New handbook for standardised measurement of plant

New handbook for standardised measurement of plantfunctional traits worldwide

NPeacuterez-HarguindeguyAY SDiacuteazA EGarnierB S LavorelC H PoorterD P JaureguiberryAM S Bret-Harte E W K CornwellF J M CraineG D E GurvichA C UrcelayAE J VeneklaasH P B ReichI L PoorterJ I J WrightK P RayL L EnricoA J G PausasMA C de VosF N BuchmannN G FunesA F QueacutetierAC J G HodgsonO K ThompsonPHDMorganQ H ter SteegeRMG A van derHeijdenS L SackT B BlonderU P PoschlodVM V VaierettiA G ContiA A C StaverW S AquinoX and J H C CornelissenF

AInstitutoMultidisciplinario deBiologiacuteaVegetal (CONICET-UNC) andFCEFyNUniversidadNacional deCoacuterdobaCC 495 5000 Coacuterdoba Argentina

BCNRS Centre drsquoEcologie Fonctionnelle et Evolutive (UMR 5175) 1919 Route de Mende34293 Montpellier Cedex 5 France

CLaboratoire drsquoEcologie Alpine UMR 5553 du CNRS Universiteacute Joseph Fourier BP 53 38041Grenoble Cedex 9France

DPlant Sciences (IBG2) Forschungszentrum Juumllich D-52425 Juumllich GermanyEInstitute of Arctic Biology 311 Irving I University of Alaska Fairbanks Fairbanks AK 99775-7000 USAFSystems Ecology Faculty of Earth and Life Sciences Department of Ecological Science VU UniversityDe Boelelaan 1085 1081 HV Amsterdam The NetherlandsGDivision of Biology Kansas State University Manhtattan KS 66506 USAHFaculty of Natural and Agricultural Sciences School of Plant Biology The University of Western Australia35 Stirling Highway Crawley WA 6009 Australia

IDepartment of Forest ResourcesUniversity ofMinnesota 1530NClevelandAvenue St PaulMN55108USA andHawkesbury Institute for the Environment University of Western Sydney Locked Bag 1797 Penrith NSW 2751AustraliaJCentre for Ecosystems Forest Ecology and Forest Management Group Wageningen University PO Box 476700 AA Wageningen The NetherlandsKDepartment of Biological Sciences Macquarie University Sydney NSW 2109 AustraliaLDepartment of Biological Sciences Stanford University Stanford CA USAMCentro de Investigaciones sobre Desertificacioacuten (CIDE-CSIC) IVIA Campus Carretera Nagravequera km 4546113 Montcada Valencia Spain

NInstitute of Agricultural Sciences ETH Zurich Universitaumltstrasse 2 LFW C56 CH-8092 Zuumlrich SwitzerlandOPeak Science and Environment Station House Leadmill Hathersage Hope Valley S32 1BA UKPDepartment of Animal and Plant Sciences The University of Sheffield Sheffield S10 2TN UKQNSW Department of Primary Industries Forest Resources Research Beecroft NSW 2119 AustraliaRNaturalis Biodiversity Center Leiden and Institute of Environmental Biology Ecology and Biodiversity GroupUtrecht University Utrecht The Netherlands

SEcological Farming Systems Agroscope Reckenholz Taumlnikon Research Station ART Reckenholzstrasse 1918046 Zurich Switzerland and Plant-Microbe Interactions Institute of Environmental Biology Faculty of ScienceUtrecht University Utrecht The NetherlandsTDepartment of Ecology and Evolutionary Biology University of California Los Angeles 621 Charles EYoung Drive South Los Angeles CA 90095-1606 USA

UDepartment of Ecology and Evolutionary Biology University of Arizona Tucson AZ USAVInstitute of Botany Faculty of Biology and PreclinicalMedicine University of Regensburg D-93040 RegensburgGermany

WDepartment of Ecology and Evolutionary Biology Princeton University Princeton NJ 08544 USAXCentro Agronoacutemico Tropical de Investigacioacuten y Ensentildeanza CATIE 7170 Cartago Turrialba 30501 Costa RicaYCorresponding author Email nperezcomuncoredu

CSIRO PUBLISHING

Australian Journal of Botany 2013 61 167ndash234httpdxdoiorg101071BT12225

Journal compilation CSIRO 2013 wwwpublishcsiroaujournalsajb

Abstract Plant functional traits are the features (morphological physiological phenological) that represent ecologicalstrategies and determine how plants respond to environmental factors affect other trophic levels and influence ecosystemproperties Variation in plant functional traits and trait syndromes has proven useful for tacklingmany important ecologicalquestions at a range of scales giving rise to a demand for standardised ways to measure ecologically meaningful plant traitsThis line of research has been among the most fruitful avenues for understanding ecological and evolutionary patterns andprocesses It also has the potential both to build a predictive set of local regional and global relationships between plants andenvironment and to quantify a wide range of natural and human-driven processes including changes in biodiversity theimpacts of species invasions alterations in biogeochemical processes and vegetationndashatmosphere interactions Theimportance of these topics dictates the urgent need for more and better data and increases the value of standardisedprotocols for quantifying trait variation of different species in particular for traitswith power to predict plant- and ecosystem-level processes and for traits that can be measured relatively easily Updated and expanded from the widely used previousversion this handbook retains the focus on clearly presentedwidely applicable step-by-step recipeswith aminimumof texton theory and not only includes updated methods for the traits previously covered but also introduces many new protocolsfor further traits This new handbook has a better balance between whole-plant traits leaf traits root and stem traits andregenerative traits and puts particular emphasis on traits important for predicting speciesrsquo effects on key ecosystempropertiesWehope this newhandbookbecomes a standard companion in local andglobal efforts to learn about the responsesand impacts of different plant species with respect to environmental changes in the present past and future

Additional keywords biodiversity ecophysiology ecosystem dynamics ecosystem functions environmental changeplant morphology

Received 23 November 2011 accepted 29 January 2013 published online 26 April 2013

Contents

Introduction and discussion 1691 Selection of species and individuals 17011 Selection of species17012 Selection of individuals within a species17113 Replicate measurements1722 Whole-plant traits17221 Life history and maximum plant lifespan 17222 Life form 17323 Growth form 17324 Plant height 17525 Clonality bud banks and below-ground storage

organs17626 Spinescence17727 Branching architecture 17828 Leaf area sapwood area ratio 17829 Root-mass fraction 179210 Salt resistance179211 Relative growth rate and its components181212 Plant flammability182213 Water-flux traits 1843 Leaf traits 18631 Specific leaf area 18632 Area of a leaf 18933 Leaf dry-matter content19034 Leaf thickness 19035 pH of green leaves or leaf litter 19136 Leaf nitrogen (N) concentration and leaf phosphorous

(P) concentration19237 Physical strength of leaves19338 Leaf lifespan and duration of green foliage 195

39 Vein density 197310 Light-saturated photosynthetic rate 198311 Leaf dark respiration 198312 Photosynthetic pathway 199313 C-isotope composition as a measure of intrinsic

water-use efficiency 200314 Electrolyte leakage as an indicator of frost

sensitivity 201315 Leaf water potential as a measure of water

status 202316 Leaf palatability as indicated by preference by

model herbivores203317 Litter decomposability 2054 Stem traits 20741 Stem-specific density 20742 Twig dry-matter content and twig drying time20943 Bark thickness (and bark quality) 20944 Xylem conductivity21045 Vulnerability to embolism 2115 Below-ground traits 21251 Specific root length 21252 Root-system morphology21453 Nutrient-uptake strategy2146 Regenerative traits 21561 Dispersal syndrome21562 Dispersule size and shape 21663 Dispersal potential 21664 Seed mass21765 Seedling functional morphology21866 Resprouting capacity after major disturbance218Acknowledgements220References220

168 Australian Journal of Botany N Peacuterez-Harguindeguy et al

Introduction and discussionEnvironmental changes such as those on climate atmosphericcomposition land use and biotic exchanges are triggeringunprecedented ecosystem changes The need to understandand predict them has given new stimulus to a long tradition ofstudy of the plant features (traits) that reflect species ecologicalstrategies and determine how plants respond to environmentalfactors affect other trophic levels and influence ecosystemproperties (Kattge et al 2011) There is mounting evidencethat variation in plant traits and trait syndromes (ie recurrentassociations of plant traits) within and among species isassociated with many important ecological processes at arange of scales This has resulted in strong demand forstandardised ways to measure ecologically meaningful planttraits The predecessor of the present handbook (Cornelissenet al 2003) was written to address that need by providingstandardised easily implemented trait-measurement recipes forresearchers worldwide This updated version is an extension ofthat global collective initiative with an even broader scope

The identification of general plant trait trade-offs associatedwith strategies and trait syndromes across floras taxa andecosystems has been a long-standing focus in plant ecologyand has attracted increasing interest in recent decades (egChapin et al 1993 Grime et al 1997 Reich et al 1997Cornelissen et al 1999 Aerts and Chapin 1999 Westobyet al 2002 Diacuteaz et al 2004 Wright et al 2004 Cornwellet al 2008 Baraloto et al 2010a Freschet et al 2010Ordontildeez et al 2010 Kattge et al 2011) Ample evidenceindicates that plant traits and trait syndromes significantlyaffect ecosystem processes and services (for overviews seeLavorel and Garnier 2002 Diacuteaz et al 2007 Chapin et al2008 De Bello et al 2010 Cardinale et al 2012) As aconsequence trait-based approaches are currently also gainingmomentum in the fields of agronomy and forestry (eg Brussaardet al 2010 Garnier and Navas 2012) conservation (eg Maceet al 2010) archaeobotany (eg Jones et al 2010) and at theinterface between the evolution and ecology in communitiesand ecosystems (eg Edwards et al 2007 Cavender-Bareset al 2009 Faith et al 2010 Srivastava et al 2012)

The quantification of vegetation changes in the face ofmodifications in climate at the global scale has beensignificantly improved with the use of dynamic globalvegetation models (DGVMs) (Cramer et al 2001 Arnethet al 2010) However current-generation DGVMs do notyet incorporate continuous variation in plant traits among plantspecies or types (Cornwell et al 2009) Next-generation modelscould benefit from the incorporation of functional traits and

syndromes that are simple and general enough to be assessedat the regional and global scales and yet informative enough torelate to biogeochemical dynamics dispersal and large-scaledisturbance (Ollinger et al 2008 Stich et al 2008 Dohertyet al 2010 Harrison et al 2010 Ma et al 2011)

As a consequence of this surge of theoretical and practicalinterest there has been a rapid expansion of large regional andglobal trait databases (eg Diacuteaz et al 2004 Wright et al 2004Kleyer et al 2008 Cornwell et al 2008 Chave et al 2009 Paulaet al 2009Baraloto et al 2010a Zanne et al 2010 Fortunel et al2012 Patintildeo et al 2012) The TRY Initiative (Kattge et al 2011see Box 1) is compiling a communal worldwide database of planttraits an unprecedented step in improving the capacity of thescientific community to access and utilise plant-trait informationIn this context standardisation of protocols applicable under awide range of situations and geographical contexts becomes evenmore important

In this manual we consider plant functional traits to beany morphological physiological or phenological featuremeasurable for individual plants at the cell to the whole-organism level which potentially affects its fitness (cf McGillet al 2006 Lavorel et al 2007 Violle et al 2007) or itsenvironment (Lavorel and Garnier 2002) As proposed byLavorel et al (2007) we will call the particular value ormodality taken by the trait at any place and time an lsquoattributersquoFunctional traits addressed in the present handbook rangefrom simple indicators of plant function (eg leaf nutrientconcentrations as an indicator of both potential rates ofmetabolism and of quality as food for herbivores) to plantfunctions themselves (eg palatability decomposabilitycapacity to resprout after a fire) always measured at thespecies level The traits contained in the handbook represent aset of functional traits of vascular plants that (1) can togetherrepresent key plant responses to the environment as well as keyplant effects onecosystemprocesses andservices at various scalesfrom local plots to landscapes to biomes (2) can help answerquestions of ecological and evolutionary theory as well aspractical ones related to nature conservation and landmanagement (see Box 2 for a Discussion) and (3) are in mostcases candidates for relatively easy inexpensive and standardisedmeasurement in many biomes and regions

This is a recipe book to be used in the field and in thelaboratory and contains comprehensive detailed step-by-steprecipes for direct and as far as possible unambiguous use in anyterrestrial biome To that end we have had to make hard choicesWe did not intend to provide a comprehensive list of all traits thatcould potentially be measured nor a thorough description of the

Box 1 Useful links for plant functional-trait workersTo find on-line protocols and updates related to this handbook Nucleo DiversusTools (httpwwwnucleodiversusorg)To submit corrections additions and comments to improve this handbook traitshandbookgmailcomVarious complementary protocols for specific plant (eco-)physiological as well as environmental measurements not covered in this handbook can beaccessed through the fellow project Prometheus Wiki (Sack et al 2010 httpprometheuswikipublishcsiroautiki-indexphp)To share plant functional-trait data with other researchers (both as a provider and as a recipient) TRYWorldwide Initiative and Database (Kattge et al2011 wwwtry-dborg)To calculate functional diversity metrics and indices with your trait data FDiversity Free Software Package (Casanoves et al 2011 wwwfdiversitynucleodiversusorg)

New handbook for measurement of plant traits Australian Journal of Botany 169

theory behind each trait Rather the present handbook containsconsensus traits and methods that researchers have identified asbeing useful reliable and feasible to be applied in large-scalecomparative efforts Some of them are well known and widelyused whereas for others relatively novel methods are describedParticular emphasis is given to recipes appropriate for areas withhigh species richness incompletely known floras and modestresearch budgets We give only brief ecological backgroundfor each trait with a short list of references with further detailson significance methodology and existing large datasets Themain section of each recipe contains a brief standardisedprotocol and under the heading Special cases or extras wegive pointers to interesting additional methods and parametersReaders can find complementary methods and additionaldiscussions and comments in specific associated web pages(see Box 1) Specific citations have not been included in therecipe descriptions We hope that the authors of relevantpublications (most of them cited at the end of each recipe) willunderstand this choice made for clarity and brevity and in fullrecognition of the important contribution that each of themand many additional studies have made to the theory andmeasurement procedure for each trait

This new handbook both updates theory methods anddatabases covered by its predecessor (Cornelissen et al 2003)and provides protocols for several additional plant functionaltraits especially for organs other than the leaf It has bettercoverage of (1) measurements important in less studied biomes

and ecosystems (2) floras with special adaptations and (3) plantfunctions related to carbon and nutrient cycling herbivory waterdynamics and fireWe hope that the focus on practical techniquesand streamlined trait recipes will help this handbook become auseful reference in laboratories and in the field for studies aroundtheworldWe strongly invite users to share their experienceswithus about both general issues and specific details of these protocols(seeBox 1) so that the next editionwill be an even better bed-sidetable companion

1 Selection of species and individuals

This section presents guidelines for selecting species andindividuals within species for trait measurement as well asgeneral considerations of the necessary number of replicatesIn addition suggested numbers of replicates for all traits are givenin Appendix 1

11 Selection of species

Studyobjectiveswill alwaysdeterminewhich species are selectedfor traitmeasurement For species-level analysesof trait variationand for identifying general strategies or syndromes of resourceuse or trade-offs at the local regional or global scale (eg Reichet al 1997 Westoby et al 1998 Diacuteaz et al 2004 Wright et al2004 Gubsch et al 2011) species or populations from a broadrange of environments and phylogenetic groups should be

Box 2 Why measure plant traits and which traits to measurePlant functional traits give better insight into the constraints and opportunities faced by plants in different habitats than does taxonomic identity alone(Southwood 1977 Grime 1979) They also provide understanding of how functional diversity in the broad sense underpins ecosystem processes and thebenefits that people derive from them (Chapin et al 2000 Diacuteaz et al 2007) and offer the possibility of comparing distant ecosystems with very littletaxonomic overlap (Reich et al 1997 Diacuteaz et al 2004 Cornwell et al 2008) The plant-trait approach often provides unique mechanistic insights intoseveral theoretical and practical questions although it is not necessarily less laborious or less expensive than other methods

Which traits to measure to answer which questionsNomethods handbook can answer the question ofwhat are the best traits tomeasure because this strongly depends on the questions at hand the ecologicalcharacteristics and scale of the study area and on practical circumstances For instance there is not much point in comparing multiple species forsucculencewithinwet environments or forflammabilitywithin areas that burn only very rarely whereas such datamight be useful as a reference in larger-scale studies In addition rather than setting limits to researchersrsquo curiosity this trait handbook aims at inspiring others to come upwith andmeasure traitsnot covered here including lsquonewrsquo traits to help answer exciting novel questions Some examples of additional interesting traits not covered here are in theintroductory text ofCornelissen et al (2003)Thefirst and foremost criterion in decidingwhat traits to aim for is theprocess of interest Is the intended studyabout fundamental plant or organ design in response to environmental variation in the present or about the evolution that gave rise to todayrsquos spectrum ofdesigns Is it about plant growth reproduction or dispersal over the landscapeDoes it involveplant survival in response to resources or disturbance Is themainquestionabout response toor effects onwater soil nutrientorfire regimes Is it about vegetation feedbacks to atmosphereandclimateDoes it involvethe juvenile stage the persistence of adultsDoes it involve pollinators dispersers or herbivoresDoes the target process occur above or below ground Isthe focuson coarsedifferences acrossor among regionsor continents oron subtledifferences among individualsof twoslightlydifferent local populationsAre specific ecosystem services to people assessed or predictedAll these and further types of questionswill have a direct impact on the selection of traitsAlthough there is no limit to the number of relevant traits in different research contexts a small number of traits have been considered relevant almostuniversally because they are at the core of the plant life cycle (Grime et al 1997Westoby 1998) These are plant size (usually expressed as height) seedsize (usually expressed as seedmass) and the structure of leaf tissue (often expressed as specific leaf area or leaf dry-matter content) Beyond this there aresome lsquocore listsrsquo of plant traits that are considered important for plant resource use regeneration dispersal and response to widespread disturbances (egHodgson et al 1999McIntyre et al 1999Weiher et al 1999 Lavorel andGarnier 2002Knevel et al 2003) A discussion of these is beyond the scope ofthe presentmanual and readers are referred to these papers for afirst introduction For a particular question the brief ecological background and especiallythe list of references provided for each trait should help identify the most appropriate traits to measure Logistic and financial considerations are equallyrelevant For example if resources are limited for measuring relative growth rate on hundreds of species representing a large gradient of productivity thespecific leaf areas and stem-specific densities of these speciesmight serve as less precise but still useful proxies for broadpatterns of variation in growthandvegetation productivity Similarly the choice of traits would be slightly different if the limiting factor is labour force or access to sophisticated analyticallaboratories or if the project involves an intensive one-off measurement campaign carried out by highly trained specialists or recurrent measurements bythird parties The recipes provided here including the sections on Special cases or extras should assist in making those decisions


selected For questions about evolution the choice of speciesmaybe based on the inclusion of representatives of different enoughphylogenetic groups or on other phylogenetically relevantcriteria (such as being members of particular clades) withlittle consideration about their abundance in situ In contrastwhen trying to understand how environmental variables shapevegetation characteristics or how vegetation characteristicsaffect local flows of matter and energy (eg primary andsecondary production carbon water and mineral nutrientcycling) the main criterion for species selection should belocal abundance In those cases species should be selectedthat collectively make up for ~80 of cumulative relativeabundance following Garnier et al (2004) and Pakeman andQuested (2007) (see specifics for abundance measurementsbelow) Exceptions may be made if this criterion would implymeasurements for an impracticably large numbers of specieseg communities with unusually high species richness per unitarea especially combined with a very high evenness Examplesare tropical rainforests and fynbos vegetation in which well over100 species per plot may be needed to reach the 80 biomassthreshold

In forests andother predominantlywoodyvegetation themostabundant species of the understorey may also be included (egwhen the research question relates to the whole-community orecosystem level) even if their biomass is much lower than thatof the overstorey woody species In predominantly herbaceouscommunities species contribution to a particular communitymayvarywith timeduring agrowing seasonAs afirst stepwe suggestthat the relative abundance and the traits should be measured atthe time of peak standing biomass of the community This doesnot always apply to reproductive structures which obviouslyhave to be measured when they are present and fully developedwhich sometimes does not coincide with the time of maximumvegetative growth

For comparing sites or for monitoring trends in ecosystem-level properties across environmental conditions (eg pollutionor different regional climate or fertility levels) indicator speciescan be selected on the basis of the sensitivity of their trait values tothe environmental factor of interest and their importance locallyand regionally as well as for the ease with which they can befound and identified in the field (independent of their relativeabundance) (Ansquer et al 2009 De Bello et al 2011) In thissense it may be useful to distinguish lsquovariablersquo traits frommore lsquostablersquo traits (Garnier et al 2007) Although most traitsshow some variation within species along environmentalgradients or in response to specific environmental changesthe intraspecific variation of so-called lsquostable traitsrsquo is lowcompared with their interspecific variation The reverse is thecase for so-called lsquovariable traitsrsquo which implies that theyshould preferably be measured in more than one site orcondition across the habitat range (Garnier et al 2007) Bycontrast lsquostable traitsrsquo can be measured for any representativepopulation from the entire gradient Traits known to often belsquovariablersquo include vegetative and reproductive plant heightmineral nutrient concentration in leaves onset of floweringbranching architecture and spinescence Traits that arerelatively lsquostablersquo include categorical traits such as life formclonality dispersal and pollination modes and to a lesser degreephotosynthetic type (C3 or C4) Some quantitative traits such as

leaf and stem drymatter content or leaf toughness can be lsquostablersquoalong certain gradients eg of nutrients or disturbance but notalong others eg a light gradient (cf Poorter et al 2009) Speciesmay therefore vary in which quantitative traits are stable acrossgivengradients so tests shouldbemadebefore a trait is taken tobestable for a given species (Albert et al 2010 2012 Hulshof andSwenson 2010 Messier et al 2010 Moreira et al 2012)

Appendix 1 gives a rough indication of the within-speciesvariability (coefficients of variation ie standard deviationdivided by the mean hereafter CV) for some of thequantitative traits described in the present handbook alongwith the more frequently used units and the range of valuesthat can be expected Appendix 1 summarises field datacollected in several studies for a wide range of species comingfrom different environments Because of the low number ofreplicates generally used each of the individual estimatesbears an uncertainty (and CV will likely increase as scaleincreases) however by looking at the range of CVs calculatedacross a wide range of species a reasonable estimate of thetypical within-species-at-a-site variability can be obtained Wetherefore present in Appendix 1 the 20th and 80th percentiles ofthe CV distribution

How species abundance should be measured to determine thespecies making up 80 of cumulative abundance (eg whetherto lay out transects select points or quadrats at random orsystematically or to follow a different method) is beyond thescopeof the present handbookand is extensively covered inplant-ecology and vegetation-science textbooks However it should benoted that different methods are relevant to different ecologicalquestions and associated traits (Lavorel et al 2008 see alsoBaraloto et al 2010b specifically for tropical forest) Taxon-freeapproaches that do not require species identification offer analternative to estimates of relative abundance and effectivelycapture the contribution of more abundant species These includemeasuring traits regardless of species identity along a transect(lsquotrait-transectrsquo method Gaucherand and Lavorel 2007) or forindividuals rooted nearest to random sampling points as long asthe canopy structure is quite simple (lsquotrait-randomrsquo method ndash

Lavorel et al 2008) Methods of taxon-free sampling have alsobeen applied to tropical forests being in this case strongly basedon the frequency or basal area of individual trees (Baraloto et al2010b) Trait values obtained through these methods can differfrom those obtained using the standard approach of selectingrobust lsquohealthy-lookingrsquo plants for trait measurement (seeSection 12)

12 Selection of individuals within a species

For robust comparisons across species traits should be generallymeasured on reproductivelymature healthy-looking individualsunless specific goals suggest otherwise To avoid interactionwith the light environment which may strongly depend onneighbouring vegetation often plants located in well litenvironments preferably totally unshaded should be selectedThis is particularly important for some leaf traits (seeSection31)This criterion creates sampling problems for true shade speciesfound eg in the understorey of closed forests or very close to theground in multilayered grasslands Leaves of these species couldbe collected from the least shady places in which they still look


healthy and not discoloured (see Section 31) Plants severelyaffected by herbivores or pathogens should be excluded Iffeasible for consistency among sets of measurements use thesame individual to measure as many different traits as possible

Defining lsquoindividualsrsquo reliably may be difficult for clonalspecies (see Section 25) so the fundamental unit on whichmeasurements are taken should be the ramet defined here asa recognisably separately rooted above-ground shoot Thischoice is both pragmatic and ecologically sound becausegenets are often difficult to identify in the field and in anycase the ramet is likely to be the unit of most interest for mostfunctional trait-related questions (however be aware thatsampling of neighbouring ramets may not provide biologicallyindependent replicates for species-level statistics) Individuals formeasurement should be selected at random from the populationof appropriate plants or by using a systematic transect or quadratmethod

13 Replicate measurements

Trait values are often used comparatively to classify species intodifferent functional groups or to analyse variation across specieswithin or between ecosystems or geographical regions This typeof research almost inevitably implies a conflict between scale andprecision given constraints of time and labour the greater thenumber of species covered the fewer replicatemeasurements canbe made for each species The number of individuals (replicates)selected for measurement should depend on the natural within-species variability in the trait of interest (see Section 11 for adiscussion on within-species variability) as well as on thenumber or range of species to be sampled Appendix 1 showsthe minimum and preferred number of replicates for differenttraits mainly based on common practice The most appropriatesample size depends on the purpose and scope of the studyIdeally researchers should check within-species CV at their sitebefore deciding this In broad-scale interspecific studies onemaysample relatively few plants of any given species whereas whenthe study concerns just a small number of species or a modestlocal gradient one may need to sample more heavily withineach species It is highly recommended to quantify the relativecontributions of intra- v interspecific variation A formal analysisof statistical power based on an assumed or known varianceamong individuals comparedwith that amongspeciesmeans canbe used Commonly used statistical packages generally includeroutines for power analysis as well as for variance componentanalyses (used to partition variance among different levels egspecies v individuals) Other more powerful techniques can alsobe used such as mixed models (Albert et al 2010 Messier et al2010 Moreira et al 2012)

