K .P.(1989) Patterns of community structure and morphologyof bryophytes and lichens relative to edaphic gradients in thesubarctic forest-tundraof northwesternCanada, The Bryologist 92 (4),495-512.

59. Ryan, B.D. (1988) Zonationof lichens on a rocky seashore on Fidalgo Island, Washington, TheBryologist 91 (3), 167-180.

60. Sancho, L.G., Pintado, A., Valladares, F., Schroeter8., and Schlensog M. (1997) Photosyntheticperformanceofcosmopolitan lichens in the maritime Antarctic,Bibliotheca Lichenologica 67, 197-210.

61. Sancho. L.G. and Valladares, F. (1993) Lichen colonizationof recent moraines on Livingston Island(South ShetlandI.,Antarctica),Polar Biology 13, 227-233.

62. Schroeter, 8., Olech, A., Kappen, L., and Heitland, W. (1995) Ecophysiological investigationsof Usneaantarctica in the maritime Antarctic. I. Annual microclimatic conditions and potential primaryproduction,Antarctic Science 7, 251-260.

63. Schroeter, B., Kappen, L., Schulz, F., and Sancho, L.G. (2000) Seasonal variation in the carbon balanceof lichens in the maritime Antarctic: Long-term measurementsof photosynthetic activity in Usneaaurantiaco-atra, in W. Davison, C. Howard-Williams, and P. Broady (eds.),Antarctic ecosystems:Models for wider ecological understanding, Caxton Press, Christchurch, pp. 258-262.

64. Schubert, R. (1973) Notizen zur Flechtenflora des nordlichen Mesopotamien(Irak),Feddes Repertorium .83,585-589.

65. Seaward, M .R.D. (1998) Time-space analysesofthe British lichen flora, with particular reference to airquality surveys,Folia Cryptogamica Estonica 32, 85-96.

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66. Smith, R.LL. (1994) V ascular plants as bioindicatorsof regional warming in A ntarctica,Oecologia 99,322-328 .

67. Smith, R .C. and Stammerjohn, S.E. (1996) Surface air temperaturevariations in thewesternAntarcticPeninsula region, Foundat ions fo r Ecological Research West of the Antarctic Peninsula/AntarcticResearch Series 70, 105-12 1.

68. Spellberg, I.F. (1991)Monitoring ecological changes, CambridgeUniversityPress, Cambridge.69. Takhtadjyan,A.L. (1986) Floristic regions ofthe world. CaliforniaUP ,Berkeley-LosAngeles-London.70. Trunk epiphytes (1998), in Manual f or Integrated Moni toring. UN ECE Con vention on Long-Range

Transboundary Air Pollution, InternationalCooperativeProgrammeon IntegratedMonitoring of A irPollu tion Effects on Ecosystems, ICP 1M ProgrammeCentre, Finnish EnvironmentInstitute, Helsinki,http://ww w.vyh.ftlenglintcoop/projectslicp_intlmanuaVcontents.htm

71. Watson,R.T., Zinyowera,M .C.,and Moss,R.H. (eds.) (1996) Climate change 1995. Impacts. adaptationsand mitigation ofclimate change: scientific-technical analyses. Cambridge University Press,Cambridge.

72. Watson,R.T.,Zinyowera,M .e., and Moss,R.H. (eds.) (1998) The regional impacts of climate change. Anassessment of vulnerability. Published for the IntergovernmentalPanel on ClimateChange, CambridgeUn iversityPress, Cambridge.

73. Watson,R.T.,Noble LR., Bolin, 8.,Ravindranath,N .H.,Verardo,DJ. , and Dokken,DJ . (eds.) (2000) Landuse. land-use change. and Forestry. CambridgeUniversityPress,Cambridge.

74. Weber, W.A. and Beck, H.T. (1985) Effects on cryptogamicvegetation,in G. Robinson,and E.M. Pino(eds.),El Nino en las Islas Galapagos. El evento de 1982-1983,Quito,pp. 343-361.

75. Wilske, 8. and Kesselmeier,J. (1999) First measurements of theCl- and C2-organicacids and aldehydesexchangebetween boreal lichensand the atmosphere,Plant. Cell and Environment 24 (6),725-728.

76. Zobel, K . (1988) The indicatorvalue of aset of lichen species assessed with the helpof log-linear models,Lichenologist 20 (I), 83-92.

77. Zohary,M .(1962)Plant life ofPalestine, RonaldPress,New York.

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MONITORING BIODIVERSITY AND ECOSYSTEM FUNCTION: FORESTS

S.WILL-WOLF I, P.-A.ESSEEWand P.NEITLICH 3

'Department ofBotany, University ofWisconsin, 430 Lincoln Drive,Madison, WI53706-1381 . USA ([email protected])2Department ofEcology and Environmental Science, Umea University,SE-901 87 Umea, Sweden (Per-Anders [email protected])3National Park Service, W Arctic National Parklands, 179 Front St., Suite121. Nome, AK 99762, USA ([email protected])

1.Introduction

Scientists and land managers have been concerned about the stateof forest lichens formany years. Most of the studies published during the last 100 years on lichens andpollution (e.g. reviews [49, 65] and section 1, this volume) have involved lichens ontrees, and many studies have investigated forest lichen communities. In the last 50years, concern about the loss of lichen diversity in connection with forest managementand forest fragmentation has led to many studies designed to assess patterns andmonitor trendsof lichen biodiversity in forests worldwide (e.g. reviews [2, 82]).Recognitionof the regional, continent-wide, and even global scale of adverse impactson lichen communities has fostered studies and monitoring efforts designed to assessthe impactof multiple factors adversely affecting forest lichen communities at largescales [43].

1.1. SCOPE OF THE CHAPTER AND DEFINITIONS

In this chapter we review methods for monitoring the biodiversity (includingcommunity composition and species diversity)of forest lichens, and for monitoringecosystem function with forest lichens (including maintenanceofproductivity, nutrientcycling, disturbance response, etc.) on broad scales and/or for response to multiplecauses. The word "health" referring to the degreeof maintenanceof normal, naturalecosystem function, as in "forest health" or" ecosystem health", is widely used andaccepted in some partsofthe world (e.g.[43]), but not in others, so we use the phrase" ecosystem function" for the concept in this chapter. Monitoring for both goals isconducted to document changes in the background, baseline, or"normal" range ofvariation for forest lichens, for comparison with effectsof local pollution (Section 1,this volume) or with effectsof short-term disturbances, for assessing causalrelationships, and to provide data for predictions (e.g. the future stateof biodiversityfollowing different forest management scenarios).

The definition of"community"used in this chapter is very general, referring to a

203P.L. Nimis, C. Scheidegger and P.A. Wo/seley (eds.) , Monitoring with Lichens - Monitoring Lichens . 203-222 .© 2002 Kluwer Academic Publish ers. Printed in the Netherlands.

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group of lichen species growing together (species assembly), but allowing broadinterpretation of the sizeof a community. Our usage thus includes both very strictdefinitions (e.g. the lichen community growing on shaded trunks of large oak trees incentral Europe)of a community and much broader definitions (e.g. the lichencommunity of old-growthconifer forests in Scandinavia).We do this to emphasize thatsimilar questions about relationshipsof lichen community composition to habitatvariables can be asked at several different spatial scales. For more discussionofsampledesign with respect to spatial scale, see paragraph 2.2.

Many recent reviews include broad coverageofrelevant methods (e.g. [73, 75, 82]).We include here methods that are at least semi-quantitative and are suitable forresurvey, and discuss methods specifically as they relate to monitoring forestecosystems.

We cover both forests and woodlands, with emphasis on epiphytic lichencommunities. Some discussion of ground-layer lichens is included. We excludesaxicolous lichen communities; their occurrence in forests is sporadic, their speciescomposition is often not directly linked to forest ecosystem processes, and methods forstudying them are covered in other chapters.

1.2.ECOLOGICAL ROLES OF LICHENS IN FORESTS

Lichen communities only occasionally comprise a major portionof the biomassof aforest, but they play many ecological roles in forest ecosystems [4, 18,31,39,48,64].Important among these roles are nitrogen fixation, nutrient cycling, and provisionoffood and nesting material for wildlife. Cyanolichens contribute fixed nitrogen to forestnutrient cycles; this contribution is most important in moist forests (where contributionscan reach several kglha [58]). Lichens intercept and redistribute dryfall and wetfallnutrients and pollutants in forest canopies via direct absorption and alterationofstemflow. They provide microhabitats and food for forest canopyinvertebrates (mostnotably oribatid mites and collembola), and they provide nesting material for birds andsmall mammals. In boreal and wet montane forests, lichens are an important foodsource for large and small mammals, including ungulates (e.g. deer, caribou, reindeer),and rodents such as flying squirrels and microtines [25,61].

1.3.FOREST CHARACTERISTICS IMPORTANT TO LICHENS

1.3.1.Forest macrovegetation composition and macrohabitatvariablesMost epiphytic lichens have some degreeof substrate specificity. Thus within broadregions of similar climate on the scaleof tens of thousandsof knr', the primaryinfluences on forested lichen communities are broad patternsofforest structure. Theseinclude woody species composition (especially the balance of coniferous versushardwood taxa) and large-scalepatterns in the distributionofsuccessional stages. Bothforest age and continuityof forest canopy are critical factors for thedevelopmentofepiphytic lichen communities. Forest soil lichens also vary with soil type. At the scaleof continents, forested lichen communities co-vary most closely with climatic variablessuch as precipitation, moisture status, temperature, and potentialevapotranspiration.

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1.3.2. Forest structure and microhabitat variablesForest lichens tend to be very microhabitat-specific even beyond substrate preferences.Forest structure affects lichen distribution through influence on light regime andmoisture regime. Canopy openness and position in canopy affect light and moistureregimes directly and also indirectly through densityof woody plants in subcanopylayers [47]. Gaps with low shrubby substrates or low branches on surrounding trees areimportant as diversity hotspots in moist forest climates; such microhabitats provide bothhigh moisture and direct light [51]. Forest edges differ from forest centres in both lightand wind, which affect moisture regime [15, 23, 68]. Many variables influencemicrohabitat characteristics important to lichens: substrate tree age; size and ageofgaps; standing dead snags; size, age, and quantityof downed woody debris.Distributionof lichen species and lichen biomass is heterogeneous in forests world­wide becauseofthe importanceofsmall variations in microhabitat characteristics (e.g.[28,37,54,57]).

There are many ways to partition forest structure in studies to assess its relationshipto compositionof lichen communities. Three forest layers are widely recognised. (1)The forest canopy can be defined as allof the forest that is above the reachof aninvestigator on the ground, or it can be more narrowly defined as the volume occupiedby live crownsof trees. (2) The forest trunk layer is defined by tree trunks, but alsooften includes any shrubs and branches in the same horizontal partition. The trunk layerwithin reachof the investigator on the ground is the microhabitat most frequentlymonitored in studies. (3) The ground layer includes the forest floor, often the basesoflive trees,and any long-down woody debris.

104. GOALS AND METHODOLOGY

The two different monitoring goals covered in this chapter, monitoring the diversity andcomposition of lichencommunities as "biodiversity" and monitoring forest ecosystemfunction, invite different approaches to study design. A major goal of monitoring lichenbiodiversity is to define the status and trends of change in the entire lichen community.Monitoring ecosystem function with lichens, on the other hand, focuses on lichens asindicatorsofthe statusofan entire biota or of alteration in ecosystem functions.This isoften done most efficiently with a selected subset of the lichen community that hasknown or assumed relationships to the ecosystem functions being monitored.

2.Design ofmonitoring protocols

Issuesof importance are matching survey design to goals, to available resources, and totime frame. When time, personnel, or resources for a monitoring study are scarce, thisbecomes critical, and tradeoffs in sample design and implementation must be carefullybalanced. Documentation to allow repetition of the methods is always essential.Responseof lichen communities to a particular variable often differs at different scales,and the relative importance of variables often differs with spatial scale [9] (see chapter11, this volume). Collectionofdata on environmental variables should support analysisat multiple spatial scales.

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2.1.ESTIMATING ABUNDANCE

Abundance may be estimated or measured in a variety of ways to address questionsof"how many" or" how much" for lichens (e.g. frequency, cover, and biomass for speciesor larger groupings). Strictly qualitative estimates (uncommon, common) are difficult tostandardise, so they are not so useful for repeated monitoring. Abundance classes areeasy to use and are reasonably repeatable. Many versionsof abundance or combinedabundance-coverclasses are used [71], for example theBraun-Blanquetand Dominscales or the 4-class scalesofMcCune et at. [45] and Kuusinenet al. [36].

Two common quantitative approaches to assessing abundance for lichens arepresence in multiple quadrats (frequency) and estimatesof cover [81]. Frequency inquadratsof fixed size has a long historyof use in ecological studies, but becausefrequency is dependent upon plot size, comparison between studies with different plotsizes can be misleading. Therefore, if frequency is to be an estimateof abundance, atleast one plot size (for nested plot designs) should be standardised throughout the areato be monitored, and frequency estimates should be based only on data collected for thestandard plot size.

Plotless sampling techniques, such as recording hitsof species along transects onthe ground, or around trunks (e.g. [7]), or along branches, can also be used to estimateabundance of species. These are most often implemented similarly to microplots, withseveral to many replicates per siteor macroplot.

Armlederet al. [1] estimated the abundanceof groups of pendulous("alectorioid")lichens by rating lichens on each sample tree into abundance classes by comparisonwith reference photographs, which are standardised by counting"clumps" of standardweight. This method requires that one can see tree crowns.

When cover is estimated directly or in classes, cover estimates areindependentofplot size. However, cover estimates may show substantial variation among differentobservers, so standardisation, training, and data quality assessment are essential whencover estimates are used in monitoring studies. Lichen cover can also be estimated bycalculating proportion of hits counted in many small subdivisionsofaplot (for examplethe "point frequency" methodofKuusinen [33, 34]) or proportion of hits counted alonga transect (e.g.[7]).

Estimationof lichen biomass has been a useful monitoring tool fornorth-temperateand boreal coniferous forests. Lichen litterfall can be a relatively reliable estimatorofcanopy lichen standing crop[14,42,50, 56]. This method has some limitations but isoften the best alternative for assessing epiphyte standing crop in a large numberofstands. Studies based on litterfall collection tend to be extremelypainstakingand time­consuming. The biomassofepiphytic lichens has been visually estimated from the sizeof the largest thallus on branches by McCune [40], and has been used by Esseen andRenhorn [15] for estimating abundance and biomass of pendulous lichens in the lowercanopy for large-scale rapid surveys. However, the thallus size-biomass relationshipvaries among species and type of substrate so each study should be individuallycalibrated. In large, conspicuous species such asUsnea longissima it is possible toestimate standing cropin situ by measuring the lengthofall accessible thalli [13].

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2.2.WITHIN-SITE SAMPLE DESIGN

Within-site sample design can range from estimation of abundanceof lichens withinone large plot to very complex subsampling of microplots within a macroplot based onstratification by forest layer, host tree, microhabitat, etc. The sizeofthe sample unit canbe balanced by the number of replicates to obtain meaningful data for a wide rangeofmacroplot and microplot sizes. However the kinds of data and their accuracy, and thetotal sample effort involved,will vary considerably. Macroplots represent one kind ofgeneral habitat, such as forest type or age. Microplots generally represent one specificmicrohabitat, such as treetrunk in a particular species, size class and/or aspect, branchofa particular size range, or downed woody debris in a particular size or decay class.Sizes of microplots are often chosen to be small enough that the area can be consideredto be relatively homogeneous with respect to all habitat variables relevant to lichenspecies distribution.

McCune and Lesica [46] found tradeoffs between species capture and accuracy ofcover estimates for 3 different within-site sample designs for inventoryof lichencommunities in forest plots. On average, whole-plot surveys captured a higherproportion of species than did multiple microplots, while giving less accurate coverestimates for species. The reverse was true for microplots, with lower species captureand much better cover estimates for common species.Belt transects fell in between theother two methods (Figure I). Time required to complete a survey generally was thereverse of plot size. Microplot surveys took the longest, followed by belt transects, withwhole-plot surveys the fastest. They recommended a combined strategy for completeinventoryof lichen communities: surveyof a large plot to maximise species capture,plus survey of many small microplots to increase accuracyofabundance estimates forcommon species. Studies of vegetation employing nested plots are relatively commonfor assessing spatial heterogeneity, species-area relationships and estimating totalspecies numbers across regions (e.g. [72]), and are being used more frequently instudiesof lichens. Several examples are discussed in this chapter (see also chapter 11,this volume).

Tibell [74] and Selva [66] conducted intensive qualitative searchesof large wholeplots of uneven sizes for forest standsof the same type but different ages, thendeveloped listsof lichen species indicative of old, continuously forested standsof thatforest type.

Monitoring on additional stands consistsof searching whole plots for presenceofthe indicator species, then scoring each plot for stand continuity based on the proportionofindicator species found.The method is comparable to the lAP approach (see chapter4, this volume) to monitoring pollution response. Surveys done on plotsofequal size,with equal time alloted, and with surveyors having equivalent expertise at finding theindicator species, can be validly compared over time and across investigators. Thissurvey method qualifies as a semiquantitative monitoring technique even though themethodof developing theindicator species list may not. A particular setof indicatorspecies is expected to apply to a limited geographic range and breadthof forest treespecies composition. Selva [66] found that the same lichen species have differentindicator value on hardwoods versus conifers, for instance. Investigators shouldcarefully consider the applicability of a setofindicator species before employing them.

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Ground layer, trunk layer, and canopy often require different sampling strategies.Note that we use an operational rather than a structural definitionof forest layers;" trunk layer" can include lower tree branches, and"canopy" is above the reachof aresearcher on the ground.

2.2.1. TrunkWithin-plotsample design for the trunk layer involves mainly choicesofwhether andhow finely to subdivide the sample design to balance time and effort with the desire toquantify the importance of different microhabitats. All important trunk microhabitatsshould be at least inspected in a whole plot survey, but the need to quantify differentmicrohabitats varies with the purposeofthe study and the kindof forest. Shrub stems,saplings, and twigs and branchesof trees are important trunk-layer substrates distinctfrom the trunks themselves in some forests, though the most common choice ofresearchers has been to limit sampling to tree trunks. Bark versus wood, stem sizeclassor age class, face of leaning tree, and conifer versus hardwood trees are importantvariables in temperate conifer forest[47,51]and boreal forest [16, 66, 74]. In additionto these factors, tree bark pH and surface characteristics are also important variables toexplain lichen distribution in mostbroadleafforests worldwide [24,63].

Permanent trunk microplots have been used for monitoring lichen communities (e.g.[81]), but they are most useful for short-term monitoring or studiesof succession.Trunks expand and tree canopies change, so a microplot does not necessarily remain inthe same microhabitat over long-term studies. Lichens may" move" outof fixed-areamicroplots via trunk expansion, and trunk cylinders increase surface area with time.Microplots relocated in a stratified random manner for resurveys are a better choicethan permanent microplots for long-term monitoring studies.

A plotless sampling technique--specieshits recorded along tapes placed around treetrunks at a fixed height--is used in the integrated monitoring programsof Sweden [5]and Finland [3]. This method can also be used for branch lengths within reachof theresearcher (e.g. [26]).

2.2.2. CanopyVertical differentiationof lichen microhabitats on trees is known to be very importantto lichen community composition in most kinds of forest world-wide [8, 20, 41, 47].Direct samplingof forest canopy requires fairly intense effort involving branch­pruning, tree-climbing, fellingof trees, or building structures for access to treecanopies. As a consequence, direct sampling of canopies is done at most on a verysmall subsetof the forest being studied. Thus even though muchof the lichen speciesdiversityof many forests is in the canopy, for most studies this componentof thecommunity must be assessed indirectly.

Recently fallen litter and branches are often the only available estimatorsof thecanopy componentofthe lichen community (e.g.[28,45]). The availabilityofrecentlyfallen branchesis idiosyncratic and dependent upon factors (recent storms, otherdamage, etc.) that are usually unrelated to variables correlated with lichen communitydiversity. As a consequence, data on canopy lichen composition often include a largerandom error component. However, a general estimateofabundance for lichen species

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on all recently fallen branches in amacroplotis usually worth the returnin databecauseoftheimportanceofthe forest canopy habitatto lichen diversity.

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Visual estimationofbiomass of lichens in the canopy is feasible for boreal coniferousforests (see paragraph 2.1).

2.2.3. GroundThe ground layer is highly variable and heterogeneous for occupancyof lichencommunities in many forests. It has been frequently included as a distinct importanthabitat for lichens in conifer forests (e.g.[21,46,55, 57]). Large woody debris in theground layer has emerged as a critical structural component enhancing biodiversityofseveral groups of biota, including lichens, and contributing to nutrient recycling in old­growth forests as opposed to those managed for timber production [46, 62, 76, 77].Becauseof its heterogeneity, samplingofthe ground layer should be either by whole­plot estimates or by large numbersof microplots. Line and belt transects are veryeffective in sampling downed woody debris and its associated lichen communities.

2.2.4 . Plot size and number ofreplicatesWe distinguish two kindsof plots for discussion here: macroplots and microplots.Macroplots generally correspond in size to plots used to study forest trees; theyencompass a variety of different lichen habitats. Microplots are smaller and areconsidered to encompass a single lichen microhabitat. Some sample designs includeonly one or the other typeofplot, but many sample designs include microplots nestedwithin macroplots. Lichen releve plots are generally in size rangescorrespondingtowhat are described here as microplots.

Macroplots. Macroplots for forest lichen surveys range from about 0.03 ha to about 1ha in studies and reviews cited in this chapter. When lichen monitoringprogrammesarelinked to those for forest vegetation, lichen macroplot size and shape are often chosento conform to those used for forest tree sampling. Fixed-area plots are the best choicefor monitoring studies; the only studies cited here which did not use plots of fixed sizewere those conducted for development of an indicatorspecies list [66, 74].

The numberof independent replicate macroplots in studies cited in this chaptervaries from 1 to over 100. For local studies, the numberofreplicates is often inverselyrelated to the size of the macroplot used. Studies with fewer than 3 replicates weregenerally biodiversity inventories of unique or relict forest ecosystems where more siteswere unavailable. Repeat monitoring in these cases is not as statisticallyrobust, nor areconclusions easily generalised to a large populationofforest stands, but much can stillbe learned from the continuedstudyofunique situations.

Microplots and microtransects . Microplot size, shape, and numberof replicates aretailored to the characteristicsofthe microhabitat being represented. As with macroplots,the numberof replicates is generally inversely related to the sizeofthemicroplotused.For trees and other woody substrates, trunk microplotsof 0.01-0.2 m2 and 10-25replicates, and branch lengthsof0.2-1 m and 25-100 replicates have been used by thestudies cited here. Length of trunk belt transects is usually tree circumference, so it isvariable within a study and is constrained within a range by restricting the sizeoftreessampled.Ground microplots used are in the 0.2-1.5rrr'range,with 5-60 replicates.

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McCune and Lesica ([46]; see Figure 1 in chapter 14, this volume) and Geiseret al.[21] constructed species-area curves for northwestern USA conifer forests to aid indetermining adequate sample number for microplots.

Becauseofthe strong influence of both macro- and microhabitat variables on lichencommunities, there is no particular spatial scale above about 0.001 m2 at whichmicrohabitatvariation has no effect on lichen species distribution. Bottoms of barkfurrows, edges of bark plates, and surfaceof bark plates are trunk microhabitatsfavourable for different lichen species, for instance. Thus lichen microplotsof almostany practical size incorporate at least some heterogeneity in microhabitats important tolichens, and the choice of a best microplot size for defining and sampling relativelyhomogeneous habitat units varies between investigators. The single tree is probably themost widely employed discrete "microplot" sampling unit for monitoring epiphyticlichens.

2.3. SAMPLE SITE LOCATION AND STUDY SAMPLE DESIGN

Effective monitoring of forest lichen biodiversity or monitoring lichen communities asindicatorsof forest health usually requires information on macrovegetation and siteenvironmental characteristics. Co-locating lichen study plots with macrovegetationplots in collaboration with other researchers is an efficient and economic way to acquiresuch data. Many of the monitoring studies cited here used this strategy. Design of thelichen study then is either developed around existing sample design or in collaborationwith investigators who study macrovegetation, rather than solely to address issuesoflichen ecology.

Spatial autocorrelation related to habitat structure, lichen dispersal ability, andhabitat fragmentation should be consideredin sample design. Many lichen species arenot expected to be dispersal-limited,but some are known to be and more will probablybe identified as research continues. Dispersal limitations in some lichens have resultedin different lichen species composition on coniferous forest treesof the same species,age, and having similar microhabitat characteristics, but differing in proximity to oldforest [10, 50]; differential effects persisted for at least 100 m away from an old-forestsourceofpropagules. Sillettet al. [69] showed experimentally thatLobaria oregana isdispersal-limited in young coniferous forestsofnorthwestern USA

3. Methodsfor analysis ofdata

3.1. STATISTICAL SUMMARY OF PLOT DATA

Most standard statistical textbooks cover statistical summary and testing issues relevantto study designs covered here, and the topics are considered in greater depth in Ferrettiand Erhardt (chapter 9, this volume) andWill-Wolf et al. (chapter 11, this volume).Also consult the published studies cited here. When statistical testing is conducted,nonparametric tests should also be considered. They are often more appropriate thanparametric statistical tests for community data, because assumptionsof the latter areoften violated with data on frequency or abundance of multiple species.

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Quantitative comparison of lichen communitycomposition with environmentalvariables, macrovegetation variables, or other variablesof interest can be structured intwo general ways. Continuous data can be compared using correlation or regressiontechniques. Grouped data can be compared using ANOVA or similar techniques, butblocked and/or nested analysis of variance are usually required rather than full-factorialANOVA, even when nonparametric tests are used.

3.2.DATA INTERPRETATION

Exploratory multivariate data analysis techniques such as ordination and classificationaid in sorting out factors relating to variation in community composition (Figure2; forother examples see [28, 57]). Direct gradient analysis techniques such as canonicalcorrespondence analysis are useful to compare known factorsof major concern (seechapter 11, this volume).

Several studies show that different lichen species or species groups responddifferently to forest variables. Grouping data for analysis by important guildsoflichensdiffering in function or morphology may clarify the nature of lichen response toenvironmental variables. For example, cyanolichens versus alectorioid lichens versusgreen-algal foliose lichens have shown widely divergent responses in USA PacificNorthwest forests to such variables as position in canopy, forest age, tree density, andtree species heterogeneity[41,47,56,70].

4. Monitoring biodiversityofforestlichen communities

4.1.GENERAL

Studies which monitor intensively the biodiversity of lichen communities at unique orremnant sites in local areas are important; they monitor the statusof the most naturallichen communities which remain. Equally important are studies that monitor thebiodiversityof lichen communities of forests over broad geographic areas, and addressthe responseofmultiple sites over time and with respect to managementof forests andother human modification of forest landscapes. Conclusions from these studies areapplicable to a broad rangeofforest sites. They provide information on the stateofthelarge-scale landscape context in which unique sites and hotspotsof lichen biodiversityare embedded. Baseline efforts have been completed in several areas around the worldto standardise such long-term,large-scale lichen monitoring efforts (e.g. [12, 22, 45,78]).

Studies which explicitly relate response of the entire lichen community to responseofmore easily monitored subsets can aid in calibrating useofthe subsets as indicatorsof forest ecosystem function in more frequent and widespread monitoring programs(see next paragraph).

Response of different variables representing lichen diversity and communitycomposition often vary in their relation to different causal factors and at differentspatial scales (example in Table 1), so an important componentof a biodiversity

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monitoring programme is to monitor and interpret patterns at several different spatialscales [9,II, 12].

4.2. HABITAT SUBSETS

Trunk lichens and branch lichens are the most commonly used habitat subsets formonitoring biodiversity of forest lichens. Design of rapid surveys requires selectionofan easily surveyed subsetof lichens. It is useful to include as part of large monitoringprograms a few plots in which all available microhabitats in an entire macroplot aresurveyed. This allows researchers to estimate how well the lichen community of thechosen subset, such as trunks, actually represents the biodiversityof the lichencommunity as a whole.

4.3. LICHEN GUILDS OR MORPHOLOGICAL GROUPS

The term guild is often used in ecology to refer to a groupof organisms whosefunctional roles in the community are similar; for lichens guilds are usually definedbased on morphological similarity and presumed functional similarity, as withcyanolichens. The broad morphological groups foliose, fruticose, and crustose lichenscan be considered an extensionof the guild concept. Macrolichens rather than alllichens are often the subsetofchoice for rapid survey protocols, for monitoring changesin ecosystem function, and for monitoring indicatorsof lichen biodiversity, as opposedto monitoring the conditionof all lichen biota at study sites. As with habitat subsets,calibration of a morphological subset to biodiversityofthe complete lichen communityas soon as feasible is a valuable goalofa long-term monitoring program.

For monitoringof complete community biodiversity, useof lichen functional ormorphological guilds, such as cyanolichens, alectorioid lichens, or large and smallfoliose lichens, should be ana posteriori data summary tool rather than ana priori datacollection tool.

5.Lichen communitiesas indicatorsof forestecosystemfunction

5.1. GENERAL

Lichen communities are currently used as indicators of forest ecosystem function inseveral contexts. Region-wide studies have goalsof monitoring the relationof majorfactors such as climate, air quality, and forest tree species composition to variation inlichen communities, and how these relations interact and change over time. Studies oflichens of particular forest types often have goalsof monitoring effectsof forestmanagement practices and landscape context, including a varietyof indirect humanimpacts on forest environments. Habitat alterations that have resulted in the greatestlichen community changes have included changes in forest age structure acrosslandscapes, change in species composition toward timber species ornon-fire-resistanttaxa, change in fire regime, clear-cutting versus single-tree harvesting, and roadbuilding (e.g. [9,17,19,38,50]).

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In addition, forest fragmentation,increasededge effects, and remnantretentionhaveresulted insignificantchanges to lichencommunitystructure[15, 30, 51, 60,68].

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Since the goal is often to develop indicatorsoffunction rather than response of thewhole lichen community, particularly useful and/or more easily sampled subsets of thelichen flora are almost always chosen as the monitoring targets. One should stratifysample plots with respect to particular monitoring questions, and design studies somonitoring can answer the largest-scale questions appropriately (e.g.[12]). When timeand resources are limiting, effort allocated to within-plot sampling (more microplots ormore detailed data collection) may need to be reduced to allow increased effort tosample more sites (more macroplots) to adequately address large scale monitoringquestions within the resources of the study.

5.2. INDICATORS OF FOREST ECOSYSTEM FUNCTION

One ofthe most important broad-scale ecosystem function topics currently is responseof lichens to forest management. Monitoring with selected groupsof lichens (asopposed to all species) as indicators of ecosystem function and effects on lichenbiodiversity has usually been the choice in sample design. Usually to answer mostassessment questions, large numbers of plots need to be spread over large areas andresampling over time is essential, so the reduced time and expense associated withmonitoring subsetsof lichens is critical to fundingofsuch projects. Studies are neededto validate the useof the subset as an indicatorof lichen response to the ecosystemfunction under investigation. Calicioid lichens ("pin lichens") have been shown to beparticularly useful as indicatorsof old-growth forests [27, 66, 74]. Many of thesespecies are dependent on snags and on old trees, with stable, rough bark. Because theyare difficult to find, surveysof calicioid lichens are best done by experiencedlichenologists.Tibell [74] and Selva [66] used whole-plot intensive floristic sampling todevelop a listof forest continuity indicator species including calicioid lichens; one canthen assess forest continuity of a site using just the indicator species. Lichens in the"alectorioid"morphological guild (tufted and pendulous fruticose lichens including thegenera Alectoria, Bryoria, Usnea, and others) have been found to be useful asindicators bothofforest management and of region-wide air quality in several partsofthe world. The alectorioid lichen guild is generally both more species-rich and moreabundant in older forests, on older trees, and/or where air quality is better [16, 29, 59].Variation in diversity and abundanceof epiphytic cyanolichens appears useful world­wide as an indicatorof forest ecosystem function in temperate and boreal forests.Cyanolichens are important in forest nutrient cycles; many are sensitive both topollution and to forest age and continuity (e.g. [41, 51, 70]). Lobaria spp. in general,andL. pulmonaria in particular, are sensitive to air quality as well as reliable indicatorsofspecies-rich old-growth forests with long forest continuity (e.g.[19,32,35,69]).

5.3. LARGE-SCALE MONITORING OF FOREST ECOSYSTEM FUNCTION

Large-scale monitoring programs using lichens as indicatorsof forest ecosystem func­tion have been developed in several countries in Europe and in the United States(USA).

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TABLE I . A. Species richness ofepiphytic macrolichens in relation to stage off orest development and spatialscale (stand and landscape) in managed and natural f orest landscapes (5 x 5 km} in northern Sweden . Basedon datafrom 150 circular sample plo ts (10 mradius). B. Summa ry oftwo ANO VA's testing the effect oftypeoflandscape (managed natural) and study area (Rievo, Muddus, Jelka) on species richness and abundance ofepiphytic macrolichens. Analysis on ranked data. Reproduced by permission fr om Dettki and Esseen [9j.

A : Species Richness Managed Natural

X± 1 SE n X± 1 SE nStageofforestdevelopment

Clear-cut 6.8 ± 1.5 17 5.0 ± 5.0 2Young forest 12.6 ± 0.7 20 13.5 ± 0.5 2Mature forest 16.1 ± 0.3 38 16.4 ± 0.2 71

Stand (sampleplot) level 13.0 ± 0.6 75 16.0 ± 0.3 75Landscape level 23.3 ± 1.9 3 24.3 ± 0.3 3

B: ANOVA ResultsSource DF MS F P

Species richnessType oflandscape 1 38720.7 26.23 0.000Study area 2 7065.8 4.79 0.010Type x study area 2 4394.5 2.98 0.054Error 144 1476.0

AbundanceType oflandscape I 49235.0 37.10 0.000Study area 2 14695.2 11.07 0.000Type x study area 2 2267.5 1.71 0.185Error 143 1327.3

Currentprogrammesin the Netherlands and Switzerland collect data on all speciesin microplots, and field personnel are lichen experts, while programmes inFennoscandiaand the USA collect data on macrolichens only, and some or all fieldpersonnel arenonspecialists. A newall-Europebiodiversity assessmentprojectisdesigned to collect data on all lichens in microplots for habitatsubsets nested inmacroplots; field personnel will be lichen experts. Climate and habitatvariationaresmall to moderate in the Netherlands andFennoscandiaprogrammes, moderate to largein the Swiss and all-Europe programmes, and extremely large in the USAprogramme.Geographic extent is small to large in the European programs and very large in theUSA program, whose extent is considerably larger than allofEurope [43].

In monitoring programmes in the Netherlands, investigators recorded coverofeachlichen species on up to 10 free-standing wayside trees(first priority oak and/orpoplar,second priority willow and/or elm) at sample points. Monitoring was conductedinabout 1950 and about 1970 with severalhundred sample points,and in 1977-90 with150 sample points (reviewed in [79]).Methodology is relevant for forestmonitoringstudies, even though plots were not placed in true forest habitat.Over the periodofmonitoring, lichen diversity was linked to levelsof atmospheric S02, and recoveryfollowing reduction in S02varied withbark pH.

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In Switzerland Dietrich and Scheidegger [11, 12] developed a pilot lichenmonitoring programme with explicit hierarchical structuringof locationof the 132macroplots to address monitoring questions at several geographical scales. Themonitoring network is being expanded to over 800 sample locations. Microplot sampleprotocol is similar to Netherlands studies in that cover of all lichens on trunksoftreesof different species is recorded. Trees are clustered in fixed-area macroplots, whichinclude both forested and non-forested habitats. Species diversity and abundanceoflichen guilds (subgroupsofcrustose, foliose, and fruticose species differing in dispersalmode) varied significantly between habitatclassesofmacroplots based on climate anddominant vegetation (see Figure 2ofchapterII, this volume).

In Fennoscandiamonitoring programmes, selected lichen groups are monitored onselected tree species. In Norway, coverof seven lichen groups is monitored on trunksof Pinus sylvestris on 193 permanent sites using trunk tape transects [7]. In Sweden,abundance of three groups of pendulous lichens(Alectoria sarmentosa, Bryoria spp.,and Usnea spp.)has been monitored since 1993 in several thousandpermanentplots inthe National Forest Survey [29] by measuring the maximum lengthofthe lichens in thelower canopy (>5m)ofPicea abies. Results from 1993-97 show a decline in abundanceofpendulous lichens compared to older records [67]. In Finland, abundance classof 13lichen species has been recorded on conifers in 3000 permanent plots in the NationalForest Inventory in 1985-86 [36]. All lichens increased in abundance during the period1985 to 1990, probably as a result of decreased sulphur deposition [59].

A newall-Europebiodiversity assessment project (see chapter 35, this volume) isdesigned to monitor effectsofagriculture and forestry management onbiodiversityoflichens in habitat subsets as indicatorsofecosystem function. Frequencyofall speciesin replicate microplot grids (releves) will be recorded for rocks, ground, and treespartitioned into acid versus neutral bark and large (old) and small (young) groups.Microplots will be nested in 1 ha macroplots. At eachof 6 sites in eachparticipatingcountry, 16 macroplots will be located 200 m apart in a square grid.

In the USA, lichens are being included in a nationwideprogrammeof forestecosystem monitoring conducted by the US Forest Service Forest Inventory andAnalysis Program. No one groupoftree species, forest community, or lichen species isfound across the entire country. Macrolichen community composition based on whole­plot surveys is therefore used to place each permanentplot on regional environmentalgradients, and change in positionof plots on these gradients is monitored over time[45]. No change in regional responseof lichens to air quality or climate gradients wasfound for the southeastern and northeastern regionsof USA between 1994 and 1998[52] (see chapter 34, this volume). This USA forest monitoring protocol is beingadopted in the Baltic countries and Ukraine,Eastern Europe.

5.3.1. Use ofnonspecialist personnelIn many partsof the world, there are not enough trained lichen experts to do all themonitoring that is needed, nor is there enough money to pay expert personnel to do themonitoring[6,43],so monitoringprotocols which can be performed by nonspecialistsare the best choice for such areas. This usually means that monitoring is restricted tomacrolichens; monitoringofcrustose species generally requires expert field personnel.

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The useof nonspecialists should be based upon strict training and quality controlprocedures. For instance, for the USA Forest Service Forest Inventory and AnalysisProgram, it has been determined that nonspecialists who attain a minimumof 65% ofthe species capture of expert lichenologists will achieve plot scores <10% differentthanexpert scores on air quality and climatic gradients for the same plot [44]. Training andquality control standards therefore use 65%of expert species capture as the minimumachievement level; in practice, species capture by field crew is mostly 75-90%ofexperts for the standard sample protocol in that program.

5.3.2. Minimum-effort whole-plot design as contextA useful strategy for addressing very large-scale monitoring questions is to use thesame or similar low-budget sample design as a component of most forest monitoringstudies within a large region such as Europe, North America, or northern hemisphereboreal forest. This strategy will provide context for more intensive studies and thus willallow better comparability between intensive studies in different regions, and betweenintensive studies and background conditions. The US Forest Service Forest Inventoryand Analysis Program sample design is currently being used for such a purpose in theUSA.

6.Concluding recommendations

There are several critical issues that must be considered in the design of effectivemonitoring programs.Ferretti and Erhardt (chapter 9, this volume), Noss [53], and Voset al. [80] provide a conceptual framework and general recommendations. Goal-settingis of crucial importance as it defines the whole scopeof the monitoring and theselection of both output ("final") variables and measurement variables.

Use combined macroplots and microplots for studies of lichen communitybiodiversity. This allows for estimationofspecies-area relationships and assessment ofheterogeneity in species distribution at different scales, in addition to estimationofbiodiversity within regions. Combined use of microplots and macroplots has been donein some lichen studies, and should become more common.

For use of lichens as indicatorsof either biodiversity or ecosystem function, useindicator species or subsetsof lichen communities and macroplot sampling primarily.Special tests should be performed to establish the statistical and the causal relationshipsbetween the selected indicator species and the aspectof biodiversity or ecosystemfunction for which it is considered to be indicative. Always pay attention tostratification issues in designing a study.

7.Acknowledgements

Will-Wolf wishes to acknowledge funding support from NATO to attend the LIMON workshop in August,2000. Will-Wolf and Neitlich received funding from the USDA Forest Service during the productionofthemanuscript. We thank an anonymous reviewer and the editors of this volume for helpful critique of thecontent and organisationof the chapter. John Wolf contributed many editorial suggestions that improved thereadabilityofthe text. Kandis Elliot adapted the figures.

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8.References

I. Annleder, H.M., Stevenson, S.K.,and Walker, S.D. (1992)Estimating the abundance ofarboreal foragelichens. Land Management Handbook, Field Guide Insert 7,B.C.Ministry ofForests, Victoria, B.C.

2. Bates,l .W . and Fanner, A.M . (eds.) (\992) Bryophytes and Lichens in a Changing Environment ,Clarendon Press, Oxford.

3. Bergstrom, I.,Makela, K., and Starr, M. (eds.) (\995) Integrated Monitoring Programme in Finland.First National Report. M inistryofthe Environment,Environmental Policy Department,Helsinki.

4. Boucher, V.L. and Stone, D.F. (1992) Epiphytic lichen biomass, in Carroll, G. and Wicklow, D. (eds.),The Fungal Community. Its organization and role in the ecosystem 2nd edition,Marcel Dekker Inc., pp.583-599.

5. Brakenhielm, S. and Liu, Q.H. (1995) Spatial and temporal variability of algal and lichen epiphytes ontrees in relation to pollutant deposition in Sweden,Water. Air and Soil Pollution 79, 61-74.

6. Brown, M.1., Jarman, S.1.,and Kantvilas, G. (\994)Conservation and reservation of non-vascular plantsin Tasmania, with special reference to lichens,Biodiversity and Conservation 3, 263-278.

7. Bruteig, I.E. (1993) Large-scale surveyofthe distributionand ecologyofcommon epiphytic lichens onPinus sylvestris in Norway,Annales Botanici Fennici 30, 161-179.

8. Cornelissen, l .H.C. and ter Steege, H. (\989) Distribution and ecology of epiphytic bryophytes andlichensin dry evergreen forestsofGuyana,Journal ofTropical Ecology 5, 131-150.

9. Dettki,H. and Esseen, P.-A.(1998) Epiphytic macrolichens in managed and natural forest landscapes: acomparison at two spatial scales,Ecography 21,613-624.

10. Dettki, H.,Klintberg, P.,and Esseen, P.-A.(2000) Are epiphyticlichens in young forests limited by localdispersal?, Ecoscience 7, 317-325.

II. Dietrich, M. and Scheidegger, C. (1997) Frequency, diversity and ecological strategiesof epiphyticlichens in the Swiss central plateau and the pre-Alps,Lichenologist29, 237-258.

12. Dietrich, M. and Scheidegger, C. (1997) A representative survey of frequencyofepophyticlichens at theregional and national levels and its use for the red list of Switzerland, in R. Turk andR. Zorer (eds.),Progress and Problems in Lichenology in the Nineties. Bibliotheca Lichenologica, J. Cramer, Berlin,Stuttgart,pp. 145-154.

13. Esseen, P.-A., Ericson, L., Lindstrom, H. and Zackrisson, O. (1981) Occurrence and ecologyof Usnealongissima in central Sweden,Lichenologist13, 177-190.

14. Esseen, P.-A. and Renhorn, K.-E. (1998) Mass lossofepiphytic lichen litter in a boreal forest,AnnalesBotanici Fennici 35,211-217.

15. Esseen, P.-A. and Renhorn, K.-E. (1998) Edge effects on an epiphytic lichen in fragmented forest,Conservation Biology 12,1307-1317.

16. Esseen, P.-A., Renhorn,K.-E.,and Pettersson,R.B.(1996) Epiphytic lichen biomass in managed and old­growth boreal forests:effectofbranch quality,Ecological Applications 6,228-238.

17. Frey, E. (1958): Die anthropogenen Einflusse auf die Flechtenflora und -vegetation in verschiedenenGebieten der Schweiz. Ein Beitrag zum Problem der Ausbreitung und Wanderung der Flechten,Veriiffentl. des Geobot. Inst. Rubel in Zurich (Festschrift Werner Liidi) 33, 91-107.

18. Galun, M. (ed.) (1988)CRC handbook oflichenology. Volumes I, II. and III. CRC Press Inc.,Boca Raton,Florida.

19. Gauslaa, Y. (1995) The Lobarion, an epiphyte community of ancient forests threatened by acid rain,Lichenologist27,59-76.

20. Gauslaa, Y., Ohlson, M ., and Rolstad, J. (1998) Fine-scaledistributionof the epiphytic lichenUsnealongissima on two even-aged neighbouringPicea abies trees,Journal ofVegetation Science 9,95-102.

21. Geiser, L.H., Derr, C.C., and Dillman, K.L.(\994) Air quality monitoring on the Tongass NationalForest. Methods and baselines using lichens. Report RIO-TB-46, United States DepartmentofAgriculture,Forest Service, Alaska Region.

22. Geiser, L.H., Dillman, K .L., Derr, C.C.,and Stensvold, M.C. (1994) Lichens of southeastern Alaska.Report RIO-TB-45, United States DepartmentofAgriculture, Forest Service, Alaska Region.

23. Glenn, M.G.,Webb, S.L. and Cole, M .S. (1998) Forest integrity at anthropogenic edges: air pollutiondisrupts bioindicators,Environmental Monitoring and Assessment 51, 162-169.

24. Goward, T. and Arsenault, A. (2000) Cyanolichen distribution in young unmanaged forests: a dripzoneeffect?The Bryologist 103, 28-37.

25. Hayward, G.D.and Rosentreter, R. (1994) Lichens as nesting material for northern flying squirrels in thenorthern Rocky Mountains,Journal ofMammalogy 75, 663-673.

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26. Hilmo, O. (1994) Distribution and succession of epiphytic lichens onPicea abies branches in a borealforest, central Norway,Lichenologist26,149-169.

27. Holien, H. (1996) Influenceof site and stand factors on the distribution of crustose lichens of theCaliciales in a suboceanic spruce forest area in central Norway,Lichenologist28,315-330.

28. Kantvi1as, G. and Minchin, P.R. (1989) An analysisofepiphytic lichen communities in Tasmanian cooltemperate rainforest,Vegetatio 94,99-112.

29. Kar1tun, E., Odell, G., Logren, 0 ., and Carlsson, E. (1995) Fiiltinstruktion for standortskanering avpermanenta provytor vid riksskogstaxeringen, Dept. of Soil Science, Swedish University of AgriculturalSciences,Uppsala.

30. Kivisto,L. and Kuusinen, M. (2000). Edge effects on the epiphytic lichen flora ofPicea abies in middleboreal Finland,Lichenologist32,387-398.

31. Knops, 1.M.H., Nash, T.H.III, Boucher, V.L., and Schlesinger, W.H. (1991) Mineral cycling andepiphytic lichens: implications at the ecosystem level,Lichenologist23,309-321.

32. Kondratyuk, S.Y. and Coppins, BJ. (eds.) (1998) Lobarion lichens as indicators of the primeval forestsof the eastern Carpathians. Darwin International Workshop, 25-30 May 1998, UkrainianPhytosociological Centre, Kiev.

33. Kuusinen, M. (1994) Epiphytic lichen diversity onSalix caprea in old-growth southern and middle borealforestsof Finland,Annales BotaniciFennici 31,77-92.

34. Kuusinen, M. (1996a) Epiphyte flora and diversity on basal trunksofsix old-growth forest tree species insouthern and middle boreal Finland,Lichenologist 28,443--463.

35. Kuusinen, M . (I996b) Cyanobacterial macrolichens onPopulus tremula as indicatorsofforestcontinuityin Finland,Biological Conservation 75, 43-49.

36. Kuusinen, M.,Mikkola, K, and Jukola-Sulonen, E.L. (1990) Epiphytic lichens on conifers in the 1960'sto 1980's in Finland, in P.Kauppi, P. Anttila,andK Kenttiimies(eds.),Acidification in Finland, SpringerVerlag, Berlin, pp. 397-420.

37. Kuusinen, M. and Siitonen, J. (1998) Epiphytic lichen diversity in old-growth and managedPicea abiesstands in southern Finland,Journal ofVegetationScience 9,283-292.

38. Lesica, P., McCune, B., Cooper, S.V., and Hong, W.S. (1991) Differences in lichen and bryophytecommunities between old-growth and managed second-growth forests in the Swan Valley, Montana,CanadianJournal ofBotany 69, 1745-1755.

39. Longton, R.E. (1992) The roleofbryophytesand lichens in terrestrial ecosystems, in J.W.Bates and A.M.Farmer (eds.),Bryophytesand Lichens in a ChangingEnvironment,Clarendon Press, Oxford, pp 32-61.

40. McCune,B. (1990) Rapid estimationofabundance of epiphytes on branches,The Bryologist 93, 39-43.4I. McCune,B. (1993) Gradients in epiphyte biomass in threePseudotsuga-Tsuga forestsofdifferent ages in

western Oregon and Washington,TheBryologist96, 405-411.42. McCune,B. (1994) Using epiphytelitter to estimate epiphyte biomass,The Bryologist 97,396-401.43. McCune,B. (2000) Lichen communities as indicatorsofforest health,The Bryologist 103, 353-356.44. McCune, B., Dey, J., Peck, J., Cassell, D., Heiman, K , Will-Wolf, S., and Neitlich, P. (1997)

Repeatability of community data: species richness versus gradient scores in large-scale lichen studies,TheBryologist 100,40-46.

45. McCune, B., Dey, J., Peck, J., Heiman, K., and Will-Wolf, S. (1997) Regional gradients in lichencommunitiesofthe Southeast United States,The Bryologist 100,145-158.

46. McCune, B. and Lesica, P. (1992) Thetrade-offbetween species capture and quantitative accuracy inecological inventoryoflichensand bryophytes in forests in Montana,The Bryologist 95, 296-304.

47. McCune, B.,Rosentreter, R.,Ponzetti, 1.M.,and Shaw, D.C. (2000) Epiphyte Habitats in an Old ConiferForest in Western Washington,U.S.A., The Bryologist 103(3),417-427.

48. Nash, T.H. III (ed.}(1996)Lichen Biology,Cambridge University Press,Cambridge.49. Nash, T.H. III and Wirth, V . (1988) Lichens. Bryophytes and Air Quality, Bibliotheca Lichenologica 30,

1. Cramer in der Gebruder Borntraeger Verlagsbuchhandlung,Berlin-Stuttgart.50. Neitlich, P.N. (1993) Lichen abundance and biodiversity along a chronosequencefrom young managed

stands to ancientforest. M .Sc.Thesis, UniversityofVermont.5I. Neitlich, P.N. and McCune,B. (1997) HotspotsofEpiphytic Lichen Diversity in Two Young Managed

Forests, Conservation Biology 11, 172-182.52. Neitlich, P. and Will-Wolf, S. (2000) The Lichen Community Indicator in the Forest Inventory and

Analysis FHM Program: Using Lichen Communities to Monitor Forest Health. Poster, Forest HealthMonitoring Workshop 2000, Orange Beach, Alabama, February14-17,2000.

53. Noss, R.F. 1999. Assessing and monitoring forest biodiversity: A suggested framework and indicators,Forest Ecology and Management 115, 135-146.

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54. Ojala, E., Monkkonen, M. and Inkeroinen, 1. (2000) Epiphytic bryophytes on European aspenPopulustremula in old-growthforests in northeastern Finland and in adjacent sites in Russia,Canadian Journal ofBotany 78,529-536.

55. Oksanen, J. (1988) Impactof habitat, substrate and microsite classes on the epiphyte vegetation:interpretation using exploratory and canonical correspondence analysis,Annales Botanici Fennici 25, 59­71.

56. Peck, J.E. and McCune, B. (1997) Remnant trees and canopy lichen communities in Western Oregon: aretrospective approach,Ecological Applications 7 (4),1181-1187.

57. Pharo,EJ. and Vitt, D.H. (2000) Localvariation in bryophyte and macro-lichen cover and diversity inmontane forests of WesternCanada, The Bryologist 103, 455-466.

58. Pike, L.H. (1978) The importance of epiphytic lichens in mineral cycling, The Bryologist 81,247-257.59. Poikolainen, 1., Kuusinen,M .,Mikkola, K.,and Lindgren, M.(1998) Mapping of the epiphytic lichens on

conifers in Finlandin the years 1985-86 and 1995,Chemosphere 36,1073-1078.60. Renhorn, K.-E.,Esseen, P.-A.,Palmqvist, K., and B. Sundberg. (1997) Growth and vitalityof epiphytic

lichens. I. Responses to microclimate along a forest edge-interior gradient,Oecologia 109, 1-9.61. Rominger,E.M.,Robbins,C.T.,and Evans,M.A .(1996) Winter foraging ecologyofwoodland caribou in

northeastern Washington,Journal ofWildlife Management 60, 719-728.62. Samuelsson, J., Gustafsson, L., and lngelog, T. (1994). Dying and dead trees-a review of their

importance for biodiversity, Threatened Species Unit, Swedish University of Agricultural Sciences,Uppsala.