2 Whole-plant traits

21 Life history and maximum plant lifespan

Plant lifespan (usually measured in years) is defined as the timeperiod from establishment until no live part remains of therespective individual Maximum plant lifespan is an indicatorof population persistence and is therefore strongly related toland use and climate change Lifespan is limited in non-clonalplants but may be apparently nearly unlimited in clonal plants

Maximum lifespan is strongly positively associated withenvironmental stress regimes eg low temperatures andlow nutrient availability The relationship with disturbancefrequency is mostly negative although long-lived (resprouting)clonal plants may also tolerate frequent disturbance There may bea trade-off between maximum lifespan and dispersal in time andspace Long-lived species often exhibit a short-lived seed bankand produce seeds or fruits with low dispersal potential in contrastto short-lived species which often have a very long-lived seedbank andor high dispersal potential

How to assess

(A) Life history

This simple classification distinguishes among the commontypes of timing and duration of survival behaviour of individualplants in the absence of disturbances or catastrophes

(1) Annual Plant senesces and dies at the end of its firstgrowing season (from seed) after producing seed whichmay propagate a new plant in the future (a winter annualgerminates in late summer or autumn and sohas two seasonsalthough the first may be very short)

(2) Biennial Plant grows vegetatively the first season thenflowers in the second to produce seed followed bysenescence and death of the shoot and root system

(3) Perennial The individual survives for at least threegrowing seasons(a) Monocarpicperennials After several tomany seasons

of vegetative growth the plant produces seeds thensenesces and dies

(b) Polycarpic perennials All or much of the stem androot system normally survives the harsh or dormantperiod between growing seasons stem has lateralthickening over the years(i) Herbaceous perennial Aerial shoots (and

sometimes roots) die off as growing season endsin the next season new shoots grow from aperennating organ such as a bulb corm rhizomeor lsquoroot crownrsquo (bud-bearing stem base orhemicryptophytes) near or below ground surface

(ii) Woody perennial retains from one growing seasoninto the next some living leaf-bearing shootswhich die by the end of their third season or later

Qualitative distinction between life-history classes

Aplantwith anyperennating organother than the seed is eithera perennial or a biennial (the latter only by a storage taproot)If biennial there should be individuals with a storage rootbut not an inflorescence and others with both A plant thatlacks specialised perennating organs may still be perennialby resprouting from its root-crown If so the crown willnormally carry wrinkles or scars from bud outgrowth inprevious seasons and can eventually become quite thick andevenwoody (a caudex) in contrast the root of an annual is usuallyrelatively soft and smooth its thickness extending continuouslyinto the stem A perennial in its first year of growth may resemblean annual in these respects except that perennial wild plantsusually do not flower in their first year whereas an annual always


does (many horticultural perennials however have been selectedto do so)

(B) Maximum plant lifespan quantitative assessment

In gymnosperms and angiosperms even in some non-woodyones speciesmaximum lifespan can be estimated by counting thenumber of annual rings representing annual tissue incrementsRecently a study on 900 temperate herbaceous species revealedannual rings in perennating structures in more than 80 of thespecies However the formation of annual rings can depend onhabitat conditionsAnnual ringswill be found in vegetation zoneswith clear seasonality (cold (winter) or drought seasons) such asthe polar boreal or austral temperate and even inMediterranean-type zones In the two latter climate zones annual rings maysometimes be absent In some cases annual rings may even befound in tropical species especially in regions with a distinct dryandwet seasonMaximum lifespanwithin a population is studiedin the largest andor thickest individualsData are collected fromaminimum of 10 preferably 20 individuals (replicates) In woodyspecies (trees shrubs dwarf shrubs) annual rings are determinedeither by cutting out a whole cross-section or a lsquopie slicersquo of themain stem (trunk) or by taking a core with a pole-testing drill(tree corer) It is important to obtain a rather smooth surface forclear observationTheannual rings canusuallybecountedunder adissection microscope Often a cross-section of a shoot does notrepresent the maximum age as precisely as the root collar (root-stem transition zone of primary roots) which is especially truefor most shrubs where single shoots have a limited age Wetherefore recommend digging out woody plants a bit and taking(additional) samples from the root collar In herbaceous speciesannual rings are mostly found at the shoot base or in the rootcollar and also in rhizomes Here microscopic cross-sections areessential and have to be treated first by lsquoeau de javellersquo to removethe cytoplasm and then stained (fuchsin chryosidine astrablue(FCA) alternatively astrablue and safranin) to make the annualringsvisible In somecases polarised light has proven tobeusefulto identify the annual rings Maximum lifespan of a species orpopulation is defined as the largest number of annual ringscounted among all samples (although the mean lifespan of allindividuals may be informative too)

Special cases or extras

(1) In clonal plants the identification of (maximum) lifespan ismore complicated If a ramet never becomes independentfrom the genet and will never be released from the motherplant annual rings in the tap root (eg Armeria maritimaSilene acaulis) or annual morphological markers along therhizome or stolon (eg Lycopodium annotinum Dictamnusalbus) are also a suitable tool to identify maximum lifespanof a genet In the latter case maximum lifespan can behigher if part of the rhizome or stolon is alreadydecomposed However in clonal plants where the genetconsists of more or less independent ramets genet age canbe estimated only indirectly by means of size or diameter ofa genet in relation to mean annual size increment

(2) Geophyte species especially monocotyledons maydisappear above ground for up to several years before

reappearing In such cases only permanent-plot researchwith individually marked individuals will give an ideaabout the maximum lifespan of those species

(3) Cold-climate dwarf shrubs In some of these species egthe heatherCassiope tetragona lateral annual rings are oftenveryhard to discernwhereas annual shoot-length incrementsof woody stems can be distinguished under a microscopethrough the winter-mark septa separating them and throughthe annual sequence of distances between leaf scars

(4) Life history and location Life history varies with locationand should preferably be assessed in the field rather than byreference to floras In particular many short-lived faster-growing species fall into different life-history categories indifferent regions and a fewdiffer amonghabitats evenwithinthe same region

References on theory and significance Rabotnov (1950)Schweingruber (1996) Fischer and Stoumlcklin (1997) Larson(2001) Schweingruber and Poschlod (2005) De Witte andStoumlcklin (2010)

More on methods Tamm (1972) Gatsuk et al (1980)Cherubini et al (2003) Rozema et al (2009)

22 Life form

Plant life-form classification sensu Raunkiaer (1934) is a simplebut still a useful way of functionally classifying plants Moreinformation is given in Material S1 available as SupplementaryMaterial for this paper

23 Growth form

Growth form is mainly determined by the direction and extent ofgrowth and any branching of the main-shoot axis or axes Theseaffect canopy structure including its height and both the verticaland horizontal distribution of leaves Growth form may beassociated with ecophysiological adaptation in many waysincluding maximising photosynthetic production shelteringfrom severe climatic conditions or optimising the height andpositioning of the foliage to avoid or resist grazing by particularherbivores with rosettes and prostrate growth forms beingassociated with high grazing pressure by mammals

How to record

Growth form is a hierarchical trait assessed through fieldobservation or descriptions or figures or photographs in theliterature Because we are classifying types along a continuumintermediate forms between the categories recognised here maybe encountered as well as occasional unique forms lying outsideany of these categories

(A) Terrestrial mechanically and nutritionally self-supportingplants(1) Herbaceous plants have either no or at most modest

secondary growth with stem and root tissues that arerather soft compared with typical wood(a) Rosette plant Leaves concentrated on a short

condensed section of stem or rhizome (seeCategory C under Section 25 for a definition ofrhizome) at or very close to the soil surface with aninflorescence (or single-flower peduncle) bearing


either no or reduced leaves (bracts) produced fromthe rosette axis above ground level Graminoidswhose principal photosynthetic leaves are attachedto the base of their aerial stems (eg lsquobunchgrassesrsquo) fall in this category

(b) Elongated leaf-bearing rhizomatous Thepermanent axis is an elongated rhizome thatdirectly bears photosynthetic leaves that extendindividually up into the light The rhizome canbe located either at or below ground level (egPteridiumaquilinum (bracken fern)Viola spp Irisspp) or (epiphytes) on an above-ground supportsuch as a tree branch Aerial inflorescences (orsingle-flower peduncles) with either reducedleaves (bracts) or none may grow out from therhizome

(c) Cushion plant (pulvinate form) Tightly packedfoliage held close to soil surface with relativelyeven and rounded canopy form (many alpine plantshave this form)

(d) Extensive-stemmed herb develops elongated aerialstem(s) whose nodes bear photosynthetic leavesthat are distributed nearly throughout the canopy ofthe plant except when shed from its more basalparts during later growth and lacking in distallydeveloped inflorescencesGraminoids (rhizomatousor not) with leafy aerial stems fall here

(e) Tussock Many individual shoots of a densecolony or clone grow upward leaving behind atough mostly dead supporting column topped byliving shoots with active leaves (eg the Arcticcotton grass Eriophorum vaginatum)

(2) Semi-woody plants Stem without secondary growthbut often toughened by sclerification (or alternativelywith relatively feeble soft or lsquoanomalousrsquo secondarygrowth)(a) Palmoid Bears a rosette-like canopy of typically

large often compound leaves atop a usually thick(lsquopachycaulousrsquo) columnar unbranched or little-branched stem (eg palms (Pandanus) tree ferns)Certain tropical or alpine Asteraceae such asEspeletia spp cycads Dracaena arborescentYucca spp and some Bombacaceae can beregarded as having this growth form althoughtheir stems undergo more extensive secondarygrowth (see also lsquoCorner modelrsquo within thereferences below)

(b) Bambusoid An excurrently branched (cf PointA3di in the present Section) trunk lacking orhaving only weak secondary growth is stiffenedby sclerification to support a vertically extensivesometimes tree-sized canopy (bamboos varioustall herbaceous dicots such as ChenopodiumAmaranthus and Helianthus)

(c) Stem succulent A usually leafless photosyntheticstem with extensive soft water-storage tissue andonly limited secondary growth (cacti and cactoidplants of other families most leaf succulents fall

instead into one of the subclasses of Points A1 orA3 in the present Section)

(3) Woody plants develop extensive usually toughsecondary xylem and phloem from vascular cambiumand corky outer bark from cork cambium (woody vinesare covered in Point B3 of the present Section)(a) Prostrate subshrub Long-lived woody stem

growing horizontally at ground level (examplesinclude many Arctic willows and ericoids)

(b) Dwarf shrub or subshrub with usually multipleascending woody stems less than 05m tall

(c) Shrub Woody plant between 05m and ~5m tallwith canopy typically carried by several trunks thatare usually thinner and younger than typical maturetree trunks

(d) Tree Woody plant usually gt5m tall with maincanopy elevated on a long-lived substantialusually single (but upwardly branching) trunk(i) Excurrent Single main axis (trunk) extends

up to or almost to the top with shorterascending or horizontal branches giving aconical or (in mature trees) columnar formto the crown

(ii) Deliquescent Trunk divides somewhereabove its base into two to several more orless equal branches that continue branchingupward to produce a wider more flat-toppedcrown

(e) Dwarf tree Morphology as in one of Types (i)or (ii) but substantially lt5m tall Many forestunderstorey trees but also in various climaticallyor nutritionally challenging unshaded habitatssuch as lsquopine barrensrsquo semi-deserts certaintropical cloud forests bogs and near-timberlinevegetation

(B) Plants structurally or nutritionally supported by other plantsor by special physical features(1) Epiphyte Plant that grows attached to the trunk or

branch of a shrub or tree (or to anthropogenic supports)by aerial roots normally without contact with theground (eg many tropical orchids and Bromeliaceae)

(2) Lithophyte Plant that grows in or on rocks (eg manyspecies of ferns species of Nepenthes Utriculariaforestii Cymbalaria muralis)

(3) Climber Plant that roots in the soil but relies at leastinitially on external support for its upward growth andleaf positioning(a) Herbaceous vine Usually attaches to its support

either by twining or by means of tendrils(b) Woody vine including liana Often attaches to a

support by means of aerial roots(c) Scrambler Growsup througha sufficiently dense

canopy of other plants without any means ofattachment (eg Galium spp)

(d) Strangler May start epiphytically (but becomesoil-rooted) or by climbing from ground levelHowever by secondary growth it later becomes


self-supporting and may eventually envelope theinitially supporting stem (eg certain tropical Ficusspp)

(4) Submersed or floating hydrophyte Herbaceousaquatic plant that relies on surrounding water forphysical support (Emergent hydrophytes(lsquohelophytesrsquo) mostly fall into one of the subgroupsof Point A1 in the present Section)

(5) Parasite or saprophyte obtains important nutritionalneeds directly or indirectly from other vascular plants(parasite) or from dead organic matter in the soil(saprophyte) (see Nutrient uptake in Material S2 whereother more specific forms of parasitism are covered)

References on theory significance and large datasetsCain (1950) Ellenberg and Muumlller-Dombois (1967) Whittaker(1975) Barkman (1988 and references therein) Rundel (1991)Richter (1992) Box (1996) Ewel and Bigelow (1996) Cramer(1997) Luumlttge (1997) Medina (1999) McIntyre and Lavorel(2001)

More on methods Barkman (1988 and references therein)

24 Plant height

Plant height is the shortest distance between the upper boundaryof themain photosynthetic tissues (excluding inflorescences) on aplant and the ground level expressed in metres Plant height ormaximumheight (Hmax) is themaximum stature a typical matureindividual of a species attains in agivenhabitatHmax is associatedwith growth form position of the species in the vertical lightgradient of the vegetation competitive vigour reproductive sizewhole-plant fecundity potential lifespan andwhether a species isable to establish and attain reproductive size between twodisturbance events (such as eg fire storm ploughing grazing)

What and how to measure

Healthy plants should be sampled that have their foliageexposed to full sunlight (or otherwise plants with the strongestlight exposure for that species) Because plant height is quitevariable both within and across species there are three waysto estimate Hmax depending on species size and the numberof plants and time available including the following (1) forshort species measurements are taken preferably on at least25 mature individuals per species (2) for tall tree speciesheight measurements are time-consuming and for these theheight of the five tallest mature individuals can be measuredand (3) for trees when more time is available measure ~25individuals that cover the entire rangeof their height anddiameterUse an asymptotic regression to relate height to diameter andderive the asymptote from the regression coefficients or usethe formula to calculate the height of the thickest individual inthe stand

The height to be measured is the height of the foliage of thespecies not the height of the inflorescence (or seeds fruits) orthe main stem if this projects above the foliage For herbaceousspecies this is preferably carried out towards the end ofthe growing season The height recorded should correspondto the top of the general canopy of the plant discounting any

exceptional branches leaves or photosynthetic portions of theinflorescence

For estimating the height of tall trees some options are

(1) a telescopic stick with decimetre marks and(2) trigonometric methods such as the measurement of the

horizontal distance from the tree to the observation point(d) and with a clinometer or laser the angle between thehorizontal plane and the tree top (a) and between thehorizontal plane and the tree base (b) tree height (H) isthen calculated asH= d [tan(a) + tan(b)] height estimatesare most accurate if the measurement angle is between 30degrees (easier to define the highest point in the crown) and45 degrees (a smaller height error caused by inaccuracy in thereadings) the horizontal distance between the observer andthe stem should preferably equal 1ndash15 times the tree height


(1) Rosettes For plants with major leaf rosettes andproportionally very little photosynthetic area higher upplant height is based on the rosette leaves

(2) Herbaceous For herbaceous species vegetative plantheight may be somewhat tricky to measure (if the plantbends or if inflorescence has significant photosyntheticportions) whereas reproductive plant height can be lsquosaferrsquoin this sense Additionally some authors have suggested thatthe projection of an inflorescence above the vegetative partof the plant may be a useful trait in responses to disturbanceso both of these heights should be useful to measure Otherswhile recording maximum canopy height arbitrarily use aleaf length of two-thirds of the largest leaf as the cut-off pointto estimate the position of a transition between vegetative andreproductive growth

(3) Epiphytes For epiphytes or certain hemi-parasites (whichpenetrate tree or shrub branches with their haustoria) heightis defined as the shortest distance between the upper foliageboundary and the centre of their basal point of attachment

(4) Large spreading crowns For trees with large spreadingcrowns it is difficult to estimate the height above the treestem For such individuals it is easier to measure (with anoptical rangefinder or laser) the vertical height as the distancefrom eye to a location at the crown margin that is level withthe tree top multiply this by the sine of the sighting angle tothe horizontal (as measured with a clinometer) and add thevertical height from eye level down to tree base (a subtractionif eye level is below tree base level)

(5) Dense undergrowth For vegetation types with denseundergrowth that makes the measurement of Hmax

difficult there are modified versions of the equationabove they involve the use of a pole of known height thatmust be placed vertically at the base of the tree

References on theory significance and large datasets Gaudetand Keddy (1988) Niklas (1994) Hirose and Werger (1995)Thomas (1996) Westoby (1998) Kohyama et al (2003) Kinget al (2006) Poorter et al (2006 2008) Moles et al (2009)

More on methods Korning and Thomsen (1994) Thomas(1996) Westoby (1998) McIntyre et al (1999) Weiher et al(1999)


25 Clonality bud banks and below-ground storage organs

Clonality is the ability of aplant species to reproduceor regenerateitself vegetatively thereby producing new lsquorametsrsquo (above-ground units) and expanding horizontally Clonality can giveplants competitive vigour and the ability to exploit patches rich inkey resources (eg nutrients water light) Clonal behaviour maybe an effective means of short-distance migration undercircumstances of poor seed dispersal or seedling recruitmentClonality also gives a plant the ability to form a bud bank whichcan be a very important determinant of recovery and persistenceafter environmental disturbances The bud bank consists of allviable axillary and adventitious buds that are present on a plantand are at its disposal for branching replacement of shootsregrowth after severe seasons (winter dry season fire season)or for vegetative regeneration after injury (adventitious budsthat arise after the injury which are an important means ofregeneration in some plants apparently lie outside the lsquobudbankrsquo concept) Both the characteristics of the bud bank andthe type of clonal growth exhibited by plants determine theirability to recover fromdisturbances (seeMaterial S3 for aprotocolfor Characterisation of the bud bank based on Klimeš andKlimešovaacute 2005) Clonal organs especially below-groundones also serve as storage and perennating organs a sharpdistinction between these functions is often impossible

How to collect and classify

For above-ground clonal structures observe a minimum offive plants that are far enough apart to be unlikely to beinterconnected and that are well developed For below-groundstructures dig up a minimum of five healthy-looking plants(Appendix 1) In some cases (large and heavy root systems)partial excavation may give sufficient evidence for classificationIt is best to assess clonality and bud banks near the end of thegrowing season Remove the soil and dead plant parts beforecounting buds or classifying the organs The species is consideredclonal if at least one plant clearly has one of the clonal organslisted below (see References below in the present Section fordiscussion)

Categories are then

(A) clonal organs absent(B) clonal organs present above ground including the

following(1) stolons ndash specialised often hyper-elongated horizontal

stems whose axillary bud growth and nodal rootingyields ultimately independent plants (eg strawberry(Fragaria vesca) saxifrage (Saxifraga flagellaris))

(2) bulbils ndash deciduous rooting bulblets produced fromaxillary or what would otherwise be flower buds orby adventitious bud growth on leaves (eg Cardaminepratensis Bryophyllum) analogous vegetativepropagules of bryophytes are termed gemmae and

(3) simple fragmentation of the vegetative plant body(mostly aquatic plants and bryophytes)

(C) clonal organs present below ground including thefollowing(1) rhizomesndashmore or less horizontal below-ground stems

usually bearing non-photosynthetic scale leaves (eg

many grasses and sedges) and sometimes insteadbearing photosynthetic leaves that emerge aboveground (eg Iris Viola bracken fern (Pteridium))aerial vegetative andor reproductive shoots grow upfrom axillary (or sometimes terminal) buds on therhizome most rhizomes can branch after whichdecline and decay of the portion proximal to thebranch point yields independent clonally generatedindividuals

(2) tubers and turions ndash conspicuously thickened below-ground stems or rhizomes functioning as carbohydratestorage organs and bearing axillary buds that canpropagate the plant (eg potato Solanum tuberosumJerusalem artichoke (Helianthus tuberosus)) similarorgans formed on aquatic plants are termed turions

(3) bulbs ndash relatively short below-ground stems that bearconcentrically nested fleshy scale-leaves that act asstorage organs the whole globose structure serving toperennate the plant and through growth of axillarybuds within the bulb into daughter bulbs or lsquooffsetsrsquoto multiply it vegetatively (eg tulip (Tulipa) onion(Allium))

(4) corms ndash vertically oriented globosely thickenedunderground stems that serve as storage organs andbear either scale or foliage leaves axillary or terminalbuds on the corm function for perennation and to alimited extent for clonal reproduction (eg Dahlia)

(5) tuberous roots ndash thickened roots that serve primarily forstorage but can formadventitious buds that permit clonalpropagation (eg sweet potato (Ipomoea batatas))

(6) suckers ndash shoots developed from adventitious budsproduced on ordinary non-storage roots (eg aspen(Populus tremuloides) wild plum (Prunus spp)) thesucker shoots can become independent plants once theroot connection between them and the parent is severedor dies

(7) lignotuber ndash a massive woody expansion just belowthe ground surface produced by secondary growthof the lsquoroot crownrsquo in many shrubs in fire-pronevegetation after a fire that kills the shrubrsquos aerialcanopy adventitious buds on the lignotuber grow outto regenerate the shrubrsquos canopy (see Section 66)normally not resulting in clonal multiplication and

(8) layering ndash ordinary vegetative shoots that lie on or benddown to theground there produce adventitious roots andcontinue apical growth becoming independent plantswhen their connection with the parent is severed (egblackberry and raspberry (Rubus) certain spp of spruce(Picea) and hemlock (Tsuga))

If a plant species has clonal growth (CategoriesBorCabove inthe present Section) classify it according to one or more of thefollowing categories

(1) regenerative clonal growth occurring after injury andnormally not multiplying the number of individuals aswith resprouting from a lignotuber

(2) additive (also termed multiplicative) clonal growth whichcan be either the plantrsquos normal mode of multiplication orcan be induced by environmental conditions such as high


nutrient availability and serves to promote the spread of theplant

(3) necessary clonal growth is indicated when clonality isrequired for the year-to-year survival of the plant as withmany plants that perennate from rhizomes bulbs tubers ortuberous roots and have no or weak seed reproduction

Clonal growth may fulfil more than one of these functionsin which case it may not be possible to distinguish betweenthem In some cases the functional nature of clonal growth maybe simply

(4) unknown or not evident in which case it may be recorded assuch

References on theory significance and large datasets DeKroon and Van Groenendael (1997) Klimeš et al (1997) VanGroenendael et al (1997)Klimeš andKlimešovaacute (2005)Knevelet al (2003) Klimešovaacute and Klimeš (2007)

More on methods Boumlhm (1979) Klimeš et al (1997)VanGroenendael et al (1997) Weiher et al (1998) Klimeš andKlimešovaacute (2005)

26 Spinescence

Spinescence refers to the degree to which a plant is defendedby spines thorns andor prickles Spines are sharp modifiedleaves leaf parts or stipules they also occur sometimes onfruits Thorns are sharp modified twigs or branches Pricklesare modified epidermis or cork (eg rose-stem prickles)Because spinescence is clearly involved in anti-herbivoredefence especially against vertebrate herbivores the followingtwo separate issues are critical in considering spinescence(1) the effectiveness of physical defences in preventing ormitigating damage from herbivores and (2) the cost to theplant in producing these defences Different types sizesangles and densities of spines thorns and prickles may actagainst different herbivores Although in many casescharacterisations of plant spinescence by measuring spines issufficient some researchers may decide that experiments withactual herbivores which examine the effectiveness of anti-herbivore defences are necessary eg by offering wholeshoots (with and without spines) to different animals andrecording how much biomass is consumed per unit time (seeSpecial cases or extras in Section 316)

Spines thorns and prickles can be an induced responseto herbivory meaning that some plants invest in thesedefences only when they have already been browsed byherbivores Other types of damage including pruning andfire can also induce increased levels of spinescence In

addition spinescence traits can change drastically with the ageof the plant or plant part depending on its susceptibility toherbivory For this reason spinescence sometimes cannot beconsidered an innate plant trait but rather a trait that reflects theactual herbivore pressure and investment in defence by plantsIn otherwords although there are species that always have spinesand species that never have them the spinescence of an individualplant is not necessarily representative of the potential range ofspinescence in the whole species (eg some members of Acaciaand Prosopis show a striking range of spine lengths within thesame species dependingon individuals age andpruninghistory)Spines thorns and prickles can sometimes play additional rolesin reducing heat or drought stress especially when they denselycover organs

How to measure

Spines thorns and prickles ndash summarised below as lsquospinesrsquo ndashcan either be measured as a quantitative trait or reduced to aqualitative categorical trait Data on spinescence are preferablymeasured from specimens in the field and can also be gatheredfrom herbarium specimens or descriptions in the literature Spinelength is measured from the base of the spine to its tip If a spinebranches as many do its length would be to the tip of the longestbranch Spine width measured at the base of the spine is oftenmore useful for assessing effectiveness against herbivoresand more generalisable across types of spines The number ofbranches if any should also be recorded because branches canincrease significantly the dangerousness of spines to herbivoresRatio of spine length to leaf length can also be a useful characterbecause it gives an idea of howprotected the lamina is by the spineclosest to it

Spine strength or toughness Spines are lsquosoftrsquo if whenmature they can be bent easily by pressing sideways with afinger and lsquotoughrsquo if they cannot be thus bent Spine density is thenumber of spines per unit length of twig or branch or area of leaf

Biomass allocation to spines is also an important parameter forsome research questions Its estimation is more work-intensivethan those above but still relatively simple Cut a standard lengthof stem or branch cut off all spines oven-dry and weigh leavesshoot and spines separately and estimate fractional allocation asthe ratio of spine dry weight to shoot dry weight

These quantitative trait measurements can be converted into acategorical estimate of spinescence by using the classificationproposed in Box 3

Finally to simply record the presence or absence of spinesis sufficient in some cases Bear in mind that the size structureand behaviour of herbivores vary enormously so the degree of

Box 3 Categorical estimates of spinescence

(1) No spines

(2) Low or very local density of soft spines lt5mm long plant may sting or prickle when hit carelessly but not impart strong pain

(3) High density of soft spines intermediate density of spines of intermediate hardness or low density of hard sharp spines gt5mm long plant causesactual pain when hit carelessly(4) Intermediate or high density of hard sharp spines gt5mm long plant causes strong pain when hit carelessly

(5) Intermediate or high density of hard sharp spines gt20mm long plant may cause significant wounds when hit carelessly

(6) Intermediate or high density of hard sharp spines gt100mm long plant is dangerous to careless large mammals including humans


protection provided by spine mass size and distribution can bedetermined only with reference to a particular kind of herbivoreWhen selecting the most meaningful measurements ofspinescence always consider what herbivores are relevant

References on theory significance and large datasets Milton(1991) Grubb (1992) Cooper and Ginnett (1998) Pisani andDistel (1998) Olff et al (1999) Hanley and Lamont (2002)Rebollo et al (2002) Gowda and Palo (2003) Gowda andRaffaele (2004) Agrawal and Fishbein (2006)

27 Branching architecture

Branching architecture refers to how intensively a plant branches(number of living ramifications per unit of stem length) Highlybranched plants can be better defended against vertebrateherbivores primarily by making feeding less efficient denyingaccess by herbivores to plant organs and ensuring that ifherbivores do remove growing tips there remain enough forthe plant to continue growing Conversely less branched plantscan be adapted to environments where growing tall quickly isnecessary as in a fire-prone savannah or a forest undergoing thepioneer stages of secondary succession Branching architecturecan also be adaptive in forest systems where species that utiliselow light tend to be more branched for a given height than arespecies that utilise only bright light

Although there are complicated and elegant methods forevaluating branching architecture a simple characterisationsuch as the one described below is often sufficient forunderstanding the adaptive significance of this trait Likespinescence branching architecture is a plastic trait that candiffer within a species on the basis of browsing history firehistory access to light plant vigour or disease and even waterstress Branching architecture is also variable depending onthe age and life history of the plant (see Section 11 forrecommendations related to variable traits)

How to measure

To assure measuring a branch that best represents thebranching architecture of a plant (a branch that reaches theouter part of the canopy) work backwards from a terminalleaf-bearing branch until reaching the first branch that is nowleafless at its base but bears secondary branches that have leavesThe base of this branch will be the starting point for measuring(1) the total length of the branch which is the distance from thestarting point to the tip of its longest-living terminal and (2) thenumber of ramification points that lead to living branches fromeach ramification point move towards the tip always followingthe most important branch (the main branch is often the thickestliving branch coming from a ramification point see Fig 1 for agraphic explanation) An indicator of branching architecturecalled apical dominance index (ADI) is obtained by dividingthe number of ramifications by the total length of the branch inmetres Thevalue ofADI canvary between zero (nobranching) togt100mndash1 (extremely ramified)

References on theory significance and large datasets Horn(1971) Pickett and Kempf (1980) Strauss and Agrawal (1999)Enquist (2002)Archibald andBond (2003)Cooper et al (2003)Staver et al (2011)

More on methods Fisher (1986)

28 Leaf area sapwood area ratio

The amount of leaf area a species produces per unit cross-sectionof sapwood (the inverse ofHuber value expressed inmm2mmndash2)is crucial for both water transport (with related effects onphotosynthetic rate) and mechanical strength

What and how to collect

The ratio leaf area sapwood area (LA SA) depends stronglyon leaf phenology Furthermore there is variation betweenwet and dry seasons variation among populations of a givenspecies along moisture gradients ontogenetic trajectories forgiven individuals and within trees along a branch from trunkto tip Declining function of sapwood with age is one reason whyLA SA generally increases when one moves from larger (older)towards smaller branches Unfortunately the age-related declinein sapwood function is not always well understood can bedifficult to measure and may vary among species All thisshould be considered when designing a sampling methodologyand interpreting this trait (see Point 3 of Special cases or extras inthe present Section)

To make meaningful comparisons among species werecommend sampling terminal sun-exposed shoots from theouter canopy This means sampling terminal shoots either of acertain standard length or of a certain age (1ndash3 years) (for shootsin which terminal bud scars allow their age to be determined)This approach maximises the likelihood that all the sapwood inthe branch is still functional We recommend sampling at thepeak of the growing season when leaf area is highest At thistime LA SA should be at a maximum for the year this is similarto the efforts to measure maximum photosynthetic rate as a wayof making meaningful comparisons across species Care shouldbe taken to select shoots that have not lost leaves or parts ofleaves to mechanical damage herbivory or early senescence andabscission

2

1

3 4

5 6

branch length

startingpoint

trunk

Fig 1 Measurement of branching architecture Numbers indicateramification points to be considered for the calculation of the apicaldominance index (ie number of ramifications per meter of branch) Notethat dead branches are not considered in the index


Measuring

Leaf area sapwood area ratio can be measured at differentscales namely from whole plant to just terminal branches (andthis should be taken into consideration when scaling upmeasurements) Total leaf area of leaves distal to the collectionpoint is measured by the same method as the area of individualleaves (see Section 32) Sapwood area at the collection point ismost precisely measured with digital micrographs and image-analysis software (see Section 31 for free software) howevera calliper should work for most species in most situations Inmeasuring sapwood area care should be taken to exclude barkphloem heartwood and pith from the area measured


(1) For herbaceous species similar methods can be appliedhowever care must be taken to identify the parts of the stemthat can conduct water this distinction may not be as clear asit is within most woody species It can be quantified with adye-transport experiment (see Point 3 below in the presentSection)

(2) Seasonal changes Because cambial growth in many treescontinues well after spring flush of bud growth is completedand the final leaf area for the season is attained the LA SAratio is best measured as late in the growing season aspossible when all the seasonrsquos newly produced leavesremain attached but (for evergreens) before the seasonalabscission of older leaves has occurred

(3) In ring-porous trees the effective conductivity of xylemdrops precipitously in older sapwood sometimes within avery few annual rings For these species the conductivity ofthe sapwood (and its decline with sapwood age) can bequantified by placing the cut end of the shoot into a fairlystrong solution of a dye such as eosin and allowing thefoliage to transpire in air andafter 10ndash20min cutting a cross-section of the stem a few centimetres above its cut end andmeasuring the dye-stained area

References on theory significance and large datasets Chiba(1991) Eamus and Prior (2001) Maherali and DeLucia (2001)Maumlkelauml and Vanninen (2001) McDowell et al (2002) Prestonand Ackerly (2003) Addington et al (2006) Buckley andRoberts (2006) Maseda and Fernaacutendez (2006) Wright et al(2006) Cornwell et al (2007) Litton et al (2007)

References on meta-analysis Mencuccini (2003)

29 Root-mass fraction

Theory predicts that plants from nutrient-poor sites shouldallocate a greater fraction of new biomass to roots andmaintain a higher proportional distribution of biomass in rootsthan in shoots Distribution of biomass to roots can be simplyexpressed as the root-mass fraction (RMF synonymous to root-mass ratio RMR) identically calculated as the proportionof plant dry mass in roots Note that a true allocationmeasurement requires quantifying turnover rates as well asstanding distributions which is labour-intensive and rarelycarried out Allocation and distribution are often usedsynonymously and whether this is appropriate or not wefollow this convention herein The RMF is preferable to the

often used root shoot ratio (RSR) because the RFM is boundedbetween 0 and 1 and can be immediately interpreted andcompared whereas the RSR is unconstrained and can varyfrom a tiny to a very large number Notably root allocationcan be highly plastic across light nutrient and water suppliesSome patterns can be apparently contradictory because rootallocation can allow both greater foraging below groundwhich would be an advantage especially when resources arelow and also greater competition below ground being anadvantage when resources are plentiful In reviews ofexperimental studies including those that take an allometricapproach RMF typically decreases with increasing nitrogenavailability However other studies have reported that for fieldplants fast-growing species adapted to nutrient-rich habitatsshowed higher allocation to roots than did slow-growingspecies from nutrient-poor sites Similarly seedlings showingplastic responses to low light typically decrease their RMFwhereas plants adapted to chronic deep shade in rainforeststend to have higher RMF apparently to survive periods of lowwater and nutrient supply in competition with surrounding treesNote that some reports of differences in RMF across resourcegradients are potentially confounded by failure to account forallometry and size (see References on theory significance andlarge databases below in the present Section) AdditionallyRMF does not directly translate to a high soil resource-uptakerate Lower allocation to roots may well be compensated byhigher specific root length (see Section 51) and by higher uptakerate per allocation to root mass length or surface area

The RMF can best be used for comparative purposes ifmeasured for plants of similar mass Alternatively if plants areharvested of a range of mass allometries can be used to estimateRMF for plants of a given size

Care should be taken to harvest all the roots (see Section 5)despite the difficulty of separating roots from soil particularlyfine roots However in field studies sometimes RMF includesonly a subset of all below-ground tissues in such a case theresearcher should be clear about what is included andwhat is not


(1) Storage organs and root fractioning RMF should intheory include everything that is plant-developed (so notincluding mycorrhizae) However particular studies cansubdivide specific fractions for specific purposes (ie fineroots coarse roots crowns rhizomes (for grasses) tap roots(in trees)) to evaluate the relative proportions of each inrelation to each other andor to above-ground biomass

References on theory significance and large databases Evans(1972) Grime (1979) Aerts et al (1991) Elberse andBerendse (1993) Veneklaas and Poorter (1998) Aerts andChapin (1999) Reich (2002) Sack et al (2003) Poorter et al(2012)

210 Salt resistance

Many areas of the world including coastal ones those withpoorly drained soils in arid climates and those with poorlydesigned irrigation systems feature high concentrations of salt(gt100ndash200mM sodium chloride NaCl) Only salt-resistant


species which exhibit strategies to reduce or avoid damagingeffects of excess salt in their tissues are able to maintain viablepopulations in such areas Plants specialised for inhabiting salinesoils and often restricted to these are termed halophytes

Amongmembers of the at least 139 plant families that includehalophytes evolution has yielded multiple solutions to theproblem of excess salt in the environment involving differentbiochemical physiological structural andor phenologicaltraits Therefore rather than a single recipe for assessingsalt resistance we give several traits and measurements thattogether help identify a species as salt-resistant especially ifthese are accompanied by data on species distribution in salineareas However to positively classify a species as salt-sensitivewould be problematic from these traits alone Experimentaltesting of plant survival and growth under saline conditions isnecessary which would by no means be quick and easy forscreeningmultiple species Thus the traits described below allowa qualitative rather than quantitative assessment of salt resistanceand do not allow the clear separation of more or less salt-resistantspecies from true halophytes Hopefully this text will stimulateresearch into novel approaches and protocols for testing saltresistance more efficiently and comprehensively

Here we simplify more extensive previous classifications ofmechanisms by which plants deal with excess environmentalNaCl focusing on three common strategies Some salt-resistantplant species can limit the uptake of potentially damaging Na+

by their roots (NaCl lsquoexcludersrsquo) However many salt-resistantspecies cannot avoid significant NaCl uptake These plants caneither actively excrete excess salt or can accumulate NaCl in cellvacuoles so as to prevent toxicity to the cytosol The latter (lsquosalt-tolerantrsquo) species are often succulent with many characteristicsof drought-tolerant species Many salt-resistant species possessbiochemical mechanisms to reduce salt stress or damage inthe tissues by accumulating compatible solutes (includingsecondary metabolites) in the cytosol The salt-resistance traitsdetailed below fall into the foregoing categories except specialbiochemical adaptations that are not covered here


Selective root cation uptake Roots of many salt-resistantplant species (particularly monocots) can discriminate againstNa+ while maintaining uptake of essential potassium (K+) Thisselectivity for K+ over Na+ increases the K+ Na+ ratio in thecytosol comparedwith that in the rootingmedium Because theseratios may vary with several environmental factors includingprecipitation and evapotranspiration we suggest sampling leavesand soil on at least three different days at intervals of 2 weeksor more during the growing season but not for 5 days afterparticularly heavy or prolonged rain Collect leaves from fiveseparate plants (Appendix 1) and a soil sample from the mainfine-root zonebeloweachTheNa+ andK+concentrations of eachsample are to be determined in the laboratory by a standard assayPopular and convenient methods include atomic emissionspectrometry (EAS) also called flame photometry and atomicabsorption spectrometry (AAS) Leaf samples are to be groundin an equal mass of water which is then extracted from thehomogenate by filtration For soil add water to a dry soil until itbecomes water-saturated and then extract the liquid by suction or

vacuum filtration Na+ and K+ assays can be performed either onthe water phase or after evaporating it depending on the Na+ andK+ assay method

Calculate for each plant and associated soil sample theK Naselectivity (S) as S = ([K+][Na+])plant ([K

+][Na+])soil Amean Svalue for a species is calculated from the mean of all replicate Svalues per sampling date by taking the average of these over allsampling dates

Salt excretion Salt-excreting species eject NaCl throughspecial glands or bladders on the (usually lower) surfaces of theirphotosynthetic organs (usually leaves but in some cases stems)These glands are often visible (especially under a hand lens) assmall irregularly shapedwhite spots that are excreted salt crystalson the surface of the gland A salty taste on licking one of thesewill confirm this Some species excrete salt from their rootsAlthough this is more difficult to observe one may check forsimilar salt excretions on the surfaces of any roots uncoveredduring soil sampling Note that salt excretions on shoots or rootswill wash off during wet weather so are best sought after a dryperiod

Salt compartmentalisation Salt compartmentalisation isindicated by clear succulence of the leaves or photosyntheticstems Succulent green stems can be treated and measured as ifthey were leaves (see Special cases or extras in Section 31)Succulence leads to high leaf water content (LWC) and leafthickness (Lth) and may be quantified as the product of theseparameters (succulence (mm) =LthLWC) (see Section 33)Values gt800ndash1000mm indicate significant succulence

Strong salt-related succulence is found almost exclusively indicotyledonous species although certain salt-tolerant monocotscan be somewhat succulent eg Elytrigia juncea on beachdunes Salt-tolerant succulents show a high NaCl level in theirleaves which can distinguish them from crassulacean acidmetabolism (CAM) succulents (see Section 312 some salt-tolerant succulents are actually also CAM plants) This couldbe detected by the Na assay on leaf or stem extracts noted aboveor would be revealed very easily by measuring the electricalconductivity of such extracts (see Electrolyte leakage inSection 314) which requires only a simple widely availableconductivity meter (NaCl in solution gives a high conductivity)Qualitative evidence for this can be a combination of juicinessand noticeably salty taste when chewing the tissue This propertyhasmade somehalophytespopular ashuman food egSalicorniaspp

Special cases and extras

(1) Succulents and halophytes Many salt-tolerant succulentsare halophytes and occur only in saline environmentsexpression of the traits described above can depend on theactual salinity of the plantsrsquo soil We therefore suggestmeasuring soil salt concentrations (as described underSelective root cation uptake above within the presentSection) to accompany trait measurements Several othersalt-relatedhabitat descriptors are also relevant eg elevationand duration of daily marine inundation (if any) in saltmarshes or on beaches and location relative to the hightide mark visible as a litter belt or white patches on the soilsurface indicating salt crystals in dry areas


References on theory significance and large datasetsFlowers et al (1977 1986) Yeo (1983) Rozema et al(1985) Zhu (2001) Breckle (2002) Munns et al (2002)Vendramini et al (2002) Ashraf and Harris (2004) Flowersand Colmer (2008)

More on methods Jennings (1976) Maas and Hoffman(1977) FAO (1999) Breckle (2002) Vendramini et al (2002)

211 Relative growth rate and its components

Relative growth rate (RGR) is a prominent indicator of plantstrategy with respect to productivity as related to environmentalstress and disturbance regimes RGR is the (exponential) increasein size relative to the size of the plant present at the start of a giventime interval Expressed in this way growth rates can becompared among species and individuals that differ widely insize By separate measurement of leaf stem and rootmass as wellas LA good insight into the components underlying growthvariation can be obtained in a relatively simple way Theseunderlying parameters are related to allocation (leaf-massfraction the fraction of plant biomass allocated to leaf) leafmorphology (see Section 31) and physiology (unit leaf rate therate of increase in plant biomass per unit LA a variable closelyrelated to the daily rate of photosynthesis per unit LA also knownas net assimilation rate)


Ideally RGR is measured on a dry-mass basis for the wholeplant including roots Growth analysis requires the destructiveharvest of two or more groups of plant individuals grown eitherunder controlled laboratory conditions or in the field Individualsshould be acclimated to the current growth conditions At leastone initial and one final harvest should be carried out The actualnumber of plants to be harvested for a reliable estimate increaseswith the variability in the population Size variability can bereduced by growing a larger number of plants and selecting apriori similarly-sized individuals for the experiment discardingthe small and large individuals Alternatively plants can begrouped by eye in even-sized categories with the number ofplants per category equal to the number of harvests Byharvestingone plant from each category at each harvest each harvest shouldinclude a representative sample of the total population studiedTheharvest intervalsmayvary from less than1week in the caseoffast-growing herbaceous species tomore than 2months or longerin the case of juvenile individuals of slow-growing woodyspecies As a rule of thumb harvest intervals should be chosensuch that plants have less than doubled mass during that interval

At harvest the whole root system is excavated andsubsequently cleaned gently washing away the soil (seedetails on procedure under Section 5) Plants are divided intothree functional parts including leaves (light interception andcarbon (C) uptake) stem (support and transport) and roots (waterand nutrient uptake as well as storage) The petioles can eitherbe included in the stem fraction (reflecting support this is thepreferred option) or combined with the leaf fraction (to whichthey belong morphologically) or they can be measuredseparately LA is measured (for details see Section 31) beforethe different plant parts are oven-dried for at least 48 h at 70Candweighed

Destructive harvests provide a wealth of information butare extremely labour-intensive and by their nature destroyat least a subset of the materials being studied Alternativelyor additionally growth can be followed non-destructively forseveral individuals (~10ndash15 per treatment) by non-destructivelymeasuring an aspect of plant size at two ormoremoments in timeBy repeatedly measuring the same individuals a more accurateimpression of RGR can be obtained However RGR cannot befactorised into its components then and repeated handling maycause growth retardation Ideally the whole volume of stems(and branches) is determined in woody species (see Section 41)or the total area of leaves in case of herbaceous plants In the lattercase leaf length and width are measured along with the numberof leaves To estimate LA a separate sample of leaves (~20)has to be used to determine the linear-regression slope of leaflengthwidth

How to calculate RGR

From twoconsecutive harvests at times t1 and t2 yieldingplantmasses M1 and M2 the average RGR is calculated as

RGR frac14 ethlnM2 lnM1THORN=etht2 t1THORNIn the case of a well balanced design where plants are paired it isprobably simplest to calculate RGR and its growth parameters foreach pair of plants and then use the RGR values for each pair toaverage over the population Otherwise the average RGR overthewhole group of plants is calculated from the same equation Indoing so make sure to first ln-transform the total mass of eachplant before the averaging In the case of more than two harvestsaverageRGRcanbe derived from the linear-regression slope of ln(mass) over time

The average unit leaf rate (ULR) over a given period is

ULR frac14 frac12ethM2 M 1THORN=ethA2 A1THORN frac12ethlnA2 lnA1THORN=etht2 t1THORNwhereA1 andA2 represent the LAat t1 and t2 respectively Againthe simplest option would be to calculate ULR from each pair ofplants

Average leaf mass fraction (LMF) during that period is theaverage of the values from the 1st and 2nd harvest as follows

LMF frac14 frac12ethML1=M 1THORN thorn ethML2=M2THORN=2whereML1 andML2 indicate the leafmass at t1 and t2 respectively

Similarly average specific leaf area (SLA) is calculated asfollows

SLA frac14 frac12ethA1=ML1THORN thorn ethA2=ML2THORN=2


(1) Confounding effect of seed size Especially tree seedlingsmay draw on seed reserves for a long time after germinationInclusion of the seed in the total plant mass underestimatesthe RGR of the new seedling whereas exclusion of the seedmass will cause an overestimation of growth For largepersistent seeds the decrease in seed mass between t1 andt2 can therefore be added to M1 (excluding seed mass itselffrom M1 and M2)


(2) Ontogenetic drift Asplants changeover time they readjustallocation morphology and leaf physiology ConsequentlyLMF SLA and ULR may change with plant size and RGRgenerally decreases over time the more so in fast-growingspecies This does not devalue the use of the RGR as plantgrowth does not necessarily have to be strictly exponentialAs long as plant growth is somehow proportional to the plantsize already present RGR is an appropriate parameter thatencapsulates the average RGR over a given time periodHowever ontogenetic drift is an important characteristic ofplant growth and a higher frequency of harvestsmayprovidebetter insight into this phenomenon In comparing species ortreatments it may be an option to compare plants at a givensize or size interval rather than over a given period of time

(3) Related to ontogenetic drift shrubs and trees accumulateincreasing amounts of xylem of which large proportionsmay die depending on the species This inert mass wouldgreatly reduceRGRPrevious studies expressRGR (lsquorelativeproduction ratersquo) on the living parts of large woody plantsby treating the biomass increment over Year 1 asM1 and theincrement over Year 2 as M2 It could similarly be based onannual diameter (or volume) increments

(4) Smooth curves In the case of frequent (small) harvests aspecial technique can be applied inwhich polynomial curvesare fitted through the data This is an art in itself

References on theory significance and large datasets Evans(1972) Grime and Hunt (1975) Kitajima (1994) Cornelissenet al (1996) Walters and Reich (1999) Poorter and Nagel(2000) Poorter and Garnier (2007) Rees et al (2010)

More on methods Evans (1972) Causton and Venus (1981)Hunt (1982) Poorter and Lewis (1986) Poorter and Welschen(1993) Cornelissen et al (1996) Rees et al (2010)

212 Plant flammability

Flammability-enhancing traits are important contributors tofire regimes in (periodically) dry regions and therefore theyhave important ecological impacts (particularly on ecosystemdynamics) as well as socioeconomic and climatic consequencesThe intrinsic flammability of a plant depends on both its traitswhile alive and the effects of its leaves branches and stems afterthose organsrsquo deaths The flammability of those organs (eitherliving or dead) depends on (1) the type or quality of the tissue and(2) the architecture and structure of the plant and its organs (whichis mainly related to heat conductivity)

Note that theflammability of a given species canbeoverriddenby the flammability of the entire plant community (eg amountof litter community structure and continuity organic mattercontent of the soil) and by the particular climatic conditions(eg after a long very dry period many plants would burn quiteindependently of their flammability)

How to define and assess

Flammability (broadly defined as the propensity to burn) is acompound plant functional trait Its components vary amongauthors and disciplines Most studies are based on flammabilitymeasurements of small plant fragments in chambers in thelaboratory Although this produces highly standardised resultsit does not scale upwell towhole-shootflammabilityWepropose

a standard method for measuring flammability in which the basicarchitectural arrangement of the measured shoots is preserved(see More on methods in the present Section for reference andFig 2 for illustration) This involves a low-tech device in whichshoots up to 70 cm long are placed preheated and ignited in astandard way then the following measurements are taken

(1) maximum temperature reached during burning (in C MT)measured with an infrared thermometer from a distance of50 cm to the burning shoot

(2) burning rate (BR) a value obtained by dividing the length ofthe sample that was burnt by the burning time (in seconds) itgives an idea of how quickly flames can spread across theplant and to what extent the plant is able to carry fire and

(3) burnt biomass percentage (BB) consisting of a visualestimation of the burnt biomass (percentage intervals) theintervals are 1 = lt1 2 = 1ndash10 3 = 11ndash25 4 = 26ndash505 = 51ndash75 and 6 = 76ndash100

For calculating (overall) flammability the scores on eachcomponent must be transformed to a proportional scale withthe value 1 being assigned to a reference value In the case ofBB the value 1 was assigned to the maximum possible value forthis component (ie 6) whereas for the other components thereference value was based on the literature and the results of ourexperiments Reference values are MT = 500C BR= 1 cmndash1Standardised MT BR and BB scores are then added to obtain acompound value of flammability (rounded to two decimals) thatcould vary between 0 (no flammability) and ~3 (maximumflammability)

As an alternative to the direct measurement of flammabilityan estimation may be obtained by measuring several plantattributes that are known to influence plant flammabilityFive classes are defined for each of the attributes describedbelow (stem and twig water content canopy architecturesurface volume ratios standing litter volatile oils waxes andresins) Flammability is subsequently calculated as the average(rounded to one decimal) of the class scores for each of thefollowing individual attributes (seeTable 1 for ranges of values ofeach attribute within each class)We strongly recommend testingand calibrating this estimation against direct measurements offlammability as described above or against direct measurementsof ignitability and combustibility as described under Specialcases or extras in the present Section

(1) Water content of branches twigs and leaves Flammabilityis expected to begreater in specieswith higher leaf dry-mattercontent (see Section 33 for protocol) and higher twig dry-matter content (seeSection42 for protocol) and it is probablyalso a function of drying rate (here represented inversely bydrying time from saturation to dry equilibrium)

(2) Canopyarchitecture Plantswith complex architecture ieextensive branching tend to spread fire easily The degree(number of orders) of ramification (branching) is used here asa close predictor of canopy architectural complexity andranges from zero (no branches) to 5 (four or more orders oframification) (see Section 27)

(3) Surface-to-volume ratios Smaller twigs (ie twigs ofsmaller cross-sectional area) and smaller leaves shouldhave a higher surface-to-volume ratio (and thus faster


drying rate) and therefore bemoreflammable Since twig andleaf size tend to be correlated in interspecific comparisonsaccording to allometric rules we use leaf size here torepresent both traits A complication is that some speciesare leafless during the dry season however leaf litter is likelyto still be around in the community and affect flammabilityduring the dry season (see Section 32) However for groundfires substantial accumulation of litter of small leaves maypack densely obstructing oxygen flow and actually stronglyinhibiting fire to spread

(4) Standing litter The relative amount of fine dead plantmaterial (branches leaves inflorescences bark) still

attached to the plant during the dry season is critical sincelitter tends to have very low water content and thus enhanceplant flammability lsquoFinersquo litter means litter with diameter orthickness less than 6mm We define five subjective classesfrom no lsquofine standing litterrsquo via lsquosubstantial fine standinglitterrsquo to lsquothe entire above-ground shoot died back as onestanding litter unitrsquo

(5) Volatile oils waxes and resins in various plant partscontribute to flammability This is a subjective categoricaltrait ranging from lsquononersquo to lsquovery high concentrationsrsquoCheck for aromatic (or strong unpleasant) smells as wellas sticky substances that are released on rubbing breaking or

(m)

(e)

(g)

(h)(i )

( j ) (k )

( l )

(a)

(d )

(b )

(c )

(f )