63. Schmitt, C.K. and Slack, N .G. (1990) Host specificity of epiphytic lichens and bryophytes: a comparisonofthe Adirondack Mountains (New York) and the southern Blue Ridge Mountains (North Carolina),TheBryologist93,257-274.

64. Seaward, M.R.D. (1988) Contribution of lichens to ecosystems, in M. Galun (ed.), CRC Handbook ofLichenology. Volume ll. CRC Press Inc.,Boca Raton,Florida,pp. 107-129.

65. Seaward, M.R.D. (1993) Lichens and sulphur dioxide air pollution: field studies,Environmental Review1,73-91.

66. Selva, S.B. (1994) Lichen diversity and stand continuity in the northern hardwoods and spruce-fir forestsof northern New England and western New Brunswick, The Bryologist 97,424-429.

67. SEPA (1999) Environmental Quality in Swedish Forests, Swedish Environmental Protection Agency,Stockholm, 100 pp (in Swedish).

68. Sillett, S.C. (1994) Growth ratesoftwo epiphytic cyanolichen species at the edge and in the interiorofa700-year-old Douglas-fir forest in the western Cascades of Oregon,The Bryologist 97, 321-324.

69. Sillet, S.C., McCune, B., Peck, J.E., Rambo, T.R., and Ruchty, A. (2000) Dispersal limitationsofepiphytic lichens result in species dependent on old-growth forests,Ecological Applications 10, 789-799.

70. Sillett, S.C. and Neitlich,P.N. (1996) Emerging themes in epiphyte research in westside forests withspecial reference to cyanolichens,Northwest Science 70,54-60.

71. Stevenson, S.K.and Enns, K.A. (1993) Quantifying arboreal lichens for habitat management: a review ofmethods . British.Columbia Min istryofForestry, Victoria,B.C.,IWIFR-42 .

72. Stohlgren,TJ.,Falkner, M.B., and Schell, L.D.(1995) A Modified-Whittakernested vegetation samplingmethod,Vegetatio 117, 113-121.

73. Stolte, K., Mangis, D.,Doty,R.,Tonnessen, K.,and Huckaby,L.S.(eds.) (1993)Lichens as bioindicatorsof air quality, United States Department of Agriculture, Forest Service, Rocky Mountain Forest andRange Experiment Station, General TechnicalReport RM-224, Fort Collins, CO.

74. Tibell, L. (1992) Crustose lichens as indicators of forest continuity in boreal coniferous forests,NordicJournal ofBotany I, 427-450.

75. TUrk, R. and Zorer, R. (eds.) (1997)Progress and Problems in Lichenology in the Nineties , BibliothecaLichenologica, J.Cramer, Berlin-Stuttgart.

76. Tyrrell, L.E. (1996) National forests in the eastern region: land allocation and planning for old growth, inM .B. Davis (ed.),Eastern Old-Growth Forests. Prospects for Rediscovery and Recovery, Island Press,Washington,D.C.,pp. 245-273.

77. Tyrrell, L.E. and Crow, T.R. (1994) Structural characteristics of old-growthhemlock-hardwoodforests inrelation to age,Ecology 75,370-386.

78. Van Dobben, H.F. and DeBakker, AJ. (1996) Re-mapping epiphytic lichenbiodiversity in theNetherlands: effectsofdecreasing S02 and increasing NH3, Acta Botanica Neerlandica 45, 55-71.

79. Van Dobben, H.F.and Ter Braak,CJ.F. (1998) Effectsofatmospheric NH3 on epiphytic lichens in theNetherlands: the pitfallsof biological monitoring,Atmospheric Environment 32,551-557.

80. Vos, P., Meelis, E.,and Ter Keurs,WJ. (2000) A framework for the design of ecological monitoring

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programsas a tool forenvironmentaland naturemanagement,Environmental Monitoring and Assessment61,317-344.

81. Will-Wolf, S . (1988) Quantitativeapproaches to air quality studies, in T.H.III Nash and V. Wirth (eds.),Lichens. Bryophytes and Air Quality, BibliothecaLichenologica30, J. Cramer,Berlin-Stuttgart,pp. 109­140.

82. Will-Wolf, S.,Hawksworth,D .L., McCune, B.,Sipman, H.,and Rosentreter,R. (in press)Assessing thebiodiversityof lichenized fungi, in G.M. Mueller, G.F. Bills and M.S. Foster (eds.), Measuring andMonitoring Biological Diversity: Standard Methodsfor Fungi. SmithsonianInstitution,Washington,DC .Expectedpublicationdate: 200 I .

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MONITORING BIODIVERSITY AND ECOSYSTEM FUNCTION:GRASSLANDS, DESERTS, AND STEPPE

R. ROSENTRETER' and D. J.ELDRIDGE 2

I US Department ofInterior, Bureau ofLand Management, 1387S.Vinnell Way, Boise, Idaho, 83709, USA ([email protected])2Centre for Natural Resources, Department ofLand and WaterConservation, c/o School ofGeography, University ofNSW, Sydney,2052, Australia.

1. Introduction

During the past century, land managers have grappled with methods to assess both thecondition (health) and degreeof change (trend) of arid landscapes [77]. Traditionalapproaches have focused on the documentation of various attributesof vascular plantcommunities [34]. More recently however, Australian and American researchers haveincorporated data on biological soil crust taxa (lichens, bryophytes and cyanobacteria)in condition and trend assessmentofarid lands (rangelands) [32, 57, 78, 80] principallydue to the growing recognition that these organisms are critical for rangeland stability.In this chapter,"rangelands"refers to landscapes or vegetation communities which aredominated by native vegetation, are used predominantly for grazing, recreation ortraditional ownership (e.g. aboriginal occupation or transhumance), but excludescommercial non-native forests or areas of intensive agriculture.Lichensofnative forestsare discussed inWill-Wolf et al. (chapter 14, this volume).

Monitoring implies a regular assessmentof change through time, and is generallyviewed as being a continuing process rather than a one-timeevent. Lichens have beenused for many years for monitoring ecosystem change. However, lichens have onlyrecently been used as tools for monitoring in terrestrial systems.Terricolous (soil-borne)lichens common in rangelands are now being monitored becauseofthe perception thatthey are important components of healthy ecosystems [57,78].

Why is it important to monitor soil lichens in arid landscapes? The reason is thatarid habitats are particularly vulnerable to disturbances from humans and domesticlivestock as these areas are often in the warmer lower elevations and are easilyaccessible. Domestic livestock are typically free ranging, and their trampling has asignificant negative impact on terricolous lichens [19].Biological diversity is threatenedeverywhere [59] but arid habitats world-wide are some of the most threatened of anytype of habitat [8]. Degradation of arid habitats often causes desertification.Desertification is considered by the United Nations Environmental Programme (UNEP)to be oneofthe major environmental problemsofthis century and affects nearly 35%ofthe global land surface and almost one fifthof the world human population [8].Desertification is accompanied by a change in species composition, a general lossof

223P.L. Nimis, C. Scheidegger and P.A. Wolseley (eds.), Monitoringwith Lichens- Monitoring Lichens, 223-237.© 2002 KluwerAcademic Publishers. Printed in the Netherlands.

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biomass, lossof soil materials by wind and water erosion, all of which contribute to aloss of ecosystem productivity.

The health or ecological condition of a plant or animal community or site isfrequently difficult to quantify,and individual beneficial or "keystone" species are oftenused as the indicatorsof ecosystem health [8]. Assessing the healthof grasslands,deserts and steppe presents numerous challenges becauseof their diversity in structureand composition, the harsh environments associated with temperature extremes, theirrugged topography, and our limited knowledge of how they collectively function. Inrangelands systems, the terms "condition" and"health" have been used variously toindicate" the degree to which the integrity of the soil and ecological processes inrangeland ecosystems are maintained" [55]. Given an increasing emphasis on ecosystemfunction, composition and structure [53], we use the terms "ecosystem health" or"ecosystem function", and confine our assessment to oneof biodiversity conservationand the maintenanceofessential ecosystem processes.

Below we review studies of soil-dwelling (terricolous) lichens for assessingbiodiversity and ecosystem health. This monitoring is pertinent to woodlands,grasslands,deserts, steppe, alpine sod and tundra, though literature on alpine and tundrawas not extensively reviewed.Saxicolous lichens are treated elsewhere in this volume.

2.Monitoringlandscapesto assess lichendiversity

2.1.QUALITATIVE ASSESSMENT OF LICHEN DIVERSITY

Many studies are qualitative in nature (Table I), with often little regard to landscapeprocesses, sampling protocols or statistics.

TABLE 1. Types of surveys used for collecting information on species diversity (adapted f rom NPWS [54)).

Survey type

Standardisedsurvey

Non-standardisedsurvey

Targeted survey

Opportunistic(e.g. intuitively­controlled) survey

Description

consistenteffortand sampleselection across the survey area;quantitative

variable setoffield techniques;unevensampling across thelandscape;qualitative

sites chosen because they are likelyto be species-rich; qualitative

records collected while doing otherthings or other surveys; qualitative

Characteristics

generally cover-abundancedata; provides anobjective viewofregional-scalebiodiversity;statisticallyrobust.

generallypresence-absencedata; useful forgatheringspecies lists; limited objectivecomparisonofregional biodiversity

habitat-specificspecies likely to be found;does not provide an objective viewofregionalbiodiversity

can add to species lists(especiallyrarespecies); does not providean objective viewofregionalbiodiversity;statistics unusable

Qualitative sampling often lacks replication, robustness and representativeness, andcollections are not restricted to plots of known size and shape, or collected over fixedperiods of time. Consequently it is difficult to compare and contrast biodiversitywithinand among sites, across time, or even across broad geographic regions. Therefore thedata do not represent a true indication of biodiversity, community structure or

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ecosystem health [47]. Despite the limitationsof qualitative studies however, they areuonetheless useful in providing an indication of the likelihoodof finding a particularspecies. These studies can also be useful for targeting new areas for more detailed orstrategic studies.

Qualitative studies typically involve the collection of lichen specimens in asomewhat haphazard sequence, over landscapes which are oftenof ill-defined size andwith widely varying habitat complexity, with no specific plots, with no specific timespent searching at each collection site, and with no comparable degreeof samplingeffort [51, 65, 73, 81, 82]. Sitessampled opportunistically are often easily accessible,such as near campgrounds or on the outskirtsof cities, or they may have been chosenbecause they look interesting ecologically (e.g. ecotones on areas excluded fromgrazing). Other qualitative studies include intuitively-controlled searches [27] wherecollections of specimensare made from sites regarded as being unique or hot spotsofbiodiversity [52, 81]. Unlike quantitative studies described below, there is little controlfor differences in environmental factors such as the qualityofthe lighting, weather andtimeofday, and experienceofthe observer, and thus the resultsofthese studies may behighly biased [48].

Some ecological studies are undertaken in order to demonstrate the impactofdisturbance such as trampling by domestic livestock on lichen diversity [3, 33, 68].Whilst many of these studies are commonly found in scientific journals [68],many haveinherent weaknesses in that they do not provide species level information [31, 49, 56]and therefore do not reflect the true site diversity (Table 2). For example, often only thedominant type of organism in a soil crust is recorded such as "lichen or moss", or thedata are recorded by one known species"Lecidea decipiens" or "Lecidea sp." or as"unknownlichen sp. #1,2,3 or 4" [10].This lumpingoftaxa, morphological groups, oreven broad crust types (lichen, moss, liverwort) into a collective percentage coversuggests that a site with a greater cover has greater biological diversity. Crust cover maybe related, though weakly, to diversity. For example, studies from the Colorado plateauand Central Utah have shown that sites with an extensive lichen cover are oftencomposedofonly a few species, whilst other studies from the Columbia Basin in centralOregon and Washington have demonstrated reduced cover levels associated with agreater numberofspecies per plot [5,62, 63] (Table 2).

2.2.QUANTITATIVE ASSESSMENT OF LICHEN DIVERSITY

Soil lichens are generally measured using standard rangeland assessment techniques, i.e.quadrats placed along transects. The size of the sampling unit should be appropriate tothe size, density and spatial distribution of the lichens being studied, their habitats, andthe nature of the impact being investigated. For example, studiesof the impactofmining or recreational vehicles on lichen communities may be best examined by usingrepeated photo-points,remote sensing or aerial photography interpretation on large plotsof up to several hectares in size. For finer scale assessment, line intercept or quadratmethods are more appropriate. The size and numberof samples will be based on acompromise between statistical rigour and what is feasible.

For quantitative surveys, sampling locations are often stratified according tovegetation community, landform or soil type, and quadrats placed either randomly or at

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regular intervalsalong the transects. Stratification ensures that the relative contributionof a particular vegetation community or soil type to the study area is taken intoconsideration when the total area is measured.

TABLE 2. Diversity of lichen species or species groups reported from arid deserts, grasslands, steppe andwoodlands f rom the northern and southern hemispheres. Some of these studies included saxicolous taxa andare f rom a single location, while others are from 20 or more sample sites spread over many hundreds ofkilometers. "between 3-42 species collected per site, "recorded from six States, ' lichen species recorded on soilonly.

Location Habitat Numberofspecies Reference

New SouthWales,Australia Desert 48 14SouthwestUSA Desert 8 50Utah,USA Desert 5 38Eng land,UK Grassland/forest 72' 30GreatPlains, USA b Grassland 20 44GreatPlains,South Dakota,USA Grassland 31' 82Central Orego n,USA Steppe >150 72Wash ington, USA Steppe 49 63SouthernIdaho,USA Steppe 26 69Utah, USA Steppe 2 4Utah, USA Steppe 5 38Utah, USA Steppe 23 64Utah, USA Steppe 17 2Arizona, USA Open woodland 18 10Arizona,USA Open woodland 8 5

In shrublands and grasslands for example, the focus is frequentlyon the shrub orgrass interspaces where the majorityofunvegetated soils and hence soil lichens occur.Consequently, measurements are often restricted to the interspaces, and the data arereportedas such. One complicating feature of long-term monitoringof soil lichenscould be the turnoverof lichen habitat over long time periods.Not withstanding human­induced changes in habitatsbrought aboutby activities such as vegetation clearance androad construction, landscapes will changenaturallyover time. Accordingly, in the long­term (50-100 years),patchesofsoil crust will also likely change within permanent plotsand along transects.

Two issues need tobe consideredwhen quantitatively assessing the diversityof soillichen communities; I) the choice of measurement technique,and 2) the levelat whichthe data are collected.

2.2.1. Plot and line-intercep t techniquesPlots or quadrats are generally more practical where soils are sparsely occupied bylichens, wherelichens occur in patches, or where rare species are being studied. As invascular plant studies, quadrats maybe nested. Size and shape are importantconsiderations, and the plots need to be small enough so that the whole area underconsideration is easily visible without causing destructive trampling when the taxa arebeing closely observed. Given the small sizeof the organisms being studied and thespatial scales at which they operate« I cnr'), one sideofthe plot is often less than 50em long. Long narrow plots or small rectangular plots (20x50 em) are easier to evaluatethan larger square plots [27].

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Quadrat size and placement often vary widely among studies. In the western UnitedStates, rectangular 20x50 em plots are frequently used to monitor shrub-steppevegetation including crusts [13, 63]. Johansenet al. [38] used a 100 m line intercepttransect[II] with two levelsof smaller sub-samples,and recorded overall crust coverby species and genus in both 0.25 m2 and 20x50 em plots. Eversman [28] andRosentreter [69] used 20x50 em plots along line transectsof variable length. InAustralia, lichen diversity is commonly assessed in 0.5 m2 quadrats [14, 16, 17,23].Quadrat size may be reduced where crust cover is greater or where finer detail isrequired. For example, Rogers and Lange [68] used a quadrat sizeof 15xl5 em toexamine changes in soil crust floristics in relation to stock watering points in semi-aridand subtropical Australia. In higher rainfall rangelands in eastern Australia, Eldridge etal. [25] used small circular cores of 4.2 cnr' to sample lichens and bryophytes withinvegetated and adjacent bare patches. These small"quadrats" were used so that theywould be easily visible within a fieldofview ofa lOx dissecting microscope, enablingmeasurementsofcover-abundance to be made on a species basis.

L ine-interceptand line-point methods are used to measure the proportionofthe linecomprising various species or taxonomic groups, and are best used for densely crustedand/or species rich areas, or where individual species are intermingled [39]. The line­point method differs in that species occurring at predetermined or random points along aline are recorded.These techniques have been used on linesofvarying length in shrub­steppe habitats in the western USA [40, 62]. As soil lichens are very small, the line mustbe placed near the soil surface in exactly the same location at each measurement time.This can be achieved by placing permanent markers such as steel stakes along the routeofthe line. Where soil crusts are less continuous, a larger numberofpoints is requiredfor statistical efficiency. A variant of the line-intercept method, the short-focustelescope, has been used to record lichen and bryophyte species in Antarctic rangelands[58]. Repeat sampling by different observers using different methods can be useful forassessing species capture rates under varying sampling regime[47,48].

2.2.2. Intensity ofdata collectionMost studies of soil lichens have recorded one or moreofeither cover, cover-abundanceor species composition. Measuresof frequency for a particular species can bedetermined by calculating the percentageofoccurrenceofa particular species within alarge numberofsamples [16]. Point methods can be less subjective,but a large numberof points is often required for uncommon species and therefore diversity may beunderestimated. Generally ocular estimationof cover or cover classes by speciescaptures more species than the point-intercept method [47, 63].Ocular measurements inquadrats are well suited for studies of species richness, abundance and compositionalchange, and where broad trends are more important than precise repeatable estimatesofcover[21,63].

Different studies frequently require different levelsof effort, and the choiceofwhether to record individual species or morphological groups will depend largely on theobjectivesof the study. In general, species capture increases with increases in thecoarseness of the classification, i.e. going from species to genus to broad groupings (e.g.crustose vs. squamulose). In practice however, there is often a compromise between thefinal scale of monitoring required, and practical, financial and logistical considerations

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ofthe study.One way to increase the consistencyof measurementsof richness over time is to

lump some species that are difficult to distinguish into morphological groups [21, 62,63].Morphological groups are groups of superficially similar species that are difficult todifferentiate in the field, but which possess similar morphologies and often functionsimilarly e.g." green leafy lichens" or "gelatinous lichens" [21, 61]. In many cases,morphological groups are surrogates for functional groups [46, 60, 70]. For example,the gelatinous lichensCollema, Leptogium , and Leptochidium spp. of shrub-steppecommunities in the western USA all fix nitrogen, and provide a similar degreeofprotection on the soil surface [1, 10].

The groupingof similar species into morphological groups minimises the errorsassociated with overlooking small cryptic species or species which are frequentlyintertwined, and decreases the sampling variance. Similarly, estimates of cover orabundance are more repeatable for morphological groups than for individual species[40,63,69].Despitethe advantagesofusing morphological groups in the field [61], theresulting measurements will invariably underestimate species richness [62].

3. Lichens as indicatorsof landscapefunctionandstability

3.1. INTRODUCTION

Whilst many scientists acknowledge the close links between biological soil crusts andthe condition or healthof rangelands [42], biological soil crusts and their componentlichens and bryophytes have rarely been recorded during field-based assessment [80]. Inthe mid to late 1980s, Australian rangeland scientists pioneered a rangeoftechniques todetermine the health of landscapes which placed more emphasis on soil and landscapefunction rather than relying, as previously, on the status and conditionof the vascularplant community [76]. The resulting Soil Surface Condition (SSC) system used anumberof easily recognisablesurface features that provide a measure of the healthofthe surface. Assessment is based on the capacityofthe landscape to sustain three basicfunctions: resist accelerated erosion (stability), cycle nutrients (nutrients) and maintainwater flow (infiltration). The SSC system, which uses the coverofbiological soil crusts(including lichens) as one of its component attributes,provided a reliable estimateofthepotential productivityof the surface independentof assessmentsof the vascular plantvegetation. In the semi-arid woodlandsof eastern Australia, cover and speciescomposition of lichen and moss-dominant crusts are high on productive, stable (Class 1)surfaces, i.e. those showing little evidence of accelerated (human-induced) erosion. Onunstable, unproductive (Class 4) surfaces however, lichens and other crust biota aregenerally absent [49]. The SSC system was later refined to other landscape types [75]and to other land uses, such as mining.

Over the past decade there has been a general consensus that soil lichens andbryophytes are useful indicators of healthy stable soils, largely through the processesthey mediate [6, 20, 21, 26, 40,42, 45, 49,74, 76].While some sediment movement is anatural process in rangelands, stable soils are defined as those where surface biota suchas soil lichens restrict (reduce) the transfer of sediment at the microsite and landscape

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scale. Increasing cover and diversityof lichens are generally associated with improvedsoil health, which, in general, equates with improved soil structure and thereforeenhanced stability. Soil lichens and bryophytes have been shown to moderate the flowofwater into rangeland soils, and their importance generally increases as soils becomemore degraded [26].

In many arid and semi-arid systems, degradation is often concentrated aroundwatering points, such that sites close to watering points are generally heavily degradedwhilst those further away are in better condition. This phenomenon, termed the"piosphere"effect [43] is due to the marked impact that animals (both domestic andnative) have on the cover and compositionofplants and the conditionofthe soil surface[36]. These grazing gradients, generally in arid or semi-arid environments, illustratechanges in populations at varying distances from watering points, and provide aframework for examining the impacts of grazing and trampling on various biota [68]. Inlandscapes where lichens and bryophytes colonise the soil surface, increasing distancefrom water is generally associated with increasing crust cover [33], though this effect isnot always consistent [56]. Below we discuss attributesof terricolous lichens that havebeen used to assess the health or condition of grasslands, deserts and steppe in arid andsemi-aridlandscapes.

3.2. ATTRIBUTES FOR ASSESSING HEALTH AND ECOSYSTEM FUNCTION

3.2.1. Lichen coverCover has been used as a surrogate of rangelands health, particularly in woodlands,grasslands and shrub steppe in eastern Australia [20, 32], though cover on its own isgenerally a poor predictorofrangeland health.The reason lies in the fact that a crust ina site recovering from degradation may be dominated by cyanobacteria or cyanolichenssuch as Peltula spp., whilst similar cover levels at other healthy sites may comprisemorphologically more advanced species, and lichens characteristicof less frequentlydisturbed surfaces such as foliose or fruticose species. Clearly a system using floristicswith or without an assessment of cover would be preferable. Generally cover increaseswith decreased disturbance and hence decreased soil condition, though this is oftencomplicated by changes in the distributionofspecies (Tozer and Eldridge, unpublisheddata). Studies in western Queensland at varying distances from water points havedemonstrated that distance from water often explains a low proportionof the variance(~1O%) in crust cover.

3.2.2. Morphological groupsSome lichen species appear more tolerant of trampling than others [68].This is probablydue to differences in their morphologies, as foliose or fruticose forms seem to be moresusceptible than crustose and squamulose forms [12,15,21].Apart from their roles inassessing diversity, morphological groupsof lichens can provide valuable insights intothe health and recoveryofecosystems [20, 21]. In a studyofmore than 0.6 M km2 ofeastern Australia, Eldridge and Koen [20] found that the presence of the" yellowfoliose" morphological group comprising foliose lichens of the generaHeterodea,Xanthoparmelia and Chondropsis spp. were consistently correlated with stable,productive landscapes, i.e. landscapes with no accelerated erosion. The occurrenceof

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other groups such as "black gelatinous"(Collema spp.) was independent of landscapecondition [20]. The strong links between foliose lichens and ecosystem health probablyrelates to the fact that these lichens rarely reproduce sexually, but rely on a smallamount of fragmentation to disperse to new sites. Excessive trampling breaks the lichenup into fragments that are too small to re-establish, eventually leading to theirelimination from a site. Studies at the former weapons testing area of Maralinga in aridSouth Australia demonstrated that foliose lichens had still not returned to disturbedareas after more than 40 years of recovery [18].

3.2.3. Measures integrating cover and morphological groupIn an effort to develop a simple system to determine landscape health, Eldridge andRosentreter [22] developed a Biological Soil Crust Stability Index (BSCSI) thatintegrates an assessmentofcover of lichens, bryophytes and cyanobacteria with a crudeestimateof their floristics. The index ranks morphological groupsof soil crust taxa interms of their increasing dimensionality and thus their increased ability to resist erosion[61]. In this system, crust taxa are ranked from 1(cyanobacteria= 1 dimensional)through 2 (crustose and squamulose lichens, liverworts= 2 dimensional) to 3 (mossesand fruticose lichens= 3 dimensional). The proportionof the soil surface covered byeach of the three morphological groups is multiplied by an integer ranging from 1«5%cover) to 5 (>75% cover) to obtain an average site score that indicates the percentageofmaximum landscape stability.

Studies in sagebrush steppe and semi-arid grasslands in central and northern IdahoUSA indicate a strong correlation between the BSCSI and the healthofArtemisia shrub­steppe, as assessed by the average numberof obstructions per10m of transect(R2=0.57, [74]). Similarly, the index was highly correlated with three measuresofrangeland health defined as Landscape Stability(R2=0.37; Figure 1), Biotic Integrity(R2=0.42) and Watershed Function(R2=0.41) developed for a nation-wide classificationofrangeland health [57].The BiologicalSoil Crust Stability Index has great potential asa rapid and efficient technique for monitoring erosion hazard, and temporal and spatialchanges in the qualityofplant and animal habitat in patterned sagebrush and grasslandcommunities [22].

3.2.4. Lichen size and densityIt is widely accepted that trampling reduces both the cover and diversityofsoil lichens[37,41,50,68,80].Moderate trampling destabilises lichen colonies, breaking themintosmaller colonies, but continued trampling eventually kills lichens. Anecdotal evidencetherefore suggests that sites with a stable soil surface i.e. with no accelerated erosion,would be characterised by larger lichen colonies compared with sites in poor health. Inarid South Australia, this has been tested on soil crust communities dominated bycrustose and squamulose lichens, indicating that the density and average sizeofPsoracrenata colonies increases with increasing timesince disturbance [18]

3.2.5. Lichen richness and diversityBiodiversity, as reflected by species richness, appears to be very high in arid and semi­arid regions, and the popularbeliefis that decreasing diversity of lichens leads toincreased variance in ecosystem structure and function [35]. Relationships between

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landscape function, condition and health, and lichen diversity, have generally beenconducted at three spatial scales: 1) the regional scale - many hundredsofkilometres, 2)the landscape scale, hundredsof metres, and 3) the microsite scales, metres andcentimetres.

100

90

80

70 .£

60

500 20 40 60 80

Biological soil crust index(%)

Figure 1. Relationship between the biological soil crust index and landscape stability for sagebrush steppeand grasslands in southern and central Idaho.

Recent studies along a 700km north-south rainfall gradient in the box woodlandsofeastern Australia (W. Cuddy, unpublished data) indicate significant relationshipsbetween richnessof soil crusts from sites with more than 5% soil crust cover, and anindex of landscape function as measured by a rangeof biotic and abiotic attributes(Figure 2). Most of these crusts were dominated by crustose lichens includingPsoradecipiens, Endocarpon simplicatum var. bisporum and Placynthium spp. as well asassociated mosses.

At the landscape scale, Eldridge [16] demonstrated that crustose and squamuloselichens tended to dominate intergrove areas of a patternedCallitris glaucophyllawoodland characterised by groves of trees, aligned on the contour, and separated bybare, eroded slopes devoidofvascular plants. These"runoffzones" were characterisedby soil surfaces generally indicative of degraded landscapes such as high levelsofcompaction, and low levelsof infiltration and nutrient cycling. A pioneering group oflichens comprising mainly the cyanolichenCollema coccophorum dominated sitesdevoid of vascular plants. Whilst some lichen-dominated sites are apparently degradedfrom a vascular plant perspective (Figure 3), they are essential for the redistributionofwater and nutrients to the timbered groves downslope [16]. Grazing gradient studies inwestern Queensland have shown that increasing soil health, as measured by landscapefunction analysis [75] is significantly correlated with increased number, richness and

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diversityofsoil crust lichens and bryophytes at a site, though this variedbetweensiteswithin grovesoftrees and those in the open (Tozer andEldridgeunpublisheddata).

100 • ••

80

60

~ •• • ~ • •40 • •• • • .'"• • •

200 5 10 15 20 25 30

Richness

Figure 2. Changes in species richness (a-diversity) in relation to landscape function for sites in easternAustralia. Triangles indicate sites with >5% biological crust cover, and quadrats < 5% cover. Source: Cuddy(unpublished data). The linear bestfit model (R2=0.39, P<O.OI) is indicated.

Figure 3. A structurally and fl oristically diverse soil crust community growing on a calcareous loam ineastern Australia. Up to eight bryophyte and 13 lichen species can be found within a 200 em' quadrat .

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At the microsite scale, some soil lichens show a strong affinity with particular soils.Cladonia spp.,in particular, are known to occupy bare or recovering microsites wherecompetition from vascular plants is low [14, 67], and are known to be susceptible toshading [9]. The foliose lichensHeterodea spp. are also commonly found in shadedlocations, the lichensParaporpidia glauca, Cladonia sp. andLecidea ochroleuca tendto be found in areas of higher clay and organic carbon content in open sites wherevascular plant cover is sparse [24]. Similarly some lichens such asSynalissa symphorea,Peltula spp.,Collema coccophorum and Heppia despreauxii tend to be more commonon degraded surfaces low in nutrients and plant cover [16, 23, 66, 67, 68]. The uniquestructure of cyanolichens makes them more efficient at water absorption and thereforeextends the period over which they can photosynthesise [7, 29], giving them acompetitive advantage in hostile environments.

3.3.MONITORING SOIL LICHENS :A WORD OF CAUTION

Monitoring lichens in arid landscapes requires specialised techniques. The difficulty ofidentifying arid area lichens to species level is complicated by the fact that they areoften poorly developed or sterile due to the harsh environment in which they live. Thisoften makes positive identification problematic. Marked differences in morphology ofdry area species resulting often from environmental modification [79] may necessitatethe collectionofa larger numberofvoucher specimens than would normally be requiredin more mesic environments [21]. Quadrat sizes used for vascular plants often need tobe adjusted or reduced, or the numberofsubplots increased in order to sample arid zonelichens. Plots or transects may require moistening with a fine streamofwater prior tosampling duringdry seasons in order to make lichens and associated soil organismsmore visible so that cover and richness can be more accurately assessed. Extreme careneeds to be exercised in processing voucher specimens of species growing on soil toprevent them from disintegrating over time [71].

Apart from their small size, the problem of documenting lichen diversity and coverin arid areas is hampered by the lackof a consistent, rigorous methodology. Eachinvestigator has inherent challenges in identifying and rating attributes such as cover forsmall lichens that often lack obvious morphological and colour differences[47,48,62].This often leads to wide variability in the data due to operator error.

Any soil lichen studies must take into account differences in substrates that arelikely to affect diversity. If lichens on the soil are the focusof the study, then speciesgrowing on the stems of shrubs or on small rocks should not be included in assessmentsofdiversity. For example in arid Australia saxicolous (rock-inhabiting)Xanthoparmeliaspp. often appear to occur on soil but on closer examination are found to be attached tosmall stones within the soil matrix. If total site diversity is the aimof the study, thenthese saxicolous species need to be included. The inclusionof species from a rangeofsubstrates [10] may be appropriate for comparing site or beta-diversity, but leads to aninvalid comparison of flora from one substrate between sites (Table 2). A reasonablecomparison of site and species richness can only be made with the knowledgeof thesubstrate preference of each species if a range of substrates is included. Studiesreporting species richness from more than one substrate should include some indicationofthe substrates upon which the species are found.

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Finally, the locationofsoil lichen monitoring sites needs to be well documented, assmall changes in the locationofthe transects or plots may result in large differences indiversity or cover given the high degree in variability of soil lichens within a site.Permanent markers should be located every10m along line transects to ensure thattransects are consistently read at the same location.

4.Conclusions

Regardlessof the methods used, plot size and operator skill will to a large extentdetermine the effectivenessofasampling strategy. For assessing lichen biodiversity werecommend the useof fixed-area plots at each study site, with sampling restricted to agiven time interval in order to ensure comparability of site data, particularly wherediversity indices are used. Where possible, taxa should be identified to species level.Sampling of soil lichens for assessment of ecosystem health should be carried out onpermanent fixed-area plots, with taxa amalgamated into morphological groups or keyindicator groups.

5.References

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45. Ludwig, lA . and Tongway, OJ. (1993) Monitoring the conditionof Australian arid lands: linkedplant-soil indicators, in D.H. McKenzie, D.E. Hyatt and V.J. McDonald (eds.), Ecological Indicators,Elsevier,New York, pp. 765-772.

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MONITORING LICHENS ON MONUMENTS

A .APTROOT ' and P.W.JAMES 2

"Centraalbureau voor Schimmelcultures, P.O. Box 85167 NL-3508 ADUtrecht, The Netherlands ([email protected])219 Edith Road. London W14 OSu, UK.

1. Introduction

Lichens grow naturally on all substrates, including very nutrient-poor ones. The reasonfor this is that they do not take their nutrients from the substrate,but mostly from the airand ambient water. Therefore they can survive on nutrient-poor substrates. As they arepoor competitors they tend to favour open habitats that are mostly devoidof othervegetation. This is especially true for stony substrates, and the majority of thebiodiversity on rocks usually (except when permanently submerged or heavily shaded)consistsof lichen species. The majority of lichen species are (primarily) saxicolous inmost partsof the world outside the tropics. Even in regions without natural rockoutcrops, like the Netherlands and eastern England, most lichen species are saxicolous[13]. Here, they grow naturally on artificial stony substrates, such as brick,mortar, slateand pebblesofbuildings, graveyards and megalithic monuments.

The purposeof this chapter is to attract attention to the biodiversityof lichens onmonuments, to evaluate their value for historical dating of, and to assess their potentialharmful effects on the monuments.It is meant to be an introduction to the varioustopics, with stress on monitoring techniques involving monuments. It is not aimed atproviding a complete overviewof the rich, but widely dispersed literature, which isoften published in specialistjournalson chemical aspectsofthe deterioration processes.This chapter emphasises western European work in particular. Someof the papersreferred to below (e.g. [37, 63]) contain many references from other important areassuch as southern Europe. Invaluable annotated listsof publications on lichens andbiodeterioration of stonework, with regular updates, are published by Piervittoriet al.[42,43,44].

2.Megalithicmonuments

The oldest potentially available artificial substrates for lichens are the megalithicmonuments. They are well dispersed over mostofWestern Europe, including France,Britain, Ireland, the Netherlands, Germany, Denmark and Poland. Outside this region

239P.L. Nimis, C. Scheidegger and P.A. Wolseley (eds.}, Monitoring with Lichens - Monitoring Lichens, 239-253 .

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they are exceedingly scarce, and only known from some scattered occurrences, e.g.onEaster Island.

Figure 1.A. Aptroot monitoring a menhir in Brittany (France). Photograph P.H. Hovenkamp.

Megalithic monuments in France, Spain [45], Portugal [50] and Britain are mostlymade from local (or at least regional) bedrock, but those in the Netherlands [7, 18],Germany [3], Denmark [1] and Poland are often made from erratic boulders that weretransported southward by the land-icemasses during the penultimate glacial period.

Megalithic monuments are very diverse in structure. The most common type in thesouthof Europe, especially in Brittany [20] (see also Figure 1) consistsof monolithsthat have been continually exposed since they were erected in prehistoric times. InBritain, the Netherlands, Germany and Poland, the most common structures aredolmens, comprising stones covered with slabs of rock covering the cavity (see alsoFigure 2). They were originally totally or mostly covered by sand or peat.

Lichens on megalithic monuments tend to be relatively neglected in regions withcopious outcropsof similar rock. However, they often support a lichen flora differentfrom the surrounding bedrock. A famous example is Stonehenge, on which lichenshave been recorded since the 18th century [22], which provides an inland localityofthecoastalRamalina siliquosa. In areas devoidof natural rock outcrops, they are studiedmore intensely.

The 54 megalithic monuments in the Netherlands are made from erratic graniticblocks. Although erected thousandsofyears ago, they were originally covered by sandand became exposed only about 250 years ago, when these hills were used as stonequarries for dyke stabilisation after the Shipworm(Teredo) devastated the nation'swooden sea-dykes.

They harbour 26 Red Listed species, including a few counted among the commonestlichens in adjacent countries, but also some surprising species. Rare lichens, includingspecies that have now vanished, have been recorded from these monuments since themiddleofthe 19th century [7, 12, 18].

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Figure 2. P. W. James and P. A. Wolse/ey at a do/men during the LIMON meeting.Photograph AL van Iperen.

They currently represent hot spots with up to 56 lichens per site and a total specieslist of 128. At least 14 species, viz. Aspieilia eupreogrisea, A. grisea, A. verrueigera,Fuscidea eyathoides, F. praeruptorum, Lecanora frustulosa , Lecidea promixta,Lepraria negleeta, Parmelia disjuneta, Rhizoearpon leeanorinum, Rinodinaconfragosa, Stereoeaulon dactylophyllum, S. evolutum and Umbiliearia deusta arerestricted to megalithic monuments in the Netherlands [12].

The megalithic monuments in Denmark are relatively small, but have partly (theoutsideofthe standing stones) been exposed since they were erected. Still, they are notextremely rich, with up to 26 species recorded on one site [1] and do harbour mostly thesame species as those mentioned above from the Netherlands. However, they are notstudied in much detail, as granite boulders are also otherwise a regular featureof thelandscape.

3.Ancientbuildings

In areas with remnants of ancient buildings (e.g. from Roman and Greek times) theoldest stone surfaces on monuments are walls. In areas with natural bedrockoutcroppings, they are usually built from local rock. This however is not always thecase. Many archaeological sites in the surroundingsof Rome have monuments madefrom exotic materials from distant regionsof the Roman Empire. Today, suchmonuments host a remarkably rich and diverse lichen flora; manyofthe more than 600lichen species known from this area were found on ancient monuments [36]. Many

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ancient buildings are in densely populated areas polluted with sulphur dioxide, and donot support lichens. Examples are the Acropolis in Athens and Trajan's Column inRome, which incidentally was protected by a sand cover during WW II [33]. In otherareas in and around Rome, however, lichen growth is less restricted and has recentlyeven been enhanced by eutrophication, mostly due to ammonia airpollution [53].Especially in areas which are now relatively uninhabited and/or unpolluted, old wallsofhistoric buildings are literally covered with lichens. For instance the turret-likenuraghes on Sardinia often stand out for miles due to their orangecolour fromXanthoria species (see the coverof [37], and [36]). The regular formofthe nuraghes isideal for studying the ecologyof the species, esp. as far as lightrequirementsareconcerned [62].

Lichen species present on ancient buildings do not vary from the species present onthesurroundingbedrock when it is equally available for lichen growth in the vicinity.Ancient buildings (e.g. Mayan, Aztec) in tropical regions are the obvious exception,where conditions for vascular-plant growth are so favourable that natural rockoutcroppingsare extremely sparse [30]. Even these often very large temples tend to berapidly overgrown byjungle.However, there is an exampleofa lichen species which ismore or less restricted to Inca temples, at least in Ecuador:Xanthomendoza mendozae[32].

A particularcase is thatof the mosaics that made up the floorof Roman houses,which are frequent in several archaeological areasofS Europe. The mosaics are madeup with tesseraeof different lithological nature (e.g. marble and basalt, or differentcombinationsof substrata in polychromatic mosaics), kept together with mortar. Thiscreates a complexenvironmentfor lichen colonisation which can often mask theesthetic valueofthe mosaic [28].

4.Statuesand bas-reliefs

Especially in southern Europe, statuesof calcareous rock are frequent in parksaround villas and in archaeological areas.Due to their irregular surface, they often hostcomplex mosaicsof lichen vegetation, from nitrophyticcommunitiesin the mostexposed parts (usually the headofthe statue) to specialisedcommunitiescolonisingthesurfaces protected from rain [34]. Bas-reliefs areparticularlysensitive to lichencolonisation,especially when include shallow inscriptions which are easily madeunreadable by lichen colonisation.

5.Churches,churchyardsand graveyards

The most widely available old stone surfaces are church walls. In areas with naturalbedrock outcroppings, they are often built from local rock, with relevant exceptions,such as many churchesofCentral Italy, which wereconstructedwith alternate bandsoflimestone and basalt brought from distant areas [35]. In areas without natural rockoutcrops, they are mainly built from locallymanufacturedbrick and/orimportedrocks.

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Therefore, they contribute considerably to the available variation in stone surfaces. Inthe Netherlands, which is totally devoidof natural rock outcroppings, the oldest stonechurches date from about 1100 AD and are built from imported volcanic tufa.Somewhat later, churches were built mainly from brick, but always with at least someornaments from imported rock, varying from soft marl to hard carboniferous limestoneand granite to trachyte or basalt or even iron ore, depending on fashion and availability.Stone graves in graveyards are a much younger phenomenon, as they are rarely olderthan 1650 AD. Before, wooden crosses were widely used and important people gotstone graves inside the churches.

Old churches often stand out in species richness, and can support species that areotherwise absent from the region. At least one lichen species,Lecanactishemisphaerica, is only known (and described) from churches walls in England andItaly. North-facing church walls often support a special lichen flora, e.g. supporting thisspecies (see Figure 3).

Figure 3. Warmwellchurch (Dorset), North-facing wall with Lecanactishemisphaerica,Photograph V.J. Giavarini.

Northern exposures are differing much from the other exposures, which mainlysupport ubiquitous and/or nitrophilous species. Sheltered north-facing walls are thehabitatof species likeDirina stenhammari, which can even cover extensive areas andcause a major disfigurement [25, 54] andLeproplaca chrysodeta, species which areabsent from natural environments in large partsof NW Europe. Exposed north-facingwalls, especially close to the coast, support many lichens that are usually known to bestrictly epiphytic, e.g.Lecanora chlarotera, L. compallens, L. horiza, Lecidellaelaeochroma, Opegrapha niveoatra, Pertusaria albescens and Ramalina lacera [10,57]. The recently described speciesProtoparmelia hypotremella, known fromcorticolous habitats only in the Netherlands, Belgium, France, Germany andSwitzerland, was surprisingly found for the first time in Fennoscandia on a church wallin southern Sweden [39].

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The biodiversity of churchyards can be very high: although their size is small, theycan constitute veritable hot spots.In total, 630 lichen species have so far been recordedfrom churchyards in the UK, which is a third of the total British flora [47]. Severalchurchyards have 150 to 180 species present. The highest numberof lichens found onone church in the Netherlands (only the walls, not including the churchyard) is 77 [58].To our surprise the richest churches are not situated in the coastal areas, where the oldchurches look well-covered.Instead, the churches with the highest species numbers areall close to each other in the eastern partof the country along the small IJssel River.The highest numbersof lichens found on churches in surrounding countries areconsiderably lower, e.g.21 for Denmark [1] and 28 for Luxembourg [14].

Churchyards and graveyards (cemeteries without churches) often provide a verydiverse habitat for lichens, due to the many different stone types employed for thegraves and surrounding paths and walls. Also the exposure,slope and water conditions(e.g. run-offor with temporary small pools) show much variation. In addition to stonysubstrates, cemeteries always support various trees and often also little-disturbedhabitats favourable for terricolous lichens.

There is much variation between different cemeteries in one region. Age (of thecemetery but also of the individual graves) and habitat variation are the factors thatmost influence the species richness.In general, the Roman Catholic cemeteries showmore variation (in stone types, ornaments and lichens) than Lutheran ones. Jewishgraveyards contain among the oldest graves (they are never removed, unlike Christiangraves),and consequently support much lichen growth,but often only a limited numberofspecies due to the extreme uniformity of the graves in stone type and exposure [6].

Few lichen species are restricted to cemeteries, but some species are much morefrequent in this habitat than elsewhere, often because their preferred stone substrateabounds there. Examples of typical graveyard lichens areAspicilia moenium andSarcopyrenia gibba , both preferring the exposed tops of hard limestonegraves [12].

Graveyards and individual graves are not monitored only by lichenologists forscientific reasons: caretakers of cemeteries sometimes ask for a mappingof thebiologicalvalues in termsofrare or Red Listed lichens, as has been done in Zwolle [8].They can then select certain graves for protection against clearance on these grounds, inaddition to historical or cultural considerations.

Churchyards are also ideal control sites for lichenometry as gravestones are datedand rather accurate size/age-curvescan be drawn for particular species.

Gravestones in both urban and rural areas can provide good sites for monitoringchanges in air quality, especially in the absence of other suitable substrates, e.g.roadside trees.An example is Walthamston (UK), where in 1969 only 21 species werepresent on the graves, and 51 in 2000, showing the effectsofmild eutrophication [48].

There is a marked difference in attitude towards the upkeep of churches betweenvarious countries. In much of southern and eastern Europe, Africa, Asia and America,common practice is to keep churches and graves clean and well-maintained.There evenexist recipes for how to clean graves from lichen growth, issued by the British WarGraves Commission. The opposite can be observed in partsof Britain and Ireland:abstinence from cleaning is not seen as neglect, but as an accentuation of the age.

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Consequently, relatively much research has been done on churchyard lichens in Britain(e.g. [24, 40, 64]) and there is even a special churchyard lichen mapping program [19].Through Western Europe there is a gradient with this respect from Ireland througheastern England and the Netherlands (where most churches are cleaned onlyaccidentally and only relatively few are plastered) to Denmark, Belgium [63],Luxembourg and partsof Sweden, France and Germany(Nordrhein-Westphalen),where most churches are plastered, but several dozen speciesof lichens can still befound on somewhat neglected churches. In other countries, lichens are apparentlyscarce or even absent on churches and graves, although several records exist from, forexample Spain [46, 50], Italy[35,41]and even Hong Kong [60].

6.Town walls,fortresses,houses,windmillsandcastles

In general, (town and country) houses and castles support a relatively depauperatelichen flora, as compared to churchesofsimilar age.The obvious reason is that they areoften still either in densely populated areas or rather exposed on isolated sites. Manycastles have experienced massive restoration or even rebuilding.

Fortresses and town walls, although mostlyof much younger age, often support avery rich, and partly characteristic, lichen flora. This is the resultofthe combinationofneglect, as most fortresses became redundant soon after they were finished, andofdampness due to the water from the omnipresent moats.Collema fuscovirens [II] andLecidella anomaloides [59], for example, are restricted to old town walls and fortressesin the Netherlands.

7. Industrialandwarmonuments,railwaysandurbanwastelands

Modern and industrial architecture favours concrete as building material. Although initselfa very favourable substrate for lichens, it seems to attract mostly ubiquitousand/or nitrophilous species. The same holds true for most war remains, althoughoccasionally some bunkers or tank walls from WWII have remained untouched (andnot covered by sand) to support some biodiversity. Even on the relatively recent Berlinwall some lichens were collected at the timeofthe political turnover [56].

Urban wastelands, railways and industrial areas provide an extraordinary wide rangeof substrates with and without various sourcesof pollution, notably by heavy metals.Relatively new habitats are subject to constant drastic changes [5]. Consequently, manyofthe lichens can be characterised as pioneers, including most species that are more orless restricted to urban wastelands (or their natural equivalents), like Micareaexcipulata [29]. Lichens profit in heavy metal-polluted industrial sites from the fact thatthey can cope with pollution levels that are highly toxic to most other organisms, bybinding the metal ions to secondary metabolites that are deposited inextracellularspaces. Recently, several new lichen species have been described from industrial siteswith heavy metals due to zinc smelters. A paratypeof Micarea confusa was foundgrowing on a disused car transmission belt on such a site [21], and the only paratypeof

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Pyrenocollema chlorococcum that was not found near a zinc smelter, was growingbelow a galvanised sheep grating [2]. These species, as well as other metal-tolerantlichens likeBacidia saxenii, Steinia geophana and Stereocaulon species are alsocolonising railway ballast [51]. Bacidia chloroticula is even so common on thissubstrate that its distribution in the Netherlands is mainly correlated with the railroadnetwork.

8.Historical dating: lichenometry

Lichens, especially some foliose and crustose species, show a conspicuous circulargrowth, which has led rise to names likeArctoparmelia centrifuga . Attempts have oftenbeen made to date the time of exposureof stone surfaces by measuring the yearlygrowthof lichens as compared to the diameterofthe largest individuals. This practicebecame known as lichenometry. Lichenometry uses the size/age-relationshipof lichensto date stone surfaces (see [17, 27, 65] and chapter 38, this volume). Some examplesofcase studies:

A study of neolithic stone circles at Rollright in Oxfordshire and Castlerigg inCumbria (UK) showed that stone movements in the recent past may be deduced fromthe varying sizesof lichen populations on different surfaces, and approximate datesmay be suggested for periods of change [66].Thalli ofAspicilia calcarea dating around1366 andofRhizocarpon geographicum to 1523 are reported.

Lichens have widely been utilised in dating moraines and retreatof glaciers, andalso to find out how frequent earthquakes occurred in Central Asia. Lichenometry anddendrochronology agree on the beginning of the recent glacier retreat around thePatagonian San Rafael icefield in the 1860s-70s [67]. The dating is interesting as itshows that climate warming here is proceeding synchronously with that in the northernhemisphere, a pointofmuch discussion and urgent relevance to models attempting topredict future global climates.

An attempt to use lichenometry to date the enigmatic statues on Easter Island [26]failed to some extent. The ageofthe oldest thalli was about 400 years, not the expectedmillennium or so. This could be (and was by some commentators) explained away bysuggesting that the statues had, until a century ago, been cleaned by the peopleworshipping them.

A different case was described from the Netherlands [55]. During restoration worksdead lichens were found on plaster-covered interior walls of medieval houses in theinner cityofUtrecht. The presenceofthese lichens confirmed the suspicions that thesewalls had been exposed to the outside in earlier stagesof the development of thebuildings.

Problems in past work with lichenometry have mainly concerned the effectsofgrowth, colonisation rates, aspect preferences, local effects (e.g.hypertrophication bybird perching), variable life-spans and inconsistent methodology. For instance, mostwork has so far been done withRhizocarpon geographicum, which however consistsofnumerous taxa and probably even several syrnpatric species, which might well have

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differentgrowth rates. Where aconsistentmethodologycan be appliedinvolving onespecies on one typeof substrate within a restrictedgeographicalarea, thetechniqueshows considerablepotential not only for dating in glacialenvironments,its major useto date, but also forassessingthe detailedtreatmentover the recentcenturiesofhistoricandprehistoricstructures.

9. Potentialharmanddamagecontrol

The extentofthedeteriorationof lichens on rocky surfaces depends on manyvariables,and its treatmentlies outside the scopeof this chapter. In general, lichensdominaterelativelydry stone (asopposedto mossesdominatingwetsurfaces),and lichengrowthis oftenenhancedby high humidity. The depthofbiodeteriorationby lichens is rarelymore than a fewmillimetres(obviously dependingon theporosityand textureof thestone) (e.g. see [15,16,34,37,61]).

It has beenillustrated[52] that a single thallusof Lecanora muralis can have adeleteriouseffect on an ancientmonumentin Rome by causing a blister,peelingoffseveralmillimetresof stone. The sizeof the thallus shows that thishappenedwithin ashort period (no more than 15 years). In theprevious two millenniaprobably lessdamage has been done to thesemonumentsby lichens than in the lastdecades. Someexamplesof extensivedamage caused by lichens onmonumentshave beenreported[41,46].

Endolithiclichens are aparticularcase:Theirgrowth takes placeentirelyinside therocks, usuallycalcareousones. Their ascocarpsprotrudeon the surface,leaving smallhollows as they fall; this effect is calledpitting. According to Tretiach[61] theamountofchlorophyllper unit area enclosed in rock byendolithic lichens may be the same asthat found in tree leaves, but primaryproductivityand growthare definitelysmaller(afraction of mm per year). If killed e.g. by a biocide, theoutermostlayer of theendolithiclichen (lithocortex) detaches in small scales, leaving theunderlyingsurfaceexposed. This surface, however, is extremely porousbecauseof thepenetrationof thefungal hyphae, leaving the monument defenseless against the aggression ofphysicochemicalandbiologicalagents [34].

It is a matterofappreciationwhetherlichen growth onmonumentsshouldbe treatedor not [23, 34]. Lichens will certainly not threaten tocrumblea wall. In some cases,however, thechromaticalterationproducedby lichens can causeserious aestheticdamage.The north facadeofthe Orvieto duomo in Italy is anexample[35]. The duomowas built with dark basalt andlight-colouredlimestone inalternatebands. The darkbands arecolonisedby light-colouredspecies, whereas the light-colouredbands hostorangeor dark species. The result is bizarre and may be notunpleasant,but iscertainlyfar from what the artist had in mind [34]. In other cases lichen removal is mostly aculturalmatter: A lichen-coveredwall looks fine on an oldchurchor aruinedcastle (atleast to mostpeople'seyes), and evenemphasisesthe history. A megalithicmonumentwould look new/fake/unrealisticif it was devoidof lichen growth. It is disputablewhetherlichen growth on recentlyexcavatedMayan ruins should be controlled[30].

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Lichen cover is sometimes seen as disfiguring the original design, e.g.in the case of aRoman amphitheatre in Spain [38]. On graves this is perceived variously, andespecially where the sculptures can become obliterated by the lichens, it is oftenadvocated that lichens be removed. Even worse is the situationof frescoes thatsuddenly become covered by lichens as a resultofchanging air humidity and/or quality[25].

Many treatments are being developed to remove lichens from stone [30, 37], mostof which involve chemicals hazardous to the environment. A more simple solution istreating with hot vapour (over60°C): most lichens can stand high humidity and hightemperatures, but not the combination. This treatment will cause the lichens to die andpeel off [4]. The endolithic lichens will inevitably leave scars on the surface.Obviously, the lichens can be expected to grow back again, so treatment is only usefulifthe surface is cleaned regularly afterwards.

10. Monitoring

Monitoring lichens on monuments can have different purposes:• monitoring the effectsofrestoration measures,• monitoring lichen diversity for lichen conservation purposes,• monitoring lichen growth for lichenometry,• monitoring epilithic lichens as indicatorsof pollution (not treated here, see

section I, this volume).The first point is the most relevant for practical reasons. Eliminating lichens from

statues, bas-reliefs and mosaics may be necessary to satisfy aesthetic criteria.Restoration measures however can be very expensive, and the managersof parks,archaeological areas and other monuments often ask the biologist about theeffectiveness of certain measures. This requires a close preliminary biological andecological study of the objectof restoration. Four factors should be attentivelyconsidered: species, the causesof their growth, the damage they produced, and thespeed of re-colonisation [34]. Many monuments, for instance, are colonised bynitrophytic lichens. Simply eliminating them without identifying the sourceofnitrogencompounds is useless. The same lichens will grow back in just a few years. Simplemeasures such as coverings or canalisation and deviationofrainwater can be takenif amonument is exposed to rain (or rainwater from gutters) and if the growthofbiodeteriorationagents arises from water availability. When nitrogen compounds arebrought by the wind from nearby cultivated land, a lineof trees may be planted as awindbreak. If eutrophication is due to guanodeposits,the bird population should bebrought under control or structures that prevent birds from alighting on monumentsshould be constructed, which is not always feasible. Repeated monitoringof re­colonisation after the removalof lichens may be important to evaluate the effectivenessofthe treatment [34].

Monitoringofthe lichens on monuments can be very rewarding for several reasons,but also involves some specific problems.

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A (cultural) monument can be expected to survive over long periods without majorchanges in its management. This is in strong contrast with situations in most otherhabitats, especially forests, hedges, woodland, heathlands and dune areas, wheremanagement is subject to fashion in nature conservation and more in general, substratesare short-lived. On the other hand, monuments are preserved for reasons other thantheir lichen vegetation, and may be managed in a way that is detrimental to lichens (e.g.regular cleansing of church walls and graves and climbing by tourists on megalithicmonuments). In cases where lichens develop on sites with heavy metal pollution, risksare that these sites will be cleaned for public health reasons.Attempts to preserve theseareas (which can support very special species and even near-endemics) should involvecontacting heritage or trust associations involved in preserving industrial sites.

Another challenge to studying lichens on monuments is that it requires a thoroughtaxonomic knowledgeof the lichen flora, since only minute samples for chemical andmicroscopic preparation can be taken, not good collections from nearly every speciespresent as is usually possible with extensive natural rock outcrops. Often not much isleftofthe collected samples after examination and distributionofmaterial in exsiccatesis outofthe question. A modern approach is to make digital images in the field, whichthan can be circulated to specialists or published on the Internet, as was done alreadywith an unknown lichen from churches in coastal areas in the Netherlands (onhttp://www.lichens.myweb.nl).

Monuments can support extremely rich and special lichen floras and should neverbe ignored in floristic surveysjust because they tend to be outside nature reserves,although they can be part of cultural heritage sites.For successful monitoringoflichenson monuments, contacts with the local authorities are necessary. Often they are totallyunaware of the lichen biodiversity on the monuments under their care. When alerted,they are often very willing to co-operate by refraining from unnecessary cleaning or bymaking monuments less accessible to the public (as has been done with Stonehenge,albeit for other reasons).

As long as the fieldwork is carefully documented, it can provide answers to multiplequestions, as is shown by the exampleof our megalithic monuments. The primarypurpose was the monitoring of Red Listed lichens, but various air pollution effectscould be discerned as well, because the megalithic monuments monitored were notsubjected to any other change over this period. We give one such example: In 2000 alltargeted monuments were visited for the third time after two six-year intervals. Allboulders were exhaustively examined and the individual stones on which Red Listedspecies occurred were mapped. This sounds straightforward, but many choices had tobe made. For instance, where does the monument end? The dolmens are oftensurrounded by a ring of stones, which we included in the survey, but there are alwaysother erratic blocks nearby, sometimes also in rows, which we excluded. No problem,as long as it's recorded. More worrisome is the influenceof past unsympatheticrestoration activities. Broken stones have been cemented together with concrete, andsometimes even complete brick pillars have been constructed to support the weight;missing boulders have been indicated by little concrete patches. Inclusionofall speciesgrowing on these additional substrates would seriously damage the valueofthe dataset,

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as it makes quite a difference whether aXanthoria or Physcia is growing on themonument as a result of ammonia air pollution or restoration work. Therefore, aseparate list had to be made for species growing on concrete, even when these specieswere also present on the granite. Stones completely constructedof concrete wereomitted, to avoid spending too much time on ubiquitous lichens. To make things evenmore complicated, it was observed that nitrophilous species sometimes occurred ongranite influenced byrun-offfrom the concrete, so even a third list had to be made.Only two species could not be found again, aThelocarpon and a lichenicolous fungus,probably due to their ephemeral nature. Surprisingly many species increased and otherswere even new to the monuments. The listofspecies commonly thoughtofas epiphytescontinued to grow, withParmelia borreri, P. soredians and P. revoluta among theadditions. Also the recently recognisedP. ulophylla [31] was present. There were evenspecies newly described since 1994 from corticolous habitats, like Fellhaneraviridisorediata [9]. This is a typical Dutch situation, not observed by us on similarmonuments in Germany and Denmark. Surprisingly, the listof increasing species isvery reminiscentof the listof spreading epiphytes, with Candelariella reflexa , allnitrophytes, shade-loving crusts like Fellhanera subtilis and Gyalideopsisanastomosans and manyParmelia species on the increase andLecanora conizaeoidesdecreasing. This suggests that we are here recording air pollution trends with saxicolouslichens, just like six years before at the second recording period [7, 18], which was thefirst case report where ammonia pollution has convincingly been monitored withsaxicolous lichens. The average species number per monument is steadily increasing,from about 15 in 1988 to 20 in 1994 and 28 in 2000 (with a maximumof 56 for onemonument).

Pivotal to successful monitoring remains a sophisticated, well-documented scheme,as already exists for churchyards. The schemes should be tested to make them work forthe people who are likely to use them. In order to be able to draw any significantconclusions, the baseline data should be in the same format and collected following thesame protocol. This may be simply done by photography or acetate overlays in the caseoflichenometry,or measuring diameters. In most cases this will however encompass atleast a listof species and an estimateof their abundance. Also the precise areaofresearch should be inequivocal. Obviously this is much easier with monuments, whichgenerally consistofseveral discrete elements (boulders, walls), than with natural areas,where a GPS is needed to precisely locate a small releve.If these requirements arefulfilled, monitoring lichens on monuments can be a powerful tool over large time­spans, as stone monuments are, unlike e.g. trees, likely to survive many centuriesrelatively unchanged.

11.Acknowledgements

We are thankful to Professor M.R.D.Seaward for valuable suggestions to the text.

12.References

I. Aptroot, A. (2000) A contributionto the lichen floraofWest Jutland, Denmark,Graphis Scripta 12, 24­28.

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2. Aptroot, A . and Boom, P.P.G. van den (1998)Pyrenocollema chlorococcum : a new species with achlorococcoid photobiont from zinc-contaminatedsoils and wood, Cryptogamie, Bryologie etLichenologie 19, 193-196.

3. Aptroot, A. and Brand, A.M. (1996) Lichenen van devoorjaarsexcursie 1995 naarBramsche,Niedersachsen,Buxbaumiella 39,4 I-46.

4. Aptroot, A. and Herk, K . van (1999) Algen, korstmossen en mossen op monumenten, RDMZ infoRestoratieen beheer no. 16, also on http://www.lichens.myweb.nl/

5. Aptroot, A . and Sparrius, L.B. (2000) Notes onThelocarpun citrum (Wallr.) Rossman (syn. T. herteri J.Lahm, T. vicinellum Ny!.) and a reportof T. sphaerosporum H. Magn . with pycnidia,both colonizingsandy areas recentlystrippedoftheir top soil,Lichenologist 32,513-514.

6. Aptroot, A. and Spier, L. (1996) Lichenenop de PortugeesIsraelitischeBegraafplaatste OuderkerkaidAmstel,Buxbaumiella 38, 52-54.

7. Aptroot,A ., Bakker, S. Boom, P.P.G. van den, Herk, C.M. van, and Spier,L. (1995) Lichenen ophunebedden,Buxbaumiella 38, 16-24.

8. Aptroot, A.,Bakker, S.,Herk, C.M. van, and Spier, L. (1994) Lichenenen mossen op begraafplaatseninen rond Zwolle,Buxbaumiella 33, 47-50.

9. Aptroot,A .,B rand,A .M., and Spier,L. (1998) Fellhanera viridisorediata , a new sorediatespecies fromshelteredtrees and shrubs in Western Europe,Lichenologist 30, 2 I-26.

10. Aptroot, A.,Herk, C.M. van, and Sparrius, L.B. (2000) Lichenenvan hetnajaarsweekendop Terschellingen enke1e kerkenin noordwestFriesland ,Buxbaumiella 53, 46-52.

II. Aptroot, A., Herk, C.M. van, and Spier, L.J. (1996)Naarden-Vestingvanafhetijs gezien,Buxbaum iella39,55-57.

12. Aptroot, A .,Herk, C.M. van, Dobben,H.F. van, Boom, P.P.G.van den, Brand, A.M.,and Spier, L. (1998)Bedreigde en kwetsbare korstmossen in Nederland, Buxbaumiella 46, 1-101, also onhttp://www.lichens.myweb.nl/

13. Aptroot, A ., Herk, C.M . van, Sparrius, L.B ., and Boom, P.P.G. van den (1999) Checklist van deNederlandselichenen enlichenicolefungi, Buxbaumiella 50 (I), 4-64, also on http://www.lichens.myweb.nl/

14. Aptroot, A., Sparrius, L.B ., Herk, C.M. van, and Bruyn, U. de (2001) Origin anddistributionofrecentlydescribedlichens from theNetherlands,Aktuelle Lichenolog ische Mitteilungen N.F. 5, 13-25.

15. Ascaso C.and Ollacizqueta, M .A. (1991) Structuralrelationshipbetweenlichen andcarvedstonewokofSilos monastery, Burgos, Spain, International Biodeterioration 27, 337-349.

16. Ascaso C., Galvan, J.,and Rodriguez-Pascual, C. (1982) The weatheringofcalcareousrocks by lichens,Pedobiologia 24, 219-229.

17. Beschel, R.E. (1961) Dating of rock surfaces by lichen growth andits applicationto glaciology andphysiography (lichenometry),in G.O. Rasch (ed.), Geology of the Arctic II. 1st InternationalProceedings. Arctic Geology, UniversityofToronto Press,Toronto,pp. 1044-1062.

18. Boom, P.P.G. van den, Aptroot,A, and Herk, C.M. van (1996) The lichen floraofmegalithicmonumentsin theNetherlands,Nova Hedwigia 62, 91-104.

19. Chester, T. and Palmer, K. (1994) Churchyard lichens, British Lichen Society, also onhttp://www.argonet.co.uk/users/jmgray/

20. Coppins, B J . (1971) Field meeting in Brittany,Lichenologist 5,149·169.21. Coppins, BJ. and Boom, P.P.G. van den (1995)Micarea con/usa : a new species from zinc- and

cadmium-contaminatedsoils in Belgium and theNetherlands,Lichenologist 27,81-90.22. Coppins, BJ. and Lambley, P.W. (1999) A country diary: Stonehenge, Wiltshire 200 years ago,British

Lichen Society Bulletin 85, 4 I-43.23. Dobson, F.S. (1996) Lichens on man-made surfaces. encouragement and removal, British Lichen Society,

also on http://www.argonet.co.uk/users/jmgray/mmade.htm24. Dobson, F.S. and Buckle, P. (1996) The lichensof St Paul's churchyard,Chipperfield,and surrounding

area,Transactions ofthe Hertfordshire Natural History Society 32,435-437.25. Edwards, H.G.M. and Seaward, M.R.D. (1993) Raman microscopy of lichen-substratuminterfaces,

Journal of the Hattori Botanical Laboratory 74, 303-3I6.26. Follmann, G.(1961) Estudiosliquenometricosen losmonumentosprehistoricosde la Isla de Pascua,Rev.

Universitaria (Univ. Catolica de Chile) 46, 149·154.

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27. Gallo, L.M . and Piervittori,R. (1993) Lichenometry as a method for holocene dating: limits in itsapplicationsand reliability,II Quatemario 6 (I), 77-86.

28. Garcia-Rowe,J. And Saiz-Jimenez, C. (1988) Colonizationof mosaics by lichens: the case-studyofltalica(Spain),Studia Geobotanica 8, 65-71.

29. Gilbert,O.L. (1990) The lichenfloraofurban wasteland,Lichenologist22, 87-101.30. Hale, M .E. (1984) Control of biological growths on Mayan archeologicalruins in Guatemalaand

Honduras,National Geograph ic Society Research Reports 16, 305-321.31. Herk, C.M. van and Aptroot, A. (2000) The sorediatePunctelia species with lecanoric acid in Europe,

Lichenologist 32, 233-246.32. Kondratyuk, S. and Kamefelt, I. (1997) Josefpoeltia and Xanthomendoza, two new genera in the

Teloschistaceae,Bibliotheca Lichenologica 68, 19-44.33. Monte,M .del (1991) Trajan's Column: lichensdon't live here any more,Endeavour 15 (2),86-93.34. Nimis, P.L. (2001) Artistic and historical monuments: threatenedecosystems,in The Frontiers of Life.

sect. 2: Man and the Environment, Academic Press, S.Diego, Ca.,pp.557-569.35. Nimis, P.L. and Monte, M. (1988) The lichen vegetation on the CathedralofOrvieto, in P.L. Nimis and

M . Monte (eds.),Lichens and Monuments, StudiaGeobotanica8,77-87.36. Nimis, P.L., Monte, M., and Tretiach, M. (1987) Flora e vegetazione lichenica di areearcheologichedel

Lazio,Studia Geobotanica 7,3-161.37. Nimis, P.L., Pinna, D., and Salvadori, O. (1992) Licheni e Conservazione dei Monumenti, CLUEB,

Bologna.38. Nimis, P.L.,Seaward, M.R.D.,Arino, X .,and Barreno, E. (1998) Lichen-inducedchromaticchanges on

monuments:a case-study on the RomanamphitheaterofItalica(S. Spain),Plant Biosystems 132, 53-6I.39. Nordin, A. and Hermansson, J. (1999) Floristic news from Sweden, Norway and Finland, Graphis Scripta

10, 13-20.40. Pearce, F.(1997) Is there life after death for British lichens?, New Scientist 153 (2071),7.4I. Piervittori,R .and Sampo, S. (1988)Colonizzazione lichenica su manufatti litici: la facciata della Abbazia

di Vezzolano. Asti (Piemonte),Allionia 28,93-101.42. Piervittori, R., Salvadori, 0., and Isocrono, D. (1998) Literature on lichens andbiodeteriorationof

stonework III,Lichenologist30, 263-277 .43. Piervittori, R., Salvadori, 0., and Laccisaglia,A. (1994) Literatureon lichens andbiodeteriorationof

stonework,Lichenologist 26, 171-192.44. Piervittori, R., Salvadori, 0., and Laccisaglia, A. (1996) Literature on lichens andbiodeteriorationof

stonework. II, Lichenologist28, 47 1-483.45. Prieto, B., Rivas, T., Silva, B., Carballal, R., and Lopez de Silanes,M.E. (1995) Colonizationby lichens

of granite dolmens in Galicia (NW Spain), International Biodeterioration and Biodegradation 1994,47­60.

46. Prieto, B., Seaward, M.R.D.,Edwards, H.G.M.,Rivas,T., and Silva,B. (1999) Biodeteriorationofgranitemonuments byOchrolechia parella (L.) Mass.: an FT Raman Spectroscopicstudy,Biospectroscopy 5,53-59.

47. Purvis,O .W. (2000) Lichens . British MuseumofNatural History, London.48. Purvis, O.W.,Wolseley, P.A., Reed, M .E., Wilson, P.S.,and James, P.W. (1998) Monitoring of lichen

communities as indicators of air quality in Pembrokeshire, Report to Texaco Ltd., Gulf Oil (GreatBritain) Ltd.andElf Oil (UK) Ltd., contract no. 96/77.

49. Romao, P.M.S. and Rattazzi, A. (1996) Biodeteriorationon megalithicmonuments. Study of lichens'colonizationon Tapadao and Zambujeiro Dolmens (southern Portugal), International Biodeteriorationand Biodegradation 37, 23-35.

50. Sainz de la Maza, P.B. and Alfonso, A.T. (1997) Lichen communitieson the cathedralof Leon,Aerobiologia 13, 191-197.

51. Seaward, M.R.D. (1985) A study ofsaxicolous lichens from selected sites inSouth-WestBerlin (West),Verh. Berl. Bot. Ver. 5, 121-131.

52. Seaward, M.R.D. (1988) Lichen damage to ancient monuments: A case study,Lichenologist20, 291-294.53. Seaward,M .R.D.(1989) Lichens and historicworks ofart,British Lichen Society Bulletin 64, 1-7.54. Seaward, M.R.D. and Edwards, H.G.M. (1997) Biological originof major chemicaldisturbanceson

ecclesiastical architecturestudiedby Fourier Transform RamanSpectroscopy,J. Raman Spectroscopy 28,

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691-696.55. Sipman, H.J.M. and Kliick, B . (1982) De oudste korstmossen van Nederland,De Levende Natuur 84,

183-187.56. S ipman,H.1.M. and Seaward, M .R.O. (1990) Research news and notes,International Lichenological

Newsletter 23(1),3.57. Sparrius, L.B., Aptroot, A.and Herk,C.M. van (2000) Verslag van dekorstmossenexcursienaar het zuid­

westen van Friesland,Buxbaumiella 52,3-8.58. Sparrius, L.B., Aptroot,A., Herk, C.M. van, and Spier, L. (2000) Korstmossen van Gelderland en

aangrenzend Flevoland en van soortenrijke kerkmuren in de I1sselvallei,Buxbaumiella 53, 33-4159. Spier, L., Herk, C.M. van, and Aptroot,A. (1998) Inventarisatie van mossen en korstmossen op de

stadswallen van 's-Hertogenbosch,Buxbaumiella 47, 35-39.60. Thrower, S.L. (1988)HongKong lichens, Urban Council, Hong Kong.61. Tretiach, M. (1988) Ecophysiology of calcicolous endolithic lichens, Giornale Botanico Italiano 129,

159-184.62. Tretiach, M., Monte, M., and Nimis, P.L. (1991) A new hygrophytism index for epilithic lichens

developed on basaltic nuraghes in NW Sardinia (Italy),BotanikaChronika10,953-960.63. Vanallemeersch, R. (\993)Een vegetatiekundige en ecologische studie van de epilietenvegetaties op het

Brugs Kerkhof'(Brugge, ProvoWest-Vlaanderen), Thesis,Universiteit Gent.64. Wade, A.E.(1978) Churchyard lichens in the ValeofGlamorgan, Transactions ofthe CardiffNaturalists'

Society 98,30-36.65. Webber, 0.1. and Andrews, 1.1. (1973) Lichenometry: a commentary,Arctic and Alpine Research 5, 295­

302.66. Winchester, V. (1988) An assessment of lichenometry as a method for dating recent stone movements in

two stone circles in Cumbria and Oxfordshire,1.Linn. Soc., Bot. 96, 57-68.67. Winchester, V. (2000) Lichenometry in the Patagonian Wilderness, British LichenSociety Bullettin86,1­

3.

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MONITORING MARITIME HABITATS

A .FLETCHER I and R. CRUMp 2

'Letcestershire Museums Service, Holly Hayes Envir. Resources Centre,216 Birstall Rd., Leicester LE4 4DG, UK ([email protected])2Field Studies Council, Orielton Field Centre, Pembroke, Dyfed, SA 715EZ, UK ([email protected])

1. Introduction

Maritime habitats are of considerable importance for lichens. Seashore habitats cover arange of rocky to non-rockysubstrata, principally dunes, coastal heath, saltmarsh, fenceposts and other man-made substrata, trees and scrub. Fletcher [14] reviewed the subjectextensively. More recently briefer articles have appeared by Pentecost [22] and Gilbert[15]. Rocky seashores embrace many communities, from those subject to regularinundation by tidal seawater, called marine or littoral, to supralittoral that are subject towind-borne salt spray, and terrestrial, where seawater influence is small but stillinfluential. Fletcher [9, 10] described these communities, integrating them with thedescriptive system of Lewis [20] for rocky shore biota. Keys for identification and thehabitat preferencesofBritish seashore lichens were described by Fletcher [11, 12].

Despite growing awareness of the seashore's importance for lichens, very little hasbeen done on monitoring their species and communities. This article will, therefore,deal mainly with our own work;others'work will be incorporated where relevant. Thefew monitoring projects known so far have beenof local interest, seeking to observechanges in individuals and populations. They were mainly to guide nature reservemanagement. This will be dealt with in PartA. More specialised work monitoringrecovery of lichen populations following oil pollution incidents will be reviewed in PartB. Becauseof its local interest, very little work has been published on this subject; itexists mainly as confidential reports.

It is impossible to provide a comprehensive review of results, but many reports arelisted on the British Lichen Society website (http://www.theBLS.org.uk).They will bereferred to below aspers. comm. with due acknowledgement.

This chapter will deal mainly with practical problemsof monitoring on shores inBritain and present some successful solutions.

2.PART A. Long-termmonitoringof theindividualandtheassociation

Long-term monitoringof seashore lichens appears to have started with Fletcher in thelate 1960's. However, a precedent was set by Boney [1] who cleared patches of

255P.L. Nimis, C.Scheidegger and P.A. Wolseley (eds.), MonitoringwithLichens- Monitoring Lichens. 255-266.© 2002 Kluwer AcademicPublishers. Printed in the Netherlands.

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intertidalLichina pygmaea and observed its re-colonisation by tracing outlinesofpatches on rock. The smaller patches were the most stable while larger patches changedrapidly in outline. Changes were attributed to climatic conditions. Fletcher recordedlichens manually and photographically in quadrats along transects on rocky shoresofAnglesey (as part of his MSc and PhD studies from 1967 to 1972). This work wascontinued from 1974 by the University Collegeof North Wales Coastal SurveillanceUnit (UCNWCSU) (funded by Shell UK), that recorded changes in animals and plantsfor the following 9 years. The project questioned the opinion, widely held at that time,that a"baseline"exists on rocky shores, whereby animals and plants occupy a"typical"disposition against which future observations could be compared. Results wereprivately published by the University CollegeofNorth Wales and the archive depositedwith the Countryside Council for Wales, Bangor. Briefly, the results demonstrated thatseasonal and annual changes were so great that a"baseline"was impossible to find.Most changes appeared to be cyclic and related to climatic factors. The lichenmonitoring partof the work has been continued since 1978 by Fletcher using personalfunds. Further seashore lichen monitoring work was started in 1992 on maritime rockson Skomer (O.W. Purvis, P.A. Wolseley and P.W. James) and Bardsey Island (A.Fletcher), and maritime saxicolous and terricolous lichens are now being monitored onBardsey Island National Nature Reserve (A. Fletcher). All of these projects wereinitially commissioned by the Countryside Council for Wales. But most subsequentwork now continues unfunded. Maritime shingle lichens have been monitored by A.MandRJ. Coppins [5], commissioned by Scottish Natural Heritage.

2.1.METHODOLOGY FOR MONITORING SEASHORE LICHENS

2.1.1. IntroductionThe work on seashore lichen monitoring appears to have proceeded without reference toformal definitions as set out in the introduction to this book."Surveillance"has oftenbeen taken to mean "keeping a watch", and is an informal noteofchanges occurring intime. Surveillance has been often merely anecdotal, or was backed up by photographs.However, it is not consistent in time or method, is not particularly repeatable, especiallyby others, and is not designedto answer any specific questions. "Monitoring" has beenregarded as a targeted approach to answer a specific question such as"by how much isspecies a declining or increasing?"It requires a rigorous plan to establish how resultsare to be consistent and repeatable.Numerical measurements are taken and ideally somekind of confidence limits should apply to the results."Monitoring! surveillance"programmes are relatively easy to set up but provision needs to be made for continuingthem in the future. Often funds are available initially but are not reserved for futureyears, so the programme is not kept up.

Seashore habitats present unique problems to the fieldworker. The littoral isregularly inundated by the tide, so time to work there is limited. The lower littoralcannot be reached in neap tides. Thalli are usually wet and difficult to identify andphotograph. The substratum is notoriously dangerous and uneven, so choices forplacing permanent plots can be restricted. Weather is always uncertain, so quadrats mayhave to be photographed when wet. Many attractive seashore sites are on remoteislands, so work has to be carried out in restricted periods. Lost equipment is not easily

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replaced in such places. Lichen monitoring hassometimesbeen done in the past, merelyto see what hashappenedafter an event. More recentemphasishas turned to thepracticalvalue of monitoring, especiallyregardingbiodiversitystatus or the stateofecosystemfunction, and hence the needs for sitemanagement.

Monitoring lichens is vital for Bardsey Island, formallyscheduledas a NationalNature Reserve for its bird life and saxicolous lichencommunities. The latter arepredominantlymaritime, even though the island ascends to 167m. Rabbit(Oryctolaguscuniculatus) grazing was a keymanagementagent,maintaininga short grass turf, butover the winterof 1997-8 all the estimated 14,000 animals were lost. AcombinationofMyxomatosis and Virus-borneHaemmorrhagicFever arethoughtresponsible. Lack ofrabbit grazing nowendangersbirds that feed among short turf, such as Chough(Pyrrhocorax pyrrhocorax) and Wheatear (Oenanthe oenanthe) . The decline inmaintenanceof rabbit holes is expected to affect the coloniesof Manx Shearwater(Puffinus puffinus) colonies, for which the island is renowned. Lichen monitoringnowdetects the effectsof trampling due to sheep grazing that is nowencouragedas amanagementtool in lieuofrabbits.

2.1.2 Indicator value oflichensLichens are often advocated as indicatorsof conditions such asenvironmentalfactors,habitat type, "change",etc. However, the term is rarely closely defined.Thus "maritimehabitatindicator"merely refers to a species living near the sea.It is well knownhowever, that some lichens can indicatenon-obvious environmentalconditions,forexample sulphur dioxide and more recently, high levelsof agriculturalnitrogen.Normallyone or more lichens are selected from a list but it should becautionedthat onseashores(as elsewhere), lichen species may be present fordifferentreasons[14], e.g,light level, seawater, bird lime drought. Others are merelypioneers of unstableconditions(for example.Lecanora dispersa, Catil/aria chalybeia) and are not specificto seashores. The best indicatorsof maritime influence wouldprobably include thelittoral Verrucaria and Pyrenocol/ema at one extreme, and at the other, the xericsupralittoralcommunitiesthat includeRamalina siliquosa, Rhizocarpon richardii, etc.[11, 12].

What changes are we trying to detect? Typicallymonitoringcan be used to detectchanges incommunitystructure, whetherpopulationsare increasingor decreasing,growth ratesofindividuals, and symptomsofthallus health. Some thoughtis needed asto what change is natural and what is unnatural. Formanaginghabitats we need to knowwhich changes result from natural causes and areprobablyuncontrollable,andwhichwemight wish to control.Thereforesome sites should be controls forcomparisonagainstother sites that might change. But, controls are difficult to select asexperienceshowsthat lichencommunitiesare changing at all sites.So, it becomesnecessaryat some pointto find out what" natural" patternsofchanges occur. Later we canhypothesisewhy theobservedchange isoccurringin our test site.

Finally, asmonitoringis a targeted method,one should bepreparedto stop when thequestionis answered. It is easy to continuemonitoringmerely because one has alwaysdone it(somethinginterestingmay tum up)!

2.1.3. Seashore lichen monitoring methodsVarious methods have been used for detecting change and centre mainly on counts,

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estimating changes in cover, or measuring changes in thallus size.Counts are especially useful for mobile terricolous lichens. They have been used

successfully on Skomer and Bardsey forTeloschistes jlavicans on soil (see also chapter40, this volume). Fletcher on Bardsey Island, counted every known patchofTeloschistes jlavicans, standardising the timeof search to 2hr along a ridgeof about300 m. Problems lay in determining what is a patch, as they can coalesce. Patches alsovary in size. But, patches on soil seem to reach a maximumof about 10 em thenfragment. However, it has proved impossible to use this method forHeterodermialeucomelos, which though it inhabits similar situations, is much harder to see. Fletcher(unpubl .) used a counting method on Bardsey for this species, based on that used byA.M and B.J.Coppins [5], that entails placing a cocktail stick with a paper flag besideeach lichen patch, surrounding the area with a string quadrat and photographing.

Changes in thallus cover were estimated by Wolseley and James(pers .comm.) usinga plotless sampling technique forTeloschistes jlavicans on soil, following Mueller­Dombois and Ellenberg [21]. Here a tape is laid out between two markers. Points areselected at regular intervals and four further lines laid out along 45° compass bearingsfrom the main line, making a cross.The distance of the nearestTeloschistes along thesecross lines is then measured.

Changes in cover and thallus size are usually measured in permanent plots(quadrats). Tracing thalli onto acetate sheet in the field, once popular for largecorticolousLobaria and terricolousFulgens ia transplants in terrestrial habitats [16], hasbeen largely superseded by photography. The growth of most seashore species,particularly crustose lichens, is too slow to be detected by the coarser methodoftracing.

2.104. Selection ofsites and plotsSites need to be selected carefully if they are to provide an answer to the question inmind and must be truly subject to the influence one is trying to detect. This may not beobvious until after monitoring has started. The UCNWCSU [13] had a geographicalfocus, attempting to relate changes in seashore communities to climatic factors.Twenty-nine sites were selected along a 150km stretch of the North Wales coastline.Some were taken to contrast aspect to sun and shade, exposure to wave action and rocktype.About 5-6 lichen quadrats were set up at each site in various lichen zones. Carefulexamination determined whether sites were typical of the area and whether they wouldbe safe from disturbance, but the latter was not always easy to predict, particularly theeffectsof seasonal holidaymakers. One site was partially lost through a ship runningaground. Replication of sites was needed to confirm results. This was achieved at aspecies level when the same components occur in a numberofquadrats. Replicationofsites was also felt to be essential as some sites were lost or damaged. Anotherconsideration is whether they can be accessed maybe 30 years on, when the researcheris less nimble!

Fletcher's Bardsey project took a more rigorous approach to site selection. In 1977the saxicolous floraofthe island was quantitatively sampled and the results analysed bycomputer ordination to map the island's communities. Fifty monitoring quadrats wereset up covering most of these communities, with usually at least two quadrats samplingeach community.

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The approach on seashores by British workers has been local, mainly recordingchanges in permanent plots in small geographical areas.Often, as on Bardsey Island andLoch Fleet [5], the entire populationsof species have been observed, a totalof fourthalli in the caseof Lobaria on Bardsey. But working with local plots entailsuncertainty about applying the results to a wider area.

The authors have found three sizes of quadrats to be useful for seashore lichens: 50x 50 cm for communities of large species,C/adonia, Lobaria, etc.; 30 x 20 ern for mostcrustose communities (the same proportions as 35 mm film); and, 10 x 10 em formeasuring growth ratesofcrustose species,Parme/ia, etc.

Annual changes are hard to assess using quadrats larger than 10 x 10 em simplybecause most thalli grow so slowly. Crustose seashore thalli increase by 1-2 mm perannum whileParme/ia and other foliose species may increase by 5-7 rnm. Generally, 10em quadrats have proved appropriate for measuring crustose growth rates on a monthlybasis, even for littoralVerrucaria that may grow by less than 1 mm/year.

Ten cross wiresof non-toxic stainless steel wire or nylon are useful on largerquadrats to aid making accurate counts or cover estimates. Ten-em quadrats can bemade from portions of steel tape measures that provide a useful measuring scale.

2.1.5. Positioning and marking permanent quadrats and transectsQuadrats need positioning bolts so that they can be precisely located on the rocksurface. Fletcher favoured 2 em bolts through each corner that align with drill holes inthe rock. Purvis used "picture plates" on the edge of the quadrat, that similarly insertinto bolts in the rock. Permanent terricolous quadrats are difficult to mark.It is possibleto use transponders or GPS to get close to the quadrat location, but these techniques arenot precise enough for photographing thallus size changes. The satellite technologyseems little better than using precisely drawn maps backed up by photographs for well­known areas.

Marking quadrat positions on rock is difficult. On nature reserves over-intrusivemarking has to be avoided.Elsewhere marks can attract attention from passers-by.Paintis relatively easily weathered and needs renewal and both Purvis(pers . comm.) andFletcher have relied on touching it up at each visit (usually annually). Fletcher andJones [13] used white enamel, with success even in the littoral, provided the paintedsurfaceofabout 2 em diameter was chiselled clean and it was repainted at least twiceper year. Stove enamel seems to be best as it is non-toxic. Chlorinated road markingpaint is best avoided. Occasionally paint marks can be obscured by overgrowthoffoliose thalli and have to be cleaned.

The ability to relocate quadrats is critical. Fletcher and Jones [13], aligned quadratsalong transects originating from easily found points in the supralittoral. The initial pointwas marked with a star-drilled hole of 2 em, often containing a stainless steel bolt fixedwith quick-drying silicate cement. Experiments with resin glue and 3 em lengthsofcoloured nylon 5 mm rope were abandoned as the rope soon frayed away. Drill holeswere made every metre along the transect. Lichen quadrats were marked with a 7 mmdrill hole at each corner, one being 2 cm diameter and painted white. For aestheticreasons Fletcher's Bardsey survey uses yellow enamel paint in the littoral andsupralittoral asit resemblesXanthoria in colour.In the terrestrial regions white paint isused.

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Transect lines are located from a hand-drawn, or 1:2500 "Ordnance Survey" map.The start must be lined up with known features in the background and photographed.The needsoffuture workers must be considered so that their relocation is as painless aspossible. Locations may have to be re-photographed annually as the topography canchange, especially overgrowth by shrubs such asCalluna or Ulex. A site can looksurprisingly different in sunny conditions than in rain. Accurate and copiousdocumentation is needed, especially when using photographs to zero-in on a site.

Transect methods suffer a disadvantage in that considerable damage can be done bytrampling from repeated visits. The UCNWCSU, which visited 12of their sites atmonthly intervals, noted stripping of seaweeds from the trampled sideof the transectline.

Quadrats can become overgrown by higher plants and seaweeds making them hardto find.It is advised that markers be placed well outside isolated quadrats.

2.1.6. Photographing quadratsPhotography is probably the most popular method for recording change in quadrats.Butwhile easy to set up and to repeat at intervals, it can quickly generate large numbersofimages. Analysing photographs is very time-consuming, so a large backlogofunprocessed results can develop. Identifying lichens from photographs is difficult so itis best to identify and quantify all species present in the field as soon as the quadrat isset up.

The authors' personal preferences are as follows. Single lens reflex cameras areessential, mounted on a tripod. A 50 mm lens is adequate for quadratsof30x20 em ormore, but a macro lens is necessary for smaller quadrats. Shaded natural light is betterthan sunlight and flash, that cast complex shadows. An umbrella is useful for shade insunny weather. Ring-flash,used by some workers, provides flat lighting that eliminatestexture, making it difficult to see the edgesof thalli. Weather conditions forphotography should be standardised, ideally being in dry periods from mid-morningonwards. Photographing after dewfall or too soon after rain is ill-advised as thalliexpand by several mm when wet. For long-term surveys, it is best to standardise thetime of year because thalli grow over the winter and may even shrink during thesummer (Fletcher, unpubl.) . Film type needs to be standardised if colour rendering isimportant. This will certainly be the case as computer imaging becomes moreobtainable.

Although a tripod is recommended, it cannot always be employed on uneven terrain.This means that the camera may.not be perpendicular to the quadrat and the imagebecomes distorted.Hooker and Brown [19] described a mathematical correction for thissituation.

2.1.7. Detecting change within quadratsThis aspect is possibly the most demanding and remains a great problem forphotographic monitoring.

The easiest changes to detect are visible changes in size or morphology,as thalli canreadily be seen dying back, growing, becoming parasitized, etc. Some species canchange colour, for exampleRamalina incrassata [11], that inhabits underhangs, will gored when its salazinic acid is affected by seawater after storms.Itusually recovers.

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Changes in cover were measured by Purvis(pers. comm.) using 30x20 em quadratswith steel cross wires forming a IOxiO em grid. The presenceof thalli belowintersections were recorded as frequency.The method suffers from a parallax error, thatwas reduced by Fletcher [9, 10], using an optical sightmethod. Here, perspex quadrats10 cm square were employed, scored above and below at I em intervals giving a IOxiOgrid.The above-below crosses were lined up by eye like a gunsight, and thalli below thesights were recorded. The method gives frequency estimates but suffers from ameasurableerror. Fletcher [8] measured an errorofplus or minus 4% when using a 10cm quadrat on crustose xeric supralittoral species. This degree of error could varydepending on thallus size. Later, a 50 cm frame was developed for larger-scalecommunities, having a movable perspex sheet of IOx50cm, fitting into grooves on eachsideofthe quadrat. This allowed hand access to plants beneath the quadrat. The perspexwas scored with cross lines above and below at 5 cm intervals.

Growth rates can be measured by the distance from a thallus edge to the edgeofthequadrat, or to a known marker on the rock surface, such as a knob or crack.The subjecthas been reviewed by Topham [25].

2.2.SOME RESULTS OF SEASHORE LICHEN MONITORING

Results obtained from over 30 years of seashore lichen monitoring employing nearly200 quadrats are briefly summarised below.Bardsey Island monitoring is done annuallywhile the North Wales coast is monitored approximately every 5 years now, though itwas monthly for the first 4 years.About 10,000photographs have been accumulated.

2.2.1. LittoralMarine Verrucaria grow very slowly, by up to 2 mm per annum, with most growthoccurring over autumn to spring.Lichina pygmaea can show extensive regressionofpatches, especially after periods of drought. Below the patchesHildenbrandiaprototypus (Algae), Pyrenocollema halodytes, and Verrucaria striatula are revealed.Boney [I] noted that large patches were unstable and recolonisation was slow, if at all.It seems likely that recolonisation takes place in the faceof competition with crustoselittoral species. Many examples of competition with limpets and barnacles were noted.Barnacle spat overgrowsVerrucaria mucosa but most fails to survive except increvices.Limpets make extensive grazing marks over the surfaceofmarine species.

2.2.2. Mesic SupralittoralPart of the so-called orange zone, this is dominated by crustose species such asCaloplaca marina and Lecanora helicopis . The lichens of this zone are stronglyinfluenced by light aspect. BothCaloplaca marina and C. thallincola (shaded shores)can quickly be obliterated by wave action or perhaps excessive light. Few changes havebeen noted with other species such asLecanora actophila, L. helicopis and Catillariachalybeia.

2.2.3. Submesic SupralittoralFletcher [10] noted that this was the region first colonised by foliose species andattributed this to reduced violenceof wave action. Its dominant species, Xanthoria

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parietina, grows rapidly by 5-7 mm per annum but has a quick turnover due to waveaction. Individual thalli grow to a maximum size, are then fragmented, and remnantsgrow again. So, total cover remains constant but individuals come and go.

2.2.4. Xeric SupralittoralThis is the most drought-prone area of the shore and bears manyof the mostcharacteristic maritime species. In this zone,Xanthoria parietina, Lecanora rupicolaand Tephromela atra have recently shown extensive growth whileParmelia pulla hasvaried considerably, growing or dying back depending on quadrat location.Ramalinasiliquosa can show rapid die-back, but as it has a crustose base that is very resistant toabrasion, fronds can re-grow and net populations are very stable. Species such asAnaptychia runcinata andOchrolechia parella show steady but slow growthofabout 2­4 mm per annum. Anaptychia runcinata seems to behave like a crustose species, beingstable and changing only slowly.Rhizocarpon richardii and R. geographicum haveshown some decline, possibly due to a lessening maritime influence or perhaps nitrogenpollution from the mainland.

2.2.5. Terrestrial HalophilicThis zone is dominated by lichens commonly encountered inland but which are tolerantofseawater spray. In many places overgrowthofquadrats byUlex has been noted, butCalluna has shown some regression. At one limestone site, the quadrat was overgrownby Cirsium acaule for two years but the lichens below were apparently unaffected bythe shading. On siliceousrocks on Bardsey, folioseParmelia saxatilis, P. omphalodesandP. glabratula subsp.fuliginosa have shown considerable decline probably related totrampling by sheep. Parmelia species in general seem to be unstable with a rapidturnover. Thalli reach a maximum size then abradeoff. In some places this may be dueto drought. Crustose species in this zone are generally more stable having slowergrowth. Some appear even to survive overgrowth byParmelia. For example,Rinodinaatrocinerea appeared within one season afterParmelia omphalodes had abraded off, soit apparently had been below theParmelia thalli. Dynamicsof patternof saxicolouscommunities seem to be more complex than merely a patternof succession frompioneer species on bare rock. In underhangs and on maritime buildings,invasion byDirina massiliensisf sorediata has been noted over the past 20 years. In addition, rocksbeside the main track now haveLecanora muralis and Physcia caesia , most likelytaking advantage of dust and nutrients raised by increased visitor pressure. In manyplaces, especially in rain tracks, lichens have been overgrown by algae. This used to bea spring and autumn phenomenon but seems to be occurring throughout the yearrecently.The suspected cause of this is nitrogen pollution, though its source on Bardseycan only be from the mainland via winds.

2.2.6. Terrestrial HalophobicThis zone has lichens intolerant of seawater and is found on the summitof Bardseyisland in sheltered areas.Lecanora rupicola has been increasing rapidly through theisland, dominating most maritime walls, often accompanied byTephromela atra . Thereason may be perhaps increased sunshine due to climate change or possibly nitrogen.Rhizocarpon species have been decreasing

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2.2.7. Protected speciesSpecies protectedby legislation areof particularinterest on Bardsey.Teloschistesflavicans near the summit at about 150 mdisappearedfrom a quadrat, but newlyappearedjustoutsideit. A few years later itreappearedwithin thequadratin exactly itsformer place. This suggests that tiny fragments had survivedundetected.ElsewhereTeloschistes quadrats have shown that patches reach a luxuriance and sizeofabout 2-4em, then blowoff and attach elsewhere. Remnantsof the original then regrow. Thisseems to be avagrantspecies at least on soil.Heterodermia leucomelos, also on soil,seems to beresistantto trampling, having a springy,cartilaginousthallus. But likeTeloschistes, patches seem to reach a maximum sizeofa few ern on soil, then disperse.It seems thatTelosch istes and Heterodermia may be increasing.In its largest area, aridge of about 500m length, rising from 70-130m,Teloschistes has increasedfrom 300patchesin 1992 to 2945 in 1997. Since then numbers havestabilisedatbetween2500 to3000 patches.

3. PART B. Monitoringeffectsoflarge-scaleaccidentson lichendiversityofmaritimehabitats

Monitoring the effectsof oil pollution on upper littoral andsupralittorallichencommunitiesstartedin the late1960's when 140 milesof the Cornish coast werecontaminatedby the Torrey Canyon oil spill. Pyrenocollema halodytes and Lichinapygmaea in the midlittoral were the species mostseverely damaged by oil [23].Similarly heavy oil contaminationplasteredcommunitiesof Verrucaria maura andLichina confinis but above this level oil damage was patchy. Decontaminationactivitiescan result in much more serious damage to the supralittoral lichens than thatcausedbythe oil itself, as was the case when over 1.5 million gallonsof toxic emulsifierswereapplied directly to clean Cornish beaches [23]. Brown [2], in a seriesof laboratoryexperiments, showed thatunweatheredKuwait crude oil inhibitedphotosynthesisinLichina pygmaea less than the detergent BP1002. Detergent-inducedleakage ofphotosyntheticproducts from the lichen was dueprimarilyto the effectsofsurfactants.Xanthoria parietina was even more sensitive to the detergent [3], that acted as a solventfor the orange pigment parietin. Brown [4] used systematicphotographicmonitoringinthe field to record the damage andsubsequentrecoveryof oflichen thalli(L. pygmaea)after oiling. In particular, rocks in badly oiled areas showed initial signsofrecolonisation7 years after the spill. Heconcludedthat usingdetergentsto clean oilfrom the upper shore and splash zone would lead toextensivelichenmortality.

Reporting on the effects on lichencommunitiesof an oil spill in Bantry Bay,Cullinaneet al. [7] suggested that fruticose thalliof lichens such asLichina pygmaeaand Ramalina siliquosa were worse affected by oil anddetergentthan were crustosespecies such asVerrucaria maura. Foliose Xanthoria parietina was also seriouslyaffectedshowing discolorationand necrosisof the thallus leading to a sticky messeasily removed from the rocks. The curling thallusofX parietina suggesteddamage tothe rhizinae attaching the thallus to the rock.Cullinaneet al. (7] concludedthatbecauseofa lichen'svery slow growth rateof2-5 mm per year [17] it would take many yearsfor the lichen floraofaffected areas to be replaced. While mostworking on the effects

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ofoil pollution on lichens agree that recovery is likely to be slow, and that monitoringshould continue for many years, there is little evidence that the latter has been done.Long-termstudiesof recolonisation following oil spills [18, 24] have concentrated onthe limpet-fucoid balance in the middle shore and the supralittoral has been ratherneglected.

A long term monitoring programme at two sites in Milford Haven was initiatedfollowing the "Sea Empress" oil spill in Pembrokeshire, 1996 [6].At Sawdem Point 64small (lOx20 em) quadrats, marked permanently with rawl plugs, were established ontwo old Red Sandstone ridges that had been heavily oiled by both Forties crude andbunker oil.Half of the quadrats were placed in areas that had been cleaned with high­pressure hoses and the remaining 32 were in oiled areas. The quadrats werephotographed annually. Initial results showed thatXanthoria parietina and Ramalinasiliquosa were seriously damaged by contact with oil and quickly showed necrosis anddiscolouration. CrustoseV. maura and Caloplaca marina appeared initially to be muchless susceptible, but mostC. marina had disappeared from all quadrats within 12months. High-pressure washingofexperimental areas had removed mostofthe oil andlichen thalli from the rock surface, with onlyVerrucaria maura remaining in creviceswith small traces of encrusted oil. Recolonisationofcleaned rock surfaces was unseenup to three years after high pressure hosing. By contrast, oiled quadrats were cleanednaturally by the sea within twelve months and now show good recolonisation (orregrowth from small surviving piecesof thalli) by bothCaloplaca marina andVerrucaria maura . Unfortunately, computer analysisof photographs using photo­editing software has proved impossible because of the conflict with colour shades in thebackground rock.

At West Angle Bay, a shaded vertical limestonecliffrunning from thePelvetia zoneto the grassline, was affected by Forties crude oil with an admixtureofemulsifier. Heretwelve 50x50emquadrats (six oiled and six cleaned) were permanently marked on therock using a Bosch portable hammer drill and plastic rawl plugs.An aluminium quadratwire-strung into decimetre squares was then placed at each location and photographedusing a Canon AI camera with 35-80 mm lens. Photographic monitoring makes itpossible to measure the growth rateof individual coloniesof lichens and to makeaccurate measurements of percentage cover. Initial observations indicate thatVerrucaria maura survived remarkably well in both oiled and cleaned quadrats butCaloplaca marina and C. thallincola succumbed to both oiling and cleaning. Growthrates ofVerrucaria maura have proved to be extremely slow (less thanI mm increasein radius per annum) and there has been no recolonisationofpatchesof bare rock bynew coloniesof V. maura. The first signsofregrowth ofCaloplaca marina, presumablyfrom newly established prothalli, were noted in August 2000 on the Lichen MonitoringWorkshop field trip.

4.Conclusions

It is urgently recommended that a formof widespread dissemination be found forunpublished lichen monitoring reports. When monitoring(I) a clear aim is required,define what you are monitoring for, (2) be prepared to stop monitoring when the

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questionis answered,(3) make sure that you and others can relocate sitesperhaps30 ormore years later, (4) keepequipmentsimple, cheap andreplaceable,bewareofneeds forbatteriesand other items, (5) assess the costsof copying andpreservingphotographswith regard to colour stability; CD-ROMtechnologyis anattractiveway forward.

5. Acknowledgements

In the text the following arc referred topers. comm. as sourcesof unpublishedreports; A.M.and B.J.Coppins,P.W. James, O.W. Purvis, P.A.Wolseley and R.G.Woods.

6.References

I. Boney, A.D. (1961) A note on the intertidal lichenLichina pygmaea Ag.,J. mar. bioi. Ass . U.K. 41, 123­126.

2. Brown, D.H. (1972) The effectof Kuwait crude oil and solvent emulsifier on themetabolismof themarine lichenLichina pygmaea, Marine Biology 12,309-314.

3. Brown, D.H. (1973) Toxicity studies on the componentsofan oil spill emulsifierusing Lichina pygmaeaandXanthoria parietina, Marine Biology 18,291-297

4. Brown, D.H. (1974) Field and laboratory studiesofdetergent damage to lichens at the Lizard Cornwall,Cornish Studies 2,37-40

5. Coppins, A.M. and Coppins,BJ. (1998) Loch Fleet NNR, East Sutherland (VC /07) : Lichen survey andpermanent lichen quadrats, Report for Scottish Natural Heritage, pp 214.

6. Crump, R.G.and Moore, J. (1997) Monitoring ofupper littoral lichens at Sawdern Point , A report to theCountrysideCouncil for Wales from the Field Studies Council Orielton and OPRU,UK,pp 4.

7. Cullinane, J.P.,McCarthy, P., and Fletcher, A. (1975) The EffectofOil Pollution in Bantry Bay,Mar.Poll. Bull. 6, 173-176.

8. Fletcher, A. (1972) The ecology of marine and maritime lichens on Anglesey, PhD thesis, UniversityCollegeofNorth Wales.

9. Fletcher, A. (1973a) The ecologyof marine (littoral) lichens on some rocky shoresof Anglesey,Lichenologist 5,368-400.

10. Fletcher, A. (1973b) The ecology of maritime (supralittoral)lichens on some rocky shoresof Anglesey,Lichenologist 5, 401-422.

II. Fletcher, A. (1975a) Key for the identificationofBritish marine and maritime lichens.I Sil iceous rockyshore species,Lichenologist 7, I-52.

12. Fletcher, A.(1975b) Key for the identificationofBritish marine and maritime lichens.II Calcareousandterricolous species,Lichenologist 7, 73-115.

13. Fletcher, A. and Jones, W.E. (1975) Thefirst report ofthe Coastal Surveillance Unit, UniversityCollegeofNorth Wales, Bangor, pp 109.

14. Fletcher, A . (1980) Marine and maritime lichensof rocky shores, in J.H. Price, D.E.G. Irvine and W.F.Farnham(eds.),The Shore Environment Volume 2: Ecosystems, Academic Press, London, pp. 789-842.

IS. Gilbert,O.L. (1999)Lichens, New Naturalist Library, HarperCollins Publishers, London16. Gilbert, O.L. (2001) The species recovery programme, in A. Fletcher (ed.),Lichen Habitat Management.

British Lichen Society (in press).17. Hale,M .E.(1967) The Biology ofLichens, Arnold, London.18. Hawkins,SJ. and Southward,AJ.(1992) Lessons from the Torrey Canyon Oil Spill; RecoveryofRocky

shore communities,in G.W.Thayer (ed.),Restoring the Nations Environment, pp 583-631.19. Hooker, T.N.,and Brown, D .H. (1977) A photographicmethod for accurately measuring the growthof

crustose and foliosesaxicolouslichens,Lichenologist 9,65-76.20. Lewis, J.R.(1964) The ecology ofrocky shores, English Universities Press, London.21. Mueller-Dornbois, D. and Ellenberg, H. (1974)Aims and methods of vegetational ecology , Wiley, New

York .22. Pentecost, A . (1997) Lichen and mosscommunitieson rocky coasts, in E. van der Maarel(ed), Dry

Coastal Ecosystems Part A. Ecosystems ofthe World 2C,Elsevier,Amsterdam,pp. 195-205.23. Ranwell, D.S. (1968) Lichen mortality due to Torrey Canyon oil and decontamination measures,

Lichenologist 4,55-56.

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24. Southward,AJ. and Southward, E.C. (1978) Recolonisationof rocky shores in Cornwall after useoftoxic dispersants to clean up the Torrey Canyon oil spill,J. Fish. Res. Board. Can.35, 682- 706.