Fig 2 General view of a device for measuring plant flammability in the field (reproduced with permission from Jaureguiberry et al 2011) (a) Grill (b) grillthermometer (c) temperature gauge (d) security valve (e) connection to gas cylinder (f) removable legs (g) blowtorch valve (h) blowtorch (i) burners(j) ventilation holes (k) barrel (l) removable wind protection and (m) gas cylinder See Jaureguiberry et al (2011) for technical details

Table 1 Plant flammability traitsClasses for component traits for estimating plant flammability Flammability itself is calculated as the average class value (rounded to 1 decimal) over allcomponent traits Flammability increases from 1 to 5 For this calculation twig drying time (which is probably closely negatively linked with twig dry-matter

content TDMC see Section 42) is optional

Component trait Flammability class1 2 3 4 5

Twig dry-matter content (mg gndash1) lt200 200ndash400 400ndash600 600ndash800 gt800Twig drying time (day) 5 4 3 2 1Leaf dry-matter content (mg gndash1) lt150mg 150ndash300 300ndash500 500ndash700 gt700Degree of ramification (branching)

(number of ramification orders)No branches Only 1st-order

ramification2 orders oframification

3 orders oframification


Leaf size (lamina area) (mm2) gt25 000 2500ndash25 000 250ndash2500 25ndash250 lt25Standing fine litter in driest season None as one

litter unitSome Substantial

(with dead leaves ortwigs or flaking bark)

More dead thanlive fine massabove ground

Shoot dies backentirely standing as

one litter unitVolatile oils waxes andor resins None Some Substantial Abundant Very abundant


cutting various plant parts Scented flowers or fruits are notdiagnostic for this trait


(1) Ignitability indicates how easily a plant ignites (ie starts toproduce a flame) It can be measured directly by measuringthe time required for a plant part to produce a flame whenexposed to a given heat source located at a given distanceIgnitability experiments are usually performed several times(eg 50) and the different fuels are ranked by taking intoaccount both the proportion of successful ignitions (ignitionfrequency) and the time required to produce flames (ignitiondelay) Tissues producing flames quickly in most of the trialsare ranked as extremely ignitable whereas tissues that rarelyproduce flames andor take a long time to produce them areconsidered of very low ignitability These experiments arerun in the laboratory under controlled conditions (moistureand temperature) by locating a heat source (eg electricradiator epiradiator open flame) at a given distance (fewcentimetres) from the sample Thevalues used to rank speciesaccording to ignitability depend on the type and power of theheat source on the distance of the heat source to the sampleon the shape and size of the samples and on the relativehumidity of the environment in the days before the test theseexperimental conditions should be kept constant for all trialsand samples We propose using an open flame at 420Cplacing the plant material at 4 cm from the flame A standardquantity of 1 g of fresh material is used

(2) Plant tissue heat conductivity (combustibility) can beassessed by the heat content (calorific value kJ gndash1)which is a comprehensive measure of the potential thermalenergy that can be released during the burning of the fuelIt is measured with an adiabatic bomb calorimeter using fuelpellets of ~1 g and the relative humidity of the environmentin the days before the test should be standardised aswell Evidence shows that heat content varies relativelylittle among species and is only a modest contributor tointerspecific variation in flammability

(3) Combustibility and structural variables In relation to thesurface area-to-volume ratio other structural variables havebeen used to characterise the combustibility especiallythe proportion of biomass of different fuel classes (sizedistribution) Typically the fuel classes used are thebiomass fractions of (1) foliage (2) live fine woody fuel(lt6-mm diameter sometimes subdivided in lt25 and25ndash6mm) (3) dead fine woody fuel (lt6mm) and (4)coarse woody fuel (6ndash25 26ndash75 gt75mm) The summedproportion of live and dead fine fuels (foliage and woody oflt6mm) may be the best correlate of overall surface area-to-volume ratio

(4) Fuel bulk density (= fuel weightfuel volume) and canopycompactness (ratio of fuel volume to canopy volume) havealso been used to characterise heat conductivity mainly atthe population and community levels Furthermore highlitter fall and low decomposition rate will increase thecombustibility of the community

References on theory significance and large datasets Mutch(1970) Rothermel (1972) Bond and Midgley (1995) Bond and

VanWilgen (1996) Schwilk andAckerly (2001)Gill andZylstra(2005) Scarff and Westoby (2006) Cornwell et al (2009)Pausas et al (2012)

More on methods Papioacute and Trabaud (1990) Stephens et al(1994) Valette (1997) Dimitrakopoulos and Panov (2001)Etlinger and Beall (2004) Scarff and Westoby (2006) VanAltena et al (2012) for flammability of whole shootsdescribed above see Jaureguiberry et al (2011)

213 Water-flux traits

Plants play a key role in most hydrological fluxes in terrestrialecosystems including the capture of precipitation retentionand spatial (re)distribution of water above and below groundand water loss through evaporation and transpiration Thereare considerable differences among species in the extent andmanner inwhich they influencewaterfluxes These species-baseddifferences affect not only their own growth and survival butalso influence key ecosystem processes such as growth andnutrient cycling through direct and indirect effects of moisturedistribution This section focuses on plant traits that affecthydrological fluxes external to the plants and thereforeexcludes traits related to the flow of water through plants orthe storage of water in plants (see Sections 33 and 44 amongothers) Differences in the impact on fluxes among species aremainly due to differences in plant architecture morphology andsurface features which can all be quantified The effects of litterand small plants on surface runoff and infiltration and the effect ofroots on below-ground movement of water through preferentialpathways and altered soil hydraulics can also be importanthowever they are not further discussed here

A large fraction of incident precipitation hits plant surfacesbefore reaching the ground The fate of this water can be (1) freethroughfall (2) retention followed by evaporation or uptake intothe leaf (3) release as throughfall as a consequence of drainagefrom saturated surfaces or (4) drainage via twigs and stems(stemflow) Precipitation reaches the ground as throughfall orstemflow Rainfall interception regulates the amount of waterreaching the soil (and plant roots) and may ensure a less erosivewater supply during and after heavy showers Rainfallinterception is influenced by the density of the plant crownand its ability to retain water on its surfaces Water retentiononplant surfaces is greatest in low-intensity rainfall in the absenceof wind As vegetation surfaces approach saturation furtherinterception of precipitation leads to drainage of excess water(but see fog interception under Special cases and extras in thepresent Section) Plant traits that determine the actual waterretention on plant surfaces in a given environment are surfaceangle and lsquowettabilityrsquo Hydrophobic surfaces have sharp contactangles and droplets tend to remain separate whereas hydrophilicsurfaces have small contact angles and water spreads over thesurface as a film The surface traits that most affect wettability arecuticular waxes and trichomes

How to assess

(1) Gap fraction The openness or gap fraction of a plantrsquoscrown is an important determinant of the free throughfallfraction (p) (aswell as its light interception) For each specieswe recommend taking measurements of individual plants


with minimal canopy overlap with neighbours and minimalneighbour foliage below the crown Gap fraction can beestimated photographically The lens has to be held neatlyhorizontally below the canopy and capture the entire crownbut not much more If digital pictures have enough contrastimaging software can help calculate the fraction of sky (light)relative to foliage cover (dark) within the crown outline Iftime or facilities are limited the gap fraction will be areasonable predictor of free throughfall However p is notnecessarily equal to the gap fraction measured because raindrops do not always fall strictly vertically See Special casesand extras in the present Section for directmeasurement of p

(2) Stemflow Stemflow can be quantified by attaching collarsor spirals around plant stems that channel the water runningdown the stems into a collector Usually a flexible gutter-shaped form (such as a hose cut in half) is fixed around thestem and sealed with silicone or other sealant At the bottomof the gutter a hose leads the captured water into a collectorCollars and collectors need to be able to handle large volumesofwater because stemflowconcentrates thewater running offlarge areas of foliage For a quantitativemeasure of the extentto which different plant species channel precipitation to theirstems it is important to do comparisons under similar rainfallconditions or to use rainfall simulators Stemflowvarieswithplant size and architecture because these influence eg thecapacity for water storage on stems the inclination angles ofstems and the extent of branching among other things Fromthe perspective of plant traits stemflow is best expressed as apercentage of the volume of rain falling on the plant asfollows

stemflow frac12 frac14 stemflow frac12L=ethrainfall frac12mm crown projected area frac12m2THORN 100

(3) Water retention on plant surfaces This parameter can beestimated at the whole-plant level or for plant parts such asleaves and stems The procedure involves (1) weighing theplant (or part) without surface water (2) wetting of the plant(part) and (3)weighing the plant (part) withwet surfaces Theamount of water retained can be expressed in different waysincluding water per unit plant surface area or per crownprojected area Wetting can be achieved by immersion orby simulated rainfall or fog Immersion will tend to givemaximum water retention which is seldom reached in thefield The most realistic estimates are obtained using naturalor simulated precipitation with plants in the field Designsfor rainfall or fog simulators can be found in the soil erosionand pesticide spray literature respectively Rainfall retentioncan also be estimated as total interception (rainfall minusthroughfall and stemflow) of a discrete rain event that is justlarge enough to saturate the crown in a situation of negligibleevaporation This provides estimates of retention whereweighing is impractical or impossible

(4) Leaf wettability How easily a leaf can get wet isdetermined by measuring contact angles between waterdroplets and the leaf surface Droplets on water-repellentsurfaces have greater contact angles and are more sphericalA droplet of standard volume (2ndash5mL) is pipetted onto the

leaf surface and observed from the side with a microscopeThe microscope can be fitted with a goniometer for directmeasurement of the angle or images can be obtained with acamera An alternative to the measurement of the contactangle is its calculation based on measurement of the contactarea between droplet and leaf

The sampling strategy depends on the research aims Chooseleaves randomly or select in a standardised way according to leafposition on the plant or leaf age For most leaves the upper(usually adaxial) surface is the logical surface for wettabilitymeasurements For others particularly isobilateral and needle-type leaves as well as leaves that are often exposed to inclinedrainfall fog or mist measurements on both sides of the leaf arerecommended

(5) Droplet retention ability How well water lsquosticksrsquo to a leafcan bemeasured by placing a droplet of water on a horizontalleaf surface andmeasuring the angle of inclination of the leafat which the droplet first begins to move It is useful tomeasure droplet-retention ability on the same leaves as thoseused for determination of leaf wettability


(1) Free throughfall (p) The parameter p can be estimatedgraphically as the slope of the regression line that describesthe relationship between throughfall and rainfall usingprecipitation events that are of insufficient size andintensity to cause drainage from these plants Ifprecipitation is recorded continuously observations madeimmediately after commencement of large precipitationevents can be used as well For small-scale applicationsrainfall simulators may be used for comparative studies Inmost cases free throughfall will be slightly overestimatedbecause part of rainfall striking vegetation may notbe retained because of the force of the impact or themovement of crowns Methods for quantification ofrainfall and throughfall are described in the hydrologicalliterature They typically include standard commercialrain gauges (pluviometers or pluviographs) or custom-made collectors such as funnels troughs or large sheetsContinuous measurements of precipitation utilise devicessuch as tipping-bucket mechanisms that produce output fordataloggers Variability of throughfall under plants is verylarge as a result of clustering of foliage and channellingtowards the centre of the plant or towards the outer parts of thecrown It can be useful to take these patterns into account bysampling along radii of the plant Representative (equal-area)sampling of concentric parts of the crown is achieved byfollowing the rule rn =Hn r1 where rn is the distancebetween the nth collector and the centre of the plant

(2) Drip tips These are morphological features that influenceleaf wetness and interception by accelerating drainagefrom wet leaves Water is channelled towards the long andnarrow tip at the low end of a hanging leaf which is unable toretain the accumulating water thus reducing the duration ofleaf wetness Measurement of drip-tip length involves adecision about the position of the base of the drip tip It is


recommended that this is established by drawing the tip of anormally tapering (acute or obtuse angled) tip

(3) Water absorption by leaves A certain fraction of water onleaf surfaces may be absorbed by the leaf Typically thewater on wet leaves represents ~02-mm depth Althoughmost of this water will have evaporated before it can be takenup water uptake into the leaf can be significant even in non-arid environments Water uptake is particularly efficient inplants with specialised trichomes such as certain bromeliadsA simple method to determine rates and amounts of wateruptake is by weighing

(4) Fog interception Fog consists of small water dropletsdeposited on plant surfaces through air flow rather thangravity (lsquohorizontal precipitationrsquo) Its interception can bea net gain because fog would not normally precipitate in theabsence of vegetation that captures it Fog interception can beparticularly important tomanyepiphytes or toplants fromdrysubstrates such as rocks and coastal deserts The main planttraits affecting the rate of fog interception are the surfacearea in the direction of air flow (eg cross-sectional area ofa tree crown) and the dimensions of canopy elementsNarrow structural elements with thinner boundary layersare relatively efficient in capturing fog Trichomes (seePoint 3 above in the present Section) and aerial rootscommon for epiphytes may further increase fog capture

(5) Epiphyte load which in some cases can be species-specificbut generally is not can hugely influence water interceptionof rainfall and fog and probably stemflow (increasingwater retention on the host plant and modifying waterfluxes) Wherever epiphyte load is important it should beaccounted for eg as a covariate Epiphyte load can beassessed as mass as percentage cover or as counts (forlarge epiphytes) Then their interception and retentionproperties can be quantified as described above

References on theory significanceand largedatasets Skinneret al (1989) Brewer et al (1991) Puigdefaacutebregas and Pugnaire(1999) David et al (2005) Martorell and Ezcurra (2007)

More on methods Aston (1979) Herwitz (1985) Veneklaaset al (1990) Meyer (1994) Domingo et al (1998) Brewer andNuacutentildeez (2007) Burd (2007)

3 Leaf traits

31 Specific leaf area

Specific leaf area (SLA) is the one-sided area of a fresh leafdivided by its oven-dry mass (Note that leaf mass per area(LMA) specific leaf mass (SLM) and specific leaf weight(SLW) are simply 1SLA) SLA is frequently used in growthanalysis because it is often positively related to potential RGRacross species SLA tends to scale positively with mass-basedlight-saturated photosynthetic rate and with leaf nitrogen (N)concentration and negatively with leaf longevity and Cinvestment in quantitatively important secondary compoundssuch as tannins or lignin In general species in permanentlyor temporarily (eg deserts after a rain event) resource-richenvironments tend on average to have a higher SLA than dothose in resource-poor environments although there can also beconsiderable variation in SLA among co-occurring species

Specific leaf area is a function of leaf dry-matter content (seeSection 33) and Lth (see Section 34) Both components cancontribute to SLA to different degrees depending on the habitatand the plant group in question In cool-temperate herbaceousdatasets low SLA of slow-growing species tends to be related tohigh leaf dry-matter content more than to high leaf thicknessWhen woody perennials are dominant Lth can be equallyinfluential Some species that normally grow in deeply shadedand thus presumably resource-limited micro-habitats (egOxalis) have a high SLA and low Lth In areas with severesoil-nutrient limitations slow-growing plants withsclerophyllous leaves (with thick epidermal walls and cuticleabundant sclerification high ratio of crude fibre to protein) arecommon In these low SLA is associated with high leaf dry-matter content more than with high Lth In contrast in thesucculent plants that are common in some seasonally drysubtropical to tropical areas low SLA is associated with lowleaf dry-matter content and high Lth As a consequence of thesevariations SLA and its components are often but not alwaysrelated to eachother and toproductivity gradients in a simplewayWe then recommend additional measurement of the twocomponent variables namely leaf dry-matter content and Lthas well as SLA


Select the relatively young (presumably morephotosynthetically active) but fully expanded and hardenedleaves from adult plants (unless the research is focussed on eg seedlings or expanding or senescent leaves) Whereverpossible avoid leaves with obvious symptoms of pathogen orherbivore attack or with a substantial cover of epiphylls SLAis strongly affected by light intensity Therefore for manyresearch questions it is best (giving the fairest comparisonacross individuals or species) to sample outer canopy leaves(also called lsquosun leavesrsquo) from plants growing under relativelyoptimal conditions For species that typically grow in theoverstorey take leaves from plant parts most exposed to directsunlight For true shade species (those that never grow in fullsunlight) collect leaves from the least shaded parts found (butnot from those that look light-stressed or bleached) Any rachis(stalk-likemidrib of a compound leaf) and all veins are consideredpart of the leaf for a standardised SLA measurement (butsee Special cases or extras below in the present Section for adiscussion on this and on whether petioles should be includedin the measurement) We recommend collecting whole twigsections with the leaves still attached and not removing theleaves until just before processing

Storing and processing

Plants in the field may be dehydrated to an unknown extentwhichmay lead to shrinkageof the leaves and therefore somewhatunreliable measurements of LA (see Special cases or extrasbelow in the present Section) This is more of a problem forsoft-textured high-SLA leaves than for low-SLA sclerophyllousleaves Ideally the samples (twigswith leaves attached) shouldbecut and immediately placed into test tubes or flasks with the cutend submerged in deionised water If this is not feasible wrap thesamples inmoist paper and put them in sealed plastic bags In that


case breathe into the bag before closing it to enhance CO2

concentration and air humidity which will minimisetranspirational water loss and store the bags in the darkTissues of some xerophytic species (eg bromeliads cactisome species with very small highly resinous leaves) rot veryquickly when moist and warm therefore they are better storeddry in paper bags If in doubt (eg in mildly succulent species)and if recollecting would be difficult try both moist and drystorage simultaneously anduse the dry-stored leaves in the case ofrotting of the moist-stored ones Store the collected samples in acool box or fridge (never in a freezer) in the dark until furtherprocessing in the laboratory If no cool box is available andtemperatures are high it is better to store the samples in plasticbags without any additional moisture Measure as soon aspossible after collecting and rehydrating preferably within24 h If storage is to last for more than 24 h low temperatures(2ndash6C) are essential to avoid rotting

Rehydration is preferable for most plants and essential forleaves subjected to dry storage or for lsquosoftrsquo leaves such as thosewith SLA values higher than 10ndash15m2 kgndash1 In situations wherethe rehydration procedure described above cannot be appliedstorage in sealed moist plastic bags (with or without addition ofdamp paper) for 12 h is an acceptable option although generallyyields approximately ~5 lower values than does the completerehydration method Xerophytic and especially succulent leavesshould not be rehydrated for more than 6 h whatever the storageor rehydration method used might be If this process fails werecommend collecting leaves of these species in themorning aftera rain event or a few hours after generous watering

Measuring

Each leaf (including or excluding petiole see Special casesand extras below in the present Section) is cut from the stem andgently patted dry before measurement Projected area (as in aphotograph) can be measured with specialised leaf-area meterssuch as those fromDelta-TDevices (Cambridge UK) or LI-COR(Lincoln NE USA) Always calibrate the area meter by usingpieces of known area before measuring leaves and always check(eg on the monitor) that the whole leaf is positioned flat andcompletely within the scanning area If you are to use a portableLA meter make sure that the estimation error is not too high foryour purposes by running a preliminary check against LAsscanned in the laboratory using a range of different leaf sizesImages of leaves can also be electronically captured in the field orin the laboratory and stored for later processing egwith a digitalcamera Leaves are pressed gently under a glass plate Includinga ruler or an object of known size in the image allows for sizecalibration A camera mounted on a tripod with two lampslighting from different sides and no flash gives the best resultsA third option is to determine LA with a flatbed scanner (withthe advantage that many scanners can draw their power viaUSB eg from a laptop) Scan in colour mode to obtainmaximal information for the threshold level between what is tobe considered leaf andbackgroundColoured scanswill also allowfor post hocmeasurement of other features of interest In all casesmake sure the leaves are not curled-up or overlapping Try toposition the leaves as flat as possible in the position that gives thelargest area but without squashing them to the extent that the

tissue might get damaged Cutting leaves into smaller piecesmay facilitate flattening In both cases LA can be measured withimage analysis software Freely downloadable programs areeg Leafarea (A P Askew University of Sheffield UKdownloadable from the Nucleo DiverSus toolbox see Box 1)or for more complex analyses including other plant organsImageJ (from the US National Institutes of Health httpwwwnihgov accessed 22 February 2013) and GIMP (fromthe GNU Project httpwwwgnuorg accessed 22 February2013) Transform images to the HSV colourspace for a betterseparation of leaf and background Additional details on imageprocessing can be found at Prometheus Wiki (see Box 1)

After the area measurement put each leaf sample in the oven(petioles and laminae either separately or in the same envelopeaccording to the objective of the study see Special cases or extrasbelow in the present Section) ideally at 70C for at least 72 h or at80C for 48 h (avoid higher temperatures) then determine thedry mass Be aware that once taken from the oven the sampleswill take up some moisture from the air Put them therefore in adesiccator with silica gel until weighing or else back in the ovento re-dry Weighing several tiny leaves as if they were one andthen dividing the weight by the number of leaves will generallyimprove the accuracyof theweighingWhen convertingSLA intoLMA values or vice versa always do so for each individualreplicate rather than for the average of several replicates


(1) Petioles An important issue is whether or not petiolesshould be included in SLAmeasurements The appropriatedecision depends on the research question at hand Someauthors consider that the petiole is an integral part of the leafbecause it is shed at abscission together with the leaf andbecause it provides support and a vascular system withoutwhich the leaf cannot be displayed Therefore they includepetioles in SLAmeasurements Other authors consider thatthe petiole should not be included in the SLA because themain function of the petiole is the spatial positioning andhydraulic support of the leaf thus resembling the functionof the stem whereas the main function of the leaf blade islight interception and C fixation The fraction of leaf drymass represented by the petiole varies from ~zero to almost50 therefore inclusion of the petiole may reduce thecalculated SLAdrastically Although inclusion or not of thepetiole may sometimes not be crucial within a single studyit can be a source of considerable and systematic errorwhencomparing different studies or even in certain same-sitecomparisons of species with very different leaf structuresTherefore the best (albeit more time-consuming) optionis to measure leaf blade and petioles separately so thatSLA can be calculated in both ways thereby facilitatingcomparisonswith other studiesWhen using digital imageswe suggest to scan or photograph petioles and the rest ofthe leaves of each replicate in the same image but in clearlydifferent sectors so that they can be measured togetheror separately according to the objectives of the studythen oven-dry and weigh petioles belonging to areplicate separately from the rest of the leaves from thesame replicate In general make the decision that best suits


your study objectives however specify in your publicationwhether petioles are included or not

(2) Compound leaves Sometimes species have a lower SLAbecause of their thick rachis The decision of whether toinclude the rachis or not and the practical steps in each caseare similar to those on petioles (see Point 1 above in thepresent Section) As a default option we recommendconsidering the rachis as part of the leaf for the SLAcalculation and indicating this clearly when reportingthe study Be aware than in some plants the rachis canbe more than 10 times heavier that the sum of the leafletsAnother decision tomake in the case of compound leaves iswhether to measure the SLA of a typical individual leafletor that of all leaflets taken together As in the case of rachisand petiole the decision largely depends on the objectivesof the study We recommend taking both measurementsand above all we recommend to clearly specify in thepublicationwhether the area reported is that of an individualleaflet or the whole leaf

(3) LA shrinkage Leaves decrease in size when theydesiccate Shrinkage is defined as a percentagecalculated as 100 (1 ndash dry areasaturated area)Shrinkage averages ~20 and can reach 80 for somespecies The maximum area shrinkage reflects differencesin leaf structure and is correlated with other leaf traitsincluding cuticular conductance pressurendashvolumeparameters such as modulus of elasticity and turgor losspoint leaf dry-matter content (LDMC) and Lth as well asplant growth form and deciduousness LA shrinkage isboth a potential problem (causing biases in leaf-areameasurements for herbarium material and fossils) and aneasily measured trait that reflects multiple structural andhydraulic properties of a leaf To measure this trait collectandmeasure leaves following the exact sameprotocol as forLA (Section 32) and LDMC (Section 33) Measure freshprojected LA Cut leaf into pieces in the cases when the leafis not flat This step is crucial because some leaves mayexperience very little shrinkage in area (5) so theaccuracy of the initial measurement of the saturated areais crucial Then oven-dry the leaf pressed flat in envelopesor in a plant press Measure projected LA on the dried leaftaking care that the same surface is measured This may bedifficult or impossible for some needle-like leaves andespecially thick leaves may need to be broken into severalsmaller flat pieces Calculate shrinkage using the formulagiven above

(4) Projected v total LA In lsquostandardrsquo leaves LA ismeasured as a one-sided projected LA However in non-flat leaves the projected LA is smaller than the total one-sided LA Projected LA is generally related to lightinterception whereas total LA is related to the totalamount of photosynthetically active tissue There arehowever cases such as leaf-rolling in some grassspecies where both light interception and gas exchangeare reducedClearly the choice ofmeasurement dependsonthe research question although knowledge of both wouldprovide the best insight

(5) Needle-like leaves Needle-like leaves are a specific casewhere projected and total LAs are different Projected LA

could be measured following the standard routineshowever because the leaves are generally narrow makesure that your equipment is sensitive enough to adequatelymeasure such leaves For a rough measurement you canmeasure leaf length with a ruler and leaf width with acalliper and subsequently multiply 2 lengthwidth

(6) Tiny leaves True leaves from some species (egCallitrissp) have very tiny scales closely appressed to fine softtwigs In such cases youmight treat the terminal twiglets asa leaf analogue because they are shed as a single unit

(7) Leaves of grasses and grass-like plants Usually only thelamina is considered excluding the leaf sheath Howeveras in the case of petioles (see Point 1 above in the presentSection) the decision on which measurement to takedepends on the research objectives Several species haveleaves that tend to curl or even roll up They are generallymuch easier managed by cutting leaves into shorter piecesof 5ndash10 cm

(8) Succulent and leafless plants For plants whose mainphotosynthetic organs are not true leaves take the plantpart that is the functional analogue of a leaf and treat asabove For some species with photosynthetic spines ornon-succulent stems (eg Ulex Senna aphylla) thiscould mean taking the top 2 cm of a young twig Forcacti and other succulents we recommend taking awhole leaf or equivalent (eg a cladode in Opuntia)whenever possible This sometimes poses practicaldifficulties eg a whole Agave leaf or a rib of acolumnar cactus are often too big to process or even tocollect In such cases we recommend taking severallsquopastry cutterrsquo portions (of known area) of young butfully hardened leaves (eg in Agave) or lsquoribsrsquo (in cacti)including epidermis and mesophyll on both sides plus theinternal succulent parenchyma Although this internalparenchyma does not always contain chlorophyll andtherefore some authors recommend not considering it inSLA measurements it has an essential role in the CAMmetabolism of succulent plants (see also Section 312) Theyounger stems of some rushes and sedges (EleocharisJuncus) and the lsquobranchesrsquo of horsetails (Equisetum) orsimilar green leafless shoots can be treated as leaves tooBecause many of these species occur in a range ofenvironments it is important to specify the exact methodused in each case

(9) Ferns For ferns only collect fronds (fern lsquoleavesrsquo)without the spore-producing sori often seen as green orbrown structures of various shapes at the lower side ormargin of the frond

(10) Leaves of tall trees Upper-canopy leaves of sun-exposedtrees should be preferred If these cannot be easily reachedsome workers rely on professional climbers slingshots orguns In not extremely tall trees an alternative could be toconsider exposed leaves halfway the crown length at theouter half of the crown (inner leaves are sometimes older)which could be accessedwith a pruner on an extension pole

(11) Very large leaves Once they have been placed in plasticbags large leavesmay be put in a hard-cover folder to avoidwrinkling and folding If leaves are larger than the windowof the area meter cut the leaf up into smaller parts and


measure the cumulative area of all parts Leaves with verythickveins or rachis can cast a lateral shade on theLAmeterthus overestimating the LA In the case of a thick centralvein remove with a scissor the protruding upper or lowerpart of the vein and scan the leaves without that removedpart but include it in the dry-massmeasurement In the caseof a thick rachis remove the rachis andmeasure its diameterand length halfway and calculate the rachis area as theproduct of the two Then scan the leaves without rachis butinclude the rachis in the dry mass If you want to rely onsubsamples of leavesmake sure that there is not too large ofa variation in SLA over the leaf

(12) Heterophyllous plants In the case of species with two ormore types of leaves with contrasting shape and anatomysuch as eg plants with both rosette and stem leaves collectleaves of both types in proportion to their estimatedcontribution to total LA of the plant so as to obtain arepresentative SLA value of the individual

(13) Low-tech options for the measurement of SLA and LAin the field There are situations in which taking freshleaves to the laboratory for scanning is not feasible orportable scanning devices cannot be transported to orpowered in the field site One solution in these cases isto use a digital camera (seeMeasuring above in the presentSection) Another practical and inexpensive alternative isobtaining leaf fragments of known area (eg with a punch-borer) avoiding thick veins and placing the fragments inan envelope for drying (take several punches per replicatebecause they tend to weigh very little) This method is aquick and accurate way to compare leaf laminae Howeverit overestimates SLA as compared with measurementsof whole leaves especially in the case of large leaveswith thick veins ribs and petioles Therefore SLAmeasurements obtained in this way should not becompared with or combined with those taken on wholeleaves at least not without a specific calibration Anotheralternative is to obtain plastic or paper prints or cut-outs ofthe leaves in the field and measure their area later Thismethodworkswell formedium- to large-sized leaves entireleaves and leaves that are not too narrow (eg xerophyticgrasses)

References on theory significance and large datasets Reichet al (1992 1999) Garnier and Laurent (1994) Poorter andGarnier (1999) Wilson et al (1999) Castro-Diacuteez et al (2000)Niinemets (2001)Westoby et al (2002)Diacuteaz et al (2004) Paulaand Pausas (2006) Wright et al (2007) Poorter et al (2009)Hodgson et al (2011) Blonder et al (2012) Juneau andTarasoff(2012)

More on methods Chen and Black (1992) Garnier et al(2001a 2001b) Vendramini et al (2002) Vile et al (2005)Niinemets et al (2007)

32 Area of a leaf

The area of a leaf (also called leaf area LA) is the most commonmetric for leaf size and is defined as the one-sided or projectedarea of an individual leaf expressed in mm2 (see Section 31)Interspecific variation inLAhas beenvariously related to climaticvariation geology altitude and latitude where heat stress cold

stress drought stress nutrient stress and high-radiation stress alltend to select for relatively small leaves Within climatic zonesvariation in the LAmay also be linked to allometric factors (plantsize twig size anatomy and architecture leaf number number oflateral buds produced) and ecological strategy with respect toenvironmental nutrient stress and disturbances and phylogeneticfactors can also play an important role


For the leaf-collecting protocol see under Section 31 LA israther variablewithin plants andwe recommend collecting a largenumber of replicates (ie close to the higher end of the number ofreplicates recommended in Appendix 1) For storing leaves seeSection 31

Measuring

Measure the individual leaf lamina for species with simpleleaves For compound-leaved species either the leaflet area or thewhole LA can bemeasured and the appropriate decision dependson the research question at hand For the heat balance the leafletarea is importantwhich is functionally analogous to a simple leafWhen analysing total light capture the whole leaf should bemeasured Ideally determine for compound-leaved species boththe leaflet area and whole LA because this allows one to addressmore questions and to compare the results with other studiesMeasure the laminaewith orwithout petiole and rachis accordingto the objectives of your study (seeSection31) and always reportthis in your publication Note that this whole LAmay be differentfrom the area used to determine SLA


(1) Leafless plants Because leaflessness is an importantfunctional trait record LA as zero for leafless species (notas a missing value) However be aware that these zeros mayneed tobe excluded fromcertain data analysesAlternativelysample leaf analogues (see Succulent and leafless plants inSection 31)

(2) Heterophyllous plants See Section 31(3) Ferns See Section 31(4) Leaf width This is measured as the maximum diameter of

an imaginary circle that can be fitted anywhere within a leafand is an additional trait of ecological interest related to leafsize Narrow leaves or divided leaves with narrow lobestend to have a smaller boundary layer and a more effectiveheat loss than do broad leaves with the same area This isconsidered adaptive in warm sun-exposed environmentsThere is also emerging evidence that leaf width contributesmore positively than does the area of the whole leaf to theexpression of canopy dominance

(5) Leaf number per node Leaf size is a compromise betweenfunctional and resource-use efficiency Plants are modularin construction and as a result these functions can bepartially uncoupled Species with alternate opposite andwhorled leaves frequently co-exist and leaf dry mass orarea multiplied by the number of leaves per node providesadditionally a crude estimate of the size of each growth


module This may in extreme cases be 10 times the value of asingle leaf

References on theory significance and large datasetsRaunkiaer (1934) Parkhurst and Loucks (1972) Givnish(1987) Cornelissen (1999) Ackerly et al (2002) Westobyet al (2002) Milla and Reich (2007) Niinemets et al (2007)Niklas et al (2007) Poorter and Rozendaal (2008) Royer et al(2008)

More on methods see references in More on methods ofSection 31

33 Leaf dry-matter content

Leaf dry-matter content (LDMC) is the oven-dry mass (mg) ofa leaf divided by its water-saturated fresh mass (g) expressed inmg gndash1 Using these units LWC is simply 1000-LDMC LDMCis related to the average density (fresh mass per fresh volume)of the leaf tissues In laminar leaves it is related to SLA by aformal relationship involving Lth and the average density ofthe leaf (rF) as follows LDMC=1(rF SLA Lth)Assuming that the fresh mass per volume of leaves is close to1 g cmndash3 the equation simplifies to LDMC 1(SLA Lth)LDMC therefore tends to be inversely related to SLA andLth LDMC has been shown to correlate negatively withpotential RGR and positively with leaf lifespan however thestrengths of these relationships are usually weaker than thoseinvolving SLA Litter derived from leaves with high LDMC alsotends to decompose more slowly than that from leaves withlow LDMC Leaves with high LDMC tend to be relativelytough (see Section 37) and are thus assumed to be moreresistant to physical hazards (eg herbivory wind hail)than are leaves with low LDMC Some aspects of leaf waterrelations and flammability (see Section 212) also depend onLDMCCommonly but not always specieswith lowLDMCtendto be associated with productive often highly disturbedenvironments In cases where SLA is difficult to measure (seeSection 31) LDMCmay givemoremeaningful results althoughthe two traits may not capture the same functions (this isparticularly obvious in some groups eg succulents have slowgrowth low SLA and low LDMC see also Section 31)


Follow exactly the same procedure as for Section 31 In manycases the same leaves will be used for the determination of bothSLA and LDMC As is the case for SLA LDMC may varysubstantially during the day


Similarly as for SLA except that any dry storage should beavoided (however see the case of xerophytic species in Section31) and that full rehydration beforemeasurement is compulsory

Measuring

Following the rehydration procedure the leaves are cut fromthe stem and gently blotted dry with tissue paper to remove anysurface water before measuring water-saturated fresh mass Eachleaf sample is then dried in an oven (see Section 31) and its drymass subsequently determined


Most comments for SLA also apply to LDMCReferences on theory significance and large datasets Eliaacuteš

(1985) Witkowski and Lamont (1991) Garnier and Laurent(1994) Hodgson et al (1999 2011) Wilson et al (1999)Garnier et al (2001a) Niinemets (2001) Vile et al (2005)Kazakou et al (2009) Poorter et al (2009)

More onmethodsWilson et al (1999) Garnier et al (2001b)Vendramini et al (2002) Vaieretti et al (2007) Ryser et al(2008)

34 Leaf thickness

Leaf thickness (Lth mm or mm) is one of the key componentsof SLA (see Sections 31 and 33) because SLA 1(tissuedensityLth) (where density = dry massvolume LDMC seeSection 33) Lth also plays a key role in determining the physicalstrength of leaves (see Section 37) For example leaf lsquowork toshearrsquo is (by definition) the product of Lth and tissue toughnessOptimisation theory balancing photosynthetic benefits against Ccosts of respiration and transpiration predicts that Lth should behigher in sunnier drier and less fertile habitats as well as inlonger-lived leaves These patterns are indeed often observed atleast in interspecific studies Within individuals many studieshave shown that outer-canopy lsquosunrsquo leaves tend to be thicker thanthose from more-shaded parts of the canopy Both within andamong species the strongest anatomical driver of variation in Lthis the number and thickness of mesophyll layers ConsequentlyLth is a strong driver of leafNper areaAlthough higherLth shouldlead to faster photosynthetic rates per unit LA (via a higherN area ratio) this relationship is often weak in interspecificstudies for a combination of reasons First because ofcovariance of SLA and N thicker leaves often have lower N and longer leaf-lifespan (which are associated with lowerphotosynthetic rate per unit leaf mass) Second thicker-leavedspecies may have slower CO2 diffusion (lower mesophyllconductance) via longer diffusion pathways greater internalself-shading of chloroplasts or higher optical reflectivity incombination with lower internal transmittance Thick leavesare also a feature of succulents


Followsimilar procedures as forSection31 Inmanycases thesame leaves will be used for the determination of SLA Lth andLDMC(andperhaps Section 37) For recommended sample sizesee Appendix 1


Similarly as for SLA Lth is strongly affected by LWC hencesome form of rehydration should be seriously considered asdescribed for SLA particularly if using a digital micrometerwhere any slight loss of turgor results in an underestimation

Measuring

Thickness tends to vary over the surface of the leaf generallybeing thickest at themidrib primary veinsmargins and leaf baseDepending on the research question youmay be interested in the


average thickness across the leaf or the thickness at speciallocations or of special tissues Often one measurement perleaf at a position as standard as possible within the lamina(eg at an intermediate position between the border and themidrib and between the tip and the base of the leaf avoidingimportant secondary veins) is acceptable for broad interspecificcomparisons When more precision is needed the average ofthickness measurements at several points in the lamina willbe more appropriate Another way to estimate the averagethickness over the entire leaf surface is to back-calculate itfrom the leaf volume divided by LA however it is laboriousto accurately measure leaf volume eg with a pycnometerA relatively fast approximation of whole-leaf average Lth canbe obtained by dividing leaf fresh mass by LA (which is the sameas calculating 1SLALDMC) ie by assuming that leaf freshmass and volume are tightly related This approach does not takeinto account the higher density of dry material in the leaf or thelower density as a result of intercellular spaces however as anapproximation it works well

Other approaches are needed if one wants to distinguishbetween thickness of midrib margin and intercostal regions ofthe leaf or to compare replicates at a given point on the leaf eghalf-way between the leaf base and the tip as is commonlydone One method is to measure these quantities directly fromleaf cross-sections (hand-sections) or to use image analysis(see Section 31 for free software) to calculate average Lthacross the section by dividing the total cross-sectional area bythe section width On the positive side this method enablesreasonably accurate measurements to be made On the downside soft tissuemay distort when hand-sectioned and themethodis relatively slow (eg 15min per measurement)

Probably the fastest approach is to measure Lth using a dial-gauge or a digital micrometer (or even a linear variabledisplacement transducer LVDT) Multiple measurements canbe made within quick succession and averaged to give anindicative value of Lth for the feature in question (such as egmidrib or lamina between the main veins) or region of interest(eg near midpoint of leaf) If necessary we recommendreplacing the original contact points on the micrometer withcontacts 2ndash3mm in diameter ie narrow enough to fitbetween major veins but sufficiently broad so as not to dentthe leaf surface when making measurements However for soft-leaved species such as Arabidopsis permanent deformation isdifficult to avoid


(i) Needle leaves For needle leaves that are circular in cross-section average Lth can be quickly estimated asDiameter p4 (equivalent to cross-sectional areadivided by cross-section width) Still because needleleaves typically taper towards the leaf tip severalmeasurements would normally need to be made

References on theory significance and large datasetsClements (1905) Givnish (1979) Parkhurst (1994) Enriacutequezet al (1996) Knapp and Carter (1998) Smith et al (1998)Wilson et al (1999)Green andKruger (2001)Niinemets (2001)Diacuteaz et al (2004)

MoreonmethodsWitkowski andLamont (1991)Garnier andLaurent (1994) Shipley (1995) Wright and Westoby (2002)Vile et al (2005) Poorter et al (2009) Hodgson et al (2011)

35 pH of green leaves or leaf litter

The pH of green leaf tissue and of senesced leaves (leaf litter)measured by grinding up the tissue and extracting it with distilledwater varies substantially among species The variation is at leastpartly intrinsic (presumably genetic) because this pH can differgreatly amongdifferent species growing in the same soil (and alsoduring the day in CAM plants see below under Special cases orextras in the present Section) and is robust to differences in soilchemistry (including pH) Ground leaf-tissue pH integrates theeffects of many compounds and processes in the leaf that affectits exchange capacity for H+ ions However some substancesare particularly strong determinants of leaf-tissue pH Highconcentrations of metal cations (calcium magnesiumpotassium) will give high pH whereas high concentrations oforganic acids and of C-rich secondary metabolites (chemical-defence compounds) such as tannins will tend to give a lowerpH The latter may explain why leaf-tissue pH tends to becorrelated (negatively) with important biogeochemical traitssuch as C concentration or C N ratio and (positively) withSLA (Section 31) Green leaf-tissue pH correlates positivelywith digestibility making it an important predictor of palatabilityto herbivores which may actually lsquotastersquo acidity Differences inleaf-tissue pH among species tend to persist during senescencemaking leaf-litter pHaworthwhile trait also It canbe a reasonableproxy for litter decomposability because substrate pH can beimportant to decomposers Shifts in species composition amongspecies that differ in leaf-litter pH can drive changes in the pH ofthe litter layer and of the soilrsquos organic horizon For exampleplanting pines on high-pH soils dominated bybacteria can via theleaf litter lead to more acidic soil organic matter within decadesand a decomposer community dominated by fungi Whethergreen leaves or senesced leaves or both should be measureddepends on the question to be addressed however green leavesshould be used if in doubt and if decomposition is not the mainissue

Physiological caveat

The cells of any plant tissue consist of three or moremembrane-delimited compartments that can and usually dohave different internal pH values Therefore the pH of thetype of crude water-extracted homogenate or ground materialused for this method although it can be useful as an ecologicaltrait must not be mistaken for the actual pH of the leaf as awhole or any part thereof It will at best be a weighted averageof the pH values of the various compartments of the leafpossibly modified by reactions that occur between componentsof different compartments including vacuoles when they aremixed together If leaves are oven-dried before grinding loss ofcompartmentalisation under conditions favouring pH-modifyingreactionswill have occurred longbefore the tissue is ground Stillthe pH measured this way has been shown to also represent thepH of green leaves Because in leaf litter metabolic processeshave already stopped unlike in living leaves litter pH values


should reflect the actual situation in the litter at the time ofsampling


For green leaves see Section 31 for the collecting and storingprocedure before processing If leaves are small make sureenough of them are collected so as to get enough material forthe analysis Initial leaf rehydration is not necessary

Processing and storing

As a default option any petiole or rachis should be removedbefore pH analysis however see Special cases or extras inSection 31 Fresh green leaves can simply be ground orchopped as noted below Leaves collected for leaf SLAanalysis can instead be used after being oven-dried to obtaintheir biomass (see Section 31) and subsequently stored air-drybecause pH values obtained from oven-dried leaves are largelycomparable to those from fresh leaves Air-dried leaves or leaflitter are ground as for leaf N analysis (see Section 36) and storedair-dry until the analysis

Measuring

Add distilled or de-mineralised water to the ground leafsample to give an 8 1 volume ratio of water to leaf sampleShake the samples in a laboratory rotary shaker for 1 h thencentrifuge until there is a clear separation of the sediment and thesupernatant The supernatant can then be measured for pH byusing any of a wide range of laboratory pH meters as long ascalibration is adequate (using buffer solutions of pH 4 and pH 7)If samples are small in volume we recommend adding 12mL ofwater to 015mL of ground leaf material in a 25-mL Eppendorftube then following the above procedure A thin SenTix 41electrode connected to an Inolab level 2 pH meter (both WTWWeilheim Germany) fits nicely inside such Eppendorf tubes


(1) Additional measurements on fresh leaves Althoughmeasurements on dried ground leaves tend to match thoseon fresh leaves fairly well for various specific purposes itmay be of interest to (additionally) measure fresh leaves thecell contents of which have remained intact until shortlybefore measurement For instance this may be useful tofollow diurnal changes in leaf pH to indicate possible CAM(see Section 312) Related to this in comparisons of CAMplants with other plants for leaf pH leaves of CAMplants arebest collected in the afternoon ie well after nocturnal acidaccumulation Grinding fresh leaves does not always workwell because solidsmay stick to the surfacesor to the balls in aball mill making cleaning between samples laboriousInstead we recommend chopping the leaf sample into~1-mm-diameter pieces with a razor blade or an automaticchopper that gives comparable fragmentation This should becarried out immediately before shaking and centrifuging

References on theory significance and large datasets Zinke(1962) Marschner (2012) Finzi et al (1998) Cornelissen et al(2006 2011) Freschet et al (2010)

More on methods Cornelissen et al (2006 2011)

36 Leaf nitrogen (N) concentration and leaf phosphorous (P)concentration

Leaf N concentration (LNC) and leaf P concentration (LPC) arethe total amounts of N and P respectively per unit of dry leafmass expressed in mg gndash1 (or sometimes as dry-leaf mass)Interspecific rankings of LNC and LPC are often correlatedAcross species LNC tends to be closely correlated with mass-basedmaximum photosynthetic rate andwith SLA High LNC orLPC are generally associated with high nutritional quality to theconsumers in food webs However LNC and LPC of a givenspecies tend to vary significantly with the N and P availability intheir environments The LNC LPC (N P) ratio is sometimesused as a tool to assess whether the availability of N or P is morelimiting for plant growth Actively N-fixing species eg manylegumes tend to have higher LNC LPC ratios than other plantsgrowing at the same site


See Section 31 for the leaf-collecting procedures Initialrehydration is not necessary Petiole or rachis are often cut offbefore LNC and LPC analysis but are included in some othercases See under Section 31 for discussion of when and whetherto consider them Oven dry at 6070C for 72 h Leaves used forLA or SLA analysis can be also used to measure LNC and LPCprovided drying temperature has not been higher than 70C Forreplication see under Section 31 butmake sure that enough totalleaf material per replicate is collected according to the analyticalmethod and equipment to be applied (~2 g drymatter per replicatefor N and 5 g for P in the case of acid digestion 02 g for N in thecase of combustion techniques see below under the presentSection)


After oven-drying the leaves store the material air-dry and inthe dark until use to a maximum of 1 year Grind each replicateseparately Manual grinding with mortar and pestle is an optionfor small numbers of samples but is not recommended for largeones (repetitive strain injury) Effective inexpensive mechanicalgrinders are available Samples may also be ground by shakingthem with steel balls in individual plastic vials on a roller millwhich is an efficient way to grind many samples at once Avoidinter-sample contamination by cleaning the grinder or steel ballscarefully between samples Use a ball mill for small samples Drythe ground samples again for at least 12 h before analysis

Measuring

Several techniques are available to measure LNC and LPCin ground plant material Macro- or micro-Kjeldahl (acidic)digestion followed by colorimetric (flow-injection) analysis(using different reagents for N and P) has been widely usedWet acidic digestion followed by formation of bluephosphomolybdenum complex from orthophosphate is a moreprecise method for measuring total P Alternatively you canmeasure P by inductively coupled plasmandashoptical emissionspectroscopy (ICPndashOES) Kjeldahl digestion for N analysis isincreasingly being replaced by methods that employ acombination of combustion analysis converting organicmatter into N2 and CO2 followed by mass spectrometry or gas


chromatography These combustion techniques provideconcentrations of both N and C in the leaf and if carried outwith automated N analysers are generally less labour- andchemical-intensive than are Kjeldahl analyses Combustiontechniques also generally recover more N than do Kjeldahlanalyses because some N fractions (eg NO2

ndash NO3ndash and

some cyclic N compounds) do not react in Kjeldahl analysisHowever we believe that all of these standard methods shouldgive reasonably accurate LNC and LPCWe recommend runninga standard reference material with known LNC and LPC alongwith the samples


(1) Leafless and heterophyllous plants See Section 31

References on theory significance and large datasets Chapin(1980) Field and Mooney (1986) Lambers and Poorter (1992)Aerts (1996)Cornelissen et al (1997)Grime et al (1997)Reichet al (1997 2010)Aerts andChapin (1999)Wright et al (2004)

MoreonmethodsAllen (1989)Anderson and Ingram (1993)Horneck and Miller (1998) Temminghoff and Houba (2004)

37 Physical strength of leaves

Physically stronger leaves are better protected against abiotic(eg wind hail) and biotic (eg herbivory trampling)mechanicaldamage contributing to longer leaf lifespans Physicalinvestment in leaf strength is a good indicator of C investmentin structural protection of the photosynthetic tissues It also tendsto have afterlife effects in the form of poor litter quality fordecomposition Because leaves have different strength propertiesaccording to the direction in which the force is applied thephysical strength of the leaves can be defined and measured indifferent ways The three most common measured properties areforce to tear (Ft) work to shear (Ws) and force to punch (Fp) Ft isthe force needed to tear a leaf or leaf fragment divided by itswidth expressed in Nmmndash1 Note that Ft has been previouslyreferred to as lsquoleaf tensile strengthrsquoWs sometimes called lsquoforceof fracturersquo reflects the mean force needed to cut a leaf or leaffragment at a constant angle and speed expressed in N or itsanalogue Jmndash1 Fp is the force needed to force a punch through aleaf or leaf fragment (expressed in Nmmndash1) BothWs and Fp arestrongly influenced by Lth (see Section 34)


For the selection and collecting procedure see Section 31 forrecommended sample size see Appendix 1


Follow the procedure described within Section 31 and storeleaves in a cool box or fridge Rehydration is not indispensablealthough it may be desirable for accurate measurement of LthMeasure as soon as possible after collecting certainlywithin 72 hfor species with soft leaves Tougher leaves tend to keep theirstrength for several days If this is not possible (eg if sampleshave to be sent to distant locations) an alternative is to air-dry thesamples by putting them between sheets of absorbing paper in aplant press immediately after collecting In this case rehydrationis needed before measuring Toughness of fresh and rehydrated

leaves is well correlated for sclerophyllous leaves and grassleaves in the cases of both Ft and Ws

Measuring

For fresh samples proceed tomeasurement straight away Forair-dried samples first rehydrate by wrapping in moist paper andput in a sealed plastic bag in the fridge for 24 h (gentle sprayingmay be better for some xerophytic rotting-sensitive species seeSection 31) Here we describe threemethods that have producedgood results and for which purpose-built equipment is availableIf you have the choice we recommend measuring the propertythat is most closely related to the process of interest In the case ofherbivory by vertebrate grazers Ft is likely to be the mostmeaningful property However if the focus is on chewinginsects or trampling by mammals work to shear tests wouldbe the best approach (Table 2)

(1) Tearing (tensile) tests

Force to tear (Ft) can be easily and inexpensively measuredwith a simple apparatus that includes a 0ndash3-kg-rangedynamometer (Fig 3a) To proceed cut a leaf fragment fromthe central section of the leaf but away from the midrib (centralvein) unless the latter is not obvious (eg some Poaceae orLiliaceae) or the leaf is too small for doing so without using amagnifying lens The length of the fragment follows thelongitudinal axis (direction of main veins) The width of theleaf or leaf fragment depends on the tensile strength and tends tovary between 1mm (extremely tough species) and 10mm (verytender species) Whenever possible we recommend usingfragments with a length width ratio between 5 and 10 tomake sure that force is applied along its main axis Measurethe exact width of the leaf sample before placing it in theapparatus Place it perpendicular to the edges of the clampsThen fix both ends of the sample with the clamps Try to do thisgentlywithout damaging the tissues A thin piece of rubber addedto the edges of the clamps could help Very small leaves verytough and slippery leaves and slightly succulent leaves may beclamped tightly without much tissue damage by using strongdouble-sided tape Then pull slowly with increasing force untilthe leaf tears Watch the dynamometer to read the force at themoment of tearing For unit conversion remember that1 kg = 981N Divide the total force by the width of the leaffragment to obtain Ft Express the result in Nmmndash1 There aresome more sophisticated instruments to measure Ft such asthe 5542 (Instron Canton MA USA) or (with adaptations)Mecmesin Ultra Test Tensiometer (Mecmesin Slinfold UK)

Leaves too tender to provide an actual measurement withthe apparatus are assigned an arbitrary tensile strength of zeroFor leaves too tough to be torn first try a narrower sample(down to 1mm if necessary and possible) If still too toughthen tensile strength equals the maximum possible value inapparatus (assuming sample width of 1mm) In the case ofhighly succulent leaves (or modified stems) which would besquashed if clamped into the apparatus carry out themeasurements on epidermis fragments

(2) Shearing (cutting) tests

At least five instruments have been used to measure work toshear (Ws) They all measure how much work is required to cut a