25. Topham, P.B. (1977) Colonisation, growth and succession. in M.R.D. Seaward (ed.),Lichen Ecology,Academic Press, London,pp. 31-68.

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Section3

METHODS FOR MONITORING LICHENS

editedby

PatriciaA. WOLSELEY and DavidJ.HILL

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METHODS FOR MONITORING LICHENS

An Introduction

P.A.WOLSELEyl and D.1.HILL 2

IBotany Department, The Natural History Museum, Cromwell Rd,London SW7 5BD, UK ([email protected])2School of Biological Sciences, University of Bristol, Bristol. UK(D.J.Hil/@bristol.ac.uk)

The third sectionof this volume presents methods that have been used to monitorlichens, as individual thalli, species or communities. These have been written asexperimental protocols with aims, materials, procedures, examplesof results,applications and limitationsofthe method, with the intention that these could be used inother situations where monitoring is required. The methods include some that requireconsiderable technical equipment and expertise, and some relatively simple fieldmethods, with examples from a rangeofcountries and conditions.The emphasis of themethods outlined may shift with a change in human activities; in some partsof theworld industrial pollution is still an enormous problem while in others eutrophicationfrom intensive agriculture has replaced it (see chapters 5 and 21, this volume). InEurope we are monitoring the declineof rare and endangered species, but in tropicalcountries it is often difficult to define rare species and preliminary data are urgentlyneeded before we can begin to assess the effectsof logging or other environmentalchanges on lichens. The range of problems requires flexibility and adaptability in boththe monitoring technique and the sampling method, which are both well described inSections I and 2. The arrangement of the methods largely follows thatofsubjects in thefirst two sectionsofthe book.

The use of organisms as surrogates for assessing the impactof environmentalfactors has been widely used in pollution monitoring. Within an ecosystemit is oftendifficult and expensive to measure the environmental variables and easier to measurethe signal from an identified indicator and use this to estimate the environmentalcondition. These methods all require a traditional experimental approach and thedevelopmentof an hypothesis which is tested. In situations where there are nopredictions of changes the designing of a rigorous sampling method allows thecollectionofdata that can be analysed to detect causal relationships at a later date. Forexample the lAP method described by Astaet al. has been designed to detect changesin the lichen community, and as it is based on frequency of lichens in unit areas, can beused in a wide rangeofconditions and geographical areas. In regions where the flora isknown, Loppiet al. have devised a scale to estimate deviation from naturality that hasbeen applied in a range of bioclimatic units in Italy. The analysisofwhole trunk data

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sets from roadside trees in the Netherlands has revealed shifts in taxa that are tolerantofacidification to taxa that are nitrophilous. van Herk has shown that this is consistentwith increasing levelsofammonia associated with intensive animal husbandry. Lichenson twigs have been used by Wolseley to assess changes over short periodsof time,where annual growth rings are used to estimate the ageofyoung twigs, and changes inlichen communities along a branch used to demonstrate changes in ambientatmospheric conditions at a very local scale. The latter two methods are based on alllichen species present and so require some knowledgeofregional lichen floras.

Accumulationof trace elements in lichen thalli is now widely used to identifysources of pollutants. Outlines for the measurement of these elements in epiphyticlichens are provided by Bargagli and Nimis. However this assumes that it is possible tocollect lichens in the areas under investigation whereas in highly polluted areas, whichmay not be possible in areas where the lichen flora is already lost. Mikhailova describesa technique for transplanting lichens in areas that are already lichen deserts for similarstudies. The preparationof samples for analysis often requires special laboratoryfacilities and methods, and this has been considerably expanded by Rusu in her paperon sample preparation for instrumental analysis. Spiro et a/. use sulphur isotopes inlichens to identify sourcesofpollution. However all these methods require destructionof the lichens used for investigation. Vorobeichik and Mikhailova have correlatedhealth and diversity of epiphytic lichen communities with background levelsofcontaminants in the environment, in order to use lichens as ongoing indicatorsofenvironmental conditions.

The health and conditionofthe photobiont is an important indicator of the healthofthe lichen, and a technique for measuring chlorophyll and phaeophytin in order toassess the rateof degradation of chlorophyll is described by Boonpragob. Thedrawbackofthis method is that it is destructive, uses considerable amountsofmaterial,and can only be used on a limited selectionof species. The useof chlorophyllfluorescence to detect photobiont activity in lichen thalli in the fieldis described byJensen and Kricke, and will permit us to follow the growth and fateof an individualthallus over a periodof time. The measurementof bark pH has played a role indetecting acidificationoftrunks and the advent of portable surface electrodes has madefield measurement more practical (Kricke). However occasional or repeatedmeasurements still do not provide a picture of the rangeof variation experienced bylichens on a trunk in both wet and dry periods of the year, orof the rangeofvariationfrom twig to trunk, so that the measurement of the responseof the lichen to localconditions provides valuable evidence as in Purviset al.

Although lichens are widespread in a great range of habitats and climates, some havebecome specialists associated with microniches that develop over time, particularlywithin habitats where there is ecological stability or continuityofconditions. In BritainEurope and Canada species associated with old-growth forests where there has beenecological continuity have been used as a basis from which to develop indices in orderto assess sitesof conservation importance. This method is described by Rose andCoppins and includes the basic lists and calculationsof indices used for deciduouswoodlands in lowland Britain. Manyof these lichen specialists are associated withveteran trees which provide an ancient bark surface and a varietyof niches that havedeveloped over time. Selva has used the Caliciales, a widespread family often

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associated with particular niches in ancient woodlands, to develop diversity indices forhardwood and coniferous forests in North America. Bothof these methods depend onspecialists and training specialists in finding and identifying the targeted species.Another approach has been used over extensive areasofthe USA using macrolichens toassess changes in forest ecosystems and comparing this with air quality, climatic andmanagement conditions. This project has trained non specialist recorders and testedtheir results against specialists results. It has also detected significant variation acrossbioclimatic regions, but no difference over time since the project began in 1993.Lichens are widespread on a range of substrata in managed habitats, but their diversityand survival will vary with the typeofmanagement practised. In order to evaluate theeffectsof management on lichen communities at a landscape level Scheideggeret at.have devised a sampling method to assess lichen diversity on corticolous, saxicolousand terricolous substrata under a range of management conditions. This method will beapplied in a numberofEuropean countries in diverse climates and conditions, coveringa great range of species and habitats, and producing very large data sets for analysis. Inthese situations appropriate computer software is an important aid in expanding lichenmethodology and application.

This is practicable in areas where the lichen flora is well known, but there are largeareas of theearth'ssurface which support lichen communities that are still poorlyknown. In wet eucalypt forestsof Tasmania Kantvilas and Jarman have recordedspecies associated with habitat niches and estimated their contribution to the diversityof these forests. This study has shown the high diversity of these eucalypt forests aswell as highlighting the restricted distributionofrare and local species and the problemsin identificationof taxa. Theirs is a base-line surveyof plots prior to logging fromwhich assessmentsofchanges following logging in both taxa and microhabitats will bemade. Tropical forests are disappearing fast and the pressure to assess biodiversity andthe effectof management on biodiversity has led to developmentofmethodology forsurveying cryptogamic communities in plots established in a rangeof managementconditions. Using a standardised sampling technique frequencyof lichen taxa inquadrats, on tree boles and in plots can be estimated in order to compare and contrastthe effectsofmanagement (Wolseley). In these tropical forests it is not always possibleto identify lichens to species or to assess lichen rarity, so that the useof associatedcharacters and frequency at several levels has provided a useful wayof assessingchanges in forest condition. From this it is clear that lichens are valuable indicatorsofforest diversity and health, and that there are also taxa associated with niches that arelost on conversion to plantation or agriculture

Crustose lichens grow slowly, mainly in two dimensions and this has been used inlichenometry as a meansof estimating the age of a substratum since exposure.McCarthy describes simple techniques for using this method. The processingofphotographic images has been widely used to monitor changes in individual thalli withtime, but the adventof the digital camera and computerised image handling andprocessing has dramatically altered the useofthese images. Purviset al. describe theiruse to measure area and quantify more precisely changes in lichens within a quadrat.The advances in molecular science have also had an effect on our understandingofthedual nature of lichens and genomic structure of populations which may relate to theirsurvival in the ecosystem. A provenance clone experimentundertaken onLobaria

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pulmonaria is described by Walser and Scheidegger. This project shows how much westill have to learn about lichen genetics that may relate to conservationofrare and localspecies.

Red Lists for lichens are still far behind those devised for flowering plants or largerfauna, but once rare or vulnerable species are identified, there is now a statutoryobligation to monitor the existing populations in many countries. For lichens this maypresent recorders with many problems ranging from inaccessibilityof the site, todefining an individual or a population, and then estimating changes over time. A verysimple methodof assessing changes in a populationof Teloschistes described byWolseley and James, can be used in a varietyofhabitats and scales. Red listed speciesexist in association with other species which may be (or become) common or invasive,so that it is important to assess changes in the whole lichen community if we are todetect changes that may be associated with lossofa vulnerable or endangered species.Aptroot and Sparrius describe a project in the Netherlands to monitor all speciesassociated with Red listed species in permanent plots. Lichen communities may respondrapidly to shifts in climatic conditions and Insarov describes a method for surveyinglichens in areasof natural vegetation, where other factors are minimised, to detectlarge-scale changes in climate.

Much ofthe methodology included in this section has beenpublishedas accounts inpapersofjournalsof restricted circulation or in unpublished reports. We hope that thepublicationofthese practical accounts will stimulate the wider useoflichen monitoringand its application to a range of environmental problems, and in geographical areaswhere there is as yet little or no environmental monitoring. The widespread occurrenceof lichens enables their use as biomonitorsof environmental conditions, but it shouldnot obscure the fact that loss of habitat puts manyof the more specialised lichens atrisk, and that the monitoring of these species is an important aid in understanding andconserving biodiversity. In areas where rapid lossofhabitatis taking place there is anurgent need to establish basic surveillanceofboth species and environmental conditionsin order to develop appropriate monitoring of lichens and lichencommunities. Thismay present a taxonomic problem that we cannot underestimate, and it will hamper thecontinuing developmentofbiomonitoring with lichens, as well as our assessmentofanimportant componentof global biodiversity. Monitoring programmes require acontinuing effort of on-site observance and recording, for which the trainingof localobservers is a prerequisite.

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MAPPING LICHEN DIVERSITY AS AN INDICATOR OF ENVIRONMENTALQUALITY

1. ASTA 1, W. ERHARDT 2

, M . FERRETTe , F. FORNASIER 4, U.

KIRSCHBAUM s, P. L. NIMIS 6, O. W . PURVIS 7

, S. PIRINTSOS 8, C.

SCHEIDEGGER 9, C.VAN HALUWYN IO

, V .WIRTH II

(Lab. EcosystemesUniv., 2233 r. Piscine BP 53X, F-38041 Grenoble.2UMEG, Grossoberfeld 3,D-76135Karlsruhe.3LINNAEA AmbienteSri. Via G. Sirtori 37./-50137 Firenze.4ANPA. via Brancati28./-00100 Roma.SFachhochschule Giefien-Friedberg, Wiesenstrasse 14, D-35390 Giej3en.6Dept. Biology, Univ. Trieste. via Giorgieri 10./-34127Trieste.7The NaturalHistoryMuseum. CromwellRoad, UK-SW75BD London.8Dept. Biology.. Univ. Crete. PiO'Box 2208. GR-71409Heraklion.9WSL. Swiss Fed. ResearchInstitute, CH-8903 Birmensdorf.IOLab. Botanique, Fac Sc.Pharmac. Biologiques. B.P. 83, F-59006Lille.IIStaatl. Museumfiir Naturkunde, Erbprinzenstr. 13.D-76133Karlsruhe.

In the last decades, several methods were proposed for assessingenvironmentalquality- mainly air pollution - on the basisof lichen data (seechapter4, this volume). At theend of the 80s thepredictivityof 20 different methods with respect to instrumentalpollution data was tested in Switzerland using multiple regression [I, 5]. The highestcorrelation was found with the sumof frequenciesof lichen species within a samplinggrid of 10 units positioned on the trunksof free-standing trees. This method wasimmediately and widely adopted in several other countries, esp. Italy and Germany,with some modifications, chiefly concerning the sizeof the sampling grid. Since 1987,hundredsofstudies were carried out with this approach, which led to itsstandardizationin the formofguidelines both in Germany [13], and in Italy [7].

The bioindicationmethod presented here is largely based on the Swiss approach [1,5], and on the German and the Italian guidelines [7, 13], with several importantmodificationswhich were agreed upon during a meetingof the authors in Rome(November 2000), sponsored by the Italian National Agency for theEnvironment(ANPA). The main modifications concern several elementsof subjectivityin thesampling process which were present in bothof the original guidelines. They regardmainly the locationofsampling sites, the selectionofsample trees, and thepositioningofthe sampling grid on the trunks (see also chapter 9, this volume).

More attention is also paid to vegetational data as a sourceof information forinterpretinglichen diversity patterns, as in thephytosociologicalapproach largelyfollowed in France [11].

273P.L. Nimis, C. Scheideggerand P.A. Wolseley (eds.), Monitoring with Lichens - MonitoringLichens. 273-279.© 2002 KluwerAcademic Publishers. Printed in the Netherlands.

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1. Aims

• To provide a repeatable and objective method for assessing lichen diversity values(LDV) and for describing vegetation on the barkoftrees, based on the frequencyofoccurrenceoflichen species on a defined portionofthe trunks.

• To outline zones of different environmental quality.

2.Method

2.1.SAMPLING DESIGN

Figure 1. Scheme for shifting thesampling unit when suitable conditionsare not found at the originallyidentified unit (UCP 0). Numbering ofquadrants corresponds to shiftingpriority (after Ferrelli [3]).

2

4

3

56

Several sampling designs are possible, depending on the aimsof the study, on itsgeographical scale, on the characteristics of the survey area, and on the availableresources. As a general rule, any elementof subjectivity must be avoided in theselection of sample trees andof monitoring sites (hereafter referred to as"samplingunits").A survey ofthe effects of environmental alteration calls for an even distributionof sampling units, which can be best obtained by locating them according to ageographical grid (see chapter 9,this volume).The following procedure is suggested:1.Delimit the geographical area to be sampled.2.Carry out a preliminary survey on the availabilityofsuitable trees before deciding on

the tree species and size of the sampling units, in order to avoid many units with notrees.

3. Select a geographicalgrid for obtaining an even locationofsampling units across thegeographical area. Sampling units are located at the intersection pointsof thegeographical grid.

4. Choose a sampling area ranging from0.25 km2 (for large-scale studies) to 1km2 (forsmall-scale studies) which must then be used throughout the survey.The shapeofthesampling unit can be rectangular, quadrat, orcircular.

5. Establish the numberof trees (x) to besampled in each unit according to theavailability of suitable trees (see samplingprocedure), and project requirements.Recommended ranges are 4-8 trees forstudies where a rather imprecise estimateoflichen data is sufficient, 9-12 when a greaterprecision is required.

6. If fewer thanx trees are available in any area,use the following standard procedure to shiftthe unit to be sampled:• Identify the 8 sampling units adjacent to

the original.• Move to the unit north and then clockwise

until the sampling requirementsof x treesare met (Figure 1).

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• In the absence ofx trees in any unit, the sampling unit is omitted.7. When in a sampling unit the suitable trees are more than the number chosen for

sampling, there is the need to ensure a documented and statistically sound selectionof trees. Different procedures can be adopted, provided they are unbiased (non­subjective). Statistically, the best solution would be to select the trees randomly.This, however, may cause several practical problems. Otherwise, the followingprocedure is suggested (see Figure 2):• Define the centre of the sampling unit, and divide the unit into four sectors,• Number the sectors clockwise from 1 to 4, starting from the upper right sector,• First, for each sector look to the three suitable trees which are closest to the

centreofthe unit. Two cases may arise:1) There are at least three suitable trees per sector; this is the ideal situation, and12 trees can be sampled.2) There are some sectors with less than 3 suitable trees and others with morethan 3 trees. In this case come back to the first sector with more than 3 trees andselect new trees until the number of 12 is reached; if not, move to the next sectorand so on, until the number of 12 is reached.

In both cases,the trees closest to the centreofthe unit must be selected.

•4 1

•EB

••• •·0 •3 0 2

00 )0000

e Trees selectedin the rU'5t operation

o Trees selectedin the second operation

o Trees not selectedin the tithen SlIney

@Centre orthe.ampliJ'll'mi.

Figure2.Exampleofselectionoftrees in a sampling unit.

2.2.SAMPLING PROCEDURE

1. Select treesof the same species, or with similar bark properties (e.g. pH, waterstoring capacity, nutrient content) [14]. Selected trees must be free-standing(well­lit), with girths >70 cm and near straight trunks (inclination <100 from vertical).

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Trees subjectto damage ordisturbancefrom liming/fertiliser,grazing animalsetc.shouldbe avoided.

2. Attach the foursamplingladders (each having five IOxiO emcontiguousquadrats)to the trunk at thecardinalpoints, so that the upper edgeofthe ladder is 1.5 m abovethehighestpointof the ground (Figure 3). A shift of 200 max clockwiseis allowedfor individual ladders, to avoid partsof the trunk which are notsuitable forsampling.

3. Avoid thefollowingsituations:• damagedor decorticatedpartsofthe trunk,• parts with knots,• partscorrespondingto seepage tracks after rain,• parts with >25% coverofbryophytes.At least3 laddersofthe grid should be placed, or the tree should bediscarded.

4. Record all lichen species in each ladder and theirfrequencyin the 5quadratsof theladder (nr. of quadrats in which a species occurs). The list of species with theirfrequencyvaluesin a single ladderconstitutesa releveoflichenvegetation.

5. Each sampled tree should begeoreferenced,to permit repetitionsof the study(monitoring).On a local level, the methodproducesreliable results also with areducedset of

species[13]. This,however,is not suggestedhere, in viewof thepotentialapplicationofthis methodthroughoutEurope.

3. Dataanalysis

3.1.CALCULATION OF LICHEN DIVERSITY VALUES (LDVs)

1. Within eachsamplingunit, sum the frequenciesofall lichen species at eachcardinalpoint on each tree (i).Thus, for each tree there are four SumsofFrequencies

SF iN, SF iE, SF iS, SF iW2.Next, for each cardinalpointthearithmeticmeanof the SumsofFrequencies(MSF)

for samplingunit j are calculated:MSF Nj = (SFINj+SF 2Nj+SF3Nj+SF4N j+.....+SFnNj)/n

Where:MSF: Mean ofthesumsoffrequenciesofall treesofunitj at a given cardinal pointSF : Sum offrequenciesofall the species found at one cardinal pointoftree iN, E, S, W: north, east, south, westn: numberofladderswith a given exposure in unitj

The LichenDiversityValue ofsamplingunitj(LDV j) is the sumofthe MSFs ofthecardinal points

3.2. MAPPING LICHEN DIVERSITY

Maps ofLDVs can beconstructedin two different ways:

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1. The sampling units are plotted onto thegeographicalarea map. Their LDV valuesare assigned to classesof lichen diversity (see below). The sampling units arecolouredaccording to the respective class.

s

Figure 3.Sampling grid composed offour ladders each with 5 contiguous quadrats .

2. Programs of automatic mapping can be used. Theseprograms calculateinterpolationsfrom adjoining points and require carefulconsiderationas towhetherthe geomorphologyof the survey area and thesamplingdensity permit the useofsuch algorithms(see chapter 9, this volume).For the subdivision of LDV values into classes which are suitable formappingwe

refer to the VDI guidelines [13].

3.3.DETERMINATION OF ENVIRONMENTAL ALTERATION

• Where regional scalesof deviation from"natural" conditions areavailableLDVresults can be used to assessmagnitudeofalteration (seechapter20, thisvolume).

• In absenceofa regional scale, use thedifferencebetween maximum andminimumLDVs within the survey area to create a scale with which to detect a local patternofenvironmentalalteration,However the magnitudeof deviation from naturalitycannotbe assessed.

3.4.INTERPRETATION OF RELEVE DATA

• Use multivariateanalysis to distinguish groupsof species withsimilar ecological

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behaviour, and groupsof'releveswith a similar floristiccomposition(communities)e.g. nitrophytic/non-nitrophyticto reveal patternsof eutrophication/acidification[12].

• Investigate the effectofaspect on lichen distribution.• Determine local sensitivity values for lichens in relation to atmospheric conditions

to map air quality patterns [4,9, 10].• If available for the survey area, use ecological indicatorvalues,e.g. for pH to reveal

acidification, etc. [2, 6, 8, 14].• Use average frequenciesofselected indicator species in eachsamplingunit to make

distribution mapsofthose species, in the same way as suggested for the LDVs.

4. DataQualitycontrol

• Investigationsperformed according to this method require personnel with thenecessary expertise. Standardsofquality assurance should be followed, and NationalAuthoritiesshould ensure that operators are properly trained and inter-calibrated.

• Several sampling designs are possible, depending on the aimsof the study, on itsgeographical scale, on the characteristicsof the survey area, and on the availableresources. As a general rule, any elementof subjectivity must be avoidedin theselectionofsample trees andofmonitoring sites.

5.Application

This method supplies information on the long-term effectsof air pollutants,eutrophication, anthropization and climatic changes on sensitive organisms.It can beapplied in the vicinityof an emission source to provideproofof the presenceof airpollution and to detect its causes, or, on a larger scale, for detecting patterns and hot­spots of environmental stress. Its repetition at the same sites permits to monitor theeffectsofenvironmental changes.

6. Limitations

In areas where trees are infrequent it is not possible to use this method. LDVs shouldnot be uncritically compared among floristically and climatically very different areas. Inparticularlydry areas (e.g. partsof the Mediterranean region) the method cannot beapplied.

7.References

I. Ammann, K., Herzig, R., Liebendoerfer,1., and Urech, M. (1987) Multivariatecorrelationofdepositiondata of 8 different air pollutants to lichen data in a small town inSwitzerland,in Advances inAerobiology. Birkhauser,Basel, pp. 401-406.

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2. Dietrich, M . and Scheidegger,C. (1996) Diversitatund Zeigerwertevon epiphytischen Flechten derhaufigstenBaumarten: Ein methodischer Ansatzzur Beurteilungvon Umweltveranderungen im Wald undim Freiland,Botan ica Helvetica 106,85-102.

3. Ferretti, M. (2000) Design e procedure di assicurazione di qualita dei dati per una rete nazionale per itritevamento della qualita dell 'aria mediante i'indice di biodivers ita lichenica, Rapporto Finale Inc.ANPA, Roma ,00/AMB/58-3050-75.

4. Hawksworth, D.L. and Rose, F. (1970) Qualitative scale for estimating sulphur dioxide air pollution inEngland and Wales using epiphytic lichens,Natur e 227, 145-148.

5. Herzig,R. and Urech, M. (1991) Flechten als Bioindikatoren. Integriertes biologisches Meftsystem derLuftverschmutzungfur das Schweizer Mittelland, BibliothecaLichenologica43,Cramer,Berlin-Stuttgart:

6. Nimis P.L. (2000) Checklist of the Lichens oj Italy 2.0, Universityof Trieste,Dept.of Biology, IN2 .0/2,http://dbiodbs.univ.trieste.it/

7. Nimis, P.L. (1999) Linee guida per la bioindicazione degli efTetti dell'inquinamento tramite labiodiversitadei licheniepifiti, inC. Piccini and S.Salvati(eds.),Att i Workshop Biomonitoraggio Qualitadell 'Aria sui terr itorio Nazionale, ANPA , Ser.Atti,Roma, pp 267-277.

8. Nimis,P.L.and Martellos,S. (2001)Testingthe predictivityof ecologicalindicator values.A comparisonof real and"v irtual" releves ofJichen vegetation,Plant Ecology (in press).

9. Trass,H. (1973) Lichensensitivity to air pollutionand index of poleotolerance (I.P.),Folia CryptogamicaEstonica 3, 19-22.

10. Van Dobben, H.F. and Ter Braak, J.F. (1999) Ranking of epiphytic lichen sensitivity to air pollutionusing survey data:a comparison ofindicator scales,Lichenologist 31, 27-39.

I I. Van Haluwyn, C. and Lerond,M. (1988) Lichenosociologieet qualite del'air; protocole operatoire etlimites, Cryptogamie, Bryologie et Lichenologie 9, 313-336.

12. Van Herk, C.M. (1999) Mapping of ammonia pollution with epiphytic lichens in the Netherlands,Lichenologis t 31,9-20.

13. VOl (1995). Richtlinie 3799. Blatt l: Ermittlung und Beurteitung phytotoxischer Wirkungen vonImm issionen mit Flechten: Flechtenkartierung, VOl, Dusseldorf.

14. Wirth, V. (1992). Zeigerwerte von Flechten, in H. Ellenberg (ed.), Zeigerwerte von Pflanzen inMiue leuropa, ScriptaGeobotanica18, 215-237.

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IDENTIFYING DEVIATIONS FROM NATURALITY OF LICHEN DIVERSITYFOR BIOINDICA TION PURPOSES

S. LOPPI 1, P. GIORDANI 2

, G. BRUNIALTI 2, D. ISOCRON0 3 and R.

PIERVITTORI 3

IDepartment of Environmental Sciences, University of Siena, Via P.A.Mattioli 4,1-53100 Siena, Italy (loppi@unisUt)2DIP.TE.RJS., University of Genova, C.so Dogali lm, 1-16136 Genova,Italy (serrato@csita .unige.it)3Department of Plant Biology, University of Torino, Viale P.A. Mattioli251-10125 Torino, Italy ([email protected] [email protected])

The results of many bioindication studies can be interpreted in termsofdeviations from"normal/natural"situations (see chapter 1, this volume). The definitionof "normality"or "naturality"is a very difficult one. Here, we shall consider as"natural"those areaswhich are free from heavy anthropization, and from long-distance transportofimportantpollution loads. Species richness, cover and frequencyof epiphytic lichens areobviously different in different bioclimatic areas, which requires the developmentofdifferent interpretation scales. The methodology presented here has been developedwithin the frameworkofa national programme sponsored by the Italian EnvironmentalAgency (ANPA) , to monitor the diversityof epiphytic lichens countrywide, followingthe approach proposed by Astaet al. (chapter 19, this volume).

1.Aims

• To provide a standardised protocol for determining lichen diversity (LD) that wouldoccur in a bioclimatic area (BA) based only on climate and other natural factors(maximum naturality).

• To establish interpretative scales of deviation from naturality in order to compareLD values in different BAs for biomonitoringpurposes.

2.Method

2.1.PROCEDURE

• Establish roughly homogeneous BAs, taking into account the main climaticparameters affecting lichen colonisation, such as humidity, temperature,

281P.L. Nimis, C.Scheidegger and P.A. Wolseley (eds.), Monitoring with Lichens- Monitoring Lichens, 281-284.© 2002Kluwer Academic Publishers. Printed in the Netherlands.

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precipitationetc., and vegetation types such as potential vascular vegetation,distribution patternsofbioclimaticindicator species, etc.

• Determine maximum naturality eithera) using previously collected data or b) newdata collected in natural areas.

2.2.DATA COLLECTION

It is out of the scopeof this chapter to suggest a sampling protocol to measure LDvalues. However, LD data should be collected using a single sampling method or ifmore than one method is used, they should be directly comparable.An example is givenin chapter 19,this volume.

3.DataAnalysis

3.1.DETERMINATION OF MAXIMUM NATURALITY WITH EXISTING DATA

If sufficient LD data (at least 100 releves) are available for each substrate in each BA,the proper interpretative scale for that substrate in that BA can be determined (e.g."Quercus of the dry Mediterranean BA","Quercus of the humid sub-MediterraneanBA", "Tilia ofthe dry sub-Mediterranean BA", etc.).The data set should be statisticallyrepresentative of all LD values for that substrate in that BA, and data stratificationaccording to the rangeofanthropogenic impacts is recommended (e.g.data collected innatural, rural, urban and industrial areas).If the data set does not have a homogeneous(gaussian) distribution (e.g. too many data collected in altered areas), it is necessary toextract a stratified randomly selected sub-sample to achieve a normal distributionofthedata (applicable where original data are more than 100 releves).Prepare a table for each substrate in each BA with:

• Numberofobservations,• Maximum LD value scored,• LD value of the 98° percentileofthe frequency distribution,• Averageof LD values ~ the 98° percentile of their frequency distribution. The

latter is the theoretical maximum naturalityofLD for that substrate in that BA.

3.2.DETERMINATION OF MAXIMUM NATURALITY WITH NEW DATA FROMNATURAL AREAS

In the absenceofexisting data, LD maximum naturality for each substrate in each BAcan be estimated from new data (at least 10 releves) collected in natural areas. Thearithmetic meanofLD counts provides a first approximationof 100% naturality for thatsubstrate in that BA. This value will require progressive updating with ongoingresearch.

Characteristicsof each natural area should be defined, especially in relation toanthropogenic alteration such as industrial complexes, urban areas, vehicular traffic,intensive agriculture, coppiced woods etc., or natural sourcesof alteration such asgeothermal vents, volcanoes, etc. If natural areas are not present in the BA under study,

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data fromsimilaror neighbouringBAs can be used.

3.3. INTERPRETATIVE SCALES

Substratashowing comparablevalues of maximumnaturality(± 10%) in a given BAcan be comparedusing the sameinterpretativescale. Thedifferentclasses of thesescales are based on progressivepercentagesdeviationsfrom thehighest LD values(naturality)obtainedin each BA.A 5-class scale ofdeviationfrom naturalityis suggestedfor large-scalebiomonitoringsurveys(Table I); inmoredetailedstudies, furthersub-classescan be added.

TABLE 1. Scale ofdeviation from naturality.

4.Workedexample

% deviationfromnaturalconditions

10075-9950-7525-500-25

Interpretation

LichendesertAlteration

Semi-alterationSemi-naturality

Naturality

In orderto compareLD values in differentbioclimaticareas of Italy and todevelopinterpretativescalesof deviationfrom naturality, the~ 98° percentileof LD frequencydistributionhas recentlybeenadopted[2]. An exampleis given for Tilia and deciduousQuercus (commonlyused trees in lichenmappingstudies in Italy) from theTyrrhenianBA of Italy (Table 2). The LD wascalculatedas the sumof frequenciesof epiphyticlichensin a samplinggrid of30x50 emdividedinto 10 unitsof 15x10 em,followingthemethodologyproposedby Nimis [3]. Data from bothphorophyteswere combinedsettingat 100naturalityin termsof LD values and a scaleofdeviationfrom naturalitywas estimated(Table3). For theevergreenoak Quercus ilex 36 releves wereperformedin two natural areas. The meanofthe LD valueswas 61.2, so that thenaturalityfor thistree was set to 60. The scaleofdeviationfrom naturalityfor this tree isshown in Table3. These scales wereappliedin Siena (Tuscany,centralItaly) where 10 Tilia and 10Quercus ilex trees weresampledin a single station. The meanLD was 86±13 for Tiliaand 56±17 for Quercus ilex. Accordingly,thestationwas classifiedas 'natural' for bothphorophytes.

TABLE 2. Number ofreleves (N), maximum LD value (Max), LD value ofthe 98°percentile of the frequencydistribution (98°),average ofLD values .? the 98°percentile (.? 98°), for Tilia and deciduous Quercus treesfrom the Tyrrhenian side ofItaly.

TiliaQuercus (dec.)

N15431343

Max132133

8696

100106

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TABLE 3. Scale for the interpretation ofLD values on Tilia,deciduous Quercusand Quercus ilextrees fromthe Tyrrhenian side ofItaly.

lichenAlteration

Semi- semi-naturalitydesert alteration naturality

Tilia - Quercus (dec.) 0 1-25 25- 50 50-75 >75Quercus ilex 0 1-15 15- 30 30-45 >45

5.Data Quality control

• Training and intercalibrationof the operators involved in data collection arenecessary to assess the reliability and consistencyof the data (for detailsofprocedures see [I]).

• Progressive updating of maximum naturality determination with ongoing researchwill provide a meansofquality evaluation.

6.Application

This method can be used widely in roughly homogeneous bioclimatic areas.

7.Limitation

Where existing data are not available, the absenceof specific substrata in natural areasor the absence of natural areas themselves will prevent the formulationof a scaleofnaturality.

8.References

I. Brunialti, G., Giordani, P., Isocrono, D., and Loppi, S. (2001) Evaluation of data quality in lichenbiomonitoring studies:the Italian experience,Environmental Monitoring and Assessment (in press).

2. Loppi, S., Giordani, P., Brunialti, G., Isocrono, D., and Piervittori, R. (2001) A new scale for theinterpretationoflichen biodiversity values in the Tyrrhenian side of Italy,Bibliotheca Lichenologica (inpress).

3. Nimis, P.L. (1999) Linee-guida per la bioindicazione degli effetti dell'inquinamento tramite labiodiversita dei licheni epifiti, in C. Piccini and S. Salvati (eds.),Proc. Workshop "Biomonitoraggiodella qualita dell 'aria sul territorio Nazionale", Roma, 26-27 November 1998, ANPA, Roma, pp. 267­277.

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EPIPHYTES ON WAYSIDE TREES AS AN INDICATOR OFEUTROPHICA nON IN THE NETHERLANDS

C. M. VAN HERK

Lichenologisch Onderzoekbureau Nederland (LON), Goudvink 47, NL­3766 WK Soest, The Netherlands ([email protected])

The present method was developed in 1989, following recognition thatammoniapollution was a major threat to vegetation (forests, heaths, bogs, fens, mires), soil anddrinking water in The Netherlands (seechapter5, this volume). It has been testedextensivelyover the last 12 years in most partsofthe country [5].

1.Aim

To map and monitor the spatial patternsofammonia (NH3) pollution, usingabundanceofselected nitrophytic and acidophytic species.

2.Method

2.1.EQUIPMENT AND MATERIALS

• Ordnance survey map or area with X-Y- co-ordinates,• Tape measure for tree girth.

2.2.PROCEDURE

1. Select 10 roadside treesofthe same species, size and age, with acid bark and well-littrunks, preferablyQuercus robur, but otherQuercus spp., Fagus sylvatica andPinus spp. may be used, depending on the mean levelofNH3 pollution and the areaconcerned. Populus x canadensis, Salix spp. andFraxinus excelsior (trees withslightly acid or neutral bark) may be used at low NH3 pollution rates [5].Ulmus andTilia are not used.

2. Map all trees using X- Y- co-ordinates, and recordtopographicdetails.3. Record habitat type, tree species, tree girth(preferablybetween10-25 dm).4. Record all lichen species or at least the lichensmentionedunder"dataprocessing"

occurringup to 2 m for each tree.5. Recordabundanceofeach species per site using the followingabundancescale:

285P.L. Nimis , C. Scheidegger and P.A. Wolseley (eds.), Monitor ing with Lichens - Monitoring Lichens, 285-289.

© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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(l) only one thallus present;(2) more thalli on one tree(3) present on 2-5 trees, less thanldmvtree(4) present on 2-5 trees,more thanldmvtree(5) present on6-10 trees, less thanldmvtree(6) present on6-10 trees,more than1dmvtree.

In practice only the distinction between class 1, 2, 3 and 5 vs. 4 and 6 is important. Aspecies is considered to cover more than1drnvtreeif this applies for at least two outoften trees, and the total cover is at least10 dm2at all ten trees together.

2.3.DATA COLLECTION AND HANDLING

Species are scored as nitrophytes (reacting positively to NH3) or acidophytes (sensitiveto NH3) .

The following lichens are considered to be nitrophytes:Caloplaca citrina, C.holocarpa, Candelariella aurella, C. rejlexa, C. vitellina, C. xanthostigma, Lecanoramuralis, L. dispersa (incl.L. hagenii), Phaeophyscia orbicularis, P. nigricans, Physciaadscendens, P. caesia, P. dubia, P. tenella, Rinodina gennarii, Xanthoria candelaria,X calcicola, X parietina andX polycarpa.

Lichen species scored as acidophytes are:Cetraria chlorophylla, Chaenothecaferruginea, Cladonia spp. (all taken together),Evernia prunastri , Hypocenomycescalaris, Hypogymnia physodes, H. tubulosa, Lecanora aitema, L. conizaeoides, L.pulicaris, Lepraria incana, Ochrolechia microstictoides, Parmelia saxatilis,Parmeliopsis ambigua, Placynthiella icmalea, Platismatia glauca, Pseudeverniafurfuracea, Trapeliopsisjlexuosa, T. granulosa and Usnea spp.(all species).

Total nitrophytic species are included in NIW (derived from Dutch"NitrofieleIndicatie Waarde") and total acidophytic species are included in AIW ("AcidofieleIndicatie Waarde") .

NIW (and AIW) are defined as the mean numberof nitrophytic (or acidophytic)species found per tree, thus results from all trees are added and the average number ofnitrophytes/acidophyteson one tree is calculated for each site. Species covering morethan 1 dm2(abundance class 4-6), however, count as double at all trees where present.Note that common species present on all trees add much more to NIW or AIW(2.0points) than species present in small numbers on one outoftentrees only(0.1point).

3.Worked example

Over one year at c.100 sites measurements of ambient NH3 air concentationwerecarried out; and the results were compared with the lichen composition [7]. A strongcorrelation between NIW and NH3 became apparent (Figure 1). There was nocorrelation between NIW and sulphur dioxide, probably due to the very low mean S02level (ca.51lglm3) at present in the Netherlands.

Approx.50% ofthe Netherlands has been mapped using NIW and AIW.Large scale

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NIW patterns[5, Figure 1] as well as detailed patterns in NIW [5, Figure 1] show astriking resemblancewith respect toammoniaair concentrationcalculations[2] and topatternsbased oncorroborativeinformation e.g. distance to livestock farms.

403510 15 20 25 30

ammonia concentration (average/5x5 knr)

e/~'~

Va

.//=?

Ve

I- -o

o

10

12

Figure 1. The Nitrofiele Indicatie Waarde (NIW) as a function ofNH3concentration (llglm3, mean/yr), bothaggregated to an average per square of5 x 5 km', Linear regression analysis is performed. Total number ofsquares: 21, total number ofsites: 104. Variance (~) accountedfor by all squares: 89.8% (p<0.05).

NIW and AIW methods revealedcomplementaryresults, i.e. where NIW is high,AIW is usually low and reverse(r= -0.64, p< 0.0001). High NIW values (NIW 10-12utilising Q. robur) were only recorded in areas with a high cattle density, e.g. areaswhere largenumbersofpigs are kept in stables all year round. High AIW values (AIW10-12, Q. robur) appearedto be restricted to sites at least 10km distance from largecattleconcentrations. Between 1989-1999 mean NIW valuesincreasedfrom 2.7 to 3.9and mean AIW decreasedfrom 2.8to 2.0 [7]. Changes inindividualspecies between1990 and 1995 are described by van Herk and Aptroot [9]. Results are now used by theDutch governmentfor an emission reductionprogramme.

A cycle of5 years formonitoringpurposesseems to beappropriate,as many speciesappear to show distinct changes during this time. For example, in an areamappedin1994 and 1999 with 140 lichen speciesoccurringin 688 sites, 51 lichenspeciesshoweda significantincrease (Wilcoxen Matched Pairs) and 9 species showed asignificantdecline [6].

Other results include the descriptionof several species new to science, and thedistinctionbetweenPunctelia subrudecta s.str.andP. ulophylla [1, 8, 10].

4. Data Quality control

The ecologicalbehaviourof the speciesmentionedin paragraph2.3was checkedwithmultiple regressionanalysis [3, 4] using NH), NH4 (both following [2]),S02' N02(measurementstaken from National Instituteof public Health andEnvironmental

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Protection, RIVM) and tree girth as independent variables. The nitrophytic speciesshowed a significant positive correlation with NH3 the acidophytic species showed asignificant negative correlation with NH3. Some species occurring at very lowfrequencies were excluded from the statistical treatment, but as they appear to occuronly in sites with very high NIW (nitrophytes) or very high AIW (acidophytes) they areincluded in the indicator list. The abundance values of four common species(P.orbicularis, X parietina, H. physodes and E. prunastri) at different levelsof NH 3

pollution are shown in Van Herk [7].

5. Application

Widely applicable in countries where wayside trees are present, although it may benecessary to specify other tree species (e.g.Pinus in the Mediterranean), or to use otherappropriate indicator lichen species.

As all species are recorded on each tree, this method may be used:• to monitor the effectsofS02;• to obtain base-line information for air pollution studies;• to calculate trends for all common species; and• to monitor threatened (Red Listed) lichens.

When using lAP-like methods, this method may be implemented relatively easily byusing the grid counts as a measure for the species' frequencies insteadofthe frequencieson a rowof 10 trees. A modified application in twig increment studies[II] may bepossible as well.

6.Limitations

This method is dependent on the presenceofwayside trees that are not affected by localconditions, e.g. dunged trees, trees close to farms, or trees with bark wounds. In built­up areas dust, street dirt and dogs may obscur results. Exhaust fumes have so far notshown to obscure results, but some care must be exercised in urban areas, especiallywhen tree species with neutral or basic bark are used. Trees in dense forest standsshould be avoided. Strongly heterogeneous sites (young and old trees taken together,big differences in exposure to wind and sunlight) will give erratic results.

Only results obtained from a single tree species can be compared to each other. Ingeneral, trees with basic bark have more nitrophytic species than those with acid bark.The reverse holds for acidophytic species. Differences between tree species may becalculated when NH) air pollution levels are known.

The method may be used in atlantic, continental and boreal climate regimes. In thewarm and dusty climatesof the Mediterranean regions nitrophytic species may be anatural constituentoflichen communities on acid bark.

7. References

I. Aptroot, A. and Van Herk, C.M. (1999) Lecanora barkmaneana, a newnitrophiloussorediatecorticolous

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lichen from the Netherlands,Lichenologist31,3-8.2. Asman, W.A.H.and Van Jaarsveld, J.A. (1990)A variable-resolution statistical transport model applied

for ammoniaand ammonium, RIVM report no.228471007, Bilthoven.3. Van Herk, C.M. (1991) Korstmossen a/s indicator voor zure depositie, Basisrapport, Provincie

Gelderland,Amhem.4. Van Herk, C.M. (1993) Korstmossen en zure depositie in Drenthe en Friesland, Hoofdrapport, Provincie

Drenthe& Provincie Friesland, AssenlLeeuwarden.5. Van Herk, C.M. (1999) Mapping of ammonia pollution with epiphytic lichens in the Netherlands,

Lichenologist31, 9-20.6. Van Herk,C.M .(2000)Monitoringvan ammoniakmet korstmossen in Overijsselin /999, LON onbehalf

of Provincie Overijssel, Zwolle.7. Van Herk, C.M. (2001) Bark pH and susceptibility to toxic air pollutants as independent causesof

changes in epiphytic lichen composition in space and time,Lichenologist33 (5), in press.8. Van Herk, C.M. and Aptroot, A. (1999) Lecanora compallens and L. sinuosa, two new overlooked

corticolous lichen species from western Europe,Lichenologist 31,543-553.9. Van Herk, K. and Aptroot, A. (1998) Recoveryofepiphytic lichens in the Netherlands,British Lichen

Society Bulletin82, 22-26.10. Van Herk, K. and Aptroot, A. (2000) The sorediatePunctelia species with lecanoric acid in Europe.

Lichenologist32, 233-246.II . Wolseley, P.A.and Pryor, K .V. (1999) The potential of epiphytic twig communities onQuercuspetraea

in a Welsh woodland site (Tycanol) for evaluating environmental changes,Lichenologist31,41-61.

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USING LICHENS ON TWIGS TO ASSESS CHANGES IN AMBIENTATMOSPHERIC CONDITIONS

P.A.WOLSELEY

TheNatural HistoryMuseumDepartmentofBotany. CromwellRd.London SW7 5BD. UK ([email protected])

This method was first developed inPembrokeshireWest Wales [3, 4] in order todeterminethe effectofchanges in land use on epiphytic lichencommunitiesin regionswhere native trees are frequent.

1.Aims

• To use lichen communities on twigsof deciduous trees to assessenvironmentalconditions and changes with time.

• To provide a standardised sampling procedure to comparecolonisation andestablishmentoflichens in different locations.

• To test the associationoflichenswith surrounding environmental conditions.

2.Method

2.1.EQUIPMENT AND MATERIALS

• 20 m tape,• labels and markers,• recording sheets,• xl0 hand lens or magnifying glass,• Ordnance survey mapofselected area.

2.2.PROCEDURE

1. Select siteswithin an area where either a rangeoflandmanagementpracticesare inoperation or across a rangeofenvironmental gradients andgeographicalareas [3,4].

2. Select a boundary with accessible and exposed twigs (preferablyof the same treespecies) where a 20 m length can be established.

3. Use a tape to delimit 20 m on the ground beneath the canopy margin(if revisits areplanned use aconvenientpermanent marker).

291P.L. Nimis, C.Scheidegger and P.A. Wolseley(eds.), MonitoringwithLichens- MonitoringLichens, 291-294.© 2002 Kluwer AcademicPublishers. Printed in the Netherlands.

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4. Use random numbers to establish 10 points along the 20 moftape, select the nearestbranch vertically above the point, and attach a label to allow identificationof theproximal twig.If a twig is selected twice,discard it and use the next random numberso that 10 different twigs are selected for sampling. Indicate twigs associated with anindividual tree and record bark pH with a surface electrode wherepossible.

5. Mark the 5thgirdle scar on the main lichen twig, excluding side branches (Figure I).

~,/girdle scar side branch~ &. lenticels / .

~I~~-~-- _g'zscar---- , .. •••. • :-;-~ ---T~£neyea~s:ro~th~ --"-~-

Figure I. Marking the 5'h girdle scar on a twig.

6. Starting at the older 5+years partof the twig (older lichens are easier to identify),record the lichen present by species, including other epiphytes such as the green algaDesmococcus, bryophytes and fungi (without a lichen thallus). Record lichens on 1­5 years checking the young bark for signsof endophloedal species where onlyfruiting bodies are apparent. Where initial stages are not possible to identify recordas genus e.g.Usnea sp.,Hypogymnia sp.Parmelia sp.

7. Identify specimens using [5] or NHM websitewww.nhm .ac.uk/ botany/lichen/twig.Recording forms and sulphur and nitrogen tolerant scales are included on the latter.

8. Record environmental conditions including boundary type, aspect, altitude, exposureand land use (including woodland, arable,pasture, farmstead, road, houses, industry)in four compass directions.

9. Remove branch labels.

2.3.DATA COLLECTION AND HANDLING

I . For each site use records for 10 or more branches with 2 or more samples (annualincrementoftwig substrate or groupsofincrements e.g.1-5,6+).

2. Assess species diversity by site, by branch, and by sample unit, and speciesfrequency within a site.

3. Enter data and perform multivariate analysis to identify clustersof associatedspecies within the species matrix. Using Principal Co-ordinate Analysis (PCO) andMulti Dimensional Scaling (MDS) superimpose environmentalparameterssuch aspollution or climate data on ordination to determine factors affecting lichencommunities in sites and on branches.

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3.Workedexample

Lichen data collected fromQuercus petraea twigs sampled along woodland marginsadjacent to 4 areas with different management regimes were analysed usingpea onGENSTAT [I], and showed: -greatest dissimilarity on the first axis between samplesfrom an undisturbed woodland glade margin(Usneetum subfloridanae dominant) andsamples from old pasture(Physcietum adscendentis dominant). Samples from themoorland edge fall in between these groups. Samples from the woodland edge adjacentto improved pasture extended along the 2nd axis showing some similarity with otherexposed moorland and pasture margins (Figure 2).

0.60 R

0."

0.30

.-.0,16 ~~~)

0.00

~.15

-0.415 -

-0.60 ,_ . _ .._L .... .L-._.l-_ L-0.60 -0 .45 -O.SO -0.15 0.00 0.15 0.30

Figure 2. peA oflichen datafrom Quercuspetraeatwigs on woodland glade margins (b). moorland edge (a),old pasture (d). and improved grassland (c)(from [4J).

Two Way Indicator Species Analysis (TWINSPAN) was performed on a data set fromtwigs along a transect through agricultural and urban areas of Pembrokeshire [2].Results showed communities with indicator species characteristicofyoung twigs in lessimproved areas withArthopyrenia punctiformis and Cyrtidula quercus distinguishedfrom those in intensive agriculture dominated byScoliciosporum chlorococcum andPhyscia tenella, and thoseofmore eutrophic pasture margins withXanthoria parietinaand Physcia aipolia distinguished from acid uplands withHypogymnia physodes andEvernia prunastri.

4.DataQualitycontrol

Lichens on twigs may present an additional problem in that they are not always easy toidentify in their early stages when no fruiting bodies are present. Reference materialneeds to be collected and identified in the lab. This method has been developed by

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lichenologists in the UK, and species includedIn the publication and website areappropriate to the westofBritain.

5. Application

In many parts of the UK hedgerows, isolated trees and woodlands are still an integralpart of the countryside and are adjacent to a range of land management types.Graveyards usually have trees or hedgerows available and are publicly accessible.It isalso appropriate in areas where lichens are still frequent or are recolonising as on theedge of conurbations. Permanent site markers allow conditions to be monitored alongthe same boundary, even if twigs are lost in winter storms or eaten by grazing animals.

6.Limitations

• In areas where a lichen desert has been created by past activitiesofeither pollutionor land management the study may be unrewarding due to the absenceof lichenpropagules. However many urban areas that were formerly lichen deserts are nowcolonised by lichens tolerant of eutrophication, and these areas may produceinteresting results.

• Availabilityof native tree species in a wide rangeof conditions with accessibleedges that allow sampling of exposed twigs.

• Availabilityofnear-natural conditions as a control site e.g. woodland glades that areprotected from adjacent environmental conditions of agriculturally managed land.

7.References

1. Genstat 5 Committee (1993)Genstat 5 Release Reference Manual. Clarendon Press, Oxford.2. Malloch,AJ.C. (1985) VESPAN: Fortran Programsfor Handling and Analysis ofvegetation and Species

Distribution. University of Lancaster, Lancaster.3. Purvis, O.W., Wolseley, P.A., Reed, M.E ., Wilson, PJ., and James, P.W. (1998)Monitoring of lichen

communities as indicators ofair quality in Pembrokeshire, Report to Texaco Ltd,GulfOil (Great Britain)Ltd andElf Oil (UK) .

4. Wolseley, P.A. and Pryor, K.V . (1999) The potential of epiphytic twig communities on Quercus petraeain a Welsh woodland site(Tycanol) for evaluating environmental changes,Lichenologist 31, 41-61

5. Wolseley, P.A.,James, P.W.,and Alexander, D. (2001) Key to lichens on twigs. AlDGAP Field StudiesCouncil Publications, Montford Bridge, Shrewsbury, SY4lHW .

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GUIDELINES FOR THE USE OF EPIPHYTIC LICHENS AS BIOMONITORSOF ATMOSPHERIC DEPOSITION OF TRACE ELEMENTS

R. BARGAGLI ' and P.L. NIMIS 2

IDipartimento di Scienze Ambientali, Universita di Siena. Via P.A.Mattioli 4,I-43100 Siena. Italy ([email protected])2Dipartimento di Biologia, Universita di Trieste, Via Giorgieri 10, I­34127 Trieste, 1taly ([email protected])

1.Aim

To set out guidelines for sampling and preparation of autochtonous epiphytic lichensfor the analytical determination of trace metals, which may be used to enhance thescientific and legal acceptabilityoflichen biomonitoring and its cost efficiency. Mostofthis chapter is based on the Italian guidelines proposed by Nimis and Bargagli [9].

2.Procedure

2.1. SAMPLING DESIGN

Sampling must ensure that:• each lichen thallus should have the same probability of being selected from a total

populationofthe same species,• collected samples suffer no alteration in chemical composition from the original

population.To achieve representative sampling, sampling sites may be located according to anappropriate sampling plan, which should minimize field time and laboratory expenseswithout sacrificing important statistical information. We refer chapter 9, this volume,for assistance with sampling design which is suited tobioaccumulationstudies.

2.2. TAKING SAMPLES

1. Sample fruticose or foliose broad-lobed species only. Depending on thelatitude/altitudeof the study area, climate, and the levelsof atmosphericmycophytotoxic pollutants, the most frequently used species in Europe are:Parmelia spp., Xanthoria parietina, Hypogymnia physodes, Pseudeverniafurfuracea, Parmotrema spp. Fruticose lichens (e.g.Ramalina and Usnea) havebeen used more rarely, such as in Israel [6].

295P.L. Nimis, C. Scheidegger and P.A. Wolseley (eds.). Monitoring with Lichens - Monitoring Lichens. 295-299 .«:l2002 Kluwer Academ ic Publ ishers. Printed in the Netherlands.

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2. Use epiphyticspecieswheneverpossible, because tree bark provides arelativelyhomogenoussubstrateand the thalli are rather looselyconnectedto it.

3. Sample the same species within one survey, as there isevidenceof interspecificvariation in theabsorptionofsome metals [10].

4. Sample within a relatively short time period, and at least a week after heavyprecipitations.

5. Collect from trees which satisfy the following conditions:• trunks with inclination not higher than 10°,• withoutevident signsofdisturbance,• surfaceswithoutstem-flow tracks,• far from woundsofthe bark,• with growthofbryophytesnot higher than 25%

6. Sample lichens from all around the trunk, unless the scopeofthe study is to detectthe effectsof wind in the dispersionof metals, in which case anappropriatesampling design should be developed.

7. Sample at a heightof more than 1 m above the ground, to avoidterrigenousandcaninecontamination.

8. Detach thalli with a steel knife and place them inenvelopesof metal-freefilterpaper.

9. Sample at each station at least six individual thalli and from at least threedifferenttrees, to obtain a mixture reflecting the averagecontaminationofthe site.

10. Record for each station, the following information:• exact locationofthe station andofthe single trees,• speciesoftree(s)andoflichen,• circumferenceofthe trees at breast height,• diametersofthe sampled thalli,• health conditionsof the sampled thalli (1: decoloration,2: mechanical

damages, 3: presenceofnecrotic parts),• soil type and/or land use.

2.3.PREPARATION AND ANALYSIS OF SAMPLES

1. Air dry samplestransportedto the laboratory in a cleanenvironment(residualwatercontentof 2-3%). If a desiccatoris used, thetemperatureshould not exceed40°C to avoid the lossofvolatile substances.

2. Store samples inpaperbags for the shortestpossible time afterbeing air-dried.Two basic practical rules are:

• to avoidcontaminationof samples (with theequipment,containersof bypersons),and,

• to avoid anyvolatilisationof chemical compoundsas a resultof microbialactivity oroverheating.

3. Sort dry lichen samples under a binocularmicroscopeto removesenescentor deadtissues andextraneousmaterials such as piecesof bark, other lichen species,mosses and large soil particles. Samples should not be washed as there isevidencethatwashingmay result in the lossof not only particles but alsoabsorbedcations

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(seechapter6, this volume).4. Select for analysis the clean peripheral partsof the sampledthalli (e.g. to 2 mm

from the margin inXanthoria, to 4 mm in largeParmelia species), as there isevidence that the older (central) partsof the thalli tend to have higherconcentrationsof most metals [3, 10]. The numberof sampledthalli should beenoughto obtain, after this operation, at least 200 mgofmaterial.

5. Homogenise to obtain arepresentative(homogeneous)sample for analyticaldeterminations. There are nostandardisedproceduresfor samplehomogenisation;in general, these depend on thequantity/numberofsamples to behomogenised.Asgrindingand milling may lead tocontaminationof samples, stainless steel, agata,zirconium oxide and Teflon devices should be used.

6. Store dried andground lichen samples in brown glass bottles or in clean Tefloncontainers(provided with a Teflon insert topreventmoisture uptake),in theabsenceof light. Often samples may fractionateaccordingto grain-size,and thebottles should be shaken before takingsubsamplesfor analyticaldeterminations;still better, a Teflonball should be put into eachcontainerwhen initially filled, tofacilitatesubsequentre-hornogenisation,

7. Determineelementconcentrationsin samples digested with a smallamountofreagents(ofhigh purity grade), in closed Teflon vessels (the ratioofvessel surfaceto sample should be as small aspossible) at medium or highpressurewithmicrowaveheating.

8. Element concentrations should be determined by atomic absorptionspectrophotometry, multi-elementtechniques such as ICP-AES and ICP-MS orInstrumentalNeutronActivationAnalysis (INAA), using standardprocedures(seechapter25, this volume).The useofa standard reference material (e.g.CRM 482­BCR, European Commission)is recommended[1, 2, 7].

2.4. DATA ANALYSIS AND INTERPRET AnON

1. Performmultivariateanalysis(classificationandordination)ofthe matrixofmetalsand stations to detect clusters of metals with the samepatternsof deposition,clusters of stations with similarcontamination,and eventual gradientsofcontamination.

2. Detect provisional correlationsbetween metalconcentrationswith simple linearregression;correlationsmay beeventuallyindicativeofa commonorigin.

3. Only use programs for automaticmapping of patternsof metal concentrationswhen thegeomorphologyof the survey area is flat, and thesampling density ishigh enoughas tojustifyextrapolationfrom a sampling point to another.

4. Compare metal concentrationsin lichens withbackground values in order tointerpret data in termsof environmentalalteration.An example of a scale ofnaturality/alterationdevelopedfor Italy by Nimis& Bargagli [9] is in Table 1.

5. When backgroundvalues are not available, express the results asdeviationsfromthe minimum value within the survey area. In this case, however, thesamplingdesign should include some stations located far frompollutionsources, in order toobtain baseline values.

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6. In interpreting the results, clearly identify and distinguish anyperturbationsandtrends attributable to human impact from intrinsic biological variability and/orinputsofelements from natural sources.

TABLE 1. Scales proposed by Nimis and Bargagli [9] for interpreting metal concentrations (I'-g/g dry wt.} inlichens in terms ofenvironmental alteration (deviation from natural backgrounds) . The scales are based onthe statistical analysis ofthousands ofmeasurements referring to Italy.

Al As Ba Cd Cr Cu Fe Mn Ni Pb V Zn1- VerYhieh naturalitv <350 <0.2 <3.3 <0.2 <1.2 <7.0 <290 <20 <1.0 <4.0 <0.63 <30

- Hizh naturalitv 600 0.6 6.0 0.4 2.2 10.0 500 25 2.0 10.0 1.7 40- Middle naturalitv 1000 1.2 10.0 0.8 4.0 15.0 800 35 3.0 25.0 3.1 65- Low nat./alteration 1600 1.9 18.0 1.4 6.0 25.0 1200 60 5.0 55.0 5.1 94- Middle alteration 2500 2.4 25.0 2.0 9.0 34.0 1500 90 6.0 80.0 6.7 115- Hizh alteration 3200 3.0 35.0 2.6 16.0 53.0 1800 140 8.0 108.0 9.3 155- VerYhizh alteration >3200 >3.0 >35.0 >2.6 >16.0 >53.0 >1800 >140 >8.0 >108.0 >9.3 >155

Vlax Italv 8390 5.53 787 9.04 60.5 161 4276 685 34.4 494 15.00 358

3.Limitationsand applications

1. Limitations in the proposed method are that it cannot be used:• in areas with a scarcity of free-standing trees,• in very polluted areas with scarcityofmacrolichens,• to extrapolate the deposition rateof metals from metal concentrations in

lichen thalli.2. Applications are in the:

• rapid production at low costsof a deposition map with a high samplingdensity,

• detectionofdeposition patterns and high-spots of metal deposition,• monitoringofmetal deposition in time, at intervalsofalleastone year.

3. Advantages include reduction in data variability by:• using only epiphytic lichens (reductionofbias introduced by differences in

the substrata),• useofthe same species (no variability between species),• useofa mixtureofthalli (reductionofintraspecific variability),• sampling under standard conditions (reduction of variability due to micro­

environmental conditions),• sampling of the peripheral partsofthe thalli only (reductionofwithin-thallus

variability in metal contents),• the useof only foliose and fruticose lichens ensures easier sampling and

preparation, as they have less contact with the substrate than crustose lichens.For use of saxicolous or epigeic lichens (e.g. C/adonia, Cetraria andUmbi/icaria) to monitor airborne metal deposition in Arctic and Antarcticregions see references [4, 5, 8].

4.References

I. A1fassi,Z.B.(ed.)(1994) Determination ofTrace Elements. VCH, Weinheim.2. Anders, a.v.and Kim, J.I. (1977) Representative sampling and the proper useof reference materials,

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Journal ofRadioanalytical Chemistry 39, 435-445.3. Bargagli, R. (1998) Trace Elements in Terrestrial Plants. An Ecophysiological Approach to

Biomonitoring and Biorecovery, Springer-Verlag,Berlin.4. Bargagli, R.,Sanchez-Hernandez, J.C.,and Monaci, F. (1999) Baseline concentrationsofelements in the

Antarctic macrolichenUmbilicaria decussata, Chemosphere 38,475-487.5. France, R. and Coquery, M. (1996) Lead concentrations in lichens from the Canadian high Arctic in

relation to the latitudinal pollution gradient,Water, Air, and Soil Pollution 90, 469-474.6. Garty, J. and Hagemeyer, 1. (1988) Heavy metals in the lichenRamalina duriaei transplanted at

biomonitoring stations in the region of a coal-fired power plant in Israel after 3 years of operation,Water, Air and Soil Pollution 38, 311-323.

7. Markert,B. (1996) Instrumental Element and Multi-Element Analysis of Plant Samples . Methods andApplications , John Wiley& Sons, Chichester.

8. Nieboer, E. and Richardson, D.H.S. (1981) Lichens as monitorsof atmospheric deposition., in S.J.Eisenreich (ed.),Atmospheric Pollutants and Natural Waters, Ann Arbor Sci., Ann Arbor, pp. 339-388.

9. Nimis,P.L.and Bargagli, R. (1999) Linee guida perl'utilizzodi licheni epifiti come bioaccumulatori dimetalli in traccia, in C. Piccini and S. Salvati (eds.),Atti Workshop Biomonitoraggio Qualita dell 'Ariasui Territorio Nazionale, ANPA, Ser .Atti 2, Roma, pp. 279-289.

10. Nimis, P.L., Andreussi, S., and Pittao, E. (2001) The performanceof two lichen species asbioaccumulators of trace metals,The Science ofthe Total Environment , 275,43-51.

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TRANSPLANTED LICHENS FOR BIOACCUMULATION STUDIES

1.MIKHAILOV A

Institute ofPlant and Animal Ecology UD RAS, 8 Marta Str., 202, 620144Ekaterinburg, Russia ([email protected])

1.Aims

• To assess pollutant deposition and/or accumulation over a fixed exposure period.• To assess temporal variation in pollutant deposition and in theaccumulation

capacity of lichens.• To compare the accumulation capacityoftransplanted and indigenous thalli in order

to reveal possible adaptationsofthe latter to polluted environment.• To determine threshold levelsof toxic substances in a thallus (by simultaneous

assessmentofmorphological,physiological and ultrastructural damage).

2.Method

Transferofhealthy thalli from unpolluted localities to polluted ones by two methods:• (A) Transplant thalli in good health to habitats which are similar to those where theywere collected, taking care to keep their vitality.• (B) "Lichen -bag" method, where lichens are transplanted regardlessoftheir vitalityand adaptation to new habitats.

Transplantationstudies usually involve species which are easily collected,transplanted and observed,i.e. relatively large foliose (Hypogymnia or Parmeliaspecies) or fruticose (genusRamalina) lichens. Generally, epiphytes are used morefrequently, though epigeic and epilithic lichens have no seriousdisadvantages. Epigeicspecies(Cladonia , Cladina) are more suitable for (B) than for (A) technique.

2.1.COLLECTION OF SAMPLES FOR TRANSPLANTATION

In collecting samples, the following points are important:• (A) Collect lichens together with their substrate using a sharp knife or drill, but

avoiding damage to thalli [4, 6]. Epiphytes can be collected on twigs or bark pieces.• (B) Collect lichen thalliofsimilar sizeofthe selected species without any substrate.• Collect thalli from the same substrate, as its chemicalcompositioncan influence that

oflichens.• Collect enough material for the planned numberof replicates x numberofsampling

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dates, and allow 10% extra to cover losses not related to pollution (damage byanimals, etc.). In addition, allow for determinationof baseline chemicalcomposition.

• Minimise the period between collecting and transplanting as much as possible(preferably, no longer than 2-3 days). Samples must be kept air-dried in bags toprevent any contamination.

2.2. TRANSPLANTA nON

• (A). Samples of epiphytic lichens can be attached to natural vegetation (trees,shrubs) or to artificial posts. The latter way is recommended when no suitable hosttrees can be found. Any kind of plastic string is appropriate for attachmentoftwigswith lichens. Non-toxic, water-resistant glue must be used to fix transplants on barkpieces to a new phorophyte.Any metallic meansofattachment should be avoided.Ifartificial posts are used, insert bark "plugs" onto respective recesses in woodenplates attached to posts [4, 6]. Samples of epilithic lichens should be attached to therespective rocks.It is important to transplant thalli into microclimatic and ecologicalconditions similar to the original ones (including type of substrate - for epiphytes,the same tree species - height above ground, orientation, etc.).

• (B) Place thalli in bags of plastic net or in perforated plastic containers [1].Containers can be established on the ground, and bags can be hanged on trees.For both A and B, the thalli should be transplanted also in the original collection site

to estimate the effect of the transplantation procedure.

2.3.COLLECTION OF TRANSPLANTS FOR THE ANALYSES

Determine the appropriate exposure time considering the aimof the study, thecomposition and rate of emissions, and the expected lichen damage. In order to detectpollutant fall-out a single transplant for a short period is appropriate. Other aims mayrequire repeated transplants for a short period each, or single transplants for longperiods with multiple collections. Usually, exposure periodsof 1-3 months are suitable.Long exposure periods are not recommended as they can lead to the death of thalli andtheir subsequent loss due to detachment from the substrate. The first samples should betaken not later than a month after starting the experiment. Samples must be immediatelyair-dried and transported in bags avoiding any contamination.

2.4. LABORATORY ANALYSES

Sample preparation and measurements of required elements are performed usingappropriate analytical techniques (see chapter 25, this volume).

3.Workedexamples

Examples of use of transplantedHypogymnia physodes as accumulator of differentpollutants are given below.

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BranchesofPinus mugo covered byH. physodes were transplanted to the vicinityofthe steelwork in Denmark [3]. Transplants were exposed for 7 months, and after eachmonth a partofthe samples was taken.ConcentrationsofCd, Cr, Cu, Fe, Mn, Ni, Pb, Vand Zn were analyzed in both lichens and bulk precipitation. The amountsof metalsaccumulatedin lichens were found to be linearly correlated with the deposition from theatmosphere.

BranchesofPicea abies withH. physodes were transplanted to thesurroundingsofafertilizer plant and a strip mine in Central Finland [2].Accumulation of fluoride,sulphur and plant nutrients N, P, K, Mg and Ca were found to depend on the distancefrom the emission source. Within 4 to 6 months, elementconcentrationsin transplantsreached the levels measured in indigenous lichens at the same sites. Seasonal variationin accumulationrates and ultrastructural damageofthalli are discussed.

H. physodes growing on twigsof Larix was transplanted to the vicinityof a pointsource of ammonia (a pig farm) in Denmark [5]. Two-fold increaseof a nitrogencontent was found after 4 monthsofthe exposure in the locality closest to the farm.

4. DataQualitycontrol

Results can be acceptedifthalli transplanted to background conditions show no signsofinjury. Where chemical contentof these transplants alterssignificantlycomparedtobaseline content, the situation should be investigated to determine possible causes, e.g.contaminationduring transplanting procedure, seasonal changesconnected withdifferent precipitation rates or other factors which were not taken intoconsiderationatthe startofthe experiment.

5.Application

This method can be used in areas where lichens weredepauperatedby air pollution, butcommon species are available for transplantion from similar habitats.Transplantationofuniform material in required amounts allows greater flexibility inexperimentaldesignandplacementofsample plots compared to sampling naturally occurring thalli.

6.Limitations

The accumulationofpollutants by a thallus is not linearly dependent onpollutantfall­out. For example, a different damageof transplanted thalli in differently pollutedlocalities leads to different accumulation rates (differentaccumulationcapacityofdeadand living partsof a thallus). Moreover, transplants in highly polluted localities canreach a saturation level whereas thalli in other localities continue theaccumulation.

7.References

I. Kauppi, M. (1976) Fruticose lichen transplant technique for air pollution experiments, Flora 165,407­414.

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2. Palomliki, V.,Tynnyrinen, S., and Holopainen, T. (1992) Lichen transplantation in monitoring fluorideand sulphur deposition in the surroundings ofa fertilizer plant and a strip mine at Siilinjlirvi,AnnalesBotanici Fennici 29, 25-34.

3. Pilegaard, K. (1979) Heavy metals in bulk precipitation and transplantedHypogymnia physodes andDicranoweisia cirrata in the vicinityofaDanish steelworks,Water, Air and Soil Pollution 11,77-91.

4. Schonbeck, H. and van Hut, H. (1971) Exposure of lichens for the recognition and the evaluationofairpollutants, inIdentification and Measurement of Environmental Pollutants, Proceedings of theInternational Symposium, Ottawa, Ontario, June 1971, National Research Council of Canada, Ottawa,pp. 329-334.

5. Sechting, U.(1995) Lichens as monitors of nitrogen deposition,Cryptogamic Botany 5,264-269.6. Swieboda,M. and Kalemba, A. (1978) The lichenParmelia physodes (1.) Ach. as indicator for

determination ofthe degreeofatmospheric air pollution in the area contaminated by fluorine and sulphurdioxide emission,Acta Societatis Botanicorum Poloniae 47,25-40.

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SAMPLE PREPARATION OF LICHENS FOR ELEMENTAL ANALYSIS

A. M. RUSUDepartment ofAnalytical Chemistry, University ofCluj-Napoca, 11Arany Janos, 3400 Cluj-Napoca, Romania ([email protected])

Several differentmethods exist for the determination of trace elementsin lichensamples. Amongst these are: neutron activation analysis [5, 6, 9,II , 15], X-rayfluorescence(XRF) [4, 17], and variousspectrometrictechniques including flameatomic absorptionspectrometry(FAAS [11, 18]), graphite furnace orelectrothermalvaporization(GF- or ET-AAS [6, 9, 16]), inductively-coupledplasmaatomic emissionspectrometry(lCP-AES [9, 13]),and ICP massspectrometry(lCP-MS [9 ,3, 10, 12]).

1. Aims

• Preparationof lichen samples for chemical analysis in order to quantifyelementsattrace andultratraceconcentrationsusing widely availablespectrometrictechniques.It is essential to use methods that yield reliable andreproducibleresults so thatanalytical data fromdifferent laboratories examining similar material arecomparable.

• Selection of the analyticaltechniquemost appropriatefor the elementsto bemeasuredin lichen material.Furtherinformation on samplepreparation and theanalysis of plant and otherenvironmentalmaterials can be found in more generalreferencetexts (see e.g. [1-2, 7]).

2. Methods

Samples are normally introduced in liquidfonn for analyses by thespectrometrictechniques. The speed and accuracyof an analysis depends on the choiceofdecompositionmethod as well as the useofappropriatereagents for sample dissolution.Pretreatmentoflichen samples will vary with:• the elements to be determined (e.g. volatility,solubilityin the reagents used),• elementconcentrations,• the analyticaltechniqueused.For spectrometricanalysis the effectsofthe samplepreparationproceduremust be takeninto accountin the calibrationcurve. Blank samples must bepreparedin a manneridentical to that used for the samples to ensure that potential sourcesof contamination

305P.L. Nimis, C. Scheideggerand P.A. Wolseley(eds.), Monitoring with Lichens- MonitoringLichens. 305-309~ 2002 KluwerAcademic Publishers. Printed in the Netherlands.

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are revealedand compensatedfor. Each stepof the preparationproceduremust befollowedpreciselyfor samples and blanks alike.

Decompositionof lichens anddissolutionofthe traceelementsthey contain impliescompleteoxidationofthe organic matrix.This is generallyaccomplishedby wet ashingusing strongly oxidizing acids. Because ICP-AES is a multi-elementcomplementtoAAS much ofthe samemethodologyapplies to thecollection,storage,and digestionoflichens. The followingproceduresfor lichen dissolution are recommendedfor thewidely available spectrometricinstruments(AAS, ICP-AES, and ICP-MS) and arebasedon personalexperience[13, 14] andpublisheddata [3, 9]. In bothprocedurestheweight of sampletaken for analysis and thevolumesof reagentsused dependon theconcentrationof the analyte in the lichen. This largely depends onwhetherthe lichenwas collectedfrom apollutedor anunpollutedarea.

2.1.PROCEDURE USING MICROWAVE DIGESTION (FAAS, ICP-AES, ICP-MS)

1. Carefullyclean the lichen thalli under abinocularmicroscopewithoutwashing.2. Dry thesampleat 40° C for 8 hours.3. Grind the dried material in an agate mortar.4. Place 250 mg(accuratelyweighed)ofpowderedlichen into adigestionvessel.5. Add 7 mlofRN03 (65% m/v) and 3 mlofHzOz (30% m/v).6. Heat the vessel in amicrowaveoven accordingto the followingprogramme:

i. 250 Watt (W) 5 min.,ii. 600 W 1 min.,iii. 0 W 1min.,iv. 300 W 3 min.

7. Removethe vessel and cool it in awaterbath.8. Wash the cooledsolutioninto a 50 mlvolumetricflask and bulk it tovolumewith

deionisedwater. The solid material (siliceousmaterialundissolvedin nitric acid)remainingafter digestion settles quickly to thebottomofthe vessel.

9. Filterofftheinsolubleresiduethrough anappropriatefilterpaper.10.Treat samplescontainingsilicates with 7 mlof RN0 3 (65% m/v), 3 mlof HzOz

(30% m/v) and 0.2 ml HF (40%) to ensurecompletedigestionand totalrecoveryofthe metals (e.g. Si, Ba, AI, Cr, Zr, Th, Ce, Rb, etc.). Digest them under themicrowaveconditionsgiven above and dilute the digest to aknown volume withdeionisedwaterso that the analyteconcentrationsfall within theworking range ofthe chosentechnique[3].

2.2.MICROW AVE DIGESTION (GF-AAS)

Dissolve 100 mg of dry sampleby microwavedigestionwith 1 ml of concentratedRN0 3 (65% m/v) and 1 mlof HzOz (30% m/v) in a highpressureteflon vessel.Aniridium or zirconium matrix modifier is necessaryfor thedeterminationof volatileelements(As, Cd, Pb, Zn) in agraphitefurnace [16].

2.3.OPEN VESSEL PROCEDURE

This procedureis recommendedfor laboratorieswithouta microwavedigester.

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1. Place 500 mg (accurately weighed)ofdried material in a borosilicate test tube, add14 mlofHN03 (65% m/v) then fit a reflux bulb and digest in at 50°C overnight.

2. Raise the temperature slowly to 120°Cfor 3 hours.3. Cool the solution and add 6 mlofH202 (30% m/v).4. Reheat the solution to 50°C for 30 minutes, then increase the temperature slowly to

120°Cand hold at that temperature until brown fumes no longer appear.5. Cool the resulting solution, transfer it to a 50 ml volumetric flask and bulk to

volume with deionised water.6. Filteroffthe insoluble residue through an appropriate filter paper.

2.4.MERCURY DETERMINATION

Using the cold vapor technique performed with an automatic mercury analyzer.1. Decompose the sample in the analyzer furnace. The decomposition products flow

through a catalyst to a gold amalgamator where the mercury is collected.2. Heat the amalgamator to 700°C to release elemental Hg, which is detected and

measured by AAS [16].

3. DataQualitycontrol

Replicate analysis is recommended for analytical quality control. Take one randomlyselected sample through the entire analytical procedure in duplicate, for every 10samples. The quality of analytical data may be characterized by precision, accuracy andbias [8]. Verificationof the accuracy of an analytical procedure may be accomplishedusing certified reference materials (CRMs) or by recoveries from spiked samples.• The method can be tested to avoid systematic errors such as lossof volatile

compounds and incomplete digestion, using CRMs. These may also be used to testthe final stepofthe analytical determination,esp.the choiceofthe analytical line forinterference-free measurement and background correction. To improve the quality oftrace element measurements in lichens, the Community Bureauof Reference hasprepared lichen standard reference materials (CRM 482 -Pseudevernia furfuracea;TP 24 -Evem ia prunastri; and TP 25 - Parmelia sulcata) [9].

• Recoveries from spiked samples may be used to test a method when CRMs are notavailable. Appropriate volumes (ul)ofstock standard solution are added to samplesprior to digestion, and the results for spiked and unspiked samples are compared todetermine the amountofspike recovered.

4.Application

The quantitative measurementof trace elements in lichens and other organisms hasbecome a major aspectof environmental diagnostic and monitoring programmes.Element concentrations in lichens are easily measured using the most appliedspectrometric techniques, even in samples from unpolluted areas.

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5.Advantagesand limitations

No single analytical technique is ideally suited to all sample matrices and analyteconcentrations. Selectionofthe most appropriate technique requires that several criteriabe taken into account, including sensitivity and detection limit, accuracy, precision,scope, selectivity, and also the time and cost per analysis. All of the methods haveadvantages and disadvantages.• Neutron Activation methods offer several advantages including high sensitivity,

minimal sample preparation and easy calibration. Disadvantages include the needfor large and expensive equipment and special facilities for the safe handling anddisposalofradioactive materials.

• Generally, the XRF method is non-destructive and multi-element analyses can becompleted within a few minutes. Disadvantages include poorer sensitivity thanvarious optical methods and the high cost of instrumentation.

• AAS methods are simple, convenient, and widely applicable: I) Flame AAS hashigh specificity, but refractory elements such as B, W, Zr and Ta cannot bedeterminedbecause the flame is not hot enough. 2) Graphite furnace offers greatersensitivity than flame AAS and requires a smallervolume of sample solution but haslower precision and is considerably more susceptible to matrix effects. The maindisadvantages of AAS are that itis slow relative to ICP and XRF because only oneelement can be determined at a time.

• ICP-AES and ICP-MS have rapid (often simultaneous) multi-element capability,operational simplicity, long dynamic concentration ranges and both permit thedetermination of refractory elements becauseofthe high temperature of the plasmasource (8000K). ICP-MS has the capability for isotopic measurement and is verysensitive. The major disadvantage is high sensitivity, which may require excessivedilutionofsample solutions, and the interference caused by polyatomic ions.A comparison of the detection limits achievable using various spectrometric

techniques shows that these are complementary rather than competitive (ICP-AES 1­IO0f.1gr1, ICP-MS O.OI-O.lf.1grl, FAAS 1-I000f.1grt, GF-AAS O.OI-If.1gr1). However,this may not reflect realistically the results obtained from routine analytical work.Reliable measurements may often be 1-10 fold above these levels (the sample matrixinfluences the true limits of detection). GF-AAS provides superior limitsofdetection toFAAS and ICP-AES but has similar limitsofdetection to ICP-MS. Refractory elementswhich are not very sensitiveby FAAS are generally easily determined by ICP-AES orICP-MS. Concentration measurements in lichen samples are typically made at ppb levelusing ICP-MS or GF-AAS and at ppm level using ICP-AES or FAAS.

6. References

1. Alfass i,Z.B. (ed.) (1994) Determination of trace elements. VCH, Weinheim.20 Bargagli,R. (1998) Trace elements in terrestrial plants : an ecophysiolog ical approach to biomonitoring

and biorecovery, Springer-Verlag, Berlin.30 Bettinelli, M; Spezia, So, and Bizzarri, G. (1996) Trace elementdeterminationin lichensby ICP-MS ,

Atomic Spectroscopy 17 (3),133-141.

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4. Calliari, I., Caniglia, G., Tollardo, A.M., and Callegaro, R. (1995) EDXRS studyof lichens asbiomonitorsand effectofwashing procedure on element concentration, X Ray Spectrometry 24 (3), 143­146.

5. Ila, P. (1988) Multi-elementanalysis oflichenby instrumentalneutron-activationanalysis,J. Radioanal.Nucl. Chem. 120 (2), 247-252.

6. Jones, K.C ., Peterson, PJ., Davies, B .E.,and Minski, MJ. (1985) Determinationof silver in plants byflameless atomic absorptionspectrometryand neutron activation analysis, Int. J. Environ . Anal. Chem.21 (1-2),23-32.

7. McKenzie, H.A. and Smyth, L.E. (OOs.) (1988) Quantitative Trace Analysis of Biological Materials,Elsevier,Amsterdam.

8. Miller, J.e.and Miller, J.N.(1984) Statistics for Analytical Chemistry, John Wiley& Sons, Chichester.9. Quevauviller, Ph., Herzig, R., and Muntau, H. (1996) Certified reference materialof lichen (CRM 482)

for the quality controlof trace elementbiomonitoring,The Science of the Total Environment 187, 143­152.

10. Reimann, C., Halleraker, Jo. H., Kashulina, G., and Bogatyrev,I. (1999) Comparison of plant andprecipitationchemistry in catchments with different levelsof pollution on the Kola Peninsula, Russia,The Science ofthe Total Environment 243/244, 169-191 .

I J. Reis, M.A., Alves, L.C., Freitas, M .C., van as, B ., and Wolterbeek, H.Th. (1999) Lichens (Parmeliasulcata) time response model toenvironmentalelementalavailability, The Science of the TotalEnvironment 232, 105-115.

12. Rodrigo, A.,Avila, A., and Gomez-Bolea, A. (1999) Trace metal contents inParmelia caperata (L.) Ach.compared to bulk deposition, throughfall and leaf-wash fluxes in two holm oak forests in Montseny (NESpain),Atmospheric Environment 33,359-367.

13. Rusu, A.M., Bartok, K., D in, V., Purvis, O.W., and Dubbin, W. (2000) Trace elementdeterminationbyICP -AES for routine multielement analysisof lichen and soil samples forenvironmentalpollutantscontent studies,Stud. Univ. BB. Chem. XLV (in press) .

14. Rusu, A.M ., Bartok, K .,Purvis, O.W., and Dubbin, W. (2000) Pilot assessmentofcontaminantelementsin soils and cryptogam plants from emissions from an ore processing plant,Zialna region, Romania,Stud.Univ. BB. Chem. XLV (in press) .

IS . Saiki, M., Chaparro, C.G., Vasconcellos, M.B.A., and Marcelli, M.P . (1997) Determinationof traceelementsin lichens by instrumentalneutron-activationanalysis,J. Radioanal. Nucl. Chem. 217 (1), 111­115.

16. Scerbo, R., Possenti, L., Lampugnani, L., Ristori, T., Barale, R., and Barghigiani,C. (1999) Lichen(Xanthoria parietina) biomonitoringof trace elementcontaminationand air qualityassessmentinLivomo Province(Tuscany, Italy), The Science ofthe Total Environment 241,91-106.

17. Schmeling, M., All, F., Klockenkaemper,R., and Klockow, D. (1997) Multi-elementanalysis by totalreflection x-ray fluorescence spectrometry for the certificationof lichen research material, Fresenius'Journal ofAnalyt ical Chemistry 357 (8), 1042-1044.

18. Tarhanen,S.,Metsarinne, S., Holopainen, T., and Oksanen, J. (1999) Membranepermeabilityresponseoflichen Bryoria fuscescens to wet deposited heavy metals and acid rain, Environmental Pollution 104,121-129.

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SULPHUR ISOTOPES IN LICHENS AS INDICATORS OF SOURCES

B. SPIRal, 1.MORRISSOW and O.W. PURVIS 3

'Isotope Geosciences Laboratory, Kingsley Dunham Centre. Keyworth,Nottingham NG12 5GG. UK.2Micromass Ltd, Floats Road, Wythenshawe, Manchester M23 9LZ, UK.3The Natural History Museum. Cromwell Rd. London SW7 5ED. UK([email protected])

Gaseous atmosphericsulphur compoundsare damaging to many lichen species andbecauselichens absorb S and other elements largely from theatmosphere, analysis oflichensprovidesan indicationofatmospheric034S. Harmful elevatedlevels ofS02 arenormallydue to local emissions and result inincreasedaccumulationby lichens - anactive, energy-dependentprocess. As many anthropogenicsources have isotopiccompositionsthat aredifferentfrom the regionalbackgroundand the uptake from theair is notassociatedwith isotopic fractionation,determining 034S providesa powerfulsourceindicator.

1.Aim

To determinesulphurisotopes in lichens tissues and to use the034S values to trace theorigin ofsulphurtransportedas atmosphericgases,wetdepositionandparticles.

034S is defined as the ratioof the numberof 34S atoms to 32S in thesamplesaccordingto the formula:

=

2.Methods

2.1.EQUIPMENT AND MATERIALS

Lichen samples with habitat, location and dateofcollection.Either a dual inlet massspectrometeryor byon-linepreparation-continuousflow massspectrometry.They can be obtained from:• Micromass Ltd, Floats Road,Wythenshawe,Manchester,M23 9LZ, UK .• TheromoquestLtd,AdvancedMS Division, Headquarters,Barkhausenstrasse2,

311P.L.Nimis, C. Scheidegger and P.A. Wolseley (eds.). Monitoring withLichens- Monitoring Lichens. 311-315© 2002KluwerAcademicPublishers. Printedin the Netherlands.

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Bremen, D-28197, Germany.• PDZ EuropaLtd, Hill Street, ElworthSandbach,CheshireCW11 3JE, UK .The other facilities,equipmentand materials are indicated in theprocedurebelow.

2.2.PROCEDURE

Collect samplesofat least 1 gconsistingofmore than6 individualthalli,accordingtorecommendedprocedures(see chapter 23, this volume) tominimisevariationthroughbiologicalvariation andheterogeneoushabitats.

Laboratoryanalysis: The classical method consists of:1) Preparationofthe analyte(BaS04).2) PreparationofSOzfor analysis.3) Mass spectrometricanalysis.

In recent years continuous flow mass spectrometersenabled constructionofintegratedanalytical systems where all three parts are carried outconsecutivelyon line(see 2.2.3 below).

2.2.1 Preparation ofthe analyte (BaSOJfrom lichen samplesTwo methods are described [4, 5]:• Place ground lichen sample in thesuspendedboatof a cylindricaloxygen bomb.

Onto the bottom partof the bomb pour distilled water andHzOz. Add BaCl zsolution. Close bomb, fill with oxygen and operate bomb. The lower partof thebomb will contain the aqueous solution withBaS04precipitate.

• Oxidise the lichen with theEschka mixture (Na2:MgO 1:2) followed bycombustionat 900°C and finally precipitationof BaS04 by additionof BaCl zsolution.

2.2.2 Preparation ofSO]from BaSO, in a vacuum line has several variantsThe method is based on reactionofmeasurementofthe yieldofBaS04with either CuO[2], or VzOs [7]. A mixtureof BaS04 with eitherof these metaloxides and quartzpowderare reacted at 1150°C in a tube furnace attached to a vacuum line.Along withthe samples, SOz is alsopreparedfrom laboratorystandards, normallytwo gas aliquotsarepreparedfor eightunknowns.The combustionis followed by:1. cryogenictransferofthe SOzgas through the vacuum line,2. reductionofminor S03 to SOz in acopperfurnace,3. cryogenic trappingofHzO,4. separationofCOz,the purified SOz,5. collectionin acollectionvessel or breakseal for massspectrometricanalysis.

2.2.3 Mass spectrometric analysisEither:• Carry out analysisof SOz by dual inlet massspectrometryalternatingbetweenthe

sample gas and reference gasinflow from variable volumereservoirs into thesource. Obtain aprecisionof +/- 0.10/00. Determinethe 34S/3ZS (66/64rn/z)of thesample with respect to the ratio in the reference gas. Calibrate thereferencegas

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against the VCDT scale using reference materials (see paragraph 4).Or:• Use a continuous flow isotope-ratiomass-spectrometerwith an on-line system for

the generation and purificationofS02 which is connected directly to the sourceofthe mass spectrometer. Each run consists of samples and laboratory standards.Load aliquots into tin capsules arranged in the sample holder to maintain a goodratio of about4:14. Introduce the reference gas in pulses between the samples andreference materials. Experiments with plant material were successful with aCISratioof1000:1,obtaining a precisionof0.4%0. Perform duplicate analyses.

3.Workedexamples

Investigations of0 34S in recent specimensof Usnea scabrata in Western Canada showa clear mixing line between a distinctive0 34S of a polluting source andbackgroundlevels and0 34S. The 0 34S values change with distance from the pollution source [1].Our investigationof the historical specimensof Parmelia sulcata in Britain show aremarkably similar trend which reflect a combined geographical temporal one with thesame background composition though different0 34S of the pollutant (Figure 1, dottedcircle). L ichenand bark samples from Burnham Beeches have broadly similaro34S, andthe S content varies in the order dead >healthy>bark. The0 34S values of the mostrecent (1967) samplesof P. sulcata from the historic collection have similar Sconcentrations and0 34S values as the dead specimens from Burnham Beeches,indicating that the source of S (anthropogenic fuel combustion) is similar.

Whilst the0 34S of a polluting source such as coal fired power stations, sour gasplants, or specific industries can be characterised, the "background" compositiondepends on natural and remote sources such as sea spray, oceanic dimethyl sulphonicacid, volcanic sulphur dioxide emanations or remote anthropogenic sources. The 0 34Softhe deposition may also depend on the prevailing meteorological conditions.

The highest levelof S in UK specimens was recorded in a healthy sampleofP.sulcata from Kent collected in 1967 (NHM). Other lichens known to be more sensitiveto S02 are significantly increasing at Burnham Beeches, suggesting that S02 is nolonger the major factor responsible for lichen colonisation and growth.

Whilst there is no direct relationship betweendo34S or S contentof lichens and thetimeofcollection of the UK, the lowest S and highest0 34S contents were recorded atsamples from Applecross, Rosshire, Scotland (1887) and Dunkerron, Ireland (1867),both areas remote from industrial sources.These representbackgroundconditions.

4.DataQualitycontrol

Reference materials calibrated against the (old) international standard Canyon DiaboloTroilite (o/ooCDT) are: NBS 122 +0.150/00(zinc sulphide), NBS 127+20.70/00bariumsulphate. As materialof the CDT standard is nearly exhausted a new scale VCDTVienna Canyon Diabolo Troilite is defined on the basisof IAEA-S-l (silver sulphide)

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having 0348 of -0.300/00.A second standard IAEA-8-2 (silver sulphide) is under testingand thepreliminaryrange is +21.4 to+21.70/00.A large sampleofpine needlespreparedby R. Krouse (Univ. Calgary) and used by a numberof laboratories has a 8contentof0.54% and 0 348 of 11.40/00VCDT.

350

30 0

25y = -9.2297x+ 32.3960 R = 0.3166

tJ) 20 ~ Dead Parmelia~C")

15 A Healthy Parmelia'C

• Bark10

Historical•5 0 Case & Krouse

0 •0,00 0,50 1,00 1,50 2,00

Sulphur concentration[1/5 (1/mg/g))

Figure 1. Variations in t5J4S and S content in lichens and bark substrate. From top to bottom, Usnea scabratasampled along a transect from a sour gas plant Alberta, Canada, emitting sulphur having t5J4S of +29 [I];historical Pannelia sulcatacollected at various sites in the UK between 1797 and 1967 (hb, NHM) andsamples ofP. sulcata(dead and healthy) and bark substrate collected from Burnham Beeches, W. London,July 2000. Background levels of8 34S and S are encircled.

5.Applications

• This method can be used to detect changes in sources and amounts over time.• Harmful elevated levelsof atmospheric sulphur oxides are normally due to local

emissions. As the residence timeof802 in theatmosphereis about 4 days, remotesources can also have a local influence,0 348 may discriminatebetween thebackgroundcomponentand local polluting sources[I, 3, 4, 6]. The method may beused to study both current and historical samples to determine trends in 8deposition over time and its effects on ecosystems.

• The useofsulphur isotopes in conjunction withbiochemical,ecophysiologicalandother analytical studies has great potential inunderstandingthe impactof varioussulphur compounds on lichens and in studying recovery in thepost-heavyindustrial era [8].

6. Limitations

• All partsofthis method are expensive.

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• An understandingofthe potential sources, the local and long rangetransportvectorsand the meteorologicalregime which affect thechemical reactions in theatmosphere,are needed in order to interpret the isotope results.

• In certainpolluted environmentsa greaterproportion of external particulatescontaining S (in different phases) may beadsorbed onto lichen surfaces andcomplementaryexaminationby SEMIEDS is advisable. The extentto which lichensrelease S (as H2S)as suggested by Case and Krouse [1] under highimrnissionloads,that may affect the834S ofthe residualsulphuris unclear.

• At least 1 g clean samples is normallyrequiredas detectionlimits forquantitativeanalysis by dual inlet massspectrometryis ca. 1 mg S. Continuousflow techniquesallow for smaller amounts and reducepreparationcosts. Continuous flow massspectrometryprovides greater sensitivityrequiringca 0.1 g but is subject to furtherlimitations: (a) for the analysisof BaS04 the amountof material is the limitingfactor, and, (b) for the direct analysisof lichens, on-line gas chromatographicseparationofS02 from CO2is a limiting factor.

7. References

I. Case, J.W. and Krouse, H.R. (1980) Variations in sulphur content and stableisotope composition ofvegetationnear a S04 source at Fox Creek, Alberta, Canada, Oecologia 44,248-257.

2. Coleman, M.and Moore, M.P. (1978) Direct reductionofsulfates to sulfur dioxide for isotopic analysis,Anal. Chern. 50, 1594-1598.

3. Krouse, H.R. (1977) Sulphur isotope abundance elucidate uptakeofatmosphericsulphuremissions byvegetation,Nature 265, 45-46.

4. Takala, K . Olkkonen, H., and Krouse, H.R. (1991) Sulphur isotopecompositionof epiphytic andterricolous lichens and pinebark in Finland,Environmental Pollution 69,337-348.

5. Wadleigh, M.A. and Blake, D.M. (1999) Tracing sourcesof atmosphericsulphur using epiphyticlichens,Environmental Pollution 106, 265-271

6. Winner, W.E., Berg, V .S., and Langston Unkefer, PJ. (1980) The use of stable sulfur and nitrogenisotopes in studies ofplant responses to air pollution , in P.W.Rundel (ed.),Stable isotopes in ecologicalresearch, Springer, Berlin.

7. Yanagisawa,F and Sakai, H. (1983) Thermaldecomposition ofbariumsulphate-vanadiumpentaoxide­silica glass mixtures for preparationof sulphur dioxide in sulphur isotope ratiomeasurements,Anal.Chern. 55,985-987

8. Zhao,FJ., Spiro, 8., Poulton,P.R., and McGrath,S.P. (1998) Useofsulphur isotope ratios to determineanthropogenic sulphur signals in a grassland ecosystem,Environmental Science and Technology 32,2288-2291.

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ESTIMATION OF CRITICAL LEVELS OF AIR POLLUTION (METALS) ONTHE BASIS OF FIELD STUDY OF EPIPHYTIC LICHEN COMMUNITIES

E.VOROBEICHIK and I. MIKHAILOVA

Institute ofPlant and Animal Ecology UD RAS, 8 Marta Str., 202, 620144Ekaterinburg, Russia ([email protected])

This method is the developmentof a classical toxicological technique for estimatingtoxicometric parameters (LDIO, LD so, LD so, etc.) based upon dose-response models(survival of ith species vs concentrationof jth toxic substance under laboratoryconditions). There are a numberof similar examplesof well-established methods inecological toxicology [4].It is well known that under natural conditions biotic andpollution parameters are highly variable both in space and time.This variability is takeninto account in the method described below.

1.Aim

To estimate the safe levels of metal pollution for epiphytic lichen communities.

2.Method

2.1. EQUIPMENT AND MATERlALS

• Soil collecting equipment (well-polished steel shovel, plastic bags or containers),• Apparatus for measuring metal contentof soil and litter (or snow) samples (flame

atomic absorption spectrometry - FAAS, or inductively-coupled plasma atomicemission spectrometry - ICP-AES),

• Equipment for description of epiphytic lichen communities (gridsof appropriatekind, see below),

• Computer; statistical software with non-linear estimation module (e.g., Statisticafor Windows,SAS, SPSS, S-Plus) .

2.2. PROCEDURE

I . Carry out a reconnaissance study around a point emission source to provide a roughmappingofthe stateoflichen communities (indicating, at least: lichen desert zone,strong damage, transitional and background zones).

317P.L. Nimis, C. Scheideggerand P.A. Wolseley(eds.),MonitoringwithLichens- MonitoringLichens. 317-321© 2002 Kluwer Academic Publishers. Printedin the Netherlands.

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2. Select 20-30 sample plots (25x25 m) comparable in their ecotope characteristics.Plots must represent different levelsofpollution (from background to high).

3. Describe epiphytic lichen communities by a method suitable for the region, e.g.:• determine frequency using a sampling ladder (see chapter 19, this volume) or

a sampling grid [3J,• determine projective cover using a10xi0 em or 20x20 em grid,• determine the total numberoflichen species on a certain substrate.

4. Sample an accumulative medium (such as forest litter,soil- Al-horizon- or snow)for assessment of the toxic load in each sample plot. Forest litter is preferablebecause of its high pollutant-accumulating capacity. High spatial variabilityofpollutant content in accumulative media (especially in transitional and stronglypolluted zones) should be taken into consideration to provide representativesampling. Mixed samples are recommended for soil and forest litter (e.g. fiverandom samples composed of five sub-samples each; of these five sub-samples,four are located at the comersof a I m2 quadrate and one is in the center of thequadrate).

5. Measure (by FAAS or ICP-AES -method) contentsof selected elements andcompounds which reflect deposition (or accumulation) of the overall complexofpollutants. Trace metals, such as Cu, Cd, Ni, Zn, Pb, Hg, As, Co, and Sb, arerecommended for smelting plants. When analyzing snow, the total contentofselected pollutants should be determined and converted into the valueofdeposition(mass x area" x time"). Both total content and contentof extractable forms (innitric or hydrochloric acids, ammonium nitrate, EDTA, etc.) are suitable foranalysisofsoil and forest litter (for detailed descriptionsofprocedures, see [ID.

3. Dataanalysesandinterpretation

(1)

1. Calculate an indexof the toxic load from the information on individual elements.Although a varietyof such indexes is available [5], we recommend the mean ratioofconcentration at the site to that in the background, as follows:

1 ~C ..K. =-LJ-!L

I N j=1 Cbj

where:K, is the mean toxic loadoftheith plot,Cij is theconcentrationofjth element in theith plot,Chj is theconcentrationofjth elementin thebackgroundzone,N is the numberofelements.

This index has no units, and gives an overall pictureof how much the backgroundlevel is exceeded in a certain sample plot. If the site is unpolluted,K»1.

2. Calculate lichen community parameters (lAP, LDV, projective cover, numberofspecies, etc.,see chapters 3-5, this volume).

3. Estimate a "IndexofToxic Load -lichencommunity parameter" relationship.As aregression model, the following logistic function can be recommended:

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A -aDy= a +~x + ao

l+e

319

(2)

where:y is the response (lichencommunityparameter),x is the doseofthe load (e.g.,K;from equation I),a, p, aoandA arecoefficients.

To find values for the coefficients in the equation, any methodof non-linearnumerical estimation is suitable (Marquardt method,Quasi-Newton method,Rosenbrock pattern search, etc.). In certain cases (when a parameter changes sharplyunder low pollution levels), log-transformationofK, can be recommended. Severalspecific versionsofthe equation can be derived from the same setofdata dependingon start valuesofcoefficients and initial step sizes. Take intoconsiderationwhat thecoefficientsrepresent:• A and ao are maximal and minimal valuesof a lichen community parameter,

respectively;• e determines the directionofthe parameter change with an increaseofpollution,• the magnitudeof ~ determines the sharpnessofthe change.For iterative determinationof the values for the coefficients, select the preset start

values close to expected ones. Of the specific versionsof the equation found," thebest one" should be selected, that which accounts for the maximalproportionofvariance (R2) and is closest to S pattern.

4. Calculate coordinatesofcritical points (i.e. inflectionsof the fitted curve) from thecoefficientsof "the best" fitting equation by regression to the data. Three criticalpointsof the logistic curve areofecological importance: upper, middle and lower.Their abscissae can be calculated using the following formulas:

-a+ln(2-J3) a -a+ln(2+vfJ)Xu = p , XM = -p' XL = P (3)

The upper point corresponds to the beginningof transition from the backgroundstate to thatof impact, the lower corresponds to the endof this transition. Themiddle one corresponds to 50% change from background level. Theabscissaof theupper inflection point isofgreat interest as this is a required valueofsafe pollutionlevel. Since different parametersof lichen communities differ in theirsensitivitytopollution, a rangeof safe levels can be obtained. The safe level which has theminimal valueof all the community parameters is assumed to be the safe level forthecommunityas a whole.

4. Worked example

This method was applied to data collected in the Middle Urals, in thevicinity of acopper-smeltingplant. Epiphytic lichen communitiesofbirch trunks were described in198 sample plots (10 trees per plot; the frequencyof lichen species was assessed by themethodofHerzig and Urech [3]). Three mixed samplesofforest litter were collected ineach sample plot, and concentrationsofCu, Cd, Pb and Zn (5% nitric acid extraction)were determined by FAAS (see chapter 25, this volume). All these elements wereincluded intoK; ObtainedK, ranged from 1 to 132.14.An exampleof dose-response

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curve is shown in Figure 1 (numberofspecies per baseoftrunk vs In K;). The resultingmodel (Equation 2) is:

6.15-1.00 (4)y=1+ e- 5.80+ 1.97•x + 1.00

The abscissasofcriticalpoints(Equation 3) are:-(-5.80}+ln(2-J3") -5.80 - (- 5.80}+ln(2+v'3)

Xu = 1.97 2 .2~ XM =- 1.97 =2.95, XL = 1.97 3.62

The actualK; are: exp(2.28)=9.78; exp(2.95)=19.21 ;exp(3.62)=37.38,respectively.Thus, a sharp decrease in the average numberofspecies per baseoftrunkbegins whenthebackgroundlevelofpolIution is exceeded by 9.78 times. Resultsof the analysesofdose-response relationships for lichen communities are described in more detail byMikhailova and Vorobeichik [6].

4.5

•• •

R'=O .B09

••

••

. :., ·1

• IIIIIIII

1.5 Xu 1.5 Xy 3.5 Xt.In metal concentrations in forest litter(toxic units)

•• •. ..,• •••

Figure I. Dose-response relationship "the average number of species per base ofbireh trunk (0-50 em) vs InK;" . Only highly illuminated plots are included.

5. DataQualitycontrol

Results can be acceptedif30-50% ofthe varianceis explained by the regression model,which represents the effectof the pollution, and if thedose-responsecurve has a welI­defined upper plateau,which represents the communityparameterin unpollutedsites.

6. Application

Safe polIution levels found by this method may be useful in making decisions inenvironmentalmanagement(environmental and risk assessment, see [2]). Since lichensare generalIy recognized as the most sensitive indicatorsofair polIution, safe polIutionlevels found for them may be usedas the higheststandardsofenvironmentalquality.

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7. Limitations

The method can only be applied with the conditions specified below:• The parametersof lichen communities aremonotonicallydependent on pollution

level.• The patternof this dependency is close to S-shape; or at least three clustersof

points are detectable: upper (background level), intermediate, and lower (highestpollution).

• The sample plots are establishedhomogeneouslyalong the pollution gradient.• The numberofsample plots is sufficient for non-linear regression analysis (usually,

no less than 20-30).• The sample plots are comparable in ecotope features (position in relief, forest type,

insolation, water regime, etc).

8. References

I. Carter, M.R. (ed.) (1993) Soil Sampling and Methods of Analysis, Lewis Publishers, Boca Raton,Florida.

2. De Bruin, J.H.M.and Hof, M . (1997) How to measure no effect. 4. How acceptableis the ECx from anenvironmentalpolicy point of view?,Environmetrics 8, 263-267.

3. Herzig, R. and Urech, M . (1991) Flechten als Bioindikatoren. Integriertes biologischesMesssystem derLuftverschmutzung fllr das Schweizer Mittelland,Bibliotheca Lichenologica 43, Cramer, Berlin,Stuttgart.

4. Hoeven, N. van der (1997) How to measure no effect. 3. Statistical aspects of NOEC, ECx and NECestimates,Environmetrics 8, 255-261.

5. Ott, W.R. (1978)Environmental Indices. Theory and Practice. Ann Arbor Science,Ann Arbor.6. Mikhailova, LN. and Vorobeichik,E.L. (1995) Epiphytic lichenosinusiaunder conditionsof chemical

pollution:dose-effect dependencies,Russian Journal ofEcology 26, 455-460.

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MONITORING PHYSIOLOGICAL CHANGE IN LICHENS: TOTALCHLOROPHYLL CONTENT AND CHLOROPHYLL DEGRADATION

K .BOONPRAGOB

Department ofBiology, Faculty ofScience, Ramkhamhaeng University,Bangkok 10240, Thailand ([email protected])

Chlorophyllin lichens is very sensitive to changes inenvironmentalfactorsincludingairpollution. It degrades intophaeophytinupon exposure to aciddepositionand heavymetals [10, 8, 9]. Changes in chlorophyll andphaeophytincontent can be used to assesschanges in air quality [2, 4, 6, 13]. This method can be used to detect early stagesofdamageprior to responses in growth andmorphologyof a species, so thatpreventiveandprotectivemeasures can be introduced.

1.Aim

To identify early stagesof effects of air pollution on lichens using estimatesofchlorophylla, chlorophyllb, total chlorophyll andchlorophyll/phaeophytinratios.

2. Method

2.1.EQUIPMENT AND MATERIALS

• Test tubes,• Incubatorset at65°C,UV,• Spectrophotometer,• Chemicals: acetone, calcium carbonate, dimethyl sulfoxide (DMSO), HCI,

magnesiumcarbonatepolyvinylpolypyrrolidone.

2.1.1. Lichen material and transplantationSelect, if possible, lichen taxa which do not containsecondarymetabolitesor lichenacids as they cause degradationof chlorophyll intophaeophytinduring pigmentextraction [5], or substances that mask chlorophyll absorption, i.e. parietin andanthraquinonewith broad absorption spectra in the blue region [12].Ramalina menziesiiis an exampleof a preferred lichen species without lichen acids. Most lichens do,however, contain lichen acids which can be removed by extraction withdry acetone [3].