Table 2 Types of tests commonly used for measuring leaf mechanical properties

Parameter Tearing test Shearing test Punch test

Property measured Leaf resistance to a tearing force Ft Leaf resistance to a shearing forceWs Leaf resistance to a punching force Fp

Unit Nmmndash1 Jmndash1 Nmmndash1

Apparatus Tearing apparatus (Hendry and Grime1993) universal testing machine4202 Instron High WycombeBucks UK (Wright and Illius 1995)

Leaf-cutter machine (Wright andCannon 2001) scissoring machine(Darvell et al 1996) universaltesting machine 4202 InstronHigh Wycombe Bucks UK(Wright and Illius 1995)

Universal testing machine 5542Instron Canton MA USA (Onodaet al 2008) Shimadzu DCS-5000(Lucas et al 1991)

Study objective Herbivory by mammalian grazers andother tearing herbivores (egsnails) decompositionidentification of plant resource-usestrategies

Herbivory by chewing insects andsmall vertebrates herbivore impactson vegetation (eg trampling)decomposition identification ofplant resource-use strategies

Herbivory by chewing or suckinginsects decompositionidentification of plant resource-usestrategies

(a) (b)

(c) (d )

Fig 3 Different apparati formeasuring physical strength of leavesDiagrammatic representation of the devices used for (a) tearing tests as described inHendryand Grime (1993) (b) scissoring test (prototype model from Darvell et al 1996 a very similar device is described in Wright and Illius 1995) (c) shearing testsreproduced fromWright andCannon (2001) (safety guard quick-release sample holder and electronic control unit not shown) and (d) punch tests fromAranwelaet al (1999)


leaf either with a single blade against an anvil or with a pair ofblades (lsquoinstrumented scissorsrsquo) A portable and widely useddevice for measuring the average force needed to fracture aleaf at a constant shearing angle (20) and speed is detailed inFig 3b Anon-portable apparatus for thismeasurement is showedin Fig 3c In both cases the apparatus consists of a mechanicalportion a power source and a computer in which the outputfile is recorded To proceed be sure to complete the calibrationprocedure according to the apparatus you are using Consideringthat this methodology is highly sensitive the operator must avoidany possible source of external noise (eg vibrations wind) andmust frequently clean the blade(s) with an alcohol-soaked wipeMeasure the exact thickness of the leaf sample (lamina or midrib)with a calliper before placing it in the apparatus Then placethe sample in the anvil and fix it with the clamp Leaves are cutat right angles to the midrib at the widest point along the lamina(or halfway between the base and the tip if this is difficult todetermine) In some studies the midrib may be removed so thatonly lamina tissue is tested (or lamina and midrib are testedseparately)Alternatively (and less precisely) one can remove theportion of the data that represents the midrib being cut andanalyse these data separately The procedure to calculate finalvalues can also differ between apparati (for calculation processrefer to the specific userrsquos manuals)

A calibrated copy of the apparatus described in Fig 3c isavailable for use at CNRS in Montpellier France (contact EricGarnier email garniercefecnrs-mopfr)

(3) Punch tests

Force to punch (Fp) is the resistance of the actual leaf tissues(particularly the epidermis) to rupture excluding toughnessprovided by midribs and main veins Different penetrometers(Fig 3d) have been used in the past (there is no standard design)all of which have some kind of fine-needle or a flat-ended punch(diameter ~05ndash55mm) attached to a spring-loaded balance or acounterweight (being a container gradually filled with water andweighed after penetration) The punch goes through a die with ahole in its centre A clearance of 005ndash01mm between the punchand the edgeof the hole in thedie is recommended to avoid error inmeasurements as a result of friction between the punch and thedie A sharp edge in the flat end of the punch will also avoidoverestimating toughness values resulting from compression andtension rather than shearing To standardise the force per unitfraction length one has to divide the force by the circumferenceof the punch The data are therefore expressed in Nmmndash1Consistency across the leaf tends to be reasonable as long asbig veins are avoided Three measurements per leaf are probablysufficient This test does not work well for many grasses andother monocots This method has been more widely used in thetropics Some recent studies have added the punch and die tomore sophisticated apparati designed for measuring properties ofmaterials (such as 5542 Instron)


(1) Leafless plants See Section 31(2) Leaf-tissue toughness or leaf-specific toughness This

interesting additional parameter of leaf strength can be

obtained by dividing Ft Ws or Fp by the (average)thickness of the leaf sample Measurement of Lth (inmm)for this purpose is most practically carried out with a calliperimmediately after measuring the leaf-sample width andbefore placing it in the measuring apparatus

References on theory significance and large datasets Coley(1988) Choong et al (1992) Grubb (1992) Turner (1994)Wright and Vincent (1996) Cornelissen et al (1999) Lucaset al (2000)Diacuteaz et al (2004) Read andStokes (2006)Kitajimaand Poorter (2010) Onoda et al (2011)

More on methods Hendry and Grime (1993) Darvell et al(1996) Aranwela et al (1999) Wright and Cannon (2001)

38 Leaf lifespan and duration of green foliage

Leaf lifespan (longevity) is defined as the time period duringwhich an individual leaf (or leaf analogue) or part of a leaf(see Monocotyledons below in the present Section) is aliveand physiologically active It is expressed in days monthsor years Leaf lifespan relates to the nutrient-use strategy ofa plant thus providing an indirect index of important planttraits such as potential growth rate nutrient-use efficiencyand decomposability of litter Long leaf lifespan is oftenconsidered a mechanism to conserve nutrients andor reducerespiratory costs in habitats with environmental stress or lowresource supply Species with longer-lived leaves tend to investsignificant resources in leaf protection and (partly as aconsequence) grow more slowly than do species with short-lived leaves they also conserve internal nutrients longer Thelitter of (previously) long-lived leaves tends to be relativelyresistant to decomposition

Leaf lifespan does not necessarily indicate the phenologyof growth or the proportion of the year when a plant is able toperform photosynthesis This is because leaves may senesce alltogether (as in deciduous species) or their senescence maybe spread out over a much longer period in which case a plantmay maintain several cohorts of leaves of different agessimultaneously as is the case in many temperate evergreenspecies In addition some tropical evergreen species maintaina continuous green leaf canopy with a very short leaf lifespanbecause they have a high and continuous rate of leaf productionThus to understand the ability of a plant to exploit its seasonallight environment and the timing of its growth it is also usefulto measure the duration of green leaves defined by the numberof months per year that the leaf canopy (or analogous mainphotosynthetic unit) is green This measure constitutes oneimportant component of the timing of plant growth orphenology Certain groups of competition avoiders (includingsome gap colonisers) may have very short periods of foliardisplay outside the main foliage peak of the more competitivespecies some spring geophytes manage important growth at thebeginning of the favourable season before the canopy of theseasonally green species closes in contrast many evergreenspecies have a year-round ability to photosynthesise Detailedinformation on monitoring all the aspects of plant phenology canbe obtained from several networks set up for this purposeincluding the NPN (httpwwwusanpnorg accessed 22February 2013) and Project Budburst (httpwwwneonincorgbudburstindexphp accessed 22 February 2013)


Measuring leaf lifespan

Different methods are required for taxa with different kinds ofphenological patterns and leaf demographic patterns In all caseswhere a general measure is desired select individuals and leavesby using the same criteria as in Section 31

(A) Dicotyledons Method 1 (below) is the best although it ismost labour-intensive and takes a longer time periodMethods 2ndash4 can replace Method 1 but only if thecriteria are met(1) Periodic census of tagged ormapped leaves Tracking

the birth and death of individual leaves over time atrepeated census intervals is the best although mostlabour-intensive method Tag or map individualleaves (not leafy cotyledons) when they appear for thefirst time at a census event and record periodically (atintervals of roughly 110 of the lsquoguesstimatedrsquo lifespan)whether they are alive or dead Because the censusinterval length increases relative to the mean lifespanthe accuracy of any individual measure will decreasehowever with a large sample size the estimate of themean should remain accurate The leaf-identificationinformation needs to be recorded on the leaf itself (ornearby on the branch although in an unambiguouslysystematic way) and often is done with a brief code(such as colour andor symbols) Alternatively a branchand leaf drawing ormap can bemade inwhich differentcolours are used at each census event In thismethod theposition along the branch that separates leaves producedin consecutive census intervals must be marked Leavesproduced in given census intervals are drawn in such away that their relative spatial positions are clear At thefirst census when a leaf is no longer present (or is visiblymostly or completely dead) it is crossed out on thedrawing with the colour of the present census event Foreach individual plant select two or more branches orshoots and sample all leaves on them Note that a total ofat least 40 and ideally 160 leaves per species is necessary(see Appendix 1) To achieve this we recommendincreasing the number of individuals rather than thenumber of branches or shoots per individual Calculatethe lifespan for each individual leaf and take the averageper individual plant In addition to providing the mostaccurate estimate of the mean and median leaf lifespanthis method also provides a frequency distribution andenables estimates of the variance which most othermethods do not provide

(2) Count leaves produced and died over a timeinterval Count (for each shoot or branch) the totalnumber of leaves produced and died over a time intervalthat represents a period of apparent equilibrium for leafproduction and mortality (see below within the presentSection) We recommend about eight counts over thistime interval but a higher frequency may be better insome cases Then estimate mean leaf lifespan as themean distance in time between the accumulated leaf-production number and the accumulated leaf-mortalitynumber (facilitated by plotting leaf production and leafdeath against time) This is a goodmethod if the census is

long enough to cover seasonal periodicity (so typically itneeds to be several months up to a year if seasonalperiodicity occurs) and the branch or shoot is in quasi-equilibrium in terms of leaf production and mortalityThis period can be much shorter for fast-growing plantssuch as tropical rain forest pioneers woody pioneers intemperate zone or many herbs This technique is usefulfor plants in their exponential growth phase and forplantswith very long leaf lifespan (because one gets datamore quickly) Number of individuals and leaves are thesame as in Method 1 above

(3) Observe a cohort of leaves until one-half havedied This method measures the median leaflifespan Count the number of leaves that appearbetween two census events Periodically revisit andcount the number of leaves remaining This methodis effective when many leaves are produced within ashort time period Care must be taken to make thesemeasures for multiple consecutive cohorts if seasonalor interannual variation is likely to cause shifts in themedian lifespan of different cohorts Number ofindividuals and leaves are the same as in Method 1above

(4) Counting lsquocohortsrsquo for many conifers and only somewoody angiosperms For woody angiosperms it isimportant to be very familiar with the species Thismethod is very easy and quick but can be used onlyif the species is known to produce foliage at regularknown intervals (most frequently once per year) andeach successive cohort can be identified either bydifferences in foliage properties or by scars or othermarks on the shoot or branch In that case it is simple tocount branch by branch the number of cohorts withmore than 50 of the original foliage remaining untilone gets to the cohort with less than 50 of the originalfoliage remaining and use that as the estimate of meanleaf lifespan Thisworks if there is little leafmortality foryounger cohorts and most mortality occurs in the yearof peak lsquoturnoverrsquo Many conifers especially Pinus andPicea show this pattern although some Pinus speciesmay flush more than once per year This method gives aslight overestimate because there is some mortality inyounger cohorts and usually no or very few survivorsin the cohorts older than this lsquopeak turnoverrsquo one Thismethod can also work (1) if there is some mortality inyounger cohorts and a roughly equal proportion ofsurvivors in cohorts older than the first cohort withgt50 mortality or (2) if one estimates mortalitycohort by cohort This can be tricky For instancesome conifers may appear to be missing needles(judging from scars) that were never there in the firstplace because of reproductive structures Be awarethat in Mediterranean-type climates some speciesexperience two growing seasons each year Count oneshoot (preferably the leader or a dominant shoot in theupper canopy) from at least 10 individual plants

(5) Duration of green foliage for species that produce mostof leaves in a single lsquocohortrsquo within a small time periodand shed them all within a short time period (see


Measuring duration of green foliage below in thepresent Section) This method is likely to be the leastreliable of the five described herein

(B) Monocotyledons For some monocots species thelongevity of entire blades can be measured as describedabove However given the growth habits of manymonocots(see belowwithin the present Protocol) thismay not providean estimate of green-tissue longevity that is comparableto the measures above for dicots In some grasses andrelated taxa the blade continues to grow new tissue whileold tissue becomes senescent over time making the mean ofthe whole blade lifespan much longer than the lifespan of aparticular section of the blade and therefore not particularlymeaningful as a measure of tissue longevity In such casesthe production and mortality of specific zones of the bladecan be assessed to estimate the tissue longevity with anadaptation of Method 2 above

Measuring duration of green foliage

Observe the foliage of 5ndash10 individuals of a given speciesseveral times throughout the yearWe recommend a census for allspecies in the survey at least once a month during the favourableseason (preferably including a census shortly before and shortlyafter the favourable season) and if possible oneduring themiddleof the unfavourable season The months in which the plants areestimated to have at least 20 of their potential peak-seasonfoliage area are interpreted as lsquogreenrsquomonths This census can becombined with assessment of Leaf lifespan Most species withindividual leaf lifespansgt1yearwill begreen throughout theyearNote that in some evergreen species from the aseasonal tropicsindividual leaf lifespans can be as short as a few months


(1) Leafless plants If photosynthetic tissues do not die and falloff as separate units follow Method 2 (above) for specificzones of the photosynthetic tissues as specified above formonocotyledons

References on theory significance and large datasets Chabotand Hicks (1982) Coley (1988) Reich et al (1992 1997 2004)Aerts (1995) Westoby et al (2000) Wright et al (2002 2004)Poorter and Bongers (2006)

More on methods Jow et al (1980) Diemer (1998) Craineet al (1999) Wright et al (2002) Reich et al (2004)

39 Vein density

Vein networks constrain the transport of water C and nutrientswithin the leaf Vein density the length of minor veins per unitLA (mmmmndash2) can characterise the structure of these networksVein density is a structural determinant of hydraulic conductanceand photosynthetic rateDepending on the species set consideredthe vein density may correlate with other leaf traits such as Lthstomatal density and maximum gas-exchange rates This traitshows plasticity across environments and is highly variableamong species showing both broad phylogenetic trends andpotential adaptation to resource gradients

Fresh or dried leaves may be used For the leaf collectingprotocol see Section 31 Sampling intensity should account for

known trait variation between sun and shade leaves and across thelamina of given leaves If possiblemeasurements should bemadeon sections of lamina containing no large veins For smallerleaves use the entire leaf for larger leaves a 1-cm2 section issufficient

Measuring

Soak the leaf in 5 wv NaOHndashH2O for 24ndash72 h until itbecomes transparent If theNaOHsolution turns anopaquebrowncolour during soaking replace the soaking solution Then rinsethe leaf in H2O and transfer to a 2wvNaOClndashH2O solution for5ndash30min until it becomes bleached

Rinse the leaf in H2O and transfer via dehydration series topure ethanol (eg 30 50 70 100 ethanol or 50 100ethanol each step lasting 30 s for tender leaves and 5min fortougher leaves) Stain the leaf for 15min in 1wv safranin O inethanol andor other lignin stains Destain in ethanol The leaf canbe mounted in water or glycerol on plastic transparency film orpermanently after transfer to 100 toluene in immersion oilor Permount (allow several days after mounting for toluene tofully evaporate) Veins will appear red Take particular careto ensure that all veins in the network are visible The mostnumerous and functionally important minor veins can be veryhard to see and often require the epidermis to be removed foraccurate visualisation Vein counts should be made usingmicroscope objectives of 4 for ferns and up to 40 fortropical angiosperms Photograph the venation network undera light microscope ensuring a large enough field of view (eg1ndash10mm2) Then measure the total length of veins in the imageand divide this number by the image area to obtain the veindensity using image analysis software (see Section 31) Thisimage analysis process may be semi-automated with freelyavailable software (see More on methods below in the presentSection) but the accuracy should be tested


(1) Leaf handling Leaves become delicate during processingand should bemoved carefully between solutions or solutionshould be vacuumed out of the dish so that leaves will notpuncture or tear For best results in clearing leaves of givenspecies the details of the protocol can be modified WarmNaOH may be used (although not boiling) and longerclearing times or more concentrated NaOClndashH2O solutionfor a shorter time period Small or thin leavesmay require lesssoaking time in all solutions Conversely very thick or denseleaves may require several days in the NaOH solution beforethey become transparent Hairy leaves may require removalof epidermis

(2) Sclereids In some species leaves may have sclereids thatcan be mistaken for veins and also have an importanthydraulic function Additional venation traits can bemeasured for more detailed investigations of leaf structureand function (see references below in the present Section)

References on theory and significance Uhl and Mosbrugger(1999)Roth-Nebelsick et al (2001)SackandFrole (2006)Sackand Holbrook (2006) Brodribb et al (2007 2010) Boyce et al(2009)


More on methods Dilcher (1974) Gardner (1975) Brodribband Feild (2010) Price et al (2011)

310 Light-saturated photosynthetic rate

The light-saturated photosynthetic rate (Amax) under typicalfield conditions usually expressed in mmolmndash2 sndash1 ornmol gndash1 sndash1 or both is a valuable metric as a measure (or atleast as an index) of metabolic capacity and a factor determiningaverage realised photosynthetic rate (for upper-canopy foliage)Amax scales with other structural chemical and longevityaspects of the leaf economic spectrum and along with thoseother variables enables scaling to canopy processes ofwhole ecosystems Simultaneous measures of leaf water-vapour conductance are typically made in concert with thephotosynthetic measurements

What when and how to measure

Sample young fully expanded leaves (see Section 31) Theseshould be from sunlit parts of the canopy unless specificallyfocusing on the shaded taxa of understorey Measure leavesonly if they have been in sufficiently high light just beforemeasurement (eg direct sun for 5ndash10min) to minimiseconcerns about leaf induction status or stomatal closure as aresult of shading (see Section 31 for discussion)

Because realised photosynthesis is less than maximal becauseof a host of factors including low or high temperatures limitedsoil moisture or air humidity negative leaf water potentialand sourcendashsink inhibition among others care must be takenin choosing the time of year time of day and general conditionsunder which measurements can be made Some knowledgeof gas-exchange responses of the taxa under study will beessential Do not make measurements during or just following(days to weeks) periods of severe water deficit or unusualtemperatures Do make measurements on days when soilmoisture plant water status air humidity irradiance andtemperatures are near optimal for the taxa in questionMeasurements in most ecosystems should be made at mid- tolate morning (eg from 0800 hours to 1100 hours local time)under non-limiting vapour-pressure deficits or temperaturesThis minimises the risk of sampling during midday andafternoon declines in gas-exchange rate as a result of stomatalclosure sourcendashsink inhibition or other causes If a givenmorning or mornings in general are cold relative tophotosynthetic temperature optima measurement can be madelater in the day Because most published measurements havebeen made under ambient CO2 concentrations that would berecommended If rates can also be measured under saturatingCO2 concentrations that is also useful

Any reliable leaf gas-exchange system can be used andconditions in the chamber can be either set at levels consideredoptimal or left to track the in situ conditions (whichneed to benearoptimal) If possible measure intact foliage or else leaves onbranches cut and then re-cut underwater In the latter case checkwhether given individuals fail to stay hydrated Conduct sometest comparisons of gas exchange on intact and lsquore-cutrsquo branchesto ensure the technique works for your taxa and systemMeasurements can also be made on detached foliage however

this requires even greater attention Leaves should be measuredwithin seconds to a few minutes at most after detachment andtests of intact v detached foliage should bemade for a subsampleto ensure similar rates are observed

If possible the leaf material inside the chamber should becollected (see Section 31) measured for LDMC and SLA andstored for any subsequent chemical analyses

References on theory significance and large datasets Reichet al (1992 1997 1999) Wright et al (2004)

More on methods Wong et al (1979 1985a 1985b 1985c)Reich et al (1991a 1991b) Ellsworth and Reich (1992)

311 Leaf dark respiration

Characterising leaf dark respiration (Rleaf) in a fashion thatenables comparison among species and especially among sitesand times of year is challenging given the sensitivity andacclimation of Rleaf to temperature However Rleaf undertypical field conditions is valuable because it is both a measureof basal metabolism and a rough correlate of average realisednight-time respiratory C flux Rleaf scales with other metabolicstructural chemical and longevity aspects of the leaf economicspectrum and alongwith those other variables enables scaling tocanopy processes of whole ecosystems


Sample young to medium-aged fully expanded leaves(see Section 31) to ensure negligible respiration associatedwith biosynthesis (lsquogrowth respirationrsquo) Do not makemeasurements during or soon after atypical conditions (such aseg heat or cold stress water stress) unless that is the focus ofthe research Sample foliage from parts of the canopy sunlitduring daytime unless one is specifically focussed on theshaded understorey taxa If possible measure intact leaves atnight In any case leaves must have been in the dark for ~30minto minimise variation resulting from very recently fixedphotosynthate or transient light-induced respiratory CO2 losses

Any reliable leaf gas-exchange system that can control leaftemperature can be used If possible it is best to measure intactfoliage Detached leaves should be kept moist cool (to minimiseC and water loss) and in the dark until measurement If possibletests of intact v detached foliage should bemade for a subsampleto ensure that similar rates are observed See under Section 310for any subsequent leaf handling

Leaf dark respiration (Rleaf) can bemeasured while measuringphotosynthetic rate (see Section 310) merely by turning off orshielding the chamber completely from incident light Howeverflux rates for Rleaf are roughly an order of magnitude lower thanthose forAmax and therefore the signal to noise ratio of the typicalportable photosynthesis system may be suboptimal for taxawith lower flux rates One can reduce flow rates andorincrease the amount of foliage into the chamber to alleviatethis problem however this is not always sufficient to obtainreliablemeasurements In such cases using specialised chambers(which may hold more foliage) andor choosing a standardisedtemperature that is at the high rather than low end of the candidaterange (next paragraph) can help ameliorate this problem byincreasing the flux rate


For comparative Rleaf measurements one typically choosesa standardised temperature appropriate for the site conditions(eg 25C in the tropics 20C in the temperate zone 10Cor15Cin cold boreal or summer tundra conditions) However becausecross-study comparisons are often made of taxa grown inandor measured under different temperatures instantaneoustemperature response functions can help in calibratingrespiration across temperature regimes Where possiblemeasure (subsets of) leaves at appropriate contrastingmeasurement temperatures with 10C intervals or ideally atleast four different temperatures over a 1535C range

References on theory significance and large datasets Reichet al (1998 2008) Tjoelker et al (2001) Wright et al (2004)Atkin et al (2005) Rodriguez-Calcerrada et al (2010)

312 Photosynthetic pathway

Three main photosynthetic pathways operate in terrestrial plantseach with their particular biochemistry including C3 C4 andCAM These pathways have important consequences foroptimum temperatures of photosynthesis (higher in C4 thanin C3 plants) water- and nutrient-use efficiencies andresponsiveness to elevated CO2 Compared with C3 plants C4

plants tend to performwell inwarm sunny and relatively dry andor salty environments (eg in tropical savannah-like ecosystems)whereas CAM plants are generally very conservative with waterand occur predominantly in dry and warm ecosystems Somesubmerged aquatic plants have CAM too There are obligateCAM species and also facultative ones which may switchbetween C3 and CAM depending on environmental factors(eg epiphytic orchids in high-elevation Australianrainforests) Two main identification methods are availablenamely C-isotope composition and anatomical observationsCAM can be inexpensively confirmed by verifying thatstomata are open at night and closed during the day or bymeasuring diurnal patterns of organic acids or leaf pH valuesWhich method to choose (a combination would be the mostreliable) depends on facilities or funding as well as on the aim ofthe work (eg to contrast C4 v C3 or CAM v C3) Although C-isotope composition can be affected by environmental factorsintraspecific genetic differences andor phenological conditionsintraspecific variability is small enough not to interfere withthe distinction between C4 and C3 photosynthetic pathways Inmany plant families only C3 metabolism has been found It isuseful to know in which families C4 and CAM have beenfound so that species from those families can be screenedsystematically as potential candidates for these pathways (seeMaterial S4 Table 1) Below we describe two methods which incombination provide good contrast between pathway types


Collect the fully expanded leaves or analogous photosyntheticstructures of adult healthy plants growing in full sunlight or asclose to full sunlight as possibleWe recommend sampling at leastthree leaves from each of three individual plants If conductinganatomical analysis (see under (B) Anatomical analysis in thepresent Section) store at least part of the samples fresh (seeSection 31)

(A) C-isotope analysis


Dry the samples immediately after collecting Once dry thesample can be stored for long periods of time without affectingits isotope composition If this is not possible the sample shouldfirst be stored moist and cool (see under Section 31) or killed byusing a micro-wave and then be dried as quickly as possible at70ndash80C to avoid changes caused by loss of organic matter(through leaf respiration or microbial decomposition) Althoughnot the preferred procedure samples can also be collected from aportion of a herbarium specimen Be aware that insecticides orother sprays that may have been used to preserve the specimencan affect its isotope composition

Bulk the replicate leaves or tissues for each plant then grindthedried tissues thoroughly topass througha40-mm-meshorfinerscreen It is often easier with small samples to grind all of thematerial with mortar and pestle Only small amounts of tissue arerequired for a C-isotope-ratio analysis In most cases less than3mg of dried organic material is used

Measuring

Carbon isotope ratios of organic material (d13Cleaf) aremeasured using an isotope ratio mass spectrometer (IRMSprecision between 003 permil and 03 permil dependent on the IRMSused) and are traditionally expressed relative to the Pee DeeBelemnite (PDB) standard as d13C in units of per mil (permil) ieparts per thousand After isotopic analysis the photosyntheticpathway of the species can be determined on the basis of thefollowing (see graphic explanation in Material S4 Fig 1)

C3 photosynthesis d13C 21permil to35permil

C4 photosynthesis 10permil to14permil

Facultative CAM 15permil to20permil and

Obligate CAM 10permil to15permil

Separating C3 or C4 from CAM plants is difficult on the basis ofd13C alone (for facultative CAM plants d13C values have beenfound to range as widely as from ndash14permil to ndash23permil) However as arule of thumb if d13C is between ndash10permil and ndash23permil and thephotosynthetic tissue is succulent or organic acid concentrationsare high during the night but low during the day then the plantis CAM In such cases anatomical observations and diurnalmeasurements of gas exchange or biochemical analysis wouldbe decisive (see (B) Anatomical analysis in the present Section)

(B) Anatomical analysis

C3 and C4 plants typically show consistent differences in leafanatomy best seen in a cross-section Using a razor blade ormicrotome make cross-sections of leaf blades of at least threeplants per species making sure to include some regular veins(particularly thick and protruding veins including the midrib andmajor laterals are not relevant) C3 plants have leaves in whichall chloroplasts are essentially similar in appearance andspread over the entire mesophyll (photosynthetic tissues) Themesophyll cells are not concentrated around the veins and are


usually organised into lsquopalisadersquo and lsquospongyrsquo layers parallel toand respectively adjacent to the upper to lower epidermis (seeMaterial S4 Fig 2) (vertically held C3 leaves often have apalisade layer adjacent to each epidermis and a spongy layerbetween the two palisades) The cells directly surrounding theveins (transport structures with thin-walled phloem and generallythicker-walled xylem cells) called bundle sheath cells normallycontain no chloroplasts C4 plants in contrast typically exhibitlsquoKranz anatomyrsquo viz the veins are surrounded by a distinct layerof bundle-sheath cells (Material S4 Fig 2) that are often thick-walled and possess abundant often enlarged chloroplasts thatcontain large starch granules The mesophyll cells are usuallyconcentrated around the bundle-sheath cells often as a singlelayer whose cells are radially oriented relative to the centre ofthe vein and contain smaller chloroplasts with no starch grainsThese differences can usually be identified easily underan ordinary light microscope Many plant physiology andanatomy textbooks give further illustrations of Kranz v typicalC3 leaf anatomy (see More on methods below in the presentSection)

If Kranz anatomy is observed the species is C4 If not it islikely to be C3 unless the plant is particularly succulent andbelongs to one of the families with CAM occurrence In the lattercase it could be classified as (possible) CAMManyCAM leavesdo not have typical C3 palisade or spongy mesophyll layersbut only a thin layer of more or less isodiametric chloroplast-containing cells just under their epidermis with the entire centreof the leaf consistingof large thin-walled colourless parenchymacells that store water and organic acids If living plants are withineasy reach anadditional checkcouldbe todetermine thepHof theliquid obtained by crushing fresh leaf samples in the afternoon(see Section 35) and again (with new fresh samples from thesame leaf population) at pre-dawn Because in a CAM plantorganic (mostly malic) acids build up during the night and arebroken down during the day to supplyCO2 for the photosynthesisin the leafCAMspecies showadistinctly lower pHafter the nightthan they do in the afternoon In addition C-isotope ratios canprovide further evidence to distinguish between CAM and C3 orC4 metabolism (see (A) C-isotope analysis above in the presentSection)


(1) Permanent slides or photographs and chloroplast visibilityA range of methods is available for making the microscopeslides permanent however be aware that somemay result inpoorer visibility of the chloroplastsOnemethod for retainingthe green colour of the chloroplasts is to soak the plant orleaves in a solution of 100 g CuSO4 in 25mL of 40 formalalcohol (formaldehyde alcohol) 1000mL distilledwater and03mL 10 H2SO4 for 2 weeks then in 4 formal alcoholfor 1 week subsequently rinse with tap water for 1ndash2 h andstore in 4 formal alcohol until use However material thustreated can be sectioned only by using a microtome afterembedding or freezing it in contrast to many living turgidleaves which can be sectioned free-hand by using a suitabletechnique such as sectioning a rolled-up leaf or a stackof several leaves Photomicrographs of freshly prepared

sections are an alternative way to keep records for laterassessment

References on theory significance and large datasetsOrsquoLeary (1981) Farquhar et al (1989) Earnshaw et al(1990) Ehleringer et al (1997) Luumlttge (1997) Zotz andZiegler (1997) Wand et al (1999) Pyankov et al (2000)Sage (2001) Hibberd and Quick (2002)

More on methods Farquhar et al (1989) Ehleringer (1991)Hattersley andWatson (1992)Mohr and Schopfer (1995) Beleaet al (1998) Pierce et al (2002) Taiz and Zeiger (2010)

313 C-isotope composition as a measure of intrinsicwater-use efficiency

Uptake of CO2 through stomata inevitably leads to loss of watervapour The relative magnitude of photosynthesis andtranspiration depends on several physiological morphologicaland environmental factors such that different species in differentgrowing conditions can have widely different C gain perunit water loss This quantity the ratio of the rates of netphotosynthesis and transpiration (= water use efficiencyWUE) is of great ecological interest and can be measured onshort or long time scales

On short time scales (= instantaneous) WUE is oftenmeasured with infrared gas analysis (see Section 310)However instantaneous WUE changes dramatically for agiven leaf over short time spans eg because of variable lightintensity and vapour pressure deficit This makes separatingspecies effects and environmental effects challenging Forcomparative studies we recommend taking into account theprecautions outlined in Section 310 and to calculatelsquoinstantaneous intrinsic WUErsquo the ratio of net photosynthesisto stomatal conductance This excludes the effect of differences invapour pressure on transpiration rates As CO2 and water vapourshare the same stomatal diffusion pathway but with diffusion ofwater being 16 times faster than that of CO2 intrinsic WUErelates to the CO2 gradient as follows

Intrinsic WUE frac14 A=gs frac14 ethca ciTHORN=16 frac14 ca eth1 ci=caTHORN=16where A is net photosynthesis gs is stomatal conductance ca andci are the mole fractions of CO2 in ambient air and in thesubstomatal cavity respectively

The C-isotope approach has proved extremely useful to studyWUE over longer time scales It relies on the fact thatphotosynthetic enzymes discriminate against the heavier stableisotope 13C (relative to 12C) during photosynthesis so that C inleaves is always depleted in 13C compared with that in theatmosphere The extent of the enzymersquos discrimination against13C depends on ci If ci is low relative to ca then the air inside theleaf becomes enriched in 13C and the ability of the enzymeto discriminate declines As a result the plant ends up fixing agreater proportion of 13C than a plant performing photosynthesisat ahigherci In its simplest form forC3plantsD = a+ (bndasha)cicawhere D is photosynthetic 13C discrimination a= 44permil andb = 27permil Therefore D allows time-integrated estimatesof ci ca and intrinsic WUE Note that D is calculated fromd13C (see Section 312) as follows D= (d13Cair ndash d13Cplant)


(1 + d13Cplant) which highlights the requirement for assumptionsor measurements of the isotope composition of the air

Because intrinsicWUEchanges rapidly thebulk leaf 13C 12Cratio of fixed C correlates with the cica ratio for the time periodduringwhich the C comprising the leaf was fixedweighted by thephotosynthetic flux In other words the 13C 12C represents alonger-term measure of ci ca especially reflecting ci ca duringfavourable periods


For intrinsicWUE assessment d13C is usually determined forleaves but can be determined on any plant part eg on tree ringsfor a historical record Note that in general there is fractionationbetween leaves and stems with all non-photosynthetic organsbeing more enriched in 13C than are leaves This enablesdifferentiation between growing conditions using tree rings ofdifferent ages and also means that leaves that grew indifferent years or different seasons can have different D whichhas implications for the sampling strategy Leaves at differentpositions in a tree or in a canopy can vary in D as a result ofdifferences in stomatal opening and photosynthetic capacity andalso because of differences in the isotope composition of thesource air To estimateD the isotope composition of the air needsto beknown In freely circulating air such as at the topof a canopyit is generally reasonable to assume that the isotope compositionof air is constant and equal to that of the lower atmosphere (d13Cair

ndash8permil)


Samples should be dried as soon as possible andfinely groundGrind the dried tissues thoroughly to pass through a 40-mm-meshor finer screen C-isotope ratio analysis requires only smallsamples (2ndash5mg) however it is recommended to sample andgrind larger amounts of tissue to ensure representativeness

Measuring

See Section 312 for measuring C-isotope concentrations


(1) Cellulose extracts Isotopes are sometimes analysed usingcellulose extracts to avoid variation introducedby the slightlydifferent isotope composition of other C compounds Inmostcases however the D values of the whole tissue and those ofcellulose correlate very well Shorter-term (typically at thescale of a day) studies of D have sampled recent assimilatesrather than structural C either by extracting non-structuralcarbohydrates from snap-frozen leaves or by samplingphloem sap

(2) Assumptions We reiterate here that the estimation ofintrinsic WUE from C-isotope composition involvesseveral assumptions that intrinsic WUE does notnecessarily correlate well with the actual WUE(photosynthesis to transpiration ratio) with mesophyllconductance being a particular complication and that theequation for D given above is a simplification of the theoryIt is also important to note that because of their differentbiochemistry the equation given forDdoes not apply toC4 or

CAM plants and in these groups C-isotope composition isnot useful for estimating intrinsic WUE

References on theory significance and large datasetsFarquhar et al (1989) Cernusak et al (2009)

More onmethods Ehleringer and Osmond (2000) Seibt et al(2008) Diefendorf et al (2010)

314 Electrolyte leakage as an indicator of frost sensitivity

Electrolyte leakage after freezing is an indicator of leaf frostsensitivity and is related to climate season and plantgeographical distribution Leaves of species from warmerregions andor growing at warmer sites along a steep regionalclimatic gradient have shown greater frost sensitivity than thoseof species from colder regions andor growing at colder siteswithin a regional gradient The technique described here isbased on the idea that when a cell or tissue experiences an acutethermal stress one of the first effects is disruption of membraneseliminating a cellrsquos ability to retain solutes such as ions Ionleakage from a tissue can be easily assessed by measuringchanges in electrolyte conductivity of a solution bathing thetissue The technique is suitable for a wide range of leaf types(from tender to sclerophyllous) and taxa (monocotyledons anddicotyledons) and is not affected by cuticle thickness


Collect young fully expanded sun leaves with no sign ofherbivory or pathogen damage Deciding when to collect ismore complicated The answer will depend on the questionbeing asked although in most cases collection should bestandardised across taxa Depending on the contrast of interestcollect foliage during the peak growing season (see Section 31)or preferably near the end of the season (see Special cases andextras below in the present Section) or in winter (for winterevergreen species) If a species grows along awide environmentalgradient and the objective is an interspecific comparison collectthe leaves from the point of the gradient where the species ismost abundant Ifmany species are considered try to collect themwithin the shortest possible time interval tominimise differencesresulting from acclimation to different temperatures in thefield Collect leaves from at least five randomly chosen adultindividuals of each species


Store the leafmaterial in a cool container until processed in thelaboratory (see Section 31) Process the leaves on the day of theharvest so as to minimise natural senescence processes For eachplantwith a cork borer cut four circular 5-mm-diameter leaf disks(to provide for two treatments using two disks each see belowwithin the present Protocol) avoiding themain veins For needle-like leaves cut fragments of the photosynthetically active tissuethat add up to a similar LA Rinse the samples for 2 h in deionisedwater on a shaker then blot dry and submerge two disks (or theirequivalent in leaf fragments) in 1mLof deionisedwater in each oftwo Eppendorf tubes Complete submergence is important Foreach treatment (see belowwithin the present Protocol) prepare asmany replicates (one replicate being two tubes each containing


two disks or equivalent leaf fragments) as the number of plantssampled

Measurement

Apply the following two treatments without any prioracclimation to the two leaf diskfragment samples in therespective tubes (1) incubation at 20C (or at ambienttemperature as stable as possible) for the control treatmentand (2) incubation at 8C in a calibrated freezer for thefreezing treatment Incubations should be for 14 h in completedarkness to avoid light-induced reactions

After applying the treatment let the samples reach ambienttemperature and then measure the conductivity of the solutionDo this by placing a sample of the solution from an Eppendorftube into a standard previously calibrated conductivity meter(such as the Horiba C-172 Horiba Kyoto Japan) and byrecording the conductivity Then place the Eppendorf tube in aboiling water bath for 15min to completely disrupt cellmembranes releasing all solutes into the external solutionthen re-measure its conductivity Prior to immersion puncturethe cap of each Eppendorf tube to allow relief of pressure duringboiling

Calculations

(1) Percentage of electrolyte leakage (PEL) ndash separately for thefrost treatment and the control for each individual plantreplication as follows

PEL frac14 ethes=etTHORN 100

where es is the conductivity of a sample immediately after thetreatment and et is its conductivity after boilingHighvalues ofPEL indicate significant disruption of membranes and thuscell injury the higher the PEL the greater the frost sensitivity

(2) Corrected PEL ndash the PEL of the control treatment can varyamong species because of intrinsic differences in membranepermeability experimental manipulations and differencesin injury when leaf disks or fragments are cut To controlfor these and other sources of error subtract the PEL of thecontrol treatment of each replicate from that for the freezingtreatment Corrected PEL is thus

Corrected PEL frac14 PEL in the freezing treatment

PEL in the control treatment

For calculating the mean standard deviation or standard errorfor a species the average corrected PEL for each individualplant replicate counts as one statistical observation


(1) Applicability to different plant functional types Thetechnique is not suitable for halophytes and succulents Itis not necessarily applicable to deciduous plants andhemicryptophytes because their significant frost toleranceinvolves stems and buds rather than leaves This tolerance

could possibly be tested with sections cut from stemsalthough the reliability of this has not been investigated toour knowledge as it has been for leaves

(2) Season of collection Because of the recognised wideoccurrence of autumnal acclimation in frost tolerance werecommend normally performing the procedure with leavescollected at or near the end of the growing season

(3) Incubation with dry ice A different treatment namelyincubation at about 78C (the temperature of dry ice)with the rest of the protocol the same as described abovecan be used if a freezer whose temperature can be controlledat about 8C is not available or if one wishes to detecttolerance to the kind of severe frost that can occur at highlatitudes or altitudes It would not detect tolerance to merelymild frost

(4) Acclimation The occurrence of acclimation to mild frostcould be detected using a freezer at 8C on leaf samplescollected on successive dates in summer and autumn

(5) Sensitivity tohigh temperatures The samebasic techniquewith a modification in the treatment temperature has beensuccessfully applied to leaf sensitivity to unusually hightemperatures (~40C see More on Methods below in thepresent Section)

(6) Chilling sensitivity is a physiological limitation that can beecologically important in mountains at lower latitudes andmight be detected by this technique It is usually tested for byincubation for 24 h or more at about +5C eg in an ordinaryrefrigerator Alternatively 0C in a distilled water (or rainwater) bath could be used because this will not actuallyfreeze plant tissue A chilling-sensitive tissue would leakelectrolytes after such incubationwhereas a chilling-toleranttissue should not

References on theory significance and large datasets Levitt(1980) Blum (1988) Earnshaw et al (1990) Gurvich et al(2002)

More on methods Earnshaw et al (1990) Gurvich et al(2002)

315 Leaf water potential as a measure of water status

Species facing soil water shortage can avoid water stress to adegree by dropping leaves or delay the development of waterstress in their tissues by rooting deeply or by shutting stomataand losing stored water only slowly through their cuticleAlternatively tissues may tolerate physiological desiccationThe bulk leaf water potential (YL units MPa) is a simpleindicator of leaf water status the more negative the value themore dehydrated the leaf

When measured pre-dawn the plant may have becomeequilibrated with the soil during the night and the YL maythus represent the soil water potential in the lsquoaveragersquo rootzone However recent work has shown instances ofsubstantial disequilibrium between pre-dawn YL and soilwater potential as a result of several mechanisms includingnocturnal transpiration cavitation in the xylem and osmolyteaccumulation in the cell walls Thus pre-dawnYL may be morenegative than the soilwater potential and should be used only as atentative index of soil water availability


During the dayYL will decline below the soil water potentialas a result of transpiration into the atmosphereWhenmeasured inthe dry season the midday YL can provide a useful index of thedegree of physiological drought experienced Thus theminimumvalue forYL that a plant reaches usually at midday at the driesthottest time of year can be used as an index of the tolerance towater shortage that the species (or individuals and populations)demonstrate (assuming that the plants are still healthy and notdrought-injured)


Measurement ofminimumvaluesofYL is typically carried outat the end of the hot dry season for evergreen species and inMediterraneanwinter-rain ecosystemsHowever in summer-rainecosystems the time of year at which drought stress is maximalmay not be obvious Repeated-measurements in different seasonscan help find the real minimum YL for each species

Depending on the type of pressure chamber used (see belowwithin the present Protocol) either leaves or short terminalleafy twigs should be collected Samples should be collected atmidday and as previously indicated (see Section 31) fromshoots or individuals located in the sun Leaves should havebeen exposed to direct sun for at least 30min before collection(avoid cloudy days) We recommend measuring samples as soonas possible or at least within half an hour of collecting all samples(with the number of samples dependingon the number of pressurechambers available) over a period of no more than half an hourbetween the first and last measurement Samples should becollected into sealable plastic bags into which one has justexhaled to increase moisture and CO2 to try to minimise shoottranspirationwithin thebagSamples sealed inplastic bags shouldbe kept refrigerated and in darkness (eg in a refrigerated picnicfridge or an insulated cooler box containing pre-frozen coolingbars or ice)

Measuring

The simplest way to measure leaf water potential is with apressure chamber or Scholander bomb (see diagram in Fig 4)This consists of a pressure container into which the sample (leafor terminal twig) is placed a manometer or pressure gauge tomeasure the pressure inside the chamber and as a pressure sourcea pressure tank of liquid N connected to the chamber through aneedle valve and pressure-safe (normally copper) tubingMany models with different characteristics are commerciallyavailable

A leaf or shoot is placed inside the chamber with its cut endprojecting to the exterior through the sealing port Pressure fromthe N tank is then gradually increased in the chamber When adrop of water appears at the cut end of the specimen the lsquobalancepressurersquo indicated by the gauge or manometer is recordedAssuming that the xylem osmotic potential is very low thebalance pressure represents the equilibrium water potential ofthe plant material in the chamber multiplied by ndash1 Leaf waterpotential is conventionally expressed in MPa Minimum leafwater potentials usually vary from near 0 to 5MPa but canbe lower in (semi-)arid ecosystems Extreme care should be takenwhen pressure chambers are under high pressures

References on theory significance and large datasetsHinckley et al (1978) Ackerly (2004) Bucci et al (2004)Bhaskar and Ackerly (2006) Lenz et al (2006) Jacobsenet al (2008) Bartlett et al (2012)

References on methods Scholander (1966) Turner (1988)

316 Leaf palatability as indicated by preferenceby model herbivores

Despite the vast diversity and complexity of herbivores plantsand their interactions a small number of components of leafquality affect preference by generalist herbivores in a predictableway Leaf palatability (as indicated by preference by herbivores)can be seen as an integrator of several underlying leaf-qualitytraits Additionally palatability tends to be correlated withlitter decomposability among species because both are limitedby similar constraints (eg low nutrient contents highconcentration of lignin and secondary metabolites)

One method for quantifying leaf palatability as indicatedby model herbivore preference is a cafeteria assay in whichgeneralist herbivores are allowed to feed selectively on leafsamples cut out from fresh leaves of a whole range of speciesdistributed in random positions on a feeding arena Theseexperiments can provide useful information about herbivorepreference for a broad range of plant species

Rubber gasketClean-cut surface

Compressedgas cylinder

Pressuregauge

Chamber

Lid

Fig 4 Measuring water potential with a pressure chamber A cut branch(or leaf or compound leaf) is placed inside the chamber with the cut endprotruding from the seal Once the chamber has been sealed (hermeticallyclosed) pressure is gradually applied from thegas cylinderWhen thepressurein the chamber equals the xylem pressure a drop of water appears at the cutsurface Assuming that the xylem osmotic potential is very low the balancepressure represents the equilibriumwater potential of the plant material in thechamber



Leaves are selected and collected as indicated in Specific leafarea in Section 31 preferably from at least 10 individuals perspecies Be aware that all species must be collected within 2 daysbefore the trial and stored in refrigerated and moist conditionsIf different species grow in different seasons at least two cafeteriatrials should be carried out making sure common species ofcontrasting quality are included in both for cross-calibration


All leaves are kept in sealed bags at 4ndash5C until processedOnce all leaves have been collected 10 1-cm2 samples (one fromeach individual) should be cut from each fresh leaf and randomlyplaced securing with a thin needle on a numbered grid cell on apolystyrene feeding arena (Fig 5) The surface is covered bytransparent plastic in the case of trials with snails Large veinsshouldbe avoidedunless leaves are too tiny For narrow leaves anequivalent area is reached by cutting an appropriate number of10-mm lengths from themid-leaf section and pinning these downtogether into a star shapeTiny leavesmayalsoneed to begroupedlike this (with minimal overlap) to make up to ~1 cm2 In the caseof highly succulent and aphyllous species a thin 1-cm2 fragmentof epidermis and adjacent mesophyll (relatively youngphotosynthetic tissue) is used as a leaf analogue Whilepreparing all leaf samples be sure to keep the cut leaves in awater-saturated environment (eg plastic bags containingmoistened paper towel) to preserve turgidity Includingadditional samples from a known material such as lettuce forhuman consumption popular with some of the herbivores usedfor these cafeterias (snails and slugs) can be useful to test animalbehaviour If the animals do not eat from known preferredmaterial either the animals or the feeding conditions areprobably not right

Measuring

Once all samples have been pinned onto the arena severalherbivores (~10 per 500 leaf samples) are placed in a randomposition and the arena is closed with a plastic net (Fig 5) Snails(eg Helix spp) require a cool dark and humid environment(spraying dark plastic cover) that will stimulate consumptionGrasshoppers need a dry and light and crickets a dry and dark

environment The arena should be covered with netting to avoidany escapes After herbivores have been placed consumptionis measured by direct observation after 4 8 and 12 h andsubsequently every 12 h for 3 days The LA consumed canbe estimated by eye (with 2ndash10 accuracy per sample) on thebasis of the original cut shape Actual LA may also be measuredaccurately (see Section 31) before and after the trial providedthe samples do not deteriorate during this procedure To be surethat the model herbivores do not have previous experience withthe plants included in the trials the herbivores may be bred orcollectedwhen young and raised in captivity without exposure toany of the plants included in the cafeteria experiments This and apre-trial 48-h starvation period (promoting consumption duringthe trial) are important to get unbiased results


(1) Independent feeding trials It is recommended to assessleaf palatability in at least two independent feeding trialsusing different model herbivores to cover a wider range ofpreferences by generalist herbivores Snails arerecommended for their generalist-feeding habits but theyconsume few graminoid monocots grasshoppers andcrickets are better at discriminating between leaf qualitieswithin graminoidsInstead of selecting one recording timeseveral consumption measurements can be compared withanalyse-first choice and successive choices Values of LAconsumption can be transformed into values of leaf biomassif SLA is included in the calculations (see Section 31)

(2) Palatability v accessibility Experiments can be designedto evaluate palatability v accessibility following the sametheoretical background on which palatability tests are basedFor example by offering whole shoots with and withoutspines to different animals (in this case model herbivoresshould be bigger than snails or grasshoppers) and recordinghow much biomass is consumed per unit time

References on theory significance and large datasets Grimeet al (1970 1996) Southwood et al (1986) Coley (1987)Hartley and Jones (1997) Cornelissen et al (1999) Singer(2000)

More on methods Peacuterez-Harguindeguy et al (2003)

1 2 3 4 5 6

313029282726

51 52 53 54 55 56

818079787776

101 102 103 104 105 106

131130129128

(a)

(b)

(c)

127126

Fig 5 Diagrammatic representation of a cafeteria (followingGrime et al 1996 Cornelissen et al 1999) If themodel herbivores are snails (a) the arena can beconstructed on polystyrene and (b) the numbered grid should bewrapped in plastic which is a substrate that snails like for crawling on (c) Each leaf sample shouldbe pricked on the grid avoiding the contact with the plastic so as to prevent rotting


317 Litter decomposability

Plant species exert strong control over decomposition ratesthrough the lsquoafterlifersquo effects of attributes of living plantmodules (leaves stems and branches roots) on the quality oftheir litter Shifts in species composition as a consequence ofglobal changes land use or natural succession are strong driversof changing litter decomposition rates in various biomesworldwide with feedbacks to climate through the release ofCO2 To estimate the organ afterlife effects of different specieson decomposition rates a powerful method in recent years hasbeen to comparemass losses of (leaf) litters ofmultiple species byincubating them simultaneously in semi-natural outdoorlsquocommon-garden experimentsrsquo particularly in litter bedsSpecies-specific lsquodecomposabilityrsquo thus derived integratesseveral structural and chemical traits of the leaf (or other plantpart) because it is an expressionof thequalityof litter as a substratefor microorganisms Decomposability is usually related to leafdry-matter content leaf toughness as well as N and lignincontent in most studies additionally it has been found to berelated to SLA leaf pH tannin P or base-cation content in others


Here we focus on leaf litter although the protocol can also beapplied to other plant parts with some adjustments (see underSpecial cases or extras in the present Section) Freshly senescedundecomposed leaf litter should be collected from mature plantsin thefield In species that shed their litter it canbe either collectedfrom the ground within a few days after falling or by placing aclean cloth or net beneath the tree or branch and gently shaking ituntil senesced leaves fall It is important to brush or rinse-off anysoil before further processing In species that retain dead leaves onplants or die back completely above ground cut off leaves that aresubjectively judged to have died very recently and are stillundecomposed Often such dead leaves are still shiny and indeciduous woody species may still show bright autumn coloursPetioles and rachides of compound leaves that are shed as anintegral component of the leaf litter should be collected andprocessed as such (but see discussion under Section 31) Inaphyllous species where the entire shoot functions as the greenphotosynthetic unit analogous to leaves and also senesces as aunit such units are collected as leaf litter In leaves that graduallydie from the tip down as in many monocotyledons collect thoseleaves inwhich themiddle lamina section is at the right phase iecomplete senescence without any evidence of decay For speciesin which leaves senesce sequentially in cohorts we recommendcollecting several times from the sameplants toget representativeleaf litter