If the study area is devoidoflichens, transplant a selected species into the study area

323P.L. Nimis, C. Scheideggerand P.A. Wolseley (eds.), Monitoring withLichens- MonitoringLichens. 323-326© 2002KluwerAcademic Publishers. Printed in the Netherlands.

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but take into consideration the historical reviewof its distribution. If the study area hasno record of the species to be monitored, select a control site with similar sitecharacteristics - especially climate and elevation - to the polluted sites, so that the maindifferenceis in air quality. The control site eliminates other errors that might occur dueto transplantation techniques.

Clean the lichen material before transplantation by removing any dead and damagedparts of the thallus or thalli. Gather the lichen thalli from their natural habitats stillattached to bark or branches and tie them on new hosts at the control and polluted sites.

Between wet and dry seasons - or summer and winter - air quality variesconsiderably, so that transplantation and sampling should commence at the beginningofany seasonal period in order to make it possible to observe seasonal changes in airquality.

2.1.2. Sampling lichen material1. Sample at defined time intervals, e.g. weeks, months, depending on air quality. The

greater the levelofpollution the shorter the time period should be.2. Take no less than five samples in each sampling period in order to represent any

natural variation of chlorophyll content.If the variation is high increase the samplesize.

3. Analyse the collected lichen material for chlorophyll content or percent phaeophytinwithin a few days. If the analysis cannot be performed soon after sampling, thelichens should be kept in continuously air-dry condition, under ca. 12 hours of dayand night light cycle,under ambient temperature.

2.2.PROCEDURE FOR ANALYSIS OF CHLOROPHYLL AND PHAEOPHYTIN

One ofthe most efficient solvents for extraction of chlorophylls and phaeophytins fromplant tissue including lichens is dimethyl sulfoxide (DMSO).It causes disruption of thechloroplast membrane, liberating the pigments. It does not require grinding andsubsequent centrifuging of the plant tissue [7].

Chlorophylla in DMSO shows maximum absorptionofthe red peak at 665 nm andof the blue peak at 435 nm. Upon degradation into phaeophytin, the absorption at 665nm is reduced while the peak at 435 nm is shifted to 415 nm [11]. A spectrophotometeris used to measure the optical density (OD) of the solution at wave lengthsof665 and648 nm. Chlorophylla and b content can be calculated from extinction coefficients byutilizing Arnon's equation [1]. The modificationof this procedure to lichens wasrecommended by Ronen and Galun [11]. Chlorophyll degradation into phaeophytin canbe estimated by measuring OD 435 and 415 nm.

2.2.1. Extraction ofchlorophyll and phaeophytin1. Wash 20 mg air-dry thalli six times with 3 ml ofCaC03 saturated acetone to remove

lichen acids.Then allow residual acetone to evaporate from the thalli.2. Immerse dry thalli in 5 ml DMSO containing 2.5 mg/ml polyvinylpolypyrrolidone.3. Incubate at65°C for 45 minutes in the dark to allow chlorophyll and phaeophytin to

be extracted.4. Allow extract to cool to ambient temperature then add 5 ml DMSO.

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5. Remove aliquot from the extract and measure optical densityof the solution at 665and 648 nm.

6. Measure optical density at 435 and 415 nm for estimationofchlorophyll degradationinto phaeophytin.

2.2.2. Determination ofchlorophyll contentCalculate chlorophyll contents as recommended by Barneset al. [3] using the followingequations:

Ctotal(mgll)= 20.340D648 + 7.490D665C. (mgll) = 14.850D665 - 5.140D648Cb (mgll) = 25.48 OD648 - 7.360D665

2.2.3. Preparation standard curve for estimating chlorophyll degradationThe degradationof chlorophyll into phaeophytin can be estimated by comparing theratioof OD 435/415 with the standard curve prepared from lichens collected from therelatively clean air site, or from the same site at the beginningofthe monitoring period.1. Wash 220 mg air dry thalli with 100%CaC03 saturated acetone which involves six

l-min, rinse with 30 ml of the bathing medium. Then allow residual acetone toevaporate from the thalli.

2. Immersedry thalli in 55 ml DMSO containing 2.5mgl1polyvinylpolypyrrolidone3. Incubate at65°C for 45 minutes in the dark.4. Allow to cool to ambient temperature, then add 55 ml DMSO.5. Divide the solution into 2 portions.

5.1. Keep one portion intact as the solutionofthe chlorophyll.5.2. Acidify another portion with about 330III (0.3 ml.) IN HCl for 10 minutes in

the dark: this portion is the equivalent solutionofphaeophytin.6. Neutralize the acidified portion with solidMgC0 3•

7. Prepare a seriesof mixtures by volume from 100% chlorophyll solution+ 0%phaeophytin solution, 90% chlorophyll+ 10% phaeophytin and so on to 0%chlorophyll+ 100% phaeophytin.

8. Measure optical densityofthe mixtures at 435 and 415 nm.9. Calculate the ratiosofOD435/0D415.10.Construct standard calibration graphof the ratioof chlorophyll/phaeophytinin the

mixtures plotted against the ratioofOD435/0D415.This graph is used as a standardfor estimation the extentofdegradationofchlorophyll into phaeophytin in the lichenat the polluted site.

3.Data analyses

By using statistical tests, chlorophyll content should be analyzed for:• The differences in chlorophyll contents among sampling period within sites.• The differences in chlorophyll contents among control and polluted site.• The differences in chlorophyll content among season at the control and the polluted

site.

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Degradationof chlorophyll into phaeophytin is estimated as percentage of the totalamount.

4.Application

This method can be applied to vascular plants, mosses and algae. Analysisofchlorophyll content and the proportionof phaeophytin in lichens can be related to theamount of pollutant accumulated in the lichen thallus and to the concentration of apollutant in the atmosphere, when known. If the latter two parameters were analyzed,threshold of air pollution damage to vegetation and ecosystems can be established.

5.Limitations

• This method cannot identify the pollutant, which requires analysisof lichen tissuesfor elements and/or ions.

• It cannot be used on crustose lichens. A similar technique could be used basingchlorophyll content on a unit area basis rather than mass.

6. References

I. Amon, OJ. (1949) Copper enzymes in isolated chloroplasts.Polyphenoloxidasein Beta vulgaris , PlantPhysiol. 24 (I), 1-15.

2. Balaguer, L. and Manrique, E. (1991) Interaction between sulfur dioxide and nitrate in some lichens,Environmental and Experimental Botany 31 (2), 223-227.

3. Barnes, J.D.,Balaguer,L., Manrique, E.,Elvira, S.,and Davison, A.W. (1992) A reappraisalofthe useof DMSO for the extraction and determinationof chlorophyll a and b in lichens and higher plants,Environm ental and Experimental Botany 32 (2), 85-110.

4. Boonpragob, K.and Nash,T.H.I11 (1991) Physiologicalresponsesofthe lichenRamalina menziesii Tayl.to the Los Angeles urban environment,Environmental and Experimental Botany 31 (2),229-238.

5. Brown, D.H. and Hooker, T.N. (1977) The significanceofacidic lichen substances in the estimationofchlorophyll and phaeophytinin lichens,New Phytologist 78, 617-624.

6. Garty, J.,Ronen, R.,and Galun, M . (1985) Correlation between chlorophyll degradation and the amountof some elementsin the lichenRamalina duriaei (De Not.),Environmental and Exper imental Botany 25(1),67-74.

7. Hiscox, J.D. and Israelstam, G.F. (1979) A method for the extraction of chlorophyll fromleaftissuewithout maceration,Canadian Journal ofBotany 57, 1332-1334.

8. Nash, T.H. III (1976) Lichens as indicators of air pollution,Die Naturw issenschaften 63,364-367.9. Puckett,KJ . (1976) The effect of heavy metals on some aspects of lichen physiology,Canadian Journal

ofBotany 54,2695-2703.10. Rao, D. N .and Le Blanc, F. (1966) Effects of sulphur dioxide on the lichen alga, with special reference

to chlorophyll,The Bryologist 69, 69-75.I I. Ronen, R. and Galun,M. (1984) Pigmentextraction from lichens withdimethylsulfoxide (DMSO) and

estimationof chlorophyll degradation,Environmental and Experimental Botany 24 (3), 239-245.12. Silberstein,L. and Galun, M . (1988) Spectrophotometricestimationofchlorophyll in lichens containing

anthraquinones in relation to air pollution assessments, Environmental and Experimental Botany 28 (2),145-150.

13. Von Arb, C.and Brunold, C. (1990) Lichen physiology and air pollution.I. Physiological responseofinsitu Parmelia sulcata among air pollution zone within Biel, Switzerland,Canadian Journal of Botany68,35-42.

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CHLOROPHYLL FLUORESCENCE MEASUREMENTS IN THE FIELD:ASSESSMENT OF THE VITALITY OF LARGE NUMBERS OF LICHENTHALLI

M .JENSEN and R. KRICKE

Botanik, FB 9, Universitat Essen, Universitiitsstrasse 5,D-45117 Essen,Germany ([email protected], [email protected])

1.Aim

Non-destructivemeasurementof photosyntheticactivity as an indicationof the healthand potential growthofindividual lichen thalli andoflichenpopulations.

2.Method

2.1. EQUIPMENT AND MATERIALS

• Lightproofblack foil or velvet (fromsupplierofphotographicgoods),• Distance clip (made on substratum usinghammerand nails for trunks, weights or

adhesive tape or gum for lichen on earth or rocks) to support light guide,• A fiber optic light guide.A miniature light guide (1.5 mm diam.) is easier to handle

than a normal one becauseofits lower weight but a 7 mm fibre optics light guide issuitable as well,

• Statistical software and computer for tests on results,• Distilled water to moisten thalli,• Thermometer/temperaturemeasuring device,• Lichen thalliin situ on the substratum,• A photosyntheticmeasuring instrument e.g.Walz MiniPAM. This can be obtained

from a supplier such as oneofthe following:• ADC BioScientific Ltd, Unit 12 Spurling Works,PindarRoad, Hoddesdon,

Herts, EN11 ODB, UK, phone: 44 (0)1992 445995, fax: 44 (0)1992 444567,e-mail:[email protected],internet:http://www.adc.co.uk/

• Hansatech Instruments Ltd,NarboroughRoad, Pentney, King's Lynn,NorfolkPE32 1JL, UK, phone: +44 (0)1760 338877, fax: +44 (0)1760337303,e-mail:[email protected], internet: http://www. hansatech­instruments.com!

• Heinz Walz GmbH, Eichenring 6, D-91090 Effe1trich, Germany, phone: +49­(0)9133/7765-0, fax: +49-(0)9133/5395, e-mail: [email protected],internet: http://www.walz.com

327P.L.Nimis, C. Scheideggerand P.A. Wolseley (eds.). Monitoringwith Lichens- MonitoringLichens. 327-332.© 2002KluwerAcademicPublishers. Printedin the Netherlands.

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2.2.PROCEDURE

Since dark adaptationof the samples is very important for many purposes, planningshould include a strategy to ensure a periodofpre-darkeningofthe samples. Although30 min. (or somewhat shorter)ofdarkeningof individual thalli is sufficient, it is moreeconomical to darken complete populations in the evening before(if possible) and toperform the measurements during the next day. The purposeofthis handling is to wasteas little time as possible for darkening during the dayofmeasuring. In the following, aprotocol is described for investigating large numbersofthalli within one day:1. Preparation: in the evening before the actual measurements, first moisten the thalli

by spraying with water (best: rain water) and then darken the thalli with black foil(best: black foil from photo supply stores) or black velvet.If the thalli grow on atree, envelope the whole trunk and fix the foil by a wire and by drawing-pins.

2. Take care that the foil can be removed easily and only partly. Removal (nextmorning) should expose only a few thalli to daylight. Beside the thallusof interest,darken the exposed thalli by additional piecesofvelvet (velvet is less sensitive towind).

3. Place the measuring instrument near the sample (tripod may be necessary).4. After (partial) removalof the foil from the thallus of interest, again moisten the

sample carefully and remove excess water.5. Attach or fix the "distance clip" or a similar device over the thallus (hammer and

nails for trunks, weights or adhesive tape or gum for lichen on earth or rocks).Mount one endof the fiber optic light guide within the clip and the other endwithin the measuring instrument. A miniature light guide(1.5 mm diameter) iseasier to handle than a normal one because of its lower weight. The sample must bekept in darkness, i.e.the clip and light guide setup must be darkened by additionalvelvet.If only FjFmis measured, the distance clip can be simply fixed by hand.

6. Perform your measurements (fire saturation pulses etc., see data collection andprocessing).Ifpossible,estimate or determine the water content.

7. A few thalli should be investigated more thoroughly, while they are slowlydesiccating.This control ensures the detection of (negative)supersaturationeffects.

8. Mark the measured thallus permanently by placing a numbered drawing pin (oranything similar) in the neighbourhood. This permits later identificationofindividual thalli (e.g.for photographs) and a later repetitionofthe measurement.

9. Repeat for further thalli.

2.3.DATA COLLECTION AND PROCESSING OF COMMON PARAMETERS

Modem instruments electronically store the obtained data themselves (better) or by aconnection to battery based computer (worse). The most common parameters measuredand stored areFjFm, <I>PSlh ETR and NPQ . The main features and meaningofthese 4parameters are:• FjFm: this parameter is suitable as a general vitality index. It indicates the

maximum possible efficiency of photosystem IIof the investigated sample (darkadapted). Not the real photosynthetic rate, but only the potentialperformanceofthe

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photosynthetic light reaction is monitored. This is recorded during a light"saturation pulse"of about 1 s. Low values indicate some kindof injury and lowpotential of growth.Big advantage: measurement is easy and rapid. It detectsreliably dead material (values< 0.1).Some crustose and some cyanolichens revealrelatively low values (0.5 - 0.6).The normal range for lichens is 0.6 - 0.76. Thisvitality index is independentof the ambient temperature.Restriction: darkadaptation necessary.

• cI>PSII (often also denoted asM/Fmor yield): it directly indicates the efficiencyofphotosystem II during illumination of samples (photon use efficiency), i.e. whilephotosynthesis is running. This parameter is indirectly correlated to theperformanceof CO2 fixation (see parameter ETR below). The diagnostic valueofcI>PSII + FjFm is high: high values of FjFm combined with very low valuesof cI>PSII

indicate a functional light reaction, but a disturbed dark reaction.Restriction: cI>PSII

generally decreases when light intensity increases, and the knowledgeofthe actiniclight intensity is essential. Some machines make useofstored light intensity values,that have been determined previously.

• ETR (photosynthetic electron transport rate): a calculated value (fromcI>PSII plusactinic light intensity PAR=photosynthetically active radiation). Formula: ETR=$PSII x PAR x factor. The used factor (mostly 0.42) is composedof the constants0.5 x 0.84: 0.5 because only oneof the two photosystems is concerned, 0.84,because only a proportionof the incident light energy is actually absorbed. ETRcan be used to estimate actual CO2 fixation rates, but this is not exact, and there aredeviations if compared to direct measurement of gas exchange. The minimumelectron demand per CO2 fixed is 4 but will be larger in most cases.

• NPQ ("nonphotochemical quenching"): this parameter indicates more or less theproportionofabsorbed light energy which is transformed into heat (wasted). It canbe used to monitor the degreeof photoprotection or photoinhibition whilephotosynthesis is running. High values indicate a high degreeof inhibition. Theunderlying mechanistic, molecular processes (e.g. zeaxanthin formation) are nowrelatively well known [5].Restriction: NPQ is light and temperature sensitive.These four parameters are automatically measured and calculated by modem

instruments. For reasonable measurements some instrument settings are very important.Besides intensityofmeasuring light and gain, the settingofthe intensity and durationofthe saturation flashes must be in an appropriate range.General recommendations (intensities at the lichen surface):• Saturation pulse intensity: between 2000 and 4000 umol photons m·2 S·I,

• Width of saturation pulse: 1.0 s,• Intensityof permanent (actinic) light: 50 or 100urnolphotons m·2 s',• Durationof actinic illumination: 10 min. Do not allow the pulse intensity at the

thallus surface to exceed 4000 umol photons m?S·I (mini light guide may generatevery high intensities!) because of possible light injury,

• The widthofthe saturation flash should not exceed 1 second,• Take care at low temperatures.There is danger to record incorrect Fmvalues. There

may be also differences between species.Therefore, a kinetic analysisofthe signalduring the saturation pulse may be necessary. This type of control analysis is

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possible for most instruments by the computer connected arrangement, so thiscould be done in the laboratory (but mindtemperature effects).

• For $PSlb ETR and NPQ determinations, reasonable,photosynthesis-driving("actinic") light energy has to be chosen. A light intensityof 50 or 100 umolphotons m? S-1 can be recommended, as$PSII is midrange in this case and NPQvalues < 0.5 proofthe absenceof possible photoinhibition (prerequisite: normalrange FjFm , i.e.values between 0.6 and 0.75). Within 10 minutesofillumination,normally steady state valuesof the parameters are achieved. See [1] for a morecomprehensive overview.

• Consider carefully whether simple and rapid FjFm determinations are sufficientwith the possibilityof getting a large numberof data rapidly or whether morethorough examinations are needed, although these are more time consuming andprovide less data but with all parameters.

• For the investigationof the light adaptation regimeof the samples, someinstruments offer the possibility to record "rapid light curves" automatically. Thisshould also be taken into account.

3.Workedexample

It was investigatedwhether the vitalityof small thalliof Physcia tenella (diameterbetween 2 and 4 mm) could be distinguished from thatof larger,probablyolder thalli(diameter>10 mm) growing beside them. All thalli were growing on 2 oak trees inEssen and 2poplartrees inMillheim,Germany. Measurementsofsmall and large thalliwere performed on 2 consecutive days. A Walz MiniPAM instrument was usedequipped with a normal 7 mm fiber optics light guide connected to a distance clip. Thedistance clip was pierced and attached to the bark by nails and additionally secured bywires. We used the instrument settings recommended above except an actinic lightintensityofonly 35 umol photons m-2s-l• The intensityofthe measuring light (ML) hadto be adjusted for individual thalli (level 5 - 7), and was higher for the small thalli thanfor the large ones. In order to minimize a possible actinic effect of the ML, this was setto the "burst mode". In Table 1, the resultsof this experiment are summarized.Statistical tests (performed with SigmaStat, Jandel Scientific: t-test or Mann Whitneyrank sum test) did not reveal differences between large and small thalli for theparameter FjFm- All of these values were in the rangeoftypical active thalli (Table 1).During running photosynthesis (parameter$psn), we did not detect differences either.By the naked eye, all thalli appeared healthy. Small significant differences (5 % level)were found for the parameter NPQ.But: on oak the NPQ valuesofthe small thalli weresmaller and on poplar larger (Table 1). Hence, there was no tendency at all comparingthe parametersofsmall and large thalli. Furthermore, we tested the correlation betweenthe three parameters. In 3 cases, there was a significant (Pearson product moment)correlation between the parameters FjFm and$psn , but not for the small thalli growingon oak. This possibly indicates independently varying Calvin cycle activity. Generally,we expected a negative correlation between NPQ and$psn , as an increased heat waste

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should be accompanied by a less effective photochemistry. However, this was foundonly for the large thalli growing on poplar.The reason for this finding is unclear.

TABLE 1.Fluorescenceparameters(IStd. Dev.t for small (2-4mm) and large (>10 mm) thalli oJPhysciatenella,

Type ofthaIli Nrof F)Fm et>PSII NPQ Ambientthalli temperature

SmaIl,on oak 20 0.684± 0.027 0.590 ± 0.048 0.200+ 0.098 19.3 - 20.7°CLarge,on oak 19 0.692± 0.035 0.578 + 0.030 0.263± 0.Q75 19.2 - 27.8°CSignificantdiff. (5%) - - *SmaIl,on poplar 20 0.671 + 0.023 0.515 + 0.034 0.239 + 0.061 13.5 - 16.9°CLarge,on poplar 20 0.670± 0.035 0.518 ± 0.033 0.176 ± 0.129 12.1 - 12.8°CSignificantdiff.(5%) - - *

Our original hypothesis was that the photosynthetic performance and growth ofmost small thalli could be better than thatof larger ones. This hypothesis could not beconfirmed for healthy looking thalli. The partly observed large variation of the NPQvalues cannot yet be interpreted.

4.Applications

This method can be used in qualitative and quantitative ratingof impacts frompollution. Itcan also be used to assess visible lesions in termsofremaining activity, thedeterminationofvitality indices and the diagnosisofphoto-inhibitioneffects.

5.Limitations

The method monitors only the stateofthe photobionts, not thatofthe mycobiont. Thereis no limitation concerning the lichen growth form, but:• Photosynthetic gas exchange data(C02 fixation rates) are sometimes not correlated

to photosynthetic performance as indicated by the parameter<l>PSII [2]. In prolongedmeasurements, technical problems like an instabilityof the zero-signal may be aproblem. Often the parameter<l>PSII is only used as a qualitative signal indicatingactive and inactive times [3].

• Technically, pre-darkening of the samples (see procedure) is essential for acomprehensive analysis of fluorescence signals. Moreover, dry lichens are totallyinactive. Generally, the water status is extremely important for photosyntheticperformance. Hence, for the assessment of vitality there must be an artificialincreaseof the water content to a high level (spraying with water). On the otherhand, a possible supersaturation with water [4] has to be avoided (removalofexcesswater).If low activity at high water content is found, there should be some controlmeasurements (see procedure) in order to assess a possible supersaturation effect(supersaturation may hinder CO2 diffusion). In addition, temperature affects most

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fluorescence parameters and must be always monitored. This has to be taken intoaccount especially for comparisonofseveral thalli or populations.

• Measurementsat subzero temperatures or during rain may betechnicallydifficult.

6. References

I. Bilger,W.,Schreiber,U.,and Bock,M.(1995)Determinationof thequantumefficiencyof photosystemIIandofnon-photochemicalquenchingofchlorophyllfluorescenceinthefield,Oecologia 102,425-432.

2. Green,T.G.A.,Schroeter, 8., Kappen,L., Seppelt,R.D.,andMaseyk, K. (1998)An assessmentof therelationshipbetweenchlorophylla fluorescenceand CO2 gasexchangefromfieldmeasurementson a mossandlichen,Planta206,611-618.

3. Green,T.G.A., Schlensog,M., Sancho, L.G., Winkler,J.B.,Broom, F.D., andSchroeter, B. (2001) Thephotobiont(cyanobacterialor greenalgal)determinesthepatternof photosyntheticactivitywithina lichenphotosymbiodeme:evidenceobtainedfrom in situ measurementsofchlorophylla fluorescence, Oecologia(inpress).

4. Lange,O.L.,Green,T.GA.,andReichenberger,H.(1999)TheresponseoflichenphotosynthesistoexternalCO2 concentrationanditsinteractionwiththalluswater-status,Journalof PlantPhysiology154,157-166.

5. Li, X .P.,Bjorkman,0., Shih,C.,Grossman,A.R.,Rosenquist,M.,Jansson,S., andNiyogi, K.K.(2000)Apigment-bindingproteinessentialforregulationofphotosyntheticlightharvesting,Nature403,391-395.

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MEASURING BARK pH

R. KRICKE

Institut fir Botanik, Universitiit Essen , Universitiitsstraj3e 5, D-45117,Essen, Germany (randolph [email protected])

In floristic as well as mapping studies applied to, for example, pollution monitoring,bark pH is important not only for the effect it has on the lichen distribution (see[I], andchapters 3 and 4, this volume), but also for the synergistic effect with air pollution, andneeds to be measured.Generally, trees with similar rangesofpH-valuesarecomparableregarding theircharacteristicsas phorophytes[4]. Although bark is a solid material andcannot strictly have a pH, bark pH refers to the pHofunbufferedaqueous solution incontact with the bark.

1.Aim

To determine bark pH accurately, reliably andconvenientlyfor laboratoryand fieldstudies.

2.Methods

2.1.MATERIALS

• A pH meter with a standard electrode (e.g.InLab®201, InLab®408) or asuspensionelectrode (e.g. InLab®420) or a flathead electrode (Schott 27pH, InLab®426),

• 5 ml stoppered flasks,• Grinder(coffee grinder is adequate),• 0.25 M KCI solution,• Distilled (or at least deionised water).

Suppliers: I) Schott-GerateGmbH, 1m Langgewann 5, D-65719 Hofheim. 2)Mettler-ToledoGmbH, Ockerweg 3, Postfach 110840,D-35353 Giessen, Germany.

2.2.PROCEDURE

There are two totally different methods [3]:• Laboratorymethod, in which samples arepowderedand then soaked,but it is slow.• Field method, using a flathead electrode in situ, which is much more rapid.

333P.L. Nimis, C.Scheideggerand P.A. Wolseley (eds.), MonitoringwithLichens- Monitoring Lichens.333-336.© 2002 Kluwer AcademicPublishers. Printedin the Netherlands.

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2.2.1. Measurements in laboratoryPieces as thin as possible are removed from the surfaceofthe bark on tree using a knifeor a chisel. In order to obtain comparable results the same amount of sampling materialshould be used (e.g.0.5g).The thicknessofthe sampled bark pieces is important for allestimations and the samples should be as thin as possible in order to measure only theoutermost bark layer which has closest association with the epiphytic vegetation. Thereare then two possible alternatives:• The bark is to be soaked either in distilled water or in 0.25 M KCl. A constant

volume of liquid should be used (e.g. 5 ml). After soaking forapproximately8hours (stoppered vials are recommended to prevent ingressofatmospheric CO2) at20°C or approx. 1 hour at 80°C, the pH-values can be determined by using astandard pH-electrode. To analyse the outermost bark layers only, the inner surfaceofthe bark samples can be sealed with wax [2]. Hence, protons are dissolved onlyfrom the outside during soaking.

• Alternatively the bark pieces can be ground (e.g.with a mill) and 0.5 gofpowdersuspended in 5 mlofdeionised water [8] and sealed as above. Then, after 4 hourswith occasional shaking the mixture should be filtered to measure the pHof thewater with a standard electrode. The pH value of the suspension may also beobtained by using a special electrode capableofmeasuring in suspensions.The concentrationofdissolvedprotons and hence the pH-value depends on the mass

of bark used as well as on the volume and qualityof solvent. When using a cationcontaining solvent like KCI, potassium ions are exchanged with protons in the bark andtherefore pH-values tend to be lower than those obtained using distilled or deionisedwater.

2.2.2. Field measurementsA chosen areaofsurface is sprayed with a solvent (e.g. 0.1 M KCI) to dissolve protonsfrom the bark [8]. A simple sprayer, e.g. that for flowers, can be used. The measuringspot should be sprayed till the solvent starts to run off. The flathead electrode is gentlypressed against the bark and the pH is to be measured after a few moments (20 sec. [5],3-10 min. [4]) making sureofa good contact between the electrode membrane and thebark surface.

Unlike the laboratory method described above, neither the massof bark nor thesolvent volume are defined.As the pH depends on the solvent volume and areaofbarksurface, the useof the flathead electrode was modified [6] by adding a silicone tubemounted at the endofthe electrode to provide a small cavity when pressed on the barkthat can be filled with an approximately defined volumeof liquid (0.2-0.3ml 0.25MKCI) (see Figures I and 2). The pH value can be determined after ca.3 min.

As the tested surface area as well as the solvent volume are constant, the data arecomparable from one location or tree to another.

3.Application

These techniques have been used successfully in a large number of surveys. Due todifferences in procedures, great care must be taken when comparing the data. For

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example, pH values obtained by soaking bark are slightly higher than those obtainedusing flathead electrode.

The most practicable, reliable and rapid way to estimate bark acidity is by using theflathead electrode modified with the silicone tube to standardise the amountof liquidand surface areaofbark. But this has the disadvantageof the flathead electrode beingconsiderably more expensive than a standard one.

I cable tie

I Si licone tube IHI mm

Incisionfor filling

..L

Electrode

Figure 1.Sketch ofthe modifiedflathead electrode (after [6]).

4. Limitations

Although the acidityof bark is oneof the major factors affecting lichens, it must beremembered that other bark properties like roughness, water holding capacity andnutrient content are important as well. Measuring the pH implies useof a solvent todissolve protons from the bark. Furthermore, measurementsof pH only provide dataabout the actual acidity, which can be highly influenced by external factors (e.g.geology, emission impacts). In addition, acidifying impactsof the past (e.g. sulphurdioxide immissions) will also bias the measured results, although measurementof thebuffering capacityof the bark gives more information on natural bark quality and isspecies specific.

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6.5

5.5

i 5

4.5

3.5

-0-Tilia cordata east __ l1lla cordata south-east -:O:-l1liacordata south I-+-Tilia cordata south'west --Tilla cordata est ......111ia cordata north-west-0-Tilia cordata north

~

1Za.n.. ~~ ... '\:,

~~

3

~~~~~~~~~~~~~&~~~~~~#~~

lee.

Figure 2.pH-data obtained with the modified flathead electrode. Values become constant after ca. 3 min.

5. References

I . Barkman,J.J.(1958) Phytosociology and ecology ofcryptogamic epiphytes, Van Gorcum, Assen.2. Culberson, W. (1955) The corticolous communitiesof lichens andbryophytesin the upland forestsof

northern Wisconsin,Ecological Monographs 25,215-231.3. Farmer, A .M., Bates, 1.W., and Bell, 1.N.B. (1990) A comparisonof methods for themeasurementof

bark pH,Lichenologist 22,191-194.4. Hobohm, C. (1998) Epiphytische Kryptogamen und pH-Wert - ein Beitrag zurokologischen

Charakterisierungvon Borkenoberflachen,Herzogia 13, 107-111.5. Looney, J.H.and James, P.W. (1988) Effects on lichens, in M .A. Ashmore, J.N.B. Bell and C. Garretty

(eds.), Acid rain and Britain's Natural Ecosystems, Imperial College Centre for EnvironmentalTechnology,London, pp. 13-25.

6. Schmidt, 1. (2000) Untersuchungen zur Acidittit/Basictttu der Borke ausgewiihlter Baumarten,SchriftlicheHausarbeit im Rahmen der Ersten Staatspriifung fur das Lehramt derSekundarstufeI1II,UniversitatEssen, 89 pp.(unpublished).

7. VOl (1993) VDI-Richtlinie 3799, Blatt I : Messung von Immissionswirkungen: Ermittlung undBeurteilung phytotoxischer Wirkungen von Immissionen mit Flechten-Flechtenkartierung zur Ermittlungdes Luftgiltewertes (LGW), VOl-HandbuchReinhaltung der Luft, Band I, VereinDeutscherIngenieure,Komiss ion Reinhaltungder Luft im VOl und DIN, Fachbereich Wirkungen von Staub und Gasen,ArbeitsgruppeWirkungsfestsstellungan Niederen Pflanzen.

8. Watson, M.F.,Hawksworth, D.L.,and Rose, F. (1988) Lichens on elms in the British Isles and the effectofDutch Elm Disease on their status,Lichenologist 20,327-352.

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A PHOTOGRAPHIC QUADRAT RECORDING METHOD EMPLOYINGIMAGE ANALYSIS OF LICHENS AS AN INDICATOR OFE~ONMENTALCHANGE

O. W.PURVIS 1, L. EROTOKRITOU I

, P. A.WOLSELEy l, B.

WILLIAMSON I and H. READ 2

I The Natural History Museum , Cromwell Rd, London SW7 5BD, UK([email protected])2The Corporation ofLondon , Burnham Beeches Office, FarnhamCommon , Slough SL2 3TE, UK.

1.Aims

• To assess lichen growth, health andchanges in assemblage composition byanalysing digital photographic imagesusing freely available image processingsoftware.

• To evaluate lichen distribution, age,reproductive capacityand longevity.

• To assess the impact of anthropogenicactivities andofpollutants on lichens.

Rapid advances in computer hardware,digital cameras and image analysis softwarehave allowed comprehensive studies whichwere impractical a few years ago. Digitalphotography enables visualisation of imagesin the field and rapid downloadingofimages to a computer for growthmeasurements [2, 4]. Young thalli easilymissed in the field may be magnified andfuture verificationoftaxa is also possible.

2.Materialsandmethods

2.1.EQUIPMENT AND MATERIALS

• A quadrat frame of wood or plastic,normally in the ratio of 1:1.5 equivalentto 35 mm film format with a Kodak

Figure 1. Photographic Quadrat (sheltered site B,1999) showing Kodak colour scale at base.Supported by 2 stainless steel screws permanentlyfixed to tree with a temporary nail above.

337P.L. Nimis, C.Scheidegger and P.A. Wolseley (eds.), Monitoring with Lichens - Monitoring Lichens. 337-341.«::i2002 KluwerAcademic Publishers. Printed in the Netherlands.

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colour scale and rule bars for calibration (Figure 1). There is no standard size, thisdepending on the lichens and substrate being studied. For practical purposes,quadratsof 10 x 15 em (for small lichens) and 20 x 30 cm (for larger lichens) havebeen found to be adequate.

• Signumat hammer of the Latschbacher system (http://www.latschbacher.bc.caJsignumat.html) with numbered plastic tags.

• Digital 2-3 megapixel camera, such as the Nikon Coolpix range provide excellentresolution for frames up to 20 x 30 em, are sensitive to low light levels avoiding thenecessity for flash. Some models provide extreme close focus capability permittingmacrophotography with a resolution approaching 10 urn per pixel.

• Jencons pH meter with a Gelplas flat-head electrode (see chapter 30, this volume).• Computer and Image analysis software: Scion Corporation, http://www.

scioncorp.com• Stainless steel screws (to avoid metal contamination) to mark quadrat position

permanently.• Ordnance survey map of area to estimate position or Global Positioning System

(GPS).• Flexible tape measure suitable for measuring tree girth.• Compass.

2.2.PROCEDURE

1. Select a suitable position for the quadrat and locate two stainless steel screws at thelower edgeofthe quadrat and a 3rd nail at the top to temporarily hold the quadrat inplace (Figure 1).

2. Mark trees with a coded plastic tag with unique number.3. Note the exact locationofthe tree, preferably by GPS, for future relocation.4. Photograph the location, tree and quadrat with a digital camera.5. Measure bark pH in areasin or adjacent to quadrat (x 3).6. Record tree girth, quadrat height and aspect.7. Record species in the quadrat and species list for whole tree.8. Repeat items 4-7 at selected time intervals.

2.3. DATA PROCESSING

1. Using the digital image obtained for each quadrat trace individual lichen thalliwithin and including quadrat boundaries (for scale) using the vector graphicsprogramme Corel DRAW®.

2. Correct images for parallax.3. Select colours for different species. Export as 8-bitTiF bitmaps to a freely available

image analysis shareware programme such asSCION IMAGE 4.02WIN .4. Calibrate scale with reference to quadrat size. Select individual thalli using a wand

tool or select multiple thalliof a single colour (=species) by thresholding todiscriminate areas of interest from surrounding background, based on their colourvalues. Various measurements are possible including area, centroid, perimeter, etc.as defined by the user. Results can be printed or exported to aspread-sheetor

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database.5. Correlate lichen area with a rangeof environmental parameters e.g. temperature,

rainfall, substrate, pH and pollution data to determine trends. Check for a numberof quadrats to ensure that your sample set is representative. Pollution data can beobtainedon-linefrom the UK National Air Quality Database [3].

3.Workedexample

At Burnham Beeches National Nature Reserve (Natura 2000 site), 40km west ofLondon and adjacent to a gravel working and light industrial area, 15 quadrats wereestablishedin 1994 on different trees within 70 ha. Lichen communitieswere sampledup to 3 m above the ground in the vicinityofdust monitoring gauges using a ladder.

Annual photographs over a 6-year monitoring period detecteddifferentgrowthpatterns for different species associated with changing environmental conditions [2].Two quadrats'A ' and 'B' situated only 100 m apart demonstrate site variability on alocal scale according to exposure. Thalli ofParmelia caperata and P. revoluta persistthroughoutthe monitoring period whilst there is a rapid lossof P. sulcata and theacidophilousspeciesHypogymnia physodes in the exposed road-side site (Figures 2 and3).

4.DataQualitycontrol

Routine statisticalprocedures are applied and supplemented by other methods.

8.50

-.7.50

y =-2.8098x+ 28.191R2 =0.9454

5.50 6.50

502 ppb

4.50

.. .. ..... .........---y =-1.8236x+ 18.135R2 = 0.9329

20 ,..-------------------,1816

N 14E 12 ._u 10j 8

6420+----,------,----,-----,----13.50

Figure 2. Area cover of recent colonists Parmelia caperataand P. revoluta(average of8 quadrats) over theperiod 1994-1999plotted against annual mean SO] (non-automatic monitoring site at Slough 16).SO] levelsfell on a yearly basis from 8.25 to 3.76 ppb over the same period. There is a highly significant negativecorrelation (R] = 0.945 and 0.933 respectively) between the area cover ofP. caperataand P. revolutaandmean annual SO}. This suggests that these lichens are returning in response to falling SO} levels and thattheir cover provides a direct indication ofSO}concentrations. Datafrom AEA Technology website [I}.

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1994

1998

,.1996r: .;--;:l

~iii.i1r'''-----1 A - road-sidesite

..Illllll

B - shelteredsite

IKey:

I~ Pann elia caperata

• Parmel ia sulcatn

o Damagedlichen

Figure 3. Six year monitoring period (I994-/999) demonstrating site variability. Quadrat A) road-side siteand Quadrat B) sheltered site.

5.Application

Appropriate for studying individual lichen species (little is known about the growthofmany ofour commonerspecies), rare and endangered taxa and to examineinterspecific

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competition. A uniform substrate should be chosen unless the objective is to comparegrowth on different substrata (e.g.high vs. low pH surfaces).It may be modified forrock or other hard surfaces by applying discrete and durable paint or enamel to assist inre-locating sites.

There is no standard time interval between taking photographs, this depending onthespecies'growth rate and habitat. Once a year is usually adequate in disturbed urbanhabitats and highly nutrient enriched sites typified by a higher turn-over, though thismay be extended to 2-3 years or longer in stable situationswhere crustose lichenspredominate.

6. Limitations

This method is unsuitable for irregular surfaces. Fixed photographic recording isadvisable where possible to minimise errors in parallax (e.g. by using a tripod or fixinga bracket from the camera to the quadrat).

7.References

I. AEA Technology website hosting UK National Air Quality Database, http://www.aeat.co.uklnetcen/airqual/

2. Purvis, a.w.,Bamber, R.N.,Chimonides,J., Din, V .,Erotokritou, L., Jeffiies, T.,and Jones, G. (2001)Burnham Beeches Lichen Monitoring: Phase 2. Year 2. Report for the Corporation of London by theNatural History Museum,London, 125 pp.

3. UK National Air Quality Database - see AEA Technology site, http://www.aeat.co.uklnetcenlairquaV4. Wolseley, P.A. and James, P.W. (2001) Factors affecting changes in species ofLobaria in sites across

Britain 1986-1998,Forest. Snow and Landscape Research 75 (3),319-338.

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SITE ASSESSMENT OF EPIPHYTIC HABITATS USING LICHEN INDICES

F.ROSEl and S. COPPINS 2

136St Mary's Road, Liss, Hampshire, GU33 7AH, UK.2A.M Coppins, 37 High Str., East Linton, East Lothian, EH403AA, UK.

Rose [4] established that deciduous woodlands in lowland Britain which have retainedsome degree of long-term ecological continuity support significant lichen assemblageswhich are absent or poorly representedin woods where disruption to ecologicalcontinuity has occurred to a greater or lesser degree. He concluded that these speciesrepresent a"relictflora" and developed the conceptoftheir use as" Indicator species"for grading woodlands on a scaleofincreasing or decreasing levels of past disturbance.This approach is presented here in a revised version.

1.Aims

• To establish a lichen index based on indicator species that can be used to assesslong-termecological continuity statusofdeciduous woodlands in lowland Britain,and supported where possible by independent documentary evidence.

• To use Indices to evaluate lichen and conservation interests in deciduouswoodlands and parklands in order to compare and contrast similar sites withinlowland Britain.

• To contribute towards establishing a method for grading epiphytic lichen sites fortheir conservation interest and importance, from sitesof international importance(Grade 1) to those of no lichen interest (Grade 7) [1].

2.Method

2.1.MATERIALS

• Existing large data setsoflichen records for woodlands and forests within a regiontogether with habitat descriptionsofwoodlands and forests.

• Evidence of past histories for woodlands within at least partsofthe region.

2.2. REVISED INDEX OF ECOLOGICAL CONTINUITY (RIEC)

A list of 102 lowland deciduous woodland site in Britain where lichens were recordedwas prepared [4], where possible with good historical documentation. The list

343P.L. Nimis, C.Scheideggerand P.A. Wolseley (eds.), Monitoring withLichens- MonitoringLichens. 343-348.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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encompassed woods of known long ecological continuity, together with woods knownto have been clear-felled and re-planted, and those known to be (or have been) managedas coppice. Hence, lichen floras from a rangeofwoodland history types were used.

Lichen data were compared and a listof 55 species was drawn upof species±restricted to woods of known ancient origin with some degreeof ecological continuityovertime.

The list was refined to a manageable 30 Indicator Species and formed the basis forthe RIEC [4]. Species within the RIEC are mostlyoftheLobarion pulmonariae and theLecanactidetum premneae (Table 1). The list includes species associated with old­growth forests occurring over a wide geographical range throughout lowland Britain,yet also encompasses species indicativeof old-growth forests within particulargeographical parts of the range. Hence, it includes some species which are commoninScotland but rare in southern England, and vice versa. In some cases there is flexibilityof certainspecies occurring within a given wood, e.g. whereSticta fuliginosa and S.sylvatica both occur, then only one is counted towards the overall total.

The Index is calculated as a percentage: RIEC= n/20 x 100, wheren is the numberof indicator species, and 20 the maximum numberof species expected in any'good'site.

TABLEJ. RJEC- RevisedIndexofEcologicalContinuity Maximum total- 30 - RJECis calculatedby thenumberofspeciesn/10 x J00; e.g. 6 RJECspeciesgives an RJECof30.

Arthoniavinosa Pachyphiale carneolaArthopyreniaranunculospora Pannariaconoplea

Biatorasphaeroides Parme/iacrinitaCatillaria atropurpurea Parme/iareddenda

Dege/iaatlantica!orD. plumbea!orParme/iel/a Peltigera co//inatriptophyl/a Peltigera horizonta/isDimerel/alutea Porinaleptalea

Enterographa crassa Pyrenulachlorospi/a!orP. macrospora

Lecanactislyncea Rinodinaisidioides

Lecanactispremnea Schismatomma quercicola/or Pertusariapupil/aris

Lobariaamp/issima Stenocybeseptata

Lobariapulmonaria Stictafu/iginosa!orS.sylvatica

Lobariascrobiculata Sticta /imbataLobariavirens Thelopsis rubel/a

Loxosporaelatina Thelotrema lepadinumNephroma laevigatum

2.3. NEW INDEX OF ECOLOGICAL CONTINUITY (NIEC)

Following advances in taxonomic, ecological and biogeographical knowledgeofepiphytic lichensin the 16 years since the RIEC was established, the NIEC wasdeveloped.

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The NIEC strictly covers most of lowland Britain north to southern Scotland, andtherefore encompasses a more homogeneous geographical and climatic area than theRIEC.

The NIEC is based on a list of 70 species primarily devised towards gradingwoodlands for their conservation status, rather thanjustfocusing on the "old woodland"interest. As these two interests are often linked, the NIEC in fact incorporates nearly allofthe RIEC species (Table 2).

Additional significant local or rare species not included in the base list of 70 species,are counted as"Bonus" species as they add to the overall conservation interest. The"Bonus" species are added to the NIEC total, to give an overall Index figure denoted asT. The NIEC is not intended to replace the RIEC,but to be used in conjunction with it,as the latter indicates the "ancient woodland" qualities, whilst the former has broaderapplication to assess the overall conservation importanceofa given woodland site. SiteswithT values>20 are considered to beofconservation significance.

2.4.DATA COLLECTION AND HANDLING

This technique does not depend on statistical analysis, but on the building upofcomparative data sets and of appropriate independent evidence. Much of the data is inunpublished reports manyofwhich are listed as grey literature inUnpublished Surveyson the British Lichen Society website:http://www.thebls.org.ukJ

3.Workedexamples

• RIEC [4] Useful results have accrued from the widespread useofthis index whencompiling lichen species lists of individual woodlands, given the limitationsdescribed below. The higher the Index number, the greater the likelihoodofecological continuity being a significant factor in indicating the overall highbiodiversity interest of the woodland ecosystem. The core listof 30 Indicatorspecies allows for variation in woodland type, structure, climatic and topographicalsituation, as a maximum of only 20 Indicator species is required to achieve a scoreof 100. Rose [4] found 18 woods with Index values of 100 (based on records madein the latter partofthe 20th century) outofhis listof 102 woodlands.Of these, fourare reliably backed up by documentary evidence as being in existence sincemedieval times. Thirteen woods had 0 RIEC scores. These were woods which wereeither known to have been clear-felled and replanted within the last 200 years, orold coppiced woods.

• NIEC [5] includes most of the core listof 30 RIEC species (mostlyLobarionpulmonariae and Lecanactidetum premneae), but encompasses wider ecologicalamplitude to include significant species associated with other lichen communitiesfound in additional niches (e.g.Calicion hyperelli and Usneion barbatae). The corelist of 70 NIEC species can be enhanced by the additionof recognized'Bonus'species (nationally rare or very local lichens), so that the conservation importanceofthe whole woodland ecosystem in termsofits lichen flora can be realized.

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TABLE2.NIEC - New Index ofEcologicalContinuity. Note that only one species is countedwhen alternativesor "sp. " are given. To calculate the NIEC value: total the numberof 'main' species, plus the total number of'bonus ' species. to obtain a final Index figure (T). Sites with T values of 20 or more are of conservationimportance. Note: this indexcombinesecologicalcontinuitywith wideraspectsofconservation importance.

Main sneciesAeonimia allobata LoxosnoraelatinaAeonimia octospora MeealosooratuberculosaArthoniaastroidestera Micareaalabastrites/otM. cinereaArthonia ilicina MicareapvcnidiophoraArthonia vinosa NeohromalaevieatumAnhoovrenia antecellens Neohroma oarileArthoovreniaranunculosoora Ochrolechia inversaBacidia biatorina OoeeraohacorticolaBiatoraenixantholdes OoeeraohanrosodeaBiatorasohaeroides Pachvphiale carneolaBuelliaerubescens Pannariaconoolea/otP. rubieinosaCatil/ariaatroourourea ParmeliacrinitaCetreliaolivetorum ParmeliareddendaChaenotheca stm. /excl.C.Ierrueinea) Parmeliella iamesiiCladoniacaesoiticia ParmeliellatriotoohvllaCladoniaoarasitica Peltieera collinaCollematurturaceum/otC.subflaccidum Peltieera horizontalisDeeelia atlantica/orD. plumbea PertusariamultiounctaDimerellalutea Pertusaria velataEnteroeraohasorediata Phaeozraohis so. rexcl.P. smithii)Heterodermia obscurata PhvlloosoraroseiLecanactisam lacea RinodinaisidioidesLecanactis lvncea Schismatomma niveumLecanactisoremnea Schismatomma ouercicola/orPertusaria ouoillarisLecanactissubabietina StenocvbesetnataLecanoraiamesii Sticta limbataLecanoraauercicola Sticta tulieinosa/or S.svlvaticaLecanorasublivescens StraneosooraochronhoraLetnozium cvanescens Theloosis rubellaLemoeium lichenoides Thelotrema leoadinumLetnoeium teretiusculum UsneaceratinaLobaria amplissima Usnea floridaLobariaoulmonaria Wadeana dendroeraphaLobariascrobiculata Zamenhotia coralloideaLobaria virens Zamenhofiahibemica

Bonus sneeles:thefollowinzisa selection' theirinclusionis denendentonzeozranhicalconsiderations.Anaotvchiaciliaris (Devononlv) PannariamediterraneaArthoniaanombroohila PannariasampaianaArthoniaanelica ParmeliaarnoldiiArthoniaarthonioides ParmeliahorrescensArthoniazwackhii ParmeliaminarumBacidiacircumsoecta ParmeliasinuosaBacidiasubincomma ParmeliatavlorensisCatillaria laureri ParmeliellatestaceaCaloplaca lucituea Pseudocvohellaria crocataCollema fragrans Pseudocvnhellaria intricata/orP. norvegicaCollemanierescens Ramoniaso.Collemasubnierescens Schismatomma eraohldiotdesCrvotolechia carneolutea Sohaerophorus eiobosus(S Enzland onlvlGvalectaderivata Sohaerophorus melanocamus (S Enzland onlv)Leotozium bureessii Sticta canariensis/or S.dufouriiLeotoeium cochleatum Teloschistes flavicansMeealaria erossa (S Enzland onlvl UsneaarticulataOoezraoha fumosa Zamenhofiarosei

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4.DataQualitycontrol

Data quality is dependent on the individual expertise of the lichenologists carrying outsite recording. Where necessary, critical material is sent for determination to specialistreferees.Specimens are lodged in national herbaria for future reference (BM,E, NMW).This will enable us to assess responses of lichens to changes in ecological conditionsbrought about by management or more long term effectsofchanges in climate.

5.Application

This technique can be applied in areas where there is already considerable data availableon lichen records and of historical data. Indices can be used to ascertain woodlandsofhigh conservation importance, and are being used in North America (see chapter 33, thisvolume) and in Tasmania by Kantvilas (see [2, 3], and chapter 36, this volume), and toevaluate managementof tropical forests in SE Asia (see [6], and chapter 37, thisvolume).

6.Limitations

• In areas where lichens have been mainly eradicated by former or presentatmospheric pollution (including pollutant sources such as brickworks, aluminiumsmelters, etc.),lichens cannot reliably be used as indicatorsofecological continuityor of conservation status. For example, some parkland and woodlandsof lowlandBritain have lower indicator values than would be expected from documentaryevidence.

• High nitrogen and ammonia production associated with intensive agriculture iscausing replacement of ancient woodland lichen flora by speciesoftheXanthorionparietinae, or in badly affected areas by green algaeDesmococcus spp. (seechapters 5 and 21, this volume).

• Lichen communities are generally poor in woodlands that have been managed forcoppice. This is due to direct removalofepiphytes along with coppiced stems, andalso drastic changes in light and humidity around any adjacent remaining trees.Theeffect is to disrupt ecological continuity, although the site may have been woodedover a long period.

7.References

J. Fletcher,A ., Coppins,B.1.,Hawksworth,D.L.,James, P.W.,and Rose, F. (1982)Survey and AssessmentojEpiphytic Lichen Habitats, Report by the Woodland Working Partyofthe British Lichen Society forthe Nature Conservancy Council,contractHF3/03/208.

2. KantviJas, G. (\988) Tasmanian rainforest lichen communities: a preliminary classification,Phytocoenologia 16,391-428.

3. KantviJas, G., James, P.W., and Jarman, S.1. (1985) Macrolichens in Tasmanian rain forests,Lichenologist 17,67-83.

4. Rose, F. (1976) Lichenological indicators of age and environmentalcontinuity in woodlands, in D.H.Brown, D.L. Hawksworth, and R.H. Bailey (eds.), Lichenology: Progress and Problems. AcademicPress, London, pp. 278-307.

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5. Rose, F. (1992) Temperate forest management: its effect on bryophyte and lichen floras and habitats, inl.W. Bates, and A.M. Farmer (eds),Bryophytes and Lichens in a Changing Environment, OxfordUniversity Press, pp.211-233.

6. Wolseley, P.A. (1991) Observations on the composition and distributionofthe'Lobarion ' in forestsofSouth East Asia, in D.l. Galloway (ed.),Systematics, conservation and ecology of tropical lichens,Clarendon Press, Oxford,pp.217-243.

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INDICATOR SPECIES - RESTRICTED TAXA APPROACH IN CONIFEROUSAND HARDWOOD FORESTS OF NORTHEASTERN AMERICA

S. B. SELVA

Division ofNatural and Behavioral Sci.. Univ. ofMaine at Fort Kent. 25Pleasant Street. Fort Kent. Maine 04743. USA ([email protected])

Many calicioid lichens and fungi are dependent on mature forests containing treesofdifferent ages and a varied light and humidity regime, making them good indicatorsofforest age and continuity [2,7, 9].

1.Aims

• To assess ecological continuityofforestsby using a restricted groupoftaxa that areboth widely distributed and which include species associated with old growthforests.

• To compile as complete an inventory of the calicioid species of the survey area aspossible in order to assess ecological continuityofthe site.

2.Method

2.1.EQUIPMENT AND MATERIALS

A rigid, fixed-bladeknife is essential for removing taxa from old hard wood and bark.For collecting saxicolous calicioid lichens and fungi use a rock hammer, chisel, andsafety glasses. A x10 or x20 hand lens for careful examinationof likely substrata.Collecting packets folded from 8W' by 11" sheets of paper together with writingimplement. Mostofthis equipment should be available at a forestry supplier.

2.2. PROCEDURE

2.2.1. Field methods1. Select sites within characteristic spruce-fir or northern hardwoods forests, as

defined by the SocietyofAmerican Foresters [I].2. Establish study areas within environmentally uniform habitats.3. Sample area using" intelligent meander" or Releve Analysis for Classification [4]

to locate specific microsites with which calicioid species are associated, includingstanding, decorticated hulks, stumps, and fallen logs and branches, as well as the

349P.L. Nimis, C. Scheidegger and P.A. Wolseley (eds.), Monitoring with Lichens - Monitoring Lichens. 349-352.© 1001 Kluwer Academic Publishers. Printed in the Netherlands .

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exposed heartwood of living trees. This subjective sampling method has beenadopted to ensure a high sampling intensity and collection in localized rich areas.

4. Search each living tree species for potential microsites, especially areas wherecalicioid species are not competing with other lichens and bryophytes for space.

5. Collect specimens withtheir substrate using a blade knife or hammer and chisel.6. Place specimens in packets taking care to protect fragileapothecia withtissue paper

or paper handkerchiefs.7. Label packet with site, location and substrate data and with collector and date.8. On extended collecting trips keep packets in large boxes to protect specimens from

damage.9. Collect numerous replicates to increase the chanceof capturing rarer species as

many species cannot be identified in the field [7].Field tips for calicioid lichens - Calicioid lichens and fungi are found on a wide varietyof substrata mainly associated with older trees between ground level and 2 metres.Exceptions includeSphinctrina, which typically grows parasymbiotically withPertusaria or Lecanora species, and may be found higher on thetrunk or on branches;Phaeocalicium polyporaeum , which is found on polypores; and several speciesofPhaeocalicium and Stenocybe which are restricted to the smooth barkofbranches andsaplings of Alnus. Betula. Populus, Quercus, Ilex, and Rhus. Other potentialmicrohabitats are the roots of upturned trees, cave-like "grottos" formed at the baseofolder trees, and over resin, including that formed around broken branch collars. Speciesmay also be restricted to the more porous inner bark of certain trees,down in the cracksof deeply fissured bark, on the lower surface of bark chips and flakes, and inwoodpecker holes.

2.2.2. Laboratory methods1. Assign each packet a collection number.2. Search all surfaces and edges of substrate fragments for apothecia.3. Glue bark fragments with specimens on to baseof specially designed (or

homemade) boxes and place these back in packet. This is well worth the effort,especially if anotherofyour goals is to photograph undamaged and representativespecimens.

4. Prepare squash preparationsofascocarps and note structureofasci, the morphologyand arrangementofspores, and the presence or absenceofa mazaedium.

5. Undertake spot tests on apothecia and/or thallus for all specimens with potassiumhydroxide (K), sodium hypochlorite(C), paraphenylenediamine (P), iodine (I),nitric acid(RN0 3) , and lactophenol/cotton blue.

6. Last prepare sectionofthe thallus(ifpresent) in order to determine which, if any,ofthe associated algae is indeed the photobiont [6].

7. Identify genera and species of calicioid taxa using available keys [3].

3.Worked example

The continuityof 42 spruce-fir stands and 34 northern hardwoods stands in theAdirondacks of New York, northern New England, and Maritime Canada have been

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assessed,to date, using an indexofrestrictedtaxa based on the totalnumberofcalicioidlichens and fungi found at each site[8].

Tableswerepreparedthat ranked eachofthe sites by forest type and bydecreasingtotalnumberofcalicioidtaxa collected.

When the 76 stands under investigation were arrangedaccording to decreasingnumberof totalcalicioidtaxacollectedat each site, indexvalues for the 42spruce-firsites ranged from a high of 24 to a lowof 2, and index values for the 34northernhardwoodssites ranged from a highof 21 to a lowof O. It was also determined-bycomparingRevised IndicatorsofEcologicalContinuity(RIEC) (see [5], andchapter32,this volume) and calicioid index values for the same sites - that apioneer forestharboredbetween0-2 calicioidtaxa; a seral forest >2 calicioid taxa; a young old-growthforest>I0 calicioidtaxa; and anancientforest>15 calicioidtaxa.

4. DataQualitycontrol

Expertconfirmationofspeciesidentification.

5.Application

• Widely applicableto coniferousand hardwoodforestsof thenorthernhemisphere[7, 10].

• Fewerspecies to identify, ashighestnumberrecorded,to date, atanyonesite =24.• Diversityofcalicioidspecies is highest onaccessiblepartsoftrees from base up to

2 m,allowingtotaldiversityofthis group to be assessed.

6.Limitations

• Sincecalicioidspecies do not occur within the obviouslichen-richcommunitiesontree trunks, afamiliaritywith potentialmicrositesis essentialfor finding them.Similarly, carefulattentionmust be paid to their small size, as they havefrequentlybeenoverlooked.

• Identificationrequires both amicroscopeand specialistkeys, as regionalfloras donot always include all species, and maydemandtheassistanceofan expert.

• Timeallowedfor samplinga site depends on the relative age and sizeofthe stand.Since ayoungerforest will have fewercalicioidmicrohabitatsthan anolderforestand a smaller site will have less terrain to cover than alarger one, itusuallybecomesapparentafter a six- to eight-hourday ofmeanderingfrom onepotentialmicrositeto the nextwhetheror not you haveexhaustedthe availablepool. If thestandis largerand/or older,a secondday ofsamplingmay bewarranted.

7. References

I. Eyre, F.H. (ed.) (1980) Forest cover types of the United States and Canada, Society of AmericanForesters,Washington, D.C.

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2. Hyvarinen, M.,Halonen, P.,and Kauppi, M.(\992) Influenceofstand age and structure on the epiphyticlichen vegetation in the middle-boreal forestsofFinland,Lichenologist 24,165-180 .

3. May, P.F.,Brodo, I.M.,and Esslinger, T.L. (2001) Identifying North American lichens : A guide to theliterature. http://www.herbaria.harvard.edulDatalFarlow/lichenguide/guidetoliterature.html

4. Mueller-Dombois ,D .and Ellenberg, H.(\974)Aims and methods ofvegetation ecology, New York.5. Rose, F. (1976) Lichenological indicators of age and environmental continuity in woodlands, in D.H.

Brown et al. (eds.),Lichenology: Progress and Problems. Academic Press, New York, pp 279-307.6. Selva, S.B. (1988) The Caliciales of northern Maine,The Bryologist 91,2-17.7. Selva, S.B. (1994) Lichen diversity and stand continuity in the northern hardwoods and spruce-fir forests

ofnorthem New England and western New Brunswick,The Bryologist 97, 424-429.8. Selva, S.B. (1998) Searching for Caliciales in the Adirondacks of New York, in M.G.Glennet al. (eds.),

Lichenographia Thomsoniana: North American Lichenology in honor ofJohn W. Thomson. MycotaxonLdt, Ithaca, N.Y.,pp. 337-344.

9. Tibell, L. (1980) The lichen genusChaenotheca in the Northern Hemisphere,Symbolae BotanicaeUpsaliensis 23, 1-65.

10. Tibell, L. (1992) Crustose lichens as indicators of forest continuity in boreal coniferous forests,NordicJournal ofBotany 12, 427-450.

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MONITORING REGIONAL STATUS AND TRENDS IN FOREST HEALTHWITH LICHEN COMMUNITIES: THE UNITED STATES FOREST SERVICEAPPROACH

S. WILL -WOLF

Department 0/Botany, University ofWisconsin, 430 Lincoln Drive,Madison, WI 53706-1381, USA ([email protected])

1. Aims

• To use lichen communities as biomonitorsofchange in forestecosystems,relatingto changes in air quality, climate and/or forestmanagementacross large regions andover relatively long time periods, with relatively low cost [1].

• To relate conditionof lichen communities to conditionofvascular plants as partofthe Forest Inventory and Analysis (FIA) programmeof the United States ForestService.

The lichencomponentofthe programme first assesses the initial conditionoflichencommunitiesin a region [3], and over time monitors change with respect to climate andair quality gradients, as well as responses to other anthropogenic alterations to theecosystem.

2.Methods

2.1.EQUIPMENT AND MATERlALS

• Tape measure (50 m), compass, etc.,• Maps showing FIA grid and plot locations,• Collecting equipment and paper packets for voucher specimens,• Watch or timing device.

2.2.PROCEDURE

1. Data are collected on forested plots on the permanent national FIA grid, withhexagonal grid centers 12 miles (19.3 km) apart. One permanentforestry plot israndomly located within each grid cell, and a regular subsetof grids is sampledeach year.

2. Lichen data are collected in permanent circular plotsof 0.378 ha(= 0.935 acres)centered on the centerofthe permanent forestry plot [5].

353P.L. Nimis, C.Scheideggerand P.A. Wolseley (eds.), MonitoringwithLichens- Monitoring Lichens, 353-357.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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3. Non-lichenologist field crews aretrained in macrolichen discriminationandcollectionprocedures,and must passcertificationtestsbeforecollectingdata.

4. Presenceand abundanceofmacro lichenspecieson all woody plantsin the plot areassessedusing an establishedprocedure. Note that therecordermust be able tomake distinctionsbetweentaxa, but thatidentificationof the speciesis undertakenlater by aspecialist.4.1. Within a periodof 2 hours voucherspecimensare collectedthatrepresentthe

diversityofmacrolichens in the plot as fully aspossible.Crews inspectthe fullrange of substratesand microhabitatsavailablefor macrolichens on woodyplantsabove 0.5 m from the ground,includingrecentlyfallenbranches.

4.2.The abundanceofeachseparatecollectionis estimatedusing the scalebelow.

Code Abundance1 Rare « 3 individualsin area)2 Uncommon(4-10 individualsin area)3 Common (> 10 individualsin area but less thanhalfof the boles

and brancheshave thatspeciespresent)4 Abundant(morethanhalfof boles andbrancheshave thesubject

speciespresent)

Where there isconfusion over separatingtaxa, abundancefor eachpossiblydistincttaxon collectedis rated separatelyin the field. In each bioclimaticregion supplementaldata arecollectedby expertsusing thesamefield methodsas for regulardata. Data areprocessedas for regularplots. Plots arechosentodefineregionallyimportantgradientsin climateand in airquality. Typically,plots withknowngood andpoorair qualityareestablishedin all climatezonesofaregion.

2.3.DATA COLLECTION AND HANDLING

Field samples are identifiedby lichen specialistsand abundanceof each species isassigned from field estimates.One plot recordconsists of a list of species withabundancesfor a particulardate. One regional data setconsists of a set of all plotrecords compiled for one generalbioclimaticregion in one year. In the US FIAprogram,5 bioclimaticregions have beendefinedfor the USA eastof the MississippiRiver, and 7-10 bioclimatic regionsarebeingdefinedfor theUSA in the west.Dataareenteredin standardizedformat and arearchivedin a centralfacility. Basic data areavailableon anationalweb sitewithin1-2 yearsofsampleidentification[7].

3.Data analyses

Data analysisis implementedin two phases:1. Calibrationphase: constructionand calibrationof a gradient modelof lichen

communitiesto isolateand describegradientsof interestsuch as responseto airqualityand climate,and

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2. Application phase: applicationofthe model to calculate gradient scores for regionalplots, which are then used to answer resource assessment questions, asdescribedabove.

In (I) Calibration phase: Lichen community gradients are extracted from acombined regional and supplemental data matrixof plots by lichen species, usingmultivariatemethods for data reduction. Axes are aligned with major gradients using asecond data set for plots with tree data, climate data, and air quality data. This generatesa regional gradient model, with scores on axes representing the gradientsof interest(example[3]). A generally-effectivetechnique for generating scores oncompositionalgradients, when faced with heterogeneous community data sets, is non-metricmultidimensionalscaling (NMS; implemented in software package PCORD [4]). NMSis well-suited to data that are non-normal or are on arbitrary or discontinuous scales; itcan be used both as an ordination method and as a technique for assessing thedimensionalityof a data set. Where air quality gradients are stronglyconfoundedwithclimate or topographic gradients, an air quality response gradient can be developedusing indicator species and regression techniques in addition to ordination.

In (2) Application phase: Plots in a regional data set are given gradient index scoresby fitting them to the axesof the regional gradient model developed in the calibrationphase. Program NMSCORE (software package PCORD [4]) calculatesgradientindexscores using an iterative procedure to estimate best fit to an existing model.

In all cases the gradient index scores are calculated from unitless numbersrepresentingrelative abundance (see field method)ofspecies. Comparisonsofgradientindex scores for plots and regions over time generate measuresofchange relative to thedefined regional gradients. Archived metadata include details on generationof eachgradient model, and notes on taxonomic statusof lichen species reported (updated eachyear).

4.Workedexample

Basic data have been collected for 8 USA bioclimatic regions in at least one year since1993; several summary data reports are available from a Forest Service web site [6].Gradient models are available for Southeastern (see Figure 3 inchapter14, thisvolume), Northeastern,and Colorado, USA, and will be soon for Eastern Midatlanticand NorthwesternUSA. Comparison of data for 1994 and 1998of plot scores onclimate and air quality gradients in the Southeastern andNortheasternUSA showedsignificantvariation across regions,but no difference over time (Table I).

Variations of the field protocol can be used ascost-effectiveand rapid lichencommunitymonitoringtools for many purposes. The investigatorcan record lichensseparately by forest layer and/or by tree species or category such as conifers vs.hardwoods. The investigator can estimate lichen biomass by converting abundanceclasses to numbersof thalli, then multiplying by a species- orgenus-specificsizeconversion factor. In the example below, lichen genera are given conversion factorswhich roughly approximate the surface in square centimeters which is covered bymature thalliofsome species in that genus, from an unpublished study by the author.

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Lichen Genera Size conversion factor(- = cnr')Candelaria. Hyp erphyscia 1Phaeophyscia, Physcia, Heterodermia 2Evernia, Leptogium, Melanelia, Ramalina 4Lobaria, Parmelia, Pelt igera 8

TABLE I. Lichen Indicator Trend Analysis, SE USA: A comparison ofthe 1994 and 1998 on-frame data fr omAlabama, Georgia and Virginia. Mean bias (average ofboth positive and negative deviations) implies higherspecies richness. "cooler " climate scores. and "cleaner" air quality scores in 1994 (N = 94) as compared to1998 (N = 109). Analysis of variance for the 76 resampled plots showed significantly more species/plot weref ound in 1994. However no significant difference between years was f ound for plot scores on climate and airquality gradients. suggesting no significant regional change in these factors. As expected, plots differedamong themselves for all three variables.

Paired plots, 1994 - 1998 Mean Meandeviation, Deviation <15% of AnalysisofVariance, F-ratio(P)(N = 76) bias absolutevalues aradient

Plots (df= 75) Years (df=l)

Species richness 2.5 5.18 -- ---------- 2.73 (p<O.OOI ) 13.42(p<0.00I)

Climate Gradient 1.25 17.60 71% ofplots 4.45 (p<0.00I ) O.l8(p=O.67ns)

Air QualityGradient 5.59 24.39 63% ofplots 2.53(p<O.OO I) 1.88(p=O.l7ns)

5.Data Quality control

Data must be collected within certain standards: the field crew must pass a certificationtest to be able to collect data. Remeasurements are conducted during the field season toevaluate data quality.Corrective action (retraining and retesting)is takenif standardsare not met. We have found that if the non-lichenologist trainee obtains 65% or moreofthe specialist's number of species, the gradient index scores, (see data analyses)willmostly fall within 10% ofthe expert'sscores [2]. Therefore, this 65% figure is used asan operational standard for minimum acceptable field crew performance quality.Because repeatability over time is so critical, lichen specialist trainers are also requiredto pass a trainer certification test. Lichen identification specialists all use similaridentification standards,and each submitsa voucher collection for future reference.

6.Application

This method is suitablefor establishing a biomonitoring program with lichens to assessstatus and trends in forest ecosystem response over a very large area with relatively lowcost. It is designed to be implementedwith non-lichenologist field crews, so it isprimarily suitable for use with macrolichens. Cost-effective and repeatable assessmentof broad trends areemphasized at the expense of complete species capture. Whenperformed by a lichen specialist,microlichenscan be included, and the field methodissuitablefor more precise monitoring of response. This or a similar methodology couldserve as a rapidcalibration for smaller-scaleand more precise studies in very differentgeographic regions,or using differentsmall-scale research protocols.

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7.Limitations

This method is not designed to assess total lichen biodiversity, nor to assess trends overtime for one particular place. Its strength is in statistical validity for indicating theconditionof forest ecosystems and lichen communities, and for monitoring trends overtime across large regions. The method is designed as a time-constrained plot survey bynon-specialists. Species capture is sensitive to departures from standard methods; itincreases when specialists conduct field work or when extended field time is taken. Itdecreases when field time is shortened or collecting conditions are poor (rainy, poorlight). For this reason, comparisons between surveys based solely on numberofspeciescaptured may generate misleading results.

8.References

I. McCune, B.(2000) Lichen communitiesas indicatorsofforesthealth, TheBryologist97, 396-401.2. McCune, B., Dey, J.P., Peck, J.E., Cassell, D., Heiman, K., Will-Wolf, S., and Neitlich, P.N. (1997)

Repeatabilityof community data:species richness versus gradient scores in large-scale lichen studies,TheBryologist 100,40-46.

3. McCune, B., Dey, J., Peck, J., Heiman, K., and Will-Wolf, S. (1997) Regional gradients in lichencommunities of the Southeast UnitedStates,TheBryologist 100, 145-158.

4. McCune, B. and Mefford, M.l (1999) PC-ORD. Multivariate Analysis of Ecological Data. Version 4.MJM Software Design, Gleneden Beach, Oregon, USA, http://www.ptinet.netJ-mjrn/

5. Neitlich, P. and Will-Wolf, S. (2000) The Lichen Community Indicator in the National Forest HealthMonitoring Program: Using lichen communities to monitor forest health, posterpresentedat ForestHealth Monitoring Workshop 2000, Orange Beach, Alabama,February 14-17,2000.

6. United States Forest Service (2001)Forest Inventory and Analysis/Forest Health Monitoring.http://www.na.fs.fed.uslspfo/fhm

7. United States Forest Service (2001)Monitoring Air Quality and Biodiversity with Lichen Communities,Forest Inventory and Analysis Program (FIA) Lichen Communities Indicator, USA, http://www.wmrs.eduJIichen

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BIODIVERSITY ASSESSMENT TOOLS - LICHENS

C.SCHEIDEGGER ,U. GRONER ,C.KELLER and S.STOFER

WSL, Swiss Federal Research Institute. CH-8903 Birmensdorf,Switzerland (christoph [email protected])

1.Aim

To comparelichen diversity indices within and among land-use gradients indifferentcountriesand onpredeterminedsubstrataoftrees, rocks and soil.

2. Method

2.1.EQUIPMENT FOR FIELD WORK

~Il m

Figure I. Largefrequency gridfor soilreleveswith 20 units (lOxIOem).

81--+---t---t---+---iooo:t

•••

•••••••

1 - large frequency grid with 20 unit areas, lOx10 ern each (grid size: 50x40 cm,Figure 1),4 - narrow frequency grids with one rowof5 unit areas, lOx10 em each (grid size50xIO ern, Figure 2),1 - 60 m measuringtape,I - compass ortelescope-compass,12 - signal sticks preferably with red top (1,5m high),50 m - flagging tape,1 - hand lens,2 - rubber bands (ornails) to fixing the grids,1-knife,I-hammer,2 - chisels,ca. 1000 paper bags for collected specimens.

2.2.SELECTION OF LAND-USE UNIT (LUU)

Select 6 land-use units (LUU) along a gradientof land-useintensitiesin the study areaofeach country. Each LUU is a quadratof 1 km side length.

359P.L Nimis, C. Scheidegger and P.A. Wolseley (eds.), Monitoring with Lichens - Monitoring Lichens. 359-365.~ 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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2.3.SELECTION OF SAMPLING PLOTS

Mark the centreof 16 sampling plots within each LUU as shown (Figure 3). Each plot isa circle covering I ha with 56.41 m radius from the marked centre.

10 em

EoCon

Figure 2. Four narrowfrequency grids for rock and tree releves (5 cells per grid) .

Characterisethe habitat type, disturbance intensity and frequency, as well as themanagementhistoryofeach I ha plot.

In each sampling plot 12 collecting sites are selected randomly (Table I).

1 2 3 4• • • •5 6 7 R

• • • •9 Ii W I,•13 14 15 16

• • • •..200

Figure 3.Sixteen sampling plots are marked in each LUU.

2.4.SELECTION OF COLLECTING SITES

Starting from the centreofeach sampling plot locate 12 collecting sites using a compassand a measuring tape (or infrared distance measuring device) (Figure 4). For eachofthe12 collecting sites the azimuth (given in 4000 gradation and 3600 gradation) and thedistance from the centreofthe sampling plot are given in Table I.Collectingsites are

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numberedclockwise,starting in the north(azimuth= 0) from 1 to 12. Thecentresofthecollectingsites aremarkedwith a 1,5 m high stick.

TABLE 1. Number. azimuth and distance ojthe 12 collecting sites as measured from the centre ojthe samplingplot.

AzimuthCollectingsite no.

I2345678910II12

4000 gradationo3367100133167200233267300333367

3600 gradationo306090120150180210240270300330

Distancefrom centre[m]43,7823,8011,3049,3719,9633,108,0851,7853,1726,1347,2436,41

60 N

60

Ew

-60 S

Figure 4. Sampling plot (I ha) with the 11 collecting sites. The sampling plot is defined by a circle with r =56.41m. The collecting sites are numbered clockwise starting from the north (azimuth = 0). Azimuth anddistance for the 12 collecting sites are listed in Table 1.

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2.5.SELECTION OF OBJECTS IN EACH COLLECTING SITE

At eachofthe 12collectingsites record three different releves; on soil, on rock, and ona tree.Selectionof sites for the reieves is described below and must be followed inorder to obtainconsistentdata sets.

2.5.1. Soil relevesPlace the lower right angleofthe large grid (Figure I) at the centreofthecollectingsiteso that the longer sideofthe grid is oriented to the north. Record all lichensgrowingonthe ground, on rotten wood, on shrubs and small trees (ca. 12 em diam.,up to a heightof 150 em above ground), on pebbles and rocks (size< 50x40 em) or on any othersubstrateare consideredin this reieve.This means that all lichen species which are inthe projectionof the grid up to a heightof 150 cm areconsidered.Trees (> 12 emdiam.) and boulders(size> 50x40 em) are notconsideredin this releve,If the locationofthecollectingsite coincides with a tree trunk or a saxicolous object, move the largegrid to the north until the entire grid lies outside this object.

2.5.2. Rock releveStarting from the centreof the collecting site, find thenearestsaxicolousobject with asize bigger than 50x40 cm. Place the four narrow frequency grids(Figure 2) on theobject in a way that the epilithie and terricolous(ifthesaxicolousobject ispartiallyorcompletelycovered with soil) lichen synusia can besampledas completelyas possible(withpreferencefor high speciesnumber, not for high frequency). Saxicolousobjects inthe bedsof rivers with a submerse oramphibiouslichenvegetationare excludedfromthe survey.Continuethis procedurefor collecting sites 2-12. In case no more objectscan be found in thesampling plot, mark thenumber of "missing objects" on theprotocol. Asaxicolousobject can only be observed once.

2.5.3. Tree relevesIn order tomaximisethe species diversity present in the sampling plot, and to avoidover-samplingoffrequent tree species orunder-samplingof rare orlarge-diametertreespecies(contributingto the overall lichen species richness) the followingprocedurehasbeen devised.

Define two groupsof tree species, one with acidic bark (group A), the other withmore or less neutral bark (group B). Start at the centreof collectingsite 1 and find thenearesttree which belongs to the tree species group anddiameterclass indicated inTable 2. Mark the four sectorsof the compass at 150 em above ground. Set up the 4narrow frequency grids (Figure 2) as shown in Figure 4. In generalbranches,which areinside the grid are notconsidered(but see note below). The marks define the centresofthe short sidesofthe four narrow frequency grids.Note the lichendiversityas indicatedbelow. Note "preferredobject" on the protocol. Continue thisprocedurefor collectingsites 2-12, however, a tree can only beinvestigatedonce. In case no moreobjectsoftherequired tree species group and diameter class can be found in thesamplingplot, lookfor anotherobjectof the alternativediameterclass but which belongs to therequiredtree species group. If no objectof the indicated tree species group ispresenton thesampling plot, select an objectof the alternative tree species group and therequired

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TABLE 2.Preferred trees ofcollecting sites 1-12.

Col1eeting site Nr.PreferredobjectI Group A,> 36 em2 Group A,>12< 36 em3 Group B,> 36 em4 Group B,>12< 36 em5 Group A,> 36 em6 Group A,> 12< 36 em7 Group B,> 36 em8 Group B,>12< 36 em9 Group A ,> 36 em10 Group A, >12< 36 emII Group B,> 36 em12 GroupB, > 12< 36 em

363

diameter class. Otherwise select an objectofthe alternative tree species group and thealternative diameter class. In case analternative object had to be selected, note"alternative object" on the protocol andspecify the reason (tree species group, treediameter class or both).If there is no objectavailable on the sampling plot, note thenumberof"missing objects" on the protocol.A tree can only be observed once. In the caseofcork oaks where the barkof the trunks isoften harvested, the 4 narrow frequencygrids are set up on the main branches above

the upper limit of the cork harvest. In this case, however, cork oaks should always besampled at the main branches. The precise procedure on how an alternative siteof thetree was sampled must be carefully documented.A similar procedure could also be usedin caseoftar harvest on Pine trees. As mentioned above, lichens growing on branchesare usually not considered in this method. However, if the trunks are typically denselybranched (usually fine and rather short branches, e.g.on spruce) in the parts where thegrid should be placed, also the lichens growing on branches should be considered. Inthis case, project the lichens from the branches onto the surface the grid and count thefrequencyofthe lichens. The precise procedure on how branches were considered in thereievesmust be carefully documented.

--I_---- 11 I1 I1 ..J• ,-- I

stem

N

Upper margin offrequencyladderat150em

Frequency laddersfixed wit pins

One frequencyladder in eachseetorN.E.S.W

Figure 5. The 4 frequency ladders ofa tree releve are fixed between 150 and /00 cm above ground. Thecentre ofeach frequ ency ladder is oriented to N.E. S. W. respect ively.

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2.6.RECORDING LICHEN RELEVES

Recordone protocolfor each object. Listalllichen-fonningspecieswhich occurinsidethe 50x40 em frequencygrid or inside the fourfrequencyladders. Omit lichenicolousfungi andnon-lichenisedfungi (e.g.Arthopyrenia spp.).Omitthalli,smallerthan5 mm.

The area investigatedon one object is always the same,namely the 2000 cm2

coveredby thefrequencygrid (Figure 1) or the fourfrequencyladders(Figure 2). Donot recordspecies,whichare outside this area.

For each lichenspeciescount thenumberof unit areas (lOxlO em) inwhich thelichenspeciesoccurs(valuefrom 1 to 20).

Collectvoucherspecimensofeachspeciesfor identification.

2.7.IDENTIFICATION OF LICHEN SPECIMENS

Correct identificationof specimens [5-9] may require standard microscopicalprocedures and thin layer chromatographyanalyses of the secondary chemicalcompounds[1-4]. At least onespecimenof each speciesshould be placedin a publicherbarium.

3.Data Quality control

A control surveywill becarriedout onrandomlyselectedplots in each LUU.Datawillbe used for aqualityassessmentofthe lichendiversityanalyses.

4.Application

This methodhas beendevisedto gathera statisticallyrobustestimatorof the lichendiversityin land-usegradientsfrom naturalforests toagriculturalmanagedland. It isbeyondthe scopeoftheprojecttocollectcompletespecieslists.

S. Acknowledgements

We thank Cristina Maguas, Pat Wolseley, Federico Fernandez Gonzalez, Sampsa Lornmi, Martin Schiitz,Thomas Wohlgemuthand Allan Watt forstimulatingdiscussions. We acknowledgefinancialsupportfrom theSwiss Federal Office for Education and Science(BBW 99 .0683).

6.References

I. Culberson, C.F. and Ammann, K. (1979) Standardmethodezur DiinnschichtchromatographievonFlechtensubstanzen,Herzogia 5, 1-24.

2. Culberson, C.F. and Johnson, A. (1982) Substitution of methyl tert.-butyl ether for diethyl etherinstandarzided thin-layerchromatographicmethod for lichen products,Journal oj Chromatography 238,438-487.

3. Culberson, W. and Culberson, C. (1994) Secondarymetabolitesas a tool inascomycetesystematics:lichenized fungi, in D.L. Hawksworth(ed.), Ascomycete Systematics. Problems and Perspect ives in theNineties, NATO Advanced Science Institutes Series, Plenum Press,New York, pp. 155-163.

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4. Huneck, S. and Yoshimura,1. (1996) Identification of Lichen Substances. Springer-Verlag, Berlin,Heidelberg.

5. Poelt, J. (1969)Bestimmungsschliissel EuropaischerFlechten, Cramer, Lehre.6. Poelt, J. and Vezda, A. (1977)Bestimmungsschlussel europiiischer Flechten. Ergiinzungsheft I, Cramer,

Vaduz.7. Poelt,J.and Vezda, A.(1981)Bestimmungsschlilssel europiiischerFlechten. Ergiinzungsheft Il, Cramer,

Vaduz.8. Purvis, O.W.,Coppins,BJ.,Hawksworth, D.L.,James, P.W.,and Moore, D.M. (1992)The lichen flora

ofGreat Britain and Ireland. Natural History Museum Pub\., London.9. Wirth, V.(1995)Die Flechten Baden-Wiattembergs, 1&2. Eugen Ulmer, Stuttgart.

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USING LICHENS AND BRYOPHYTES TO EVALUATE THE EFFECTS OFSILVICULTURAL PRACTICES IN TASMANIAN WET EUCALYPT FOREST

G. KANTVILAS 1 and S.J.JARMAN 2

I Tasmanian Herbarium . GPO Box 25204. Hobart, Tasmania 7001,Australia (gkantvilas @tmag.tas.gov.au)2Forestry Tasmania. GPO Box 207B, Hobart. Tasmania 7001, Australia.

A silvicultural systems trial has been established by Forestry Tasmania in the WarraLong-Term Ecological Research site in southern Tasmania to compare potentiallyfeasible alternatives to the clearfell, bum and sow system used routinely in Tasmania'swet eucalypt forest [1].The purpose is to develop indicatorsofsustainability and to testsilvicultural alternatives where habitat, aesthetics or other non-wood values have specialimportance [3]. The trial involves examining the impactof different logging andregeneration techniques on various components of the biota, including lichens andbryophytes. The vegetation isEucalyptus obliqua-dominated forest where the oldesttrees are more than 200 years old. Methods adopted for the investigationof bryophytesand lichens are outlined below.

1.Aims

• To examine the impact of different logging and regeneration techniques on thelichen and bryophyte components in wet eucalypt forest.

• To compile an inventoryofspecies inEucalyptus obliqua-dominated forest.• To evaluate the potentialofspecies as ecological indicators.

2.Method

2.1.EQUIPMENT AND MATERIALS

• 50 m and10mtape measures,• mapsofforest area.

2.2. PROCEDURE

• 50xlO m quadrats were established where possible on Forestry Tasmania'sContinuous Forest Inventory (CFI) plots, which are surveyed, relocatable, 50x20 mquadrats.

367P.L. Nimis, C. Scheidegger and P.A. Wolseley (eds.), Monitoring with Lichens - Monitoring Lichens. 367-371.(1::1 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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• Inventories of all bryophytes and lichens for each plot werecompiled bythoroughlysearchingall accessible habitats,includingtrunks, twigs and leaves toabout 2 m above the ground (thepractical,accessible height limit), and logs, rocksand soil. Fallen twigs and branches were examined to obtain floristic data fromupper levelsofthe forest (see methods described in Jarman andKantvilas[4, 5] andthose describedand tested in Goward andArsenaut[2] and McCune and Lesica[8]).

• Onlypresence/absencedata were recordeddue to thedifficulty(or impossibility)offield identification. Samplesof all species were collected for laboratoryexaminationand identificationusing standard methods. However,collectingwaskept to a minimum,recognising that survival or recoveryof species could beadversely affected byover-collectingprior to silvicultural treatment.Vouchermaterial isdepositedin theTasmanianHerbarium.It is inevitable that the identityofsome species andspecimenswill be revisedovertime.

• For the purposeof analysis, habitats were stratified to reflect keysubstratetypes.This decision was based on apreliminary field reconnaissanceagainst abackgroundofgeneral experience inTasmanianvegetation. The categoriesused insamplingwere:

a) Epiphytes;• fibrous-barkedhosts(Eucalyptus obliqua),• smooth-barkedhosts (several speciesofunderstorey trees),• papery-barkedhosts (several speciesofunderstorey trees),• living leaves (mostlyofferns).

b) Non-epiphytes.c) The forest floor, with special attention given to large logs, rocks and mounds

of inorganic soil. This additionalstratificationis particularlyrelevanttobryophytes.

• For Eucalyptus obliqua, the timber species, totalinventoriesfor tree boles to aheight of 2 m were also compiledseparatelyfor the largest andsmallesttreesrespectivelyin each of five lOx10 m subplots. The aim was toexplore therelationshipsbetween epiphytecompositionand the ageand/orsize ofthe trunk.

• A soil transectacross the forest floor, dissecting the plot lengthwise,wasestablishedto obtain frequency data. Quadratsof 25x25 em were placed onalternatesides of the transect at 50em intervals. Small specimenswere collectedfor laboratory-basedidentification. These data have yet to beanalysedbut willprovide quantitativeinformation on floristic changes on the forest floor afterharvesting and regeneration. Asimilar approach could be adopted for othersubstratesifresources are not limiting.

3.Worked example

Sampling for flora and faunacommencedin 1997, and the first coupe washarvestedin1998. Re-samplingand monitoringof harvested sites hascommencedbut no analyseson this aspectofthe work have been undertaken.

Nine plots weresurveyed. Inventoriesof species,includingseveral new records forTasmania, detaileddescriptionsof the lichen andbryophytefloras, and their ecology

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and distribution with respect to habitat type are reported by Jarman and Kantvilas [6, 7].Analysis ofeucalypt data is currently being prepared separately for publication.

The numbers of species per plot range from 60-90 for bryophytes and 37-64 forlichens,with a totalof144 bryophytes and 134 lichens recorded for the total study area.This is an unanticipated level of diversity, in view of the superficially depauperateappearance of the forest's cryptogamic flora, and underlines the importance of theseforests for plant conservation.

For the total forest, we identified a "core" flora of species occurring in at least sixplots, scattered species that occurred in 3-5 plots, and low frequency species thatoccurred in two or less plots. For each habitat, we defined a typical flora (occurring onthe same substrate in at least 3 plots) and discussed the flora in terms ofspecialistspecies that show a preference for or are confined to a single substrate, andgeneralistspecies that occur across a wide range of substrates. Smooth-barked understorey treesrepresent the richest habitat, but allof the habitats support at least some specialistspecies. Bryophytes include a large proportionof generalists, whereas many of thelichens are specialists. The specialist species are the ones that are likely to pose thegreatest challenges for forest management, because their survival depends particularlyon the maintenanceoftheir preferred habitat.

For lichens, the average number of species per plot is only 51, representing 38% ofthe total lichen flora recorded. These figures indicate that each plot is quite differentfrom the next with respect to lichen composition even though it represents the samevascular plant community. Approximately one quarter of the species occurred in 6 ormore plots, and less than onehalfoccur in three or more plots. Thus the flora consistsofmany species which are not only small and inconspicuous but are also very sporadic intheir occurrence. Although no abundance data were formally recorded, many species arenot only infrequent between the plots but are also very uncommon within the plot, beingrepresented by one orjusta few tiny thalli.

The variability of the cryptogamic flora from plot to plot means that no single plot isparticularlyrepresentativeof the whole site. Furthermore the distributionof specieswithin the forest is so patchy that sometimes a single tree can be responsible forcontributing considerably to the diversityofa plot or even the entire study area. Whilstthis may be indicativeof the nature of the forest, we also feel thatwell-developedpatchesofbryophytes and lichens may have been undersampled by strictly adhering toplot selection based on vascular plant or silvicultural criteria. Whether or not thisstrategy has impacted on our recording of species diversity and distribution is unknownat this stage and we hope to sample some additional plots in the future that may clarifythe point.

4. Data Quality control

The involvementoftrained, experienced cryptogamic botanists is necessary at all stagesofthe project. A more rapid, superficial approach, using less-experienced, non-specialistfield officers to record lichens and bryophytes may be economically attractive to afunding organisation or other commercial body but will include serious drawbacks:• only a small proportion of the total biodiversity will be recorded,

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• mainly widespread (and often weedy) species will be scored, whereas species thatare likely to provide the best ecological indicators, or be species of conservationsignificance will be overlooked,

• the amountofmaterial that needs to be collected increases (equating to damage tothe site),

• the reliability of identifications is diminished.

5. Application

In areas where there is no knowledgeof the flora or its ecology, an inventory isconsidered essential prior to the selectionof specific species or habitats as ecologicalindicators. The inventory serves as a baseline against which to monitor change, andprovides the focus and rationale for a thorough investigationof the site. It is alsoimportant when potentially rare or otherwise significant species are present.

Stratification of sampling has several advantages. It offers a meansof systematicexamination and recordingofthe flora when confronted in the field with a complexofhost trees and habitats in often uninviting and inaccessible conditions. Moreimportantly, it enables an assessment of floristic changes where forest harvesting maylead to changes in the structure and compositionof the forest, especially in theunderstorey, and the entire removalofparticular substrates such as oldgrowth trees, logsand tall stumps.

6.Limitations

There are no proven established methodologies for this type of work in wet eucalyptforest. Our method was devised to take account of the factors outlined below.• Cryptogamsofwet eucalypt forest have been poorly studied in Tasmania and the

present project represents the firstof its kind in this forest type. The flora ischaracterised by many very small species and, in the caseof the lichens, the greatmajority are crustose taxa from groups that are poorly known even on a globalscale. Devising an appropriate methodology relied heavily on basic ecological andtaxonomic information from Tasmanian vegetation types with better knowncryptogamic floras such as cool temperate rainforest.

• The vegetationofthe site impeded easy access and passage through the forest, andweather and light-levels within the forest were also limiting. These factors allimpacted on time taken in the field, and associated costs.

• Logging and any subsequent regeneration burning will remove all or mostof thevegetation and someofthe other habitats such as logs and smaller woody debris. Itwill also potentially alter the microtopography. Therefore there seemed littlepurpose in setting up permanent, revisitable plots on any moveable or removeablesubstrates.

• Bryophyte and lichen study plots were located to coincide with plots for otheraspectsof the overall project. They were not necessarily the optimum sites thatwould be selected for a cryptogamic study.

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• The numberofplots sampled (nine) was acompromisebetweenresources availableand the ideal levelofreplication.

• Costs associated with employing specialists was a consideration.In our experiencein Tasmanian vegetation,it takes a lichenologist and abryologistapproximatelyoneweek each(includingone field day) to sample and identify the colIections from asingle site. The soil transect described below may take a further 3-5 days for oneperson. These times do not take into account folIow-up taxonomic work on thespecimens, curationofthe material, nor analysisofthe data.

7.Acknowledgements

We thank RobTaylor for his support of the project, Leigh Edwards for logistic advice and Mick Brown andHumphrey Elliott for comments on the draft manuscript. The project was funded by ForestryTasmania.

8. References

J. ForestryTasmania(1998) Lowland Wet Eucalypt Forest, Native Forest SilvicultureTechnicalBulletinNo.8,Forestry Tasmania, Hobart.

2. Goward, T. and Arsenault, A. (1997) Notes on the assessmentof lichen diversity in old-growthEngelmann Spruce - subalpine fir forests, in C. HoUstedt andA. Vyse (eds.), Sicamous CreekSilvicultural Systems Project: Workshop Proceedings, 24-25 April 1996, Kamloops, British Columbia,Canada, Res. Br.,B.C.Min .For.,V ictoria,B.C.Work Pap 24/1997, pp.67-78.

3. Hickey, J.E.,Neyland, M.G., Edwards, L.G.,and Dingle, J.K. (1999) Testingalternativesilviculturalsystems for wet eucalypt forests in Tasmania, in Practising Forestry Today, Proceedingsof the 18th

Biennial Conferenceofthe InstituteofForestersofAustralia,Hobart,3-8 October 1999, pp. 136-141.4. Jarman,SJ. and Kantvilas, G. (1995) A Floristic Study of Rainforest Bryophytes and Lichens in

Tasmania's Myrtle-Beech Alliance, TasmanianNational RainforestConservationProgram Report 14,Forestry Tasmania andDepartmentoftheEnvironment,Sport and Territories, Canberra.

5. Jarman,SJ. and Kantvilas, G. (1997) Impacts of Forestry Operations on Cryptogams in Tasmania'sEucalypt Forests. Stage I. A Preliminary Assessment of Diversity. Unpublished report to theCommonwealthDepartmentofPrimary Industries and Energy, and ForestryTasmania.

6. Jarman,SJ.and Kantvilas,G. (2001) Bryophytes and lichens at the Warra LTER site. 1.An inventoryofspecies inEucalyptus obliqua forest with aGahnia/Bauera understorey,Tasforests 13.

7. Jarman,SJ. and Kantvilas, G. (2001)Bryophytesand lichens at the Warra LTER site. II . Colonisationofunderstoreyhabitats inEucalyptus obliqua forest,Tasforests 13.

8. McCune, B. and Lesica, P. (1992) Thetrade-offbetween species capture andquantitativeaccuracy inecologicalinventoryoflichensand bryophytes in forests in Montana,The Bryologist 95,296-304.

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USING CORTICOLOUS LICHENS OF TROPICAL FORESTS TO ASSESSENVIRONMENTAL CHANGES

P.A .WOLSELEY

Botany Department. TheNaturalHistory Museum. CromwellRd, LondonSW7 5BD.UK ([email protected])

The use of lichens andbryophytesas sensitive indicatorsof environmentalconditionshas beenestablishedin temperate Europe,Canadaand the USA where lichen floras arerelativelywell known (see chapters 2-4, this volume). In tropical forests lichens can bealso be used as indicators despite the fact thatidentificationto species often remains aproblem. Lichen species in a defined area are often readilydistinguishedusingmorphologicalcharactersand simple chemical spot tests.

1.Aims

• To assess the effectofmanagementconditions on corticolous lichen (andbryophyte)diversityand frequency in randomly sampled plotsoftropical forests.

• To evaluateenvironmentalchanges that areoccurringin tropical forests by usinglichencharacterfrequencies.

2.Methods

2.1.EQUIPMENT AND MATERIALS

• 2 x 100 m tapes or stringofsamelengthmarkedwith 1 m intervals,• Compass,• Flexible quadrats with 250 cm2 area in a rangeofshapes to sampleslendertrees as

well as large girthed trees e.g.12.5x20 em, IOx24.5 em. Quadrats are made in dull­colouredcard with scales (cm/mm) along each axis andlaminatedto give waterresistance,

• Aluminium or plastic tags (note: paper orcardboardis often eaten or takenif leftovernight),

• Tableofrandom numbers(waterproofed),• Knife or chisel andhammerfor collectingcryptogamand bark samples,• Paper tissue (e.g.soft toilet paper) for packingspecimens,• Permanentmarkers,• Polythenebags for short term storageofspecimens in wet conditions,

373P.L Nimis, C.Scheideggerand P.A. Wolseley (eds.), MonitoringwithLichens - Monitoring Lichens. 373-378.(!:I2002 KluwerAcademic Publishers. Printed in the Netherlands.

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374

• pH meter and buffers forcalibration,• Computerwith PCO software,• Microscopewith slides and razor blades for lichenidentificationand identification

ofphotobiont,• Reagents: 10% KOH, Calcium hypochlorite(bleach) solution,para-phenylene

diaminesolution(in waterwith 5% Na2S03) in plasticdropperbottles,whichcan betightly closed. In tropical conditions it isbetterto leave asupply in the fridge andreplenishsuppliesregularly.

2.2.PROCEDURE

2.2.1. Plot and tree selection1. Determineplot size from the terrain and forest type beinginvestigated.In accessible

and relativelyhomogeneoustropical rain forest with largeemergenttrees it ispreferableto use 100 m square plots. Wheredenserforest withsmallertrees isencountered50 m square plots can beestablished,and if the terrain is veryunevenwith markedridges use arectangularplot 100x50 m,establishingthe 100 m alongthe ridge andtakingoffsets 25 meithersideofthetape/ridge.

2. Use tapes or marked string along two sidesof the square (or the centreof therectangleon a ridge plot), and random numbers to locate 20-25 trees to besampled,(numberssampledmay vary with forest type and plot size).Althoughsmall trees canbe omittedfrom thesamplingprocedure,in tropical forests small trees may havegrownvery slowly and are oftenlichen-rich.

3. Mark trees withaluminiumor plastic tags.4. Record tree dataincluding species (wherepossible), evergreenor deciduous,

diameteror girth, and bark type.5. Estimatetree density by taking distance tonearesttree (trunk) in eachcompass

quarter,excludingtrees with small canopies. This allows a roughestimateof areaaround each tree [1]. Bark samples can be taken and dried todeterminepH in thelaboratory.

2.2.2. Lichen and bryophyte recordingDiversity within quadrat - Select areasof lichen andbryophytediversityon each trunkup to 2 m and placeappropriatequadrats (e.g. 250em') on lichen andbryophytecomponents. Record aspect and height of quadrat above ground.Record taxadistinguishedin the fieldtogetherwith % covervalue (within 100 units),reproductivestate(ascomataor typeofpropagule)and collect samplesofeach species [2].Diversity on tree - Search the restof the tree up to 2 m foradditionalspecies notcontainedin eitherquadrat.Diversity within plot - Searchthe plot for additionalhabitatsand taxa, inorderto obtainan estimateoftotal species diversity for the plot.

2.3.DATA COLLECTION AND PROCESSING

1. Label all samples collectedwith plot, quadrat and treenumberto facilitatedataentry,placingeach in a separate packet.

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375

2. Spot test specimens for colour reaction with C and K and Pd (Steiners solution)either in the field or in the laboratory, noting the reaction on the packet. This allowsfield separationofsterile crusts (e.g. crust white, sorediate, Pd+yellow).

3. Dry specimens flat, packed in tissue (or toilet paper) back to back topreventthinbarks rolling up.

2.3.1. Laboratory1. Check pH ofbark specimens using a BDH Flat tip electrode.2. Use a dissecting microscope and light microscope with high power objective, to

identify specimens to genus wherever possible, using tropical keys such as Sipmanat http://www.nmnh.si.edu/biodiversity/lichkey2.htm.

3. Provide distinguishing epithet for taxa.

2.3.2. Data entryEnter site, tree and specimen data on a data base using a field nameifno scientific nameis available.Data should be recorded at family, genus and species or taxon level so thatdiversity could be compared at all levels. Many sterile crusts aremorphologicallyandchemicallydistinct but cannot be identified. These data should beincorporatedat fieldtaxon level. Additional information should include: reproduction - soredia, isidia,folioles - photobiont type as trebouxioid, trentepohlioid orcyanobacterial,and spot testreaction.If thin layer chromatography available, check the presenceofanthraquinonesand depsides andofother lichen compounds.

3. Dataanalyses

PrincipalComponentAnalysis (PCA) can be used to establish similarityof samples inrelation to plots, tree species and other environmental factors such as altitude, climate orfire frequency and logging techniques. The frequencyof identified species on sampledtrees within plots, and thatofphotobionts in all taxa within plots should be calculated.

4.Workedexample

Lichen data from trees in forest plots in Thailand showed clear differences in speciesassociations between fire-sensitive seasonal evergreen forest and fire-tolerant deciduousdipterocarp forest and these differences could be distinguished at the family and genericlevel. Plots in fire-sensitive evergreen forest were dominated by lichen taxa inBacidicaeae,Trichotheliaceae,Arthoniaceae and Pyrenulaceae, whilst plots in fire­tolerant deciduous forest were dominated byPhysciaceae, Panneliaceae andGraphidaceae[4]. Where taxa were identified to species it was possible to detect speciesassociatedspecifically with forest type or disturbance [4, 5].

Reproductive characteristics also vary with habitat; in fire-disturbed sites isidiateand sorediate taxa are most frequent, whereas in undisturbed sites fertile species aremore frequent (Figure 1). Sites in deciduous dipterocarp sites where lichens are exposedto a hot dry period have a greater frequencyof lichens with trebouxioidphotobionts

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376

whereas sites in shaded humid evergreen forests have a greater frequencyoftrentepohlioid photobionts (Figure 2). Whole site data usingpeA ofdata from all treesshowed a continuum from evergreen forest to dry deciduous dipterocarp forest withtransitional sites where fire had caused a shift in the lichencommunitiesfrom fire­sensitive species to fire-tolerant species with low diversity.

reproduction

• fertileOsoredlate~jsjdlale

100

'" 90

-~ 80o~ 70o~ 60oS 50C8 40

~ 301-::) }

g 20r'#. i0 f-

0 '(:)</' ~,,~ &"~ <f1"b 1:/"" ~",,:> ,<,<:'

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Figure 1. Reproductive strategies of corticolous lichens in forest plots in dry dipterocarp (DDF) andseasonal evergreen forest (SEF) in northern Thailand. showing high frequency of vegetative propagules indamagedforest (DDFI and 10). and high frequency offertileforms in undisturbed plots (DDF8 and SEF 15)[3].

pholoblont• cyanobacteriao Trenteponna~ trebouxloid algae

o«.\J....<:J<;)<.:,1-<;)«;,~ "J"O ~(/,,,, 0""> 0''';plot

Figure 2.Frequency ofphotobionts ofcorticolous lichens in dry dipterocarp (DF) and evergreen (EF)forestplots in northern Thailand [3].

In Malaysia data from bryophyte and lichen quadrats in logged and unlogged plotsofthe same forest type showed a continuum between lichen and bryophyte samples and

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377

between unlogged and logged plots (Figure 3A).However when samples were identifiedby 'indicator' species there was very little overlap (Figure 3B).

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- 1·~\0 -05 0.0 0.5 1.0 1.5- 1 ·~1 .0 -0 5 0.0 0.5 1.0 1.5

-, -,A B

Figure 3. peA 's of corticolous lichen and bryophyte communities in tropical rain forest plots at Pasoh,Malaysia. A) distinguished by forest type: primary for est (50 h), unlogged disturbed forest (UP) and forestlogged 19 years ago; B) distinguished by presence ofselected indicator species; Myriotremaalbum(Ma),Myeloconis species (Ms) and Eschatagoniaprolifera(Ep) [5].

5. Data Quality control

Specimens often cannot be identified until returning to the laboratory or herbarium, sothat the building upof information on taxa collected is essential, as well as thecollectionofsmall samples for comparisonin the laboratory.

It was found useful to make reference card(s) for the field with small portionsofspecimens glued on with field name, characters used, and chemical reactionsincluded.This facilitatedfield identification.

6.Application

This method can be used to compare lichen diversity between forest types andmanagementhistory.The useoftaxonomic data at three levels allows detectionofshiftsin family, generic and taxon compositionwith changes in environmentalconditions.

The incorporationof simple data on reproduction,photobiontand where possiblechemistry allows a comparisonof adaptations to environmental conditions such asopening up of forest structure, lossof mature or single species trees, and highlightscharacteristics of taxa associated with disturbance or recovery from disturbance.It canbe used as a prerequisite to providing descriptionsof lichen communities, and toidentifying species that can be used as indicatorsofforest conditions.

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7.Limitations

• In tropical forests sampled diversityof tree trunks is a small partof the wholediversity which may be far greater in the canopy, or in other associated features suchas foliicolous species on leaves [4].

• The number of species found per plot increases with time spent searching thehabitat. In practice the time spent varies considerably with a numberof factorsincluding the accessibility of the site and day length. In tropical conditions it maynot be easy to quantify the time spent searching the plot.

8.References

I. LUcking, R. (1995)Biodiversityandconservationoffoliicolouslichensin CostaRica,in C. Scheidegger,P.A.Wolseleyand G. Thor(eds.),ConservationBiologyof LichenisedFungi,Mitteilungen der EidgenossischenForschungsanstaltfur Wald, SchneeundLandschaJt, Birrnensdorf,Switzerland.

2. Wolseley,PA, Moncrieff,C.,andAguirre-Hudson,B.(1994)LichensasindicatorsofenvirorunentalstabilityandchangeinthetropicalforestsofNorthemThailand,Global EcologyandBiogeography Letters4,116-123.

3. Wolseley, P.A. (1997) Response of epiphytic lichens to fire in tropical forests of Thailand,BibliothecaLichenologica68,165-176.

4. Wolseley, P.A. and Aguirre-Hudson,B. (1997) Fire in tropical dry forests: corticolous lichens asindicatorsof recent ecologicalchanges in Thailand,Journal ofBiogeography24,345-362.

5. Wolseley,P.A.,Ellis, L., Harrington,A., and Montcrieff,C.(1998)EpiphyticcryptogamsofPasohForestReserve, Negri Sembilan, Malaysia- quantitativeand qualitative sampling in loggedand unlogged plots,in S.S. Lee, Y.M. Dan, l.D.Gauld and1.Bishop (eds.),Conservation. Managementand DevelopmentofForestResources.Forest Research Institute,Malaysia,pp.61-83.

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LICHENOMETRY

D .McCARTHY

Department ofEarth Sciences, Brock University. St. Catharines, Ontario.L2S 3Al. Canada ([email protected])

Developedapproximately50 yrs ago [2], lichenometry has mainly been used by earthscientists to estimate the timingof(and hence monitoring)prehistoricglacial advances,landslides and othergeomorphologicalevents.The technique has beenespeciallyusefulin monitoring polar and alpineenvironmentswhere eyewitness ordocumentaryaccounts are lacking and other methodsofdating (e.g.14C analysis,dendrochronology)are unavailable or yield ambiguous results. When critically applied,lichenometrycanprovide quick, accurate, reproducible minimum ages for surfaces over aperiodof thelast three centuries. However, becauselichenometric ages are not always closeestimatesof actual age, they are termed as such and requireverificationusingdocumentarysources orindependentdating techniques to relate them to real time.There is no formula or standard practice that will ensure accuratelichenometricagesunder all circumstances. Prior to field work, potential users should review andtentatively select oneof the many sampling strategies that aredescribed[9, 10, 11].They should also examine the assumptions and criticismsof the technique recentlyreviewed by McCarthy [12].Three methods are set out below.The first is based on thebiodiversityof lichens which occur on surfaces, and because some species are latecolonisers they are also indicatorsof long term exposureofa surface. The second andthird are based on the growth ratesof individual lichen thalli which arepresumedtohavecolonisedthe surface when, or soon after, it was first exposed.

1.General Aim

To use the presence or sizeoflichen thalli (usually crustose species) as indicatorsofthetime since a substrate was exposed to the atmosphere.

2.Method 1

2.1.AIM

To assign relative ages to substrata.

379P.L. Nimis , C.Scheidegger and P.A. Wolseley (eds.), Monitoring with Lichens - Monitoring Lichens. 379-383.«:> 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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2.2.PROCEDURE

Distinction of barren (or sparsely vegetated) from lichen colonised rocks can be used asa basis for delimiting the spatial extentof floods, or identifying recently deposited,exhumed or scoured rocks. Since most lichenometrically useful species take about 20­30 years to become established, barren rock has likely been exposed for at least twodecades.Some species are late colonisers because they tend to colonise surfaces by firstbecoming established on topofcertain other species [4]. Other species appear to need aweathered surface on which to become established.The suggested procedure is as follows:I. Identify surfaces of known age that are colonised by lichens (e.g.gravestones).2. Identify the species present and record their frequencyofpresence and/or cover on

each surface.3. Construct a tableofspecies and their presence on a seriesofsurfacesofknown age.4. Identify species that are late colonisers, noting the time lapse before they are seen.5. Plot a graphofthe frequency/cover of the late colonisers against substrate age.6. Record thepresence/coverof the late colonisers on surfacesof unknown date and

readoffthe approximate date on the graph.

2.3. LIMITATIONS AND TIPS

Age assignment based on absolute differences in percentage lichen cover is not highlyrecommended - especially in the yellow-green and greyRhizocarpon communities[7].Anecdotal reports suggest that certain crustose species are restricted to surfaces thatpost-date 200 years (e.g.Rhizocarpon alpicola [6]), and some fruticose lichens can beuseful"indicators"ofold growth forest status. Accordingly, species abundance valuescan be collected along temporal gradients so as to"calibrate"the indicator status orsuccessional rankofspecies (e.g.early, mid, late successional stages [4, 14, 16]). Ageassignment based on measures of lichen diversity is best done by trained lichenologistsand should ideally use weighted indices that recognise the influenceof structuraldiversity and micro-environmental gradients on lichencolonisationand persistence.

3.Method 2

3.1. AIM

To estimate substrate ages by using direct measurementof thallus growth. The meanannual growth rateofa few thalli can be measured and extrapolated to estimate an agefor a surface that supports large thalli.

3.2.PROCEDURE

I . Select several fixed points at each thallus.2. Measure the radial growth rateof thalli in situ using calipers, trying to achieve

reproducibility to withinom mm [1], or take repeat photographs and measure from

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these [5, IS].Alternatively, fixed points can be made by using typewriter correctionfluid to make dots on the rock approximately I em from the marginofeach thallus.When the correction fluid is dry, a drafting pen and a ruler can be used to make andlabel crosshairs on each painted dot. A coat of clear urethane is then used towaterproofthe fixed points and protect them from being abraded by the calipers.

3. Calculate the mean, minimum, maximum and standard deviations for the radialgrowthofthalli in different size or age classes.

4. Divide the diameter of the largest thalli by the annual growth rate to estimate thetime since the surface was first colonised.

3.3. LIMITATIONS AND TIPS

Measurements should only be done on dry thalli on flat, stable surfaces. Calipers arenot used with species that have indistinct, fragile or hair-like margins. Directmeasurement using calipers requires steady hands, good eyesight and a significantinvestmentoftime both to establish the fixed points and do the annual measurements.Since two hands are required to position and adjust calipers, a voice activated taperecorder can be an invaluable aid.

4.Method 3

4.1.AIM

To estimate substrate ages by using indirect measurementof thallus growth. Thisapproach recognises that thallus growth may be negligible or rapid in some years butcan be assumed to be uniform over a longer period.

4.2.PROCEDURE

4.2.1. "Traditional approach ..1. Use tree-ring dating, historical documents, maps or aerial photographs to identify

several lichen-covered control surfacesofdifferent age. Make sure that all controlsurfaces have similar lithology and texture and each should cover at least 10 m2•

Search allof the substrate, but only thalli that have nearly circular outlines and arenot adjacent to moss, higher plants and/or standing water are considered.

2. Measure with a flexible plastic ruler the largest diameter (or the largest inscribedcircle or longest or shortest axis)of the largest 5-10 thalliof an identifiable lichenspecies (or aggregated species with similar appearance) on each substrateofknownage.

3. Transfer the data set to a scatterplot that has time on the X-axis and thallus size onthe Y-axis.

4. Fit an envelope curve representing the growth trend to the data by eye.5. Use the search and measurement procedure on similar substratesof unknown age

and minimum ages for the substrate are determined by matching the sizeof thelargest thallus with the corresponding age shown on the "growth curve".

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4.2.2. "Statistical approach "This approachis similarto thatdemonstratedby Matthews[10].1. Using a stratified-randomsampling,calibratea "growth curve" with five or more

quadratsoffixed size (e.g. 10 mz)located on eachofseveralsurfacesofknownage.Only habitatson a control surface that aresimilarto those found at theundatedsitesshouldbe sampled.

2. Using calipers, measurethe largestinscribedcircle or thelongestor shortestaxis ofthe 50largestindividualthalliofidentifiablespecies or speciesgroups.

3. Fit a line representingthe growth trend to the control datathrough the use ofregressionanalysis. The line can be fit to the pointsrepresentingthe single largestthalli or to the meanof the largestthalli found in eachof the 5quadratson eachcontrolsurface.

4. Repeatthis samplingand measurement procedureon surfacesof unknownage andassign substrateage bymatchingthe sizeofthe singlelargestlichen (or the meanof5, 10 or 50 largest)againstthecorrespondingage on the"growthcurve" .

5. Applications

These techniquescan be used tomonitorthe recolonisationof urban lichen desertsfollowing reductionin air pollution, monitoringthe stabilityof exposedsurfaces,andidentifyingold exposedsurfaceswhere thebiodiversityof the lichencommunitymaybe monitored.

6.Limitations

The "traditional"approachto lichenometrycontinuesto bepractisedin North Americawhere it seemsadequatefor use onmorainesofmoderatesize. It is probablythebestapproachto use onsparselyvegetatedcarbonatesurfaces(e.g. [13]),butis poorlysuitedfor use on very largemoraines."Statisticalapproaches"are wellsuitedfor use on largemorainesand are often seen in theEuropeanliterature. Recently,statisticalsmoothing(normalisation)ofthallus-sizedata has been shown toprovidehighly reproducibleandaccuratelichenometricages (e.g. [3]). Other "statisticalapproaches" rely on theassumptionthat thallus sizesnormally have aGaussian frequencydistribution.Someclaim high reproducibilityand have been used toestimateages for small clasts (e.g.[II]). Users should examinethe relevantliteraturefor a thoroughdiscussion of themany " statisticalapproaches"that are available.

7. References

I. Benedict,J.B. (1990) Experiments on lichengrowth. I. Seasonal patterns and environmental controls,Arctic and Alpine Research 22,244-254.

2. Beschel, R.E. (1950) Flechten als AltersmaBstab rezenter Moranen,Zeitschrifl fUr Gletscherkunde undGeologie. N.F. 1, 152-162. (Translated by W. Barr as "Lichens as a measure of the ageof recentmoraines",Arctic and Alpine Research S, 303-309).

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3. Bull, W.B. and Brandon, M .T. (1998) Lichen dating of earthquake-generatedregional rockfall events,Southern Alps,New Zealand, Geological Society ofAmerica Bulletin llO, 60-84.

4. Hill, OJ. (1994) The succession of lichens on gravestones: a preliminaryinvestigation,CryptogamicBotany 4, 179-186.

5. Hill, OJ. (2001) Growth, in L Kranner and R. Beckett (eds.),Methods in Lichenology, SpringerVerlag,Berlin (in press).

6. Innes,J.L.(1985) Lichenometry,Progress in Physical Geography 9,187-254.7. Innes, J.L. (1986) The useofpercentagecover values in lichenometric dating,Arctic and Alpine Research

18,209-216.8. Innes, J.L. (1988) The useoflichensin dating, in M. Galun (ed.),Handbo ok ofLichenology, CRC Press

Inc., Boca Raton, Florida, U.S.A.,pp. 75-92.9. Locke, W.W. III, Andrews, J.T., and Webber,PJ. (1979) A Manual for Lichenometry, British

Geomorphological Research Group Technical Bulletin 26, 47 pp.10. Matthews, J.A. (1975) Experimentson the reproducibility and reliabilityofliehenometric dates, Storbreen

G1etschervorfeld, Jotunheimen,Norway,Norsk Geografisk Tidsskrift 29,97-109.II. McCarroll,O. (1993) Modelling late-Holocenesnow-avalancheactivity: incorporatinga new approach to

lichenometry,Earth Surface Processes and Landforms 18,527-539.12. McCarthy, O.P.(1999) A biological basis for lichenometry?,Journal ofBiogeography 26, 379-386.13. McCarthy, O.P.and Smith,OJ. (1995) Growth curves forcalcium-tolerantlichens in theCanadianRocky

Mountains,Arctic and Alpine Research 27, 290-297.14. Rose, F. (1976) Lichenologicalindicatorsof age andenvironmentalcontinuity in woodlands, in D.H.

Brown, D.L. Hawksworth and R.H. Bailey (eds.),Lichenology: Progress and Problems, London,Academic Press, pp. 279-307.

15. Smith, R.LL. (1995) Colonizationby lichens and thedevelopmentof lichen-dominatedcommunitiesinthe maritime Antarctic,Lichenologist 27,473-483.

16. Stork, A . (1963) Plant immigration in front of retreating glaciers, with examples from the Kebenkajsearea, northern Sweden, Geografiska Annaler 45, 1-22.

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TRANSPLANTING LICHEN FRAGMENTS FOR PROVENANCE-CLONETESTS

J.-C. WALSER and C .SCHEIDEGGER

WSL, Swiss Federal Research Institute, CH-8903 Birmensdorf,Switzerland (jean-claude [email protected], [email protected])

Lichen species have generally a very broad geographical distribution and grow inecologically different habitats. We therefore expect ecotype differentiation betweendifferent populations. Transplantation experiments with vegetative diaspores and adultthalli of the threatened foliose lichensLobaria pulmonaria, Sticta sylvatica, andParmotrema crinitum have been used successfully for insitu conservationof thesespecies [2]. When transplanting lichens for conservation purposes, the design is oftenrather simple. In order to detect ecotype adaptation, more complex experimental designsare required [3]. One such design, known as a provenance test, is used throughout awide range of disciplines, e.g.forestry research.

1.Aim

To apply provenance testing to epiphytic macrolichens using initial material fromselected clones in a replicated experiment in order to compare morphological,physiological and growth parameters of lichens from different regions.

2.Method

2.1. EQUIPMENT

• Analytical balance (precision: 0.1mg),• Camera with macrolens, flash, and a tripod,• Colour films,• Aluminium staples (Stanley Bostitch STCR211506AL),• Pins and thread (tolayouta grid),• Tweezers.

2.2. EXPERIMENTAL DESIGN

Select sites for transplant experiments. The choiceof experimental design depends onthe numberofprovenances, clones and replicates.A completely randomised design hasno facilities to compensate for trends in the substrate.To allow for gradients within and

385P.L. Nimis, C. Scheideggerand P.A. Wolseley (eds.), Monitoring with Lichens- MonitoringLichens. 385-390.@ 2002KluwerAcademic Publishers. Printed in the Netherlands.

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among clonal fragments, e.g.light regime and humidity, the transplants can be split intogroups.An exampleofa 5 x 5 grid design: Select the number of:• Provenances (P= max. 4),• Clones from each provenance (C= max. 5),• Groups (G), replicates of a clone on one tree (G= 5),• Replicates (R),receptortrees(R = 2 or 3).Determine size and arrangement of transplants, e.g. a test with five clones (C= 5) fromeachof four provenances and a control (P= 4+ I) with the numberof five thalli perclone (G= 5) for each tree will need (C*P*G=) 125 fragments arranged in five groups(Figures 1 and 2). With this arrangement, there is no internal (within a group)replication but five external replications between the groups. There are usually twogradients on trees that have to be considered. In order to ensure that transplantsof aprovenance occur in each column and row per group, a more complex design wasselected (Figure I).

Group 2

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GroupEI C2 D5B4 D4 A2A4 BI C5

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BI C3 D3 A5 :;:fE2o~

E4 B3 A3 C5 !§~Di'1i~[t4~~!iiD]¥JiirB:4~~~'~EH§;R,('i~:

Figure I. A four provenances (A-D) plus control (E). five clones (e.g. Al-A5) and five groups test design. Ineach column and row every provenance occurs only once (see group I to 5 grey highlighted). The distributionon a provenance within a group was randomised.

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BI-B5

REPLICATES

El­E5

RECEPTOR

N

Figure 2. Example withfive clones ofthree provenances (AI -A5. BI-B5 and CI-C5) arranged in a 5 x 5design (X = no transplant) withfive groups on three receptor trees. £1 to £5 are controls.

2.3.PREPARATION OF THE LOBES

1. Check for sufficient source material in a populationofthe foliose species to be usedas cloning material to conduct the experiment.

2. Collect specimens to provide enough material for all transplants i.e. number ofexperimental sites (provenances, P) x numberofgroups (G) x numberofreplicates(R) + 5 references to determine the water contentof the air-dried specimens +10reserves and+ (number of groups (G) x number of replicates (R» controls. Werecommend to include the local provenance as a control (and as a treatment)in theexperiment. This will allow you to detect a loss of vitality due to possible storageand transport. The material for the control is collected from the same clones as thetreatment. Unlike the materialof the treatment, the control specimens will betransplanted immediately after collection.

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3. Cuttenninallobeswith active pseudomeristematic growth zones from each clone.The sizeofthe lobes should be as homogeneous as possible; for most species a sizeof2 x 1 em is appropriate.

4. Label the fragments with the letterofthe provenance (A, B orC) followed by thenumberoftheclone (e.g.AI, B3, etc.). Randomise the lobes and give them arunning number (e.g.Al/12or C3/17).

5. Photograph the hydrated and slightly pressed lobes on millimetre graph paper(Figure 3).

Figure 3.Hydrated and slightly pressed lobes ofLobaria pulmonariaphotographed on a millimetre graphpaper.

6. Air-dry all lobes and weigh them individually. Store the lobes individually in paperbags underdry and cool conditions and use them for transplantation within twoweeks.

7. Determine the water contentof five air dried reference lobes (105°C, 24 h) andcalculate the dry-weight of the transplanted lobes.

2.4. TRANSPLANTATION OF THE FRAGMENTS

1. Select two to three suitable acceptor trees (R) as replicates for each experimentalsite. It is likely that there is a gradient in the substrate (e.g. run-off-water or sunlight) that could have an impact on the growth or survival rateoftransplants.It isnot essential for the tree to be completely uniform, as the experimental design willhelp to remove systematic trends.

2. Select the transplantation area carefully in order to minimise ecological diversitywithin and among groups.

3. Remove all potential competitors (lichens, mosses etc.) from the experimental plotsand include a 5 em buffer zone around it.

4. Mark the upper left corner of each transplanted group with a coloured pin or anequivalent.The distance between transplants should be greater than 2 em. Hydrate

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the appropriatespecimen and fix it to the bark lobes facingdownwards,with astaple at the distal partofthe transplant.

2.5.DATA COLLECTION AND PROCESSING

1. Preliminary: group the transplants in threedifferentcategories: healthy, dead andindeterminate(Figure4).

2. After a couple of months, count thenumber of new meristems to estimatesuccessfulestablishmentand growth.

3. Quantitative analysis requires removalof the transplantsto determinethe dryweightand tophotographthem again to estimate area increase.

4. Use an appropriatestatistical method to test forsignificanceamong growth rateofdifferentprovenancesand clones.

3.Worked example

A provenance-clonetest forL. pulmonaria was establishedin British Colombia,Canada,where this foliose lichen grows inabundance.Threeprovenanceswere selectedfrom differentlife zones: intermontane, maritime, andhypermaritime[1]. Within eachprovenance, 5 clones werecollectedfrom thepopulationin order toprovideapprox. 80

Figure 4.First results at study site B after three weeks.A bar indicates a group (l to 5) and fi ve groupstogether a provenance (A to C). Transplants in goodhealth are shown in white, dead thalli in black andindeterminate thalli between good and poor health areshown in grey. No signijicant differences could befound because of the high numbers of indeterminatethalli after three weeks.

lobes from each clone. In each studysite, three trees were chosen fortransplantation,with preferencegiven toreceptortrees with youngL. pulmonariathalliestablished.

On each tree (R), we arranged 75transplants(3 provenances* 5 clones *5 groups) arranged in five (5 x 5) griddesigns (Figures 1 and 2). Clonalmaterial from location A wastransplantedto all three sites. This wasrepeated withpopulationsBand C toobtain three trees (R=3) at each site withfive groupsof all provenances. In total,450 lobes (75 x 3 x 3) were transplanted.Growth rate (as change in biomass orsize) for L. pulmonaria will bemonitoredfor the next three years. Aftera periodof3 weeks, we revisited one ofthe study sites and checked thetransplants (Figure 4). Thalli wereassigned to the following categories:healthy, if vivid when wet; dead,if thegreen colour had turned white orbrownish red. In some cases it was

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difficult to assign thalli to the first or second category, and a separate category wascreated (Figure 4).

4.Application

Cross-transplantsofL. pulmonaria from different provenances should reveal whether ornot this lichen is regionally differentiated into ecotypes with different growth anddevelopmental characteristics. In areas where fragmentationof suitable habitats hascreated isolated populations, it is important to establish whether populations displayecotype differentiation.

5.Limitation

The amount of initial material available. In the study described, five donor thalliofL.pulmonaria provided about 80 lobes. In transplanting experiments for conservationpurposes, the useofvegetative diaspores is recommended, but we have no experienceso far with this approach for provenance tests.

6.Acknowledgements

We thank Prof. Dr. Reiner Finkeldey for stimulating discussions. We acknowledge financial support from theSwiss National Science Foundation.

7.References

I. Goward, T. (1999)The lichen ofBritish Columbia. British Colombia Ministry of Forests,Victoria2. Scheidegger, C., Frey, B., and Zoller, S. (1995) Transplantationof symbiotic propagules and thallus

fragments: Methods for the conservation of threatened epiphytic lichen populations,Mitteilungen derEidgeniissischen Forschungsanstalt fiir Waldo Schnee und Landschaft 70,41-62.

3. Williams, E.R. and Matheson, A.C. (1994), Experimental design and analysis for the use in treeimprovement. Kinniburgh H.edition, CSIRO, East Melbourne.

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ASSESSING CHANGES IN DENSITY AND CONDITION OF LICHENS FORSPECIES RECOVERY PROGRAMMES

P.A. WOLSELEY ' and P.W.JAMES 2

IBotany Department. TheNatural HistoryMuseum. CromwellRd.London SW7 5BD, UK (P. [email protected])219 Edith Road. London W14 OSU. UK.

The inclusion of a number of rare or vulnerable lichens in Species RecoveryProgrammes in Britain has led to a requirement to assess changes in populationcondition, distribution and density over time.Teloschistes flavicans is a cosmopolitanfruticose species in the tropics but in Britain is now more or less restricted to coastalheaths in the south and west of the UK, especially Wales, with a northerly limit onAnglesey [1].

l.Aim

To assess the extentand current conditionof lichen populations using a repeatablesurvey method.

2.Method

This method is based on a plotless sampling method along a transect across an areaofvegetation [2], and is here adapted to monitor changes in distribution and densityof aterrestrial fruticose lichen [4].

2.1. EQUIPMENT AND MATERIALS

• 30 m or longer tape measure for transect, and shorter tape measure for distancesfrom each sampling point,

• Compass,• Coloured paint and brush,• Camera for recording location and quadrats.

2.2.PROCEDURE

1. Search the area to establish the limitsof the population to be sampled, and the

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major directionofchange (e.g. slope).2. Lay transect across the maximum extentof thepopulation, along the directionof

change, e.g.in the caseofTeloschistes jlavicans inland from sea cliffs.3. Use permanentfeatures to mark either endofthe transect andphotographthese to

allow relocationat a later date. Mark beginning and end with coloured enamelpaint.

4. Choose an interval along the sampling tape for sampling points, e.g. on a 100 mtape across a scattered populationofTeloschistes 20 m intervals were chosen.

5. From each interval locate the nearest specimenofthe chosen species in 4 quartersof the compass, and record the quadrant and the distance from the transect tape inthe same sequence for every interval.

6. Place a selected quadrat on the specimen(s) (e.g. IOxl5 em with 10 wireintersections on both axes) and estimate cover asnumberof squares occupied bythe species to be monitored.

7. Note substrate, aspect, height above ground and all associated species. Note sizeand conditionofindividualsofthe species in the quadrat.

8. Where possible set up markedpermanentquadrats to determine:• rateofcolonisation,• fateofmature thalli,• effectsofcompetition from other lichens and mosses.

2.3. DATA HANDLING

1. Calculate average distance from each transect locus in 4 quadrants to establishdistribution and densityofpopulation along the transect.

2. Calculate % cover and plot againstassociated characters, e.g.substrata, aspect anddiversity within the quadrat.

3. Repeat survey to check changes in species dominance, distribution and density.

3.Worked example

Transects acrosspopulationsof Teloschistes jlavicans wereestablishedin all locationsin Pembrokeshireespecially larger populations on Ramsey and Skomer [4].• Across the transect the highest densityof individuals occurred in the centreofthe

transect, the inland boundariesofthepopulationcorrespondingwith a change froma SW facing slope to a NE facing slope and the maritimeboundarywithin reachofsalt spray. Cover per quadrat varied throughout the transect.It was notpossibletoestablish the numberof individuals whenTeloschistes grew among foliose andfruticose lichens which obscure the holdfast.

• Preference for rock as substrate where sea mist conditions stable. Evidence ofrecolonisationon bare rock surfaces. Mainland terricolous sites haddeterioratedconsiderablysince 1995 both in extentof Teloschistes and numberof individuals.At that time no transects or quadrats were established, so it was notpossibletoquantify the loss.

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• Distribution associated with influence of humidity (sea mist) and not with aspectof substrate.Teloschistes is primarily found on south and west facing sites onRamsey, whereas on Skomer and at Manorbier it is established on NE to NWfacing sites, coinciding with sea mist and wind funnels. In all sites some are onhorizontal substrata [3].

• Teloschistes is healthy on Ramsey island and associated withRamalina andParmelia species in all quadrats, the shrubbyTeloschistes plants becominganchored among the other lichens. On Skomer and at Manorbier increase inbryophyte cover is associated with loss ofTeloschistes cover, probably throughdeterioration of thallus attachment within the bryophyte mat.

4.DataQualitycontrol

• Both endsof transect must be marked with resistant paint or other permanentmarkers to allow repeat of transect survey in the future.

• Locationofall permanent quadrats marked with red enamel paint for relocation.• Photographsof all locations and close-ups of quadrats will allowreassessmentof

data at a later date.

5.Application

This method can be used to monitor the distribution and densityof a populationoflichen species, and to assess changes in structureof the community that may beassociated with competition, with other associated or invasive species, or a change inenvironmental conditions.

6.Limitations

Although used for a fruticose species, this method could also be adapted for othergrowth forms and other substrata. The markingofpermanent location quadrats wouldrequire a different method than paint for terricolous and perhaps corticolous species.

7.References

I. Gilbert,0.1.and Purvis, O.W. (1996) Teloschistes jlavicans in Great Britain:distributionand ecology,Lichenologist 28, 493-506.

2. Mueller-Dornbois,D . and Ellenberg, H. (1974) Aims and Methods in Vegetation Ecology, Wiley &Sons,Toronto.

3. Purvis, O.W. and James, P.W. (1995) An assessment ofTeloschistesflavicans inPembrokeshire, Reportto CountrysideCouncil for Wales.

4. Wo!seley, P.A. and James, P.W. (1997) Resurvey and monitoring of Teloschistes flavicans inPembrokeshire, Report toCountrysideCouncil for Wales.

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MONITORING RED-LISTED LICHENS USING PERMANENT PLOTS

A. APTROOT 1 andL. SPARRIUS 2

lGerritvan der Veenstraat 107NL-3762 XK Soest, TheNetherlands([email protected])2Kongsbergstraat J NL-2804XV Gouda, TheNetherlands([email protected])

1.Aims

This method describes how Red-Listed lichen species can be monitored over a long periodin a fast and satisfactory way. Its advantage is that all species present in a plot are beingmonitored, including those not on the (current) Red List, so that the monitoring data can beprocessed and interpreted in various ways afterwards.

2.Method

2.1.PROCEDURE

The following steps are involved with this method:• defming and describing a plot,• making a listof(selected) species,• applying an abundance scale,• data processing and analysis.

2.2.DEFINING AND DESCRIBING A PLOT

When lichen monitoring takes place in semi-naturalto urban regions, the relevant area isoften delimited by the substratum.One can sample one tree or ten, or one or all bouldersofa certain monument, or one wall, or the whole church. Only with terrestrial releves thechoice is more open, but even here the rich areas are often semi-naturallydelimited (e.g. byvegetation boundaries, slopes, ditches, paths) and occupy overseeable areas like a fewdozen square meters. Surprisingly, there is often not much difference between species listsof4 m2 (the area preferred by many ecologists) and the whole site occupied by the targetspecies (e.g. 1000 m2) .

There is often considerable natural succession in lichen-dominated vegetation types,some of which are highly dynamic, like dune systems. Therefore, the most obvious siteselection, being the richest ones, is bound to give a decline over time, even when thesituation has not been deteriorated or even improved, because the richest sites will - aftersome time - lay outside the original plots.This problem has too often been neglected, andmany reportsofdecrease can be easily falsified by looking at other sites nearby. There are

395P.L Nimis, C.Scheidegger and P.A. Wolseley (eds .), Monitoring with Lichens - Monitoring Lichens, 395-398.II' 2002 Kluwer Academic Publishers. Printed in the Netherlands .

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sophisticated methods to circumvent this, involving complicated statistics. A well-knownexample is the stratified random site selection.Two much simplermethods are:• monitoring all the best sites plus a suiteofmediocre ones,• monitoring only the best sites at each given time.

Having themonitoringsite defined, a detailed mapof the site and itssurroundingsshould be drawn, including landmarks and distances, coordinatesof the plotcenterestimatedusing a hand-held GPS receiver, the lay-outof the recordingentities (liketrees or boulders) and the actual positionof the target species in the plot. When the sitedoes not consistofa countable numberofdiscrete entities, the site is divided into nineimaginary, equally sized blocks and a tenth block is reserved for an aberrant patch, e.g.a path or a steep margin. Some pictures takenofthe site andof the target species maypreventfutureconfusion.

2.3.LISTING SPECIES

A very importantand often neglected detail is the precisedescriptionof the speciesdelimitation.It seems logical to record all species, but what aboutomnipresentunidentifiablegreen crusts? Should one call themBacidia sp. orScoliciosporum sp., orleave them out? What about lichenicolous fungi andcorticolousfungi. There is noagreementabout the inclusionof Arthopyrenia , Mycoporum , Sarea, Peridiothelia,Hysterium and Navicella, to mention a few.Furthermore,the term"epiphytes"doesincludebryophytesas well. And in terrestrial habitats substrates additional to soil maybe present, like wood, pebbles and shrubs. On trees one may find extra species onoverhangingbranches.Itdoesn't matter what is chosen here, as long as it isindicatedinthe report, so that next time the changes are notattributableto changes in methods.When collectingfor identificationduring the fieldwork, a species should -of course ­not be competely removed.

2.4. APPLYING AN ABUNDANCE SCALE

The Braun-Blanquet scale or derivationsof it are not applicable to lichens, because theyinvolve estimating the number of individuals. But what is an individual lichen? A podetiumor a whole cushion - with or without its obvious daughter cushions - or all cushions on thesite? They may all be genetically identical. The scale in Table 1 can be used for repeatedobservations.It is an adaptationofthedaforffansleyscale, which not only takes abundanceinto account, but also spatial distribution:

TABLE I. Theabundance scaleused/or thismethod.

Scaleo1234569

DescriptionNotpresentOneindividual(thallus or colony)MoreindividualsinonerecordingunitFewindividuals on lessthanhalfoftheunitsManyindividualson lessthanhalfof theunitsFewindividuals on morethanhalfof theunitsMany individuals onmorethanhalfof theunitsPresent in unknownquantities (e.g. too small for reliabledetectionof all coloniesor foundin thecollectedmaterial)

Dafor

RareOccasionalFrequentAbundant

Dominant

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3.Dataprocessingandanalysis

The collectedfield data are stored in a database.It is alsopreferabletopublishthe entirespecies list together with the site map in amonitoringreport or in ajournal,to minimizeloss ofdata in future.The data from the database can be analyzed in various ways:• calculationoftrends for individual species as soon as time series become available.• comparison with data sets from other sources, e.g.spatial air pollution data and other

abiotic or biotic variables or Ellenberg values. Multiple regression or canonicalcorrelation analysis (CCA) can be done using computer software like SPSS andCANOCO .

Some tips for analysis:• inconspicuous lichen species and species with no quantity may be ignored when

applying statistics,especially when differentrecorders were involved,• the change in accompanying species may predict the change in the target species,

especially when the ecology of the latter is not well known.

4.DataQualitycontrol

Repeatabilityis essential, so that identical results should beobtainedif nothing haschanged, anddifferentrecorders should obtain the same results. To achieve good resultsover aperiodoftime:• Methods should be robust and clear and all details must be clearly stated.• The statusofthe target species should be known.• Taxonomic knowledgeofrecorders should be good to excellent.

5.Application

This method wassuccessfullyapplied in the Netherlandsfor monitoring red-listedlichen species in both natural (coastal and inland dunes) and artificial(granitebouldersofseadykesand megalithic monuments) habitats. See Sparriuset al. [I, 2] forexamplesofthe results.

6.Publicawareness

In order to be able to repeat the monitoring,contact with local authorities (e.g. land owners)is often advisable.If the target species is vulnerable to e.g. trampling or dependent oncertain management measures, it is not sufficient to point this out in a report or evenpublication, but it should be communicated to those responsible or the public at large.Often, but not always, the responsible authorities will co-operate, as Red Listed organismsgive added intrinsic biological value to the areas in concern.It is in the interestofboth thelichens and the lichenologists that certain target organisms get'cuddling' vernacular namesand appear on leaflets and fact sheets.

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7.Limitations

As this methodsusespresence/absencein sub-plots,it is notapplicablefor extremelysmallpopulationsand for surveyswhereonlyone speciesisconsidered.

8. References

1. Sparrius, L.B., van Herk, CoM o, Aptroot, A., and van Dobben, H.F. (2001) Landelijk MeetnetKorstmossen, Inhoudelijke rapportage 1999,Buxbaumiella 56, 1-32.

20 Sparrius, L.B., Aptroot, A .,and van Herk, C.Mo (2001) Landelijk Meetnet Korstmossen, Inhoudelijkerapportage 2000,Buxbaumiella 58, 1-44.

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A METHOD FOR DETECTING LARGE-SCALE ENVIRONMENTALCHANGE WITH LICHENS

G. INSAROV

Institute ofGlobal Climate and Ecology, 20b Glebovskaya Str., Moscow107258, Russia ([email protected])

1.Aim

To detect and measure theresponse of local lichencommumtIes to large-scaleenvironmentalchanges in e.g. climate change or airpollution.For further details see alsochapter 13,this volume.

2. Method

2.1. SAMPLINGEQUIPMENTAND MATERIALS

• Magnifying lenses x20, xlO, and x5, measuring tape and flexible plastic ruler withscale marked in millimetres, compass, plastic packs, adhesive labels, permanentmarker.

• For collecting epiphytic lichens a fixed blade knife is needed, and for epilithiclichens a I-inch wide chisel, pick-head geological hammer, and good protectiveglasses.

2.2. SITEAND PLOT SELECTION

I. Sites shouldbe chosenwithina protectedarea wherehuman impactssuch asforestry,agriculture,constructionarenon-existentor restricted,and be as far as possible fromemissionsources in order to detectthe effectsof changesin climateandlarge-scaleairpollutionon lichencommunities.

2. Select numbers of plots per site and of trees or transects per plotdependingon:availabilityof substrata,variabilityof lichencommunitycharacteristicsover the site,expectedchangesto bedetected,andlogisticalissues(seechapter9, this volume).

3. Sampling plots within the site should belong to as narrow a stratum as possible inorder to reduce the effects of spatialvariation in lichencommunitiesdue to substrate(tree speciesor rock type),altitude,slope,aspect, shadingand moistureconditions.Asfar aspossible,conditionsshould be the same for all plots, and these should bedistributedas evenlyas possiblein eachstratum.

399P.L.Nimis, C.Scheideggerand P.A. Wolseley (eds.), Monitoring withLichens- MonitoringLichens. 399-403.© 2002Kluwer AcademicPublishers. Printedin the Netherlands.

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400

2.3.SAMPLING

Figure 1. Thalli measurement on a trunk.

1.5m

,N

2.3.1. Epiphytic lichens1. Define groupsoftrees as sample plots,

with considerably less distancebetween membersof the group thanbetween plots. Avoid forest edges andmaritime situations.

2. Select mature trees with near verticaltrunks, omitting trees with severevisible damage to bark or crown.Selecttrees withouta priori information onpresence and abundanceof lichens. Toexclude effectsoftree age, trees shouldbeofthe same age and/or size over thewhole site for thefirstand subsequentobservations.

3. At a fixed height from the tree base(1.5 m recommended, 1 or 0.5 m canbe used for phorophytes with numerousdry snags), place a measuring tapearound the tree in a clockwise directionfrom north (zero) (Figure 1).

4. Starting at 0, record beginning and endof all lichen thalli intersected by theupper edge of the measuring tape.Record cover as linear distanceoccupied by each thallus. For fruticose lichens with branch width less than 1 mm notethe numberofbranches crossing each millimetre on the scale [4].

5. Takesamplesofunidentifiedspecies in thefield.

2.3.2. Epilithic lichens1. Select flat rocks or big stones preferably distributed evenly within strata over the site.

Each rock or big stone constitutes a sample plot. Omit unstable stones, and those withevident anthropogenic damage, or where snail grazingofthalli is evident. Sample plotsshould be on a similar exposureofrock on a similar slope. Noa priori information onlichen presence or abundance should be taken into account.

2. The distance among transects within a plot should be considerably less than thatamong plots.

3. Select pointsoftransect origins arbitrary within a plot. Place a 100 mm flexible plasticruler on the rock surface to be sampled in a north-south direction, so that its origin is atthe southern end. Starting at 0, record beginning and end intersectionsof all lichenthalli intersected by the edgeofthe ruler. Record cover as linear distance occupied byeach thallus. For fruticose lichens with branch length less than 1 mm note the numberofbranches crossing each millimetre on the scale [4].

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4. Take samples of unidentified species in the field.

2.4.REPEATED OBSERVATIONS

• Repeatobservationsat the same site. Theobservationintervaldependson variationoflichencommunitiesacross the site, expected changes to be detected andlogisticalissues. In practicethe intervalis betweenone and ten years.

• Selectplots and treesortransectsas describedundermethod.• Note that trees should be of the same age and/or size for allobservations.This means

that treesmay be differentfromthoseselectedfor previousobservations.

2.5.DATA COLLECTIONAND HANDLING

• Enter all data in a databaseby attributesofsite, plot, tree or transect, lichensby taxa atthe rankofspeciesand below,and thallusbeginningand end [11, 6].

• List all lichentaxa recordedin the courseof the surveyat a site.• The databaseprovidesinformationon four possiblegroupsoferrors [11]:plot and tree

(transect) numbering, thallus beginning and end, and treecircumference(= transectlength),lichencoverandintersection,and lichentaxanumbering.

3.Dataanalyses

Detaileddescriptionof data analysis is givenat [6].The essentialstepsare describedbelow.

3.1.LICHEN SENSITIVITY ASSESSMENT

Lichen sensitivity to large-scaleenvironmentalchange issite-specific.A summary tablefor sensitivityof 259 lichen species to different kindsof air pollution in the northernhemisphereis given in [7] (see Table 1).To estimatesensitivityof speciesnot found in thetable, apply proceduresofnon-uniformhierarchicalstructureddatainterpolation[5]. Eithertaxonomyor floristicregionsofthe earthcanbe used [5].

TABLE I. Sensitivity a/lichensto airpollution (from [71.withchanges).

* A ten-pointscaleIS used.Mostsensitivespeciesareassignedtenpoints.

No. Species Locality Pollution Tree BarkpH Sensitivity* Reference

17 Bacidia a) SouthBaikal S02, CO, dust, - . 7 Trass, 1985rubella H2s,CC4, No,

b) Montreal S02, CO, dust, Elm,ash Neutral 10 LeBlanc&DeHF,No, Sioover,1970

c) C & S England MainlyS02 - Neutral 6.7 Hawksworth&Rose, 1970

120 Lecidella Meirama(La Coal power Broad- - 5.7 Crespoet al., 1981euohorea Corona,Spain) station Leaf

249 Usnea Mosinee(Wis- MainlyS0 2 Pine Acid 6.2 Newberry,1974Hirta consin,USA) ..

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The estimationof lichen sensitivity to climatic factors can be conducted in oneof thefollowing ways:• Employing natural climatic gradients and interpreting distributionof lichen

communities along them [6]. Apply linear regression procedure [10] using climaticfactors as independent variables, lichen cover for each species as dependent, and usethe slope to estimate species sensitivity.

• Analysis of rangesof lichen species. Under changing climatic conditions peripheralpopulationsof species sensitive to change will disappearif these conditions becomeworse, while populationsoftolerant species will increase [5].

3.2. TREND DETECTION INDEX

Trend Detection Index (TDI) is a linear combinationof lichen species cover withcoefficients chosen to ensure maximum ability to detect global climatic trends. Thecoefficient for each species is proportional to the ratioofspecies sensitivity to varianceofspecies cover.Calculationofcoefficients, TDI value and its standard error are given in [6](see also chapter 13, this volume). To evaluate changes in TDI values over time useconventional statistics methods [10]. Indices widely used in lichenology include theweighted sumsof lichen species cover, the Indexof Poleotolerance [8], the IndicesofAtmospheric Purity (see chapter 4, this volume). TDI provides a higher detection capacityof large-scale environmental trends than anyof these indices due to the useof thecoefficients described above [6].

4.Workedexamples

• Basic surveys to detect large-scale airpollution with epiphytic lichens wereconductedin 28 nature reserves in Russia and adjacent countries[I, 3] as well as inSweden and Portugal. Samplingprocedurefor epiphyticlichens isapprovedby theInternationalCooperativeProgramme onIntegratedMonitoring of Air PollutionEffects onEcosystems(UN ECE) [9] . Itwas estimatedthat forEuropeanRussia theline-interceptmethod is about 2.5 times more effective than the"releve" (quadrat)methodofsampling [2].

• A basic survey to detect global warming has been conducted in the Negev Desert,Israel [6]. The system allows the detectionof a climate-driven change in epilithiclichen community corresponding to 0.8 °C shift in annual mean temperature (seechapter 13, this volume).

5.Applicationandlimitations

The sampling procedure can be applied in any season and in any weather condition exceptrain.The main limitations are:• It may be difficult to locate sites without human impact, that provide an even

distributionof suitable substrata, and without a significant gradient in environmentalconditions.

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• Selectionof trees for long-term epiphytic lichen monitoring in relatively xericformations (e.g. Mediterranean region) can be difficult if the numberof trees perplot is restricted and not all age classes are represented.

6.References

1. Insarov, G.E. (2001) Monitoring of epiphytic lichens exposed to background air pollution: Conservationimplications,Forest, Snow and Landscape Research (in press).

2. Insarov, G.E.,and Pchiolkin, A.V.(1983) Comparisonofdifferentmethodsoflichen measurement on atree trunk,Problems of Ecological Monitoring and Ecosystem Modelling 6, 90-100, GidrometeoizdatPublisher, Leningrad (in Russian, summary in English).

3. Insarov, G.E.,Filippova, L.M., and Semenov, S.M. (1986) The methodsof assessmentof epiphyticlichenoflora state in relation to background natural environment pollution, in Y.A. Izrael (ed.),Researchon Environmental Pollut ion and its Effects on the Biosphere, Proc. 3«1 Meeting of the InternationalWorking Group on UNESCO MAB Project No 14, 29 March-30 April 1985, Yalta,USSR,Gidrometeoizdat Publishers,Leningrad,pp. 123-131.

4. Insarov, G.E.and Pchiolkin,A.V. (1988) Measurementoffruticoseepiphytic lichens,Biological Science1, 106-109 (in Russian).

5. Insarov, G. and Insarova, I.(\996) Assessment of lichen sensitivity to climate change,Israel Journal ofPlant Sciences 44, 309-334.

6. Insarov, G., Semenov, S., and Insarova, I. (1999) A system to monitor climate change with epilithiclichens,Environmental Monitor ing and Assessment 55 (2), 279-298.

7. Insarova,1.0.,Insarov, G.E.,Brakenhielm,S.,Hultengren,S.,Martinson, P.-O.,and Semenov, S.M. (\992)Lichen sensitivity and air pollution, Swedish EnvironmentProtectionAgency, Report 4007, Uppsala, pp. I­n .

8. Trass, H. (1973) Lichen sensitivityto air pollution and index ofpoleotolerance (I.P.),Folia Cryptogamica.Estonica 3, 19-22.

9. Trunk epiphytes (1998), inManual for Integrated Monitoring, UN ECE Convention on Long-RangeTransboundary Air Pollution, International Cooperative Programme on Integrated Monitoringof AirPollution Effects on Ecosystems, ICP 1M Programme Centre, Finnish Environment Institute, Helsinki,pp.7.20-1-7.20-5,http://www.vyh.ftleng/intcoop/projectslicp_imlmanuallcontents.htrn

10. Zar,J.H.(1996)Biostatisticalanalysis. 3«1 edition,Prentice-HallInc.,New Jersey,662 pp.+ app.II. Zeltyn,SA and Insarov, G.E.(1993) A database of epiphytic lichen background monitoring, inProblems

ofEcological Monitoring and Ecosystem Modelling 15,247-263 (in Russian, summaryin English).

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Appendix

List ofparticipantsat the NATO AdvancedResearchWorkshop:

International Workshop on Lichen Monitoring (LIMON)

Orielton Field Centre (West Wales,UK) , 16-23rd August 2000

Aptroot,Andre, Centraalbureauvoor Schirnmelcultures,P.O. Box 85167,UtrechtNL­3508 AD, The Netherlands([email protected]).

Asta, Juliette,UniversiteJoseph Fourier, 2233 rue de la Piscine,GrenobleI, B.P. 53X,GrenobleF-38041, France([email protected]).

Bartok,Katalin, InstituteofBiologicalResearch Cluj,Republiciistreet 48, PO Box 229,Cluj-NapocaRO-3400, Romania([email protected]).

Benfield,Barbara, Penspool Cottage, Plymtree,Cullompton, Devon EX 15 2JY, UK([email protected]).

Bnmialti, Giorgio, DIP.TE.RIS., University of Genova, Corso Dogali 1m, 1-16136Genova, Italy([email protected]).

Coppins, Brian, The Royal Botanic Garden, Inverleith Row,Edinburgh EH3 5LR,Scotland, UK ([email protected]).

Coppins, Sandy, 37, High Street, East Linton, East Lothian, Scotland, UK.Cnunp, Robin, Field Studies Council, Orielton Field Centre, Pembroke, Pembrokeshire

SA71 5EZ ,UK ([email protected]).Cuny,Darnien,Laboratoirede Botanique, Faculte dePharmacie, Universitede Lille, 2

ProLaguesse, B.P. 83, Lille F-59006, France([email protected]).Dobson, Frank, 57 Acacia Grove, New Malden, Surrey, KT3 3BU, UK (franks@

dobson57.freeserve.co.uk).Erbardt, Walter, UMEG, Zentrum furUmweltmessungen, Umwelterhebungenund

Geratesicherheit,Grossoberfeld3, D-76135 Karlsruhe, Germany ([email protected]).

Fletcher,Anthony,LeicestershireMuseum,96 New Walk, LeicesterLE4 4DG, UK .Geiser, Linda, 4077 Research Way, P.O. Box 1148, Corvallis, Oregon, 97339, USA

(Geiser_Linda/[email protected]).Giavarini,Vince, ConsultantEcologist, Flat 2, Spring Hill, Swanage,BHI9 1E2, UK .Gionlani, Paolo, DIP.TE.RIS ., University of Genova, Corso Dogali lm, 1-16136

Genova, Italy([email protected]).Grey, Jeremy, BLS, Penmore, Perranutunve, Penzance, TR20 9NF, UK(jmgray@

argonet.co.uk).Guttova,Anna, InstituteofBotany, Slovak AcademyofSciences, Diibravska cesta 14,

Bratislava84223, Slovakia([email protected]).Hanies,David, Texacopic, Pembroke, UK.

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Haycock, Bob, The Old Home Farmyard,Stackpole,Pembrokeshire, SA71 5DQ, UK([email protected]).

Hill, David, School of Biological Sciences, University of Bristol, Woodland road,BristolBS8 1UG, UK ([email protected]).

Hilton, Barbara,Beauregard,5 Alscott Gardens,Alverdiscott,Barnstaple, EX31 3PT,Devon,UK ([email protected]).

Insarov, Gregory, Instituteof Global Climateand Ecology, 20-B Glebovskajastreet,Moscow, 107-258, Russia([email protected]).

Insarova, Irina, Biological Department,Moscow State University, Vorobjevy Gori,Moscow, 119-899, Russia([email protected]).

Isocrono,Deborah,DepartmentofPlant Biology, UniversityofTurin,v.le Mattioli 25,Torino1-10125, Italy([email protected]).

Ivanov,Dobri, BotanicalGarden,Varna 9006,Bulgaria.James,PeterW., DepartmentofBotany,The NaturalHistory Museum, Cromwell road,

London,SW7 5BD, UK .Jensen,Manfred,Universitat-Gll-Essen,Universitatsstralle5, Essen D-45117, Germany

([email protected]).Johnson,Peter,LandcareResearchNew ZealandLtd.,PrivateBag 1930, Dunedin,New

Zealand([email protected]).Kantvilas,Gintaras,TasmanianHerbarium,GPO Box 25204, Hobart,Tasmania7001,

Australia([email protected]).Kbodosovtsev,Alexander,KhersonPedagogicalUniversity,BotanyDepartment,27,40

letOktyabryaStr.,Kherson 7100,Ukraine([email protected]).Kondratyuk,Sergey, M.H . Kholodny Instituteof Botany, Tereshchenkivskastreet2,

01601 Kiev - MSP-1, Ukraine([email protected]).Kricke, Randolph,Universitat-Gli-Essen,Institutfiir Botanik & Pflanzenphysiologie,

Universitatsstralle5, Essen D-451 17,Germany([email protected]).Kudratov, Imomnazar,Tajik State National University, Rudaki street17, Dushanbe,

Tajikstan([email protected]).Lackovicova,Anna, InstituteofBotany,Slovak AcademyofSciences,Diibravskacesta

14,Bratislava84223,Slovakia ([email protected]).Lambley, Peter,English Nature, 60 Bracondale,Norwich, NRI 2BE, UK (peter.

[email protected]) .Lambrecht, Susanne, Institute of Environmental Geochemistry, University of

Heidelberg, im Neuenheimer Feld 236, Heidelberg D-69120, Germany([email protected]).

Liska, Jiri, InstituteofBotany,AcademyofSciencesoftheCzech Republic,Pruhonice,CZ-25243,CzechRepublic([email protected]).

Loppi, Stefano,Departmentof EnvironmentalSciences, Universityof Siena, via P.A.Mattioli4, 1-53100 Siena, Italy(loppi@unisLit).

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Magomedova, Margarita, Institute of Plant and Animal Ecology, 8 Marta street 202,Ekaterinburg,620144, Russia ([email protected]).

Mikhailova, Irina, Instituteof Plant and Animal Ecology, 8 Marta street 202,Ekaterinburg, 620 144, Russia ([email protected]).

Motiejunaite, Jurga, InstituteofBotany, Zaliuju ezeru 49, Vilnius, LT-2021, Lithuania([email protected]).

Nimis, Pier Luigi, Dipartimento di Biologia, Universita di Trieste, via Giorgieri 10,Trieste 1-34127, Italy ([email protected]).

Pirintsos, Stergios, Department of Biology, Universityof Crete, P.O.Box 2208,Heraklion GR-71409, Greece ([email protected]).

Purvis, Ole William, Departmentof Botany, the Natural History Museum, Cromwellroad, London, SW7 5BD, UK ([email protected]).

Rusu,Ana-Maria,University 'Babes-Bolyai' DepartmentofChemistry, Arany JanosII,Cluj-Napoca RO-3400, Romania ([email protected]).

Saipunkaew, Wanaruk, Biology Department, Chiangmai University, Huay Kaew road,Chiang Mai 50002, Thailand ([email protected]).

Scheidegger, Christoph, WSL, Swiss Federal Insititute for Forest, Snow and LandscapeResearch,BirmensdorfCH-8903,Switzerland ([email protected]).

Scholz, Peter, Institut fur Umweltfragen, Gr. Klausstrasse 11, Halle/Saale D-06108,Germany ([email protected]).

Selva, Steve, University of Maine at Fort Kent, 25 Pleasant street, Fort Kent, Maine04743, USA ([email protected]).

Signoret, Jonathan, Laboratoire de Biocatalyse, Faculte des Sciences et Tech.,Universite de Metz, UFR Scientifiques, Ile du Saulcy, Metz F-57045, France([email protected]).

Skirina, Irina, Pacific Instituteof Geography, Far East Branch, Russian AcademyofScience, 7 Radio street, Vladivostok 690041, Russia ([email protected]).

Spanius, Laurens, Kongsbergstraat I, NL-2804 XV, Gouda, The Netherlands([email protected]).

Van Haluwyn, Chantal, Laboratoire de Botanique, Faculte de Pharmacie, Universite deLille 2, Pro Laguesse, B.P. 83, Lille F-59006, France ([email protected]­lille2.fr).

Van Herk, Kok (C.M.),Lichenologisch Onderzoekbureau Nederland (LON), Goudvink47, Soest NL-3766 WK, The Netherlands ([email protected]).

Van lperen, Arien, Lichenologisch Onderzoekbureau Nederland (LON), Goudvink 47,Soest NL-3766 WK, The Netherlands.

Waterfield, Amanda, Departmentof Botany, The Natural History Museum, Cromwellroad,London SW7 5BD, UK ([email protected]).

Wheeler,David, C.C.W.,Hendy Farm, Capel Ewan, Newcastle Emlyn, Pembrokeshire,SA38 9LX, UK (d [email protected]).

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Wijeyaratlle,Chandrani, University of Sri Jayewardenepura, Dept. of Botany,Nugegoda, Sri Lanka ([email protected]).

Will-Wolf, Susan, UniversityofWisconson,DepartmentofBotany, 430 Lincoln Drive,Madison, Wisconson, 53706-1381, USA([email protected]).

Wolseley, PatriciaA., DepartmentofBotany, The Natural History Museum, Cromwellroad, London, SW7 5BD, UK ([email protected]).

Woods, Ray, Coutryside Council for Wales, Eden House, Ithon road, Llandrindod,Wells,L016 AS6l, UK ([email protected]).

Zalewska, Anna, Warmian-Mazurian University, Department of Botany, Lodzki 1,Olsztyn PL-10727,Poland ([email protected]).