Litter collections should be air-dried in open paper bags untilthey reach their equilibrium moisture content To preserve itscharacteristics litter must never be dried at high temperaturesOnce dry the litter can be stored in the same bags for up toseveral months in a relatively dry environment away fromsunlight For each species an air-dried subsample of leaf litteris weighed and again after 48 h in an oven (60C) so as tocalculate initial oven-dry weights of the samples for incubationThere are no fixed rules regarding litterbag size mesh type or

mesh material but litterbags should be made of non-degradableinert but flexible materials Typical litterbags measure from10 10 cm to 20 20 cm and are made of 1-mm polyester orfibreglass mesh Litterbag sizes may vary within one study so asto standardise the lsquorelative packingrsquo of litter inside the bags formorphologically contrasting species Finer mesh size should beused when among the litters to be compared narrow-leavedspecies are included whereas wider mesh sizes allow a morecomplete set of organisms to be involved in decomposition andhave less effect on the litter microclimate Comparing some of thesame species at different mesh sizes will help calibrate amongmethods A litterbag usually contains a standard small amount(eg 1 g) of dry litter however for some species or in someenvironments it may be necessary to increase or decrease theinitial mass per litterbag Very big leaves may have to be cut intosections with representative proportions of midrib and petiolewhereas flexible longmonocot leaves can sometimes be rolled up(if not better cut them into segments) If there are largedifferencesin initial mass among some species we recommend incubatingsome reference species at two or three different initial masses forcalibration among species afterwards It is important not to breakthe leaf litter during handling Filling the bags using funnels ortubes inserted into the bags temporarily may help here Litterbagscan be sealed with polycarbonate glue by sewing (with nylonthread) stapling (with non-rusting staples) or by using alaboratory heat-sealer Make sure a resistant label (eg plasticwith code embossed) is firmly attached to the litterbag or sealedinside

Measuring

Litterbags are incubated in a purpose-built outdoor litter bedLitter beds can range from just a rather homogeneous square in thefield cleaned from vegetation and litter with litterbags simply onthe soil surface to amuchmore elaborate bedmadebywoodenorplastic squares (with natural drainage) filled with soil thoroughlymixed with a standard or combined litters and covered withthe same litter mixture (Fig 6) Depending on the type ofstudy the composition of this mixture may be based on aparticular community or several communities however amore standardised mixture not based on local communitiesmay be used for particular purposes (see below within thepresent Protocol) In any case the composition should behomogeneous across the bed to prevent differences indecomposition rate caused by the microenvironment Tofurther account for environmental gradients within largerbeds it is recommended to divide the bed in equal statisticalblocks with one replicate of each species in a random positionin each block The litterbags are usually incubated 1ndash2 cmbelow the surface of this mixture to reduce heterogeneity inmoisture dynamics among the samples Additional controlsamples of some of the species may be treated identicallyand retrieved immediately after burial to control for loss oringression of particles during burial Spraying the sampleswith demineralised or rain water before incubation will getthem to field capacity more quickly Stretching 3ndash5-cm-meshnylon net or a metal grid over the bed may protect the samplesfrom the activities of mammals and birds Additional litterbagsfilled with pieces of plastic broadly representing litter amountand shape can be incubated during the whole period to check


for the contribution of exogeneous organic matter to thesample Retrieval dates and sampling schedule may vary firstaccording to the microclimatic conditions that will determinedecomposition rates and second to the average quality of thelitters within litterbags In moist tropical environments almost90 mass loss can be reached in less than 2 months given highlitter qualitywhereas in aridor cold environments 2yearsmaybethe minimum time needed for over 50 mass loss for the samelitter quality Two retrieval dates are usually enough to check forthe consistency of decomposability ranking across species overtime whereas five or more may be needed if decompositiondynamics are to be analysed too After retrieval samples mayeither be cleaned up and processed directly or first frozenAdhering soil soil fauna and other extraneous materials mustbe removed from the decomposed litter to the degree possibleusing eg tweezers and small brushes (Fig 6) Litter samples arethen oven-dried for 48 h at 60C then weighed Decompositionrate can be defined as the percentage of mass loss after theincubation period or as k (decomposition constant) derived

from a presumed exponential mass-loss curve over timeinvolving more than one harvest as follows

k frac14 Ln ethM t=M0THORN=t

where k= decomposition rate constant (yearndash1) M0 =mass oflitter at time 0 Mt = mass of litter at time t and t = duration ofincubation (years)

Calibration among studies

A disadvantage of the litter-bed assay is that the incubationenvironments and periods vary greatly so that mass loss or kvalues can be compared directly only within but not betweenstudies or sites We advocate the establishment of one or a fewcentralised litter beds which could simultaneously host samplesfrom multiple sites It would be better still if such litter beds orequivalent lsquocommon gardenrsquo facilities could provide close tostandardised environmental conditions from year to year eg ina controlled environment greenhouse In the absence of such

(a) (b)

unprocessed sample sample processing

(c)

Fig 6 Decomposition beds and litterbag cleaning Images of (a) decomposition bed fully coveredwith litterbags (b) litterbags coveredwith naturalmixed litterand protected by a wire mesh and (c) incubated-litterbag cleaning with brush


facility we recommend (1) additional inclusion of a few speciesfromeach range of sites in amultispecies litter bed so as to be ableto calibrate mass-loss values across multiple sites and therebyacross multiple species from these sites or (2) inclusion of littersamples of a few reference species in multiple litter beds acrosssites


(1) Microcosms Although most litter beds are situated underfield conditions litterbags can also be incubated inmicrocosms when more detailed effects of soil and litterfauna or leaching is studied or when the researcher needs tostandardise the incubation conditions for anyother reasonAsin common-garden experimentsmicrocosmscanbe sampledat certain time intervals during the process of decompositionand the incubated material can be analysed for its mass losschemical changes and biological colonisation

(2) Contamination When sand or clay contamination is higheven after removing obvious extraneous matters the samplecan be oven-dried and then sieved to remove bulk of the sandor clay fraction If contamination is too high and difficult toeliminate the incubated litter sample can be ashed for 4 h at450C and the mass loss expressed on the basis of the ash-free mass () of the initial and final litter samples (this is notappropriate for soils high in organic matter)

(3) Photodegradation One should be aware that in somedry or windy environments with high irradiance mass lossresulting fromphotodegradationor sandabrasion (in additiontomicrobial decomposition) may be substantial and this mayaffect the species rankings of mass loss in incubations underexposedconditionsorbelowa litter layer Speciesdifferencesin photodegradation at a given exposure may themselves beof interest

(4) Litter decomposabilities of other organs (fine and coarseroots fine and coarse stems) and the extent to which theseare coordinated between organs across species are also ofgreat ecological relevance A new method is available toinclude coarse woody debris together with fine litter in suchcomparative common-garden studies taking into accountdifferences in size and time scale of decomposition

References on theory significance and large datasetsCornelissen (1996) Cadisch and Giller (1997) Cornelissenet al (1999 2007) Garnier et al (2004) Austin and Vivanco(2006) Parton et al (2007) Adair et al (2008) Cornwell et al(2008) Fortunel et al (2009) Freschet et al (2012)

More on methods Taylor and Parkinson (1988) Cornelissen(1996) Robertson et al (1999) Graca et al (2005) Berg andLaskowski (2005) Freschet et al (2012)

4 Stem traits

41 Stem-specific density

Stem-specific density (SSDmgmmndash3 or kg dmndash3) is the oven-drymass (at 70C for 72 h but see Special cases or extras in thepresent Section) of a section of themain stemof a plant divided bythe volume of the same section when still fresh This trait is asynonym for lsquostem densityrsquo we distinguish it from lsquowood

densityrsquo in that SSD can also be measured on herbaceousspecies and includes the stem bark (ie secondary phloem andcork if any) which in some cases accounts for a significantproportion of the overall stem structure (see Special cases andextras in the present Section) SSD is emerging as a corefunctional trait because of its importance for the stabilitydefence architecture hydraulics C gain and growth potentialof plants Stemdensity partly underlies the growth-survival trade-off a low stem density (with large vessels) leads to a fast growthbecause of cheap volumetric construction costs and a largehydraulic capacity whereas a high stem density (with smallvessels) leads to a high survival because of biomechanical andhydraulic safety resistance against pathogens herbivores orphysical damage In combination with plant size-related traitsit also plays an important global role in the storage of C


Healthy adult plants should be selected according to previoussuggestionsDependingon theobjectives of the study either stemdensity orwood density should bemeasured (see Special cases orextras below in the present Section) For stem density removeonly the loose bark that appears functionally detached from thestem (see Section 43) Stem density can increase decrease orremain constant from pith to bark so a representative samplemust include a proportional representation of the complete stemStem density is higher at the insertion point of branches(because of tapering of vessels) and lower for branches andfor outer bark (which includes more air spaces and cork) Thesedensity gradients have important implications for the samplingprocedureCollect either awhole stemcross-sectional slice or forlarge trees a triangular sector from the bark tapering into thecentre (like a pizza slice) of cross-sectional area ~18 of the totalarea For herbaceous species or woody species with thin mainstems (diameter lt6 cm) cut with a knife or saw a ~10-cm-longsection near the base of the stem (between 10- and 40-cm height)If possible select a regular branchless section or else cut offthe branches For woody or thick succulent plants with stemdiameters gt6 cm saw out a (pizza) slice from the trunk at ~13-mheight the slice should be 2ndash10 cm thick Hard-wooded samplescan be stored in a sealed plastic bag (preferably cool) untilmeasurement Soft-wooded or herbaceous samples are morevulnerable to shrinkage so should be wrapped in moist tissuein plastic bags and stored in a cool box or refrigerator untilmeasurement

Measuring

Stem volume can be determined in at least two differentways

(1) Water-displacement method This procedure allows thevolume of irregularly shaped samples which cannot beproperly measured with the dimensional method to bemeasured easily A graded test tube or beaker largeenough to hold the entire sample is filled with distilledwater (but not completely filled to be sure that when thestem is placed within the water the liquid will not escapefrom the test tube) placed on a balance and tare the balanceThe wood sample is then completely submerged under thewater with the aid of a small-volume needle or tweezers


being careful to not touch the sides or bottom of the beakerwhich can cause variations in the weight being registeredby the balance When the wood sample is submerged theincrease on water level leads to an increase in the weightbeing registered by the balance (ie the weight of thedisplaced water) which equals the wood sample volumein cm3 (because water has a density of 1 g cmndash3) The massregistered in the balance (ie the volume of the woodsample) is quickly read and registered Tare again for eachmeasurement previously replacing water

(2) Measurement of dimensions (or dimensional method) Thevolume of a cylindrical sample can be determined simply bymeasuring its total length (L) and its diameter (D) on one ormore places along the sample using callipers If the stem isvery thin determine the diameter on a cross-section of itunder a microscope using a calibrated ocular micrometerCalculate the volume (V) of the cylinder as

V frac14 eth05DTHORN2 p L

In the case of hollow stems (eg young trees of Cecropia orsome bamboo species) estimate the diameter of the hollow andsubtract its cross-sectional area from the stem cross-sectional areabefore multiplying by L This method can be applied to sampleshaving different geometrical forms After volume measurement(by anyof themethods described above) the sample is dried in theoven Besides free water stems also contain bound water whichis removed only by drying at above 100C Samples should bedried in a well ventilated oven at 101ndash105C for 24ndash72 h (smallsamples) until a constant weight is obtained Large samples mayneed more drying time

Additional useful methods from forestry

In forest ecological studies samples are often taken with anincrement borer inwhich awoodcore is cut from thebark inwardto just beyond the centre of the stemBecause a core does not taperinward towards the centre of the stem such a sample may not beperfectly representative of the density of the stem as a wholehowever the difference from a density estimate using a sector oran entire stem section is usually probably small Large-diametercorers (12mm) are better because they cause less compactionSamples with this method are usually taken at ~13m aboveground (lsquobreast heightrsquo) After the core is extracted it can bestored in aplastic drinking strawwith the endsof the strawsealed

In the timber industry the lsquowood densityrsquo is oftenmeasured at12moisture content and density is reported as lsquoair-dry weightrsquo(ADW) (a misnomer because density is not weight but weightvolume) SSD as described in the present protocol is calledlsquooven dry weightrsquo (ODW) ADW can be transformed to ODWby using the formula ODW=0800 ADW+00134 (R2 = 099)(this formula cannot be correct for lsquodry weightsrsquo but only fordensities) We suggest that data for ODW directly measured orderived fromADWcan safely beused asSSDThis value ignoresthe contribution of the bark of a tree to its stem density howeverbecause bark usually makes up only a very small fraction of themass of a large tree trunk this error is probably unimportantexcept as noted below


(1) Oven drying and wood-specific gravity Because wood ismainly cellulose and lignin containing substantial boundwater and relatively small quantities of compounds of lowmolecular weight many wood scientists and foresters oven-dry wood samples at 100ndash110C before determining bothweight and volume They then refer to the relationshipbetween weight and volume as wood specific gravity

(2) Stems with holes Very large holes in the stem areconsidered to be air or water spaces that do not belong tothe stem tissue whereas smaller spaces such as the lumens ofxylem vessels and intercellular spaces are part of the stemtissue

(3) Wood density v stem density It might be worthwhile tomake separate density estimates for wood and bark becausethey have very different chemical and cellular compositionsphysical properties and biological functions Many treesand shrubs in savannah-type vegetation (and someMediterranean and arid species) have for example a verythick corky bark (with very low density) and often thevolume of the bark may be an important part (even 50)of the stem volume In these species most of the structuralsupport is given by the wood and wood is generally denserthan bark Thus from the support viewpoint wood density isthe important parameter

(4) Xylem density Some authors make a distinction betweenwood density (oven-dry wood density of the main trunkincluding sapwood and heartwood) from xylem density orsapwood density (measured on small ~15-cm terminalbranches of trees) Xylem density has been proposed as aproxy for the tree hydraulic architecture which in turn maylimit tree performance in terms of transpiration C exchangeand growth

(5) Plants without a prominent above-ground stem (rosetteplants grasses and sedges) Try to isolate the shortcondensed stem near ground level to which the leavesare attached and obtain its density In some plants all theleaves are attached to an underground often horizontalmodified stem called a rhizome (see Section 23) whosedensity can be determined but which does not have the kindofmechanical leaf-supporting function that an above-groundstem has Most rosette plants and basal-leaved graminoidsproduce aerial inflorescences the density of whose stem canalso be determined however this stem again usually haseither no or a reduced function in supporting photosyntheticleaves compared with that of an extensive-stemmed plant(see Section 23) If the plant has no recognisable above-ground leaf-supporting stem qualitatively recording it aslsquostemlessrsquo is probably more convenient from the point ofview of further analyses than quantitatively recording itsstem density as zero If a plant branches from ground level(eg many shrubs) select the apparent main branch or arandom one if they are all similar

(6) Dense woods When coring trees with very dense wood arope can be tied around the tree and fastened at the handle ofthe borerWhen the handle is turned around during coring therope will wrap itself around the borer increasing the tensionon the rope which helps push the borer bit into the tree


(7) Measuringother thanmain stem Ifwood samples cannot beremoved from a trunk or main stem wood from branches1ndash2 cm in diametermay be sampledMain-stemwood densityhas been found on average to equal 1411 branch wooddensity although this relationship can vary among species sousing it can sometimes introduce an error

(8) Components of stem volume (cell-wall material watergas) The volumetric fractions of cell-wall material waterand gas (lsquoairrsquo) in the stem can be calculated as follows Thevolume fractionofwater is simply thedecrease inweight (in g)on drying divided by the original volume (in cm3) of thesample The volume fraction that is cell-wall material equalsthe dry mass fresh volume divided by the density of dry cell-wall material (153 g cmndash3 for cell-wall material in dry wood)if an appreciable fraction of the stem volume is bark usingthis number may involve an error because the density ofbark cell-wall material is not necessarily the same as thatfor wood The fraction of the original volume that was gas issimply 1 minus the foregoing two volume fractions

References on theory significance and large datasets Putzet al (1983) Loehle (1988) Reyes et al (1992) Niklas (1994)Gartner (1995) Santiago et al (2004) Van Gelder et al (2006)Poorter et al (2008) Chave et al (2009) Patintildeo et al (2009)Anten and Schieving (2010)

More onmethods Reyes et al (1992) Brown (1997) Gartneret al (2004) Chave et al (2005) Swenson and Enquist (2008)Williamson and Wiemann (2010)

42 Twig dry-matter content and twig drying time

Twig dry-matter content (TDMC) is the oven-dry mass (mg) of aterminal twig divided by its water-saturated fresh mass (g)expressed in mg gndash1 Twig drying time is expressed in days(until equilibrium moisture) We consider TDMC to be a criticalcomponent of plant potential flammability particularly fireconductivity after ignition (see Section 212) Twigs with highdry-matter content are expected to dry out relatively quicklyduring the dry season in fire-prone regions TDMC should bepositively correlatedwith specificdensityor dry-matter content ofthe main stem across woody species (see Section 41) andnegatively correlated with potential RGR although this has toour knowledge not been tested explicitly


Collect one to three terminal (highest ramification-ordersmallest diameter-class) sun-exposed twigs from a minimumof five plants Twigs (or twig sections) should preferably be20ndash30 cm long If a plant has no branches or twigs take the mainstem in that case the procedure can be combined with that forSSD (see Section 41) For very fine strongly ramifying terminaltwigs a lsquomain twigrsquo with fine side twigs can be collected as oneunit


Wrap the twigs (including any attached leaves) in moistpaper and put them in sealed plastic bags Store these in a coolbox or fridge (never in a freezer) until further processing inthe laboratory If no cool box is available in the field and

temperatures are high it is better to store the samples in plasticbags without any additional moisture then follow the aboveprocedure once back in the laboratory

Measuring

Following the rehydration procedure (see Section 33) anyleaves are removed and the twigs gently blotted dry with tissuepaper to remove any surface water before measuring water-saturated fresh mass Each twig sample (consisting of 1ndash3twigs) is then first dried in an oven or drying room at 40C at40 relative humidity of the outside air or lower Every 24 heach sample is reweighed Twig drying time is defined as thenumber of days it takes to reach 95 of the mass reduction ofthe sample as a result of drying (interpolating betweenweightingsif necessary) where 100 is the final loss of mass when theweight of the sample ceases to decline further at the indicatedtemperature Continue until you are certain that a steady dryweight has been reached TDMC is defined (analogously toLDMC) as dry mass divided by saturated mass The final dryweight obtained at 40C is not the true dry mass of the twigbecause some bound water will remain within the cell walls ofthe material (and probably also in its protein) at this temperature(see Section 41) However the rather low drying temperatureadopted here comparedwith that in Section 41 is chosen so as toproduce in twigs a dry condition relatively similar to that from air-drying outdoors but which can be obtained relatively quickly


(i) Herbaceous plants For herbaceous plants the equivalentto TDMC is stem dry-matter content which can bemeasured in exactly the same way as LDMC but usingthe main stem of forbs or the leaf sheaths of grasses

References on theory and significance Bond andVanWilgen(1996) Lavorel and Garnier (2002) Shipley and Vu (2002)

More on methods Garnier et al (2001b)

43 Bark thickness (and bark quality)

Bark thickness is the thickness of the bark inmmwhich is definedhere as the part of the stem that is external to the wood or xylem ndash

hence it includes the vascular cambium Thick bark insulatesmeristems and bud primordia from lethally high temperaturesassociated with fire although the effectiveness depends on theintensity and duration of a fire on the diameter of the trunkor branch on the position of bud primordia within the bark orcambium and on bark quality and moisture Thick bark may alsoprovide protection of vital tissues against attack by pathogensherbivores frost or drought In general this trait has specialrelevance in trees or large shrubs subject to surface-fire regimesBe aware that the structure and biochemistry of the bark (egsuberin in cork lignin tannins other phenols gums resins) areoften important components of bark defence as well


Healthy adult plants should be sampled as indicated above(see Section 11) Measure bark thickness on a minimum of fiveadult individuals preferably (to minimise damage) on the samesamples that are used for measurements of SSD (see Section 41)


Measure this trait on main stem near the base between 10- and40-cm height because that is where surface fires occur (but seeSpecial cases or extras in the present Section) If you do not usethe same sample as forSSD cut out anewpieceof barkof at least afew centimetres wide and long Avoid warts thorns or otherprotuberances and removeanybarkpieces that havemostlyflakedoff The bark as defined here includes everything external to thewood (ie any vascular cambium secondary phloem phellodermor secondary cortex cork cambium or cork)

How to measure

For each sample or tree five random measurements of barkthickness are made with callipers (or special tools used inforestry) if possible to the nearest 01mm For species withfissured stems see Special cases or extras in the present SectionIn situ measurement with a purpose-designed forestry tool isan acceptable alternative Take the average per sample Barkthickness (mm) is the average of all sample means


(1) Bark quality In addition to bark thickness severalstructural or chemical components of bark quality may beof particular interest (see above within the present Protocol)An easy but possibly important one is the presence (1) vabsence (0) of visible (liquid or viscose) gums or resins in thebark

(2) Bark surface structure (texture) may determine the captureandor storage of water nutrients and organic matterWe suggest five broad (subjective) categories including(1) smooth texture (2) very slight texture (amplitudes ofmicrorelief within 05mm) (3) intermediate texture(amplitudes 05ndash2mm) (4) strong texture (amplitudes2ndash5mm) and very coarse texture (amplitudes gt05mm)Bark textures may be measured separately for the trunkand smaller branches or twigs because these may differgreatly and support different epiphyte communities

(3) Fissured stems In each sample take five randommeasurements of both the maximum (outside the fissure)and minimum (inside the fissure) bark thickness Thencalculate bark thickness as one-half the difference betweenthem

(4) Alternative height for measurements Typically in forestrysurveys bark thickness ismeasured at breast height (as DBH)Measuring at the base of the tree as suggested here hasadvantages (more related to fire resistance) and problems(often the base of the tree is deformed) An alternative canbe to make this measurement at ~50ndash60 cm (and in any casebark thickness at 50 cm is strongly related to bark thickness atbreast height) or directly at DBH

(5) Decorticating bark Decorticating bark is usuallyconsidered as standing litter so it is not included in bark-thickness measurements (however specific objectives mayimply its measurement)

(6) Bark investment The complementary measurement ofstem diameter can be useful to compare species for barkinvestments (by dividing the bark thickness by the stemradius)

References on theory and significance Jackson et al (1999)Brando et al (2012)

44 Xylem conductivity

Water transport fromsoil to leaves is critical to land-plant activityBy replacing the water lost to the atmosphere via transpirationwater transport prevents highly negative and damaging leafwater potentials from developing and permits continuedphotosynthesis The efficiency of water transport is quantifiedas the stem-specific xylem hydraulic conductivity (KS) the rateof water flow per unit cross-sectional xylem area and per unitgradient of pressure (kgmndash1 sndash1MPandash1) It can also be quantifiedas the LA-specific xylem hydraulic conductivity (KL) the rate ofwater flow per unit of supported LA per unit pressure gradient(kgmndash1 sndash1MPandash1) KL expresses the conductivity of a branchrelative to the transpiration demand of the foliage that the branchservices Xylem refers here to the conducting tissue in the stemand for trees equates to sapwood KL equals KS divided bythe ratio between LA and sapwood cross-sectional area (seeSection 28) KS is a function of the numbers and conductivityof individual xylem conduits and their interconnections via pitmembranes per unit of xylem cross-sectional area Conductivityof an individual conduit increases with the fourth power of itslumendiameter (as canbemodelled usingPoiseuillersquos lawofflowin cylindrical tubes)


The measurement may conveniently be made indoors onstem samples brought in from the field provided they are keptcool and with their ends in water The tested branches should belonger than the length of the longest conducting element in thexylem so that themeasurement includes the resistivity of both theconduits and the inter-conduit connections Conduit length variesgreatly across species gymnosperms having short tracheids(usually less than 1 cm long) whereas in some angiospermsvessel length can reach more than a metre (although it is usually10ndash30 cm see Point 1 of Special cases or extras in the presentSection) Segments ~30 cm long are commonly usedKS typicallydeclines towards the leaves because of tapering of vesselnumbers and sizes In most cases measurements are made onstems collected from the outer canopy and so they can beconsidered the minimum conductivity for the stem part of thewater-transport system

Measuring

These methods have been developed largely for the stems ofwoody plants for which the methods are simplest Analogousmethods have however been devised for herbaceous plantsleaves and roots Relatively sophisticated types of apparatusfor performing xylem-conductivity measurements have beendescribed however in many cases simple systems built fromordinary laboratory equipment can be used In the simplest case aknown pressure head is applied to push a 10mM KCl solution(use filtered degassed water) through a stemwith a known cross-sectional area and no (or with sealed-off) side branches At thedistal end of the segment a collecting container catches emergingliquid and after known time intervals its volume is determined


either directly or gravimetrically Conductivity is then calculatedas

KS frac14 J L A1 DY1

where J is the rate of water flow through the stem (kg sndash1) L is thelength of the segment (m)A is themeancross-sectional area of thexylemof the stem (m2) (to afirst approximation the averageof theareas at the two ends of the segment) and DY is the pressuredifference (MPa) between the upstream and downstream ends ofthe segment If the stem segment is held vertically during themeasurement its length (in m) divided by 10 should be added tothe applied pressure (MPa) (but not if the segment is horizontal)KS is typically reported in kgm

ndash1 sndash1MPandash1 With measurementson terminal or subterminal branch segments one can usuallyassume that A is the entire xylem cross-sectional area (all of thexylem being conductive) however with larger branches ortree trunks A would have to be the conducting xylem area ata maximum the sapwood area however often an even smallerarea is actually conductive which is not easy to determine forroutine measurements

Xylemconductance refers to the capacity of a vascular systemwith whatever length and cross-sectional area it happens to haveto transport water under a unit pressure difference Conductancecanbecalculated from the aboveequationby simplyomitting itsAand L terms


(1) Length of vessels This is needed to ensure that the length ofstem segments used for conductance measurements exceedsthat of their vessels To determine themaximum length of thevessels cut several stems in the field (see Fig 7 for furtherdetails on the procedure) In the laboratory from the upperend of one of these remove a portion such that the segmentthat remains is likely to be a little longer than the length of itslongest vessel Proceed as indicated in Fig 7

References on theory significance and large datasetsZimmermann and Jeje (1981) Brodribb and Hill (2000)Meinzer et al (2001) Zwieniecki et al (2001) Tyree andZimmermann (2002) Sperry (2003) Cavender-Bares et al(2004) Maherali et al (2004) Santiago et al (2004)Holbrook and Zwieniecki (2005) Sperry et al (2008a)

References on methods Sperry et al (1988) North and Noble(1992) Alder et al (1996) Kocacinar and Sage (2003) Sack andHolbrook (2006)

45 Vulnerability to embolism

The xylem vulnerability to embolism indicates the risk of loss ofwater transport during drought Vulnerability is expressed as thepercentageof thewater-saturatedxylemconductance that is lost atgiven stem-water potentials The water stream in the conduits isunder tension which can in some cases become as high as 100times the atmospheric pressure Any air entry into a water-conducting element will dissipate the tension in it and quicklyexpand (becoming an air embolism) effectively blocking waterflow through that element The more such emboli develop thegreater the loss of xylem conductance The ability of a species to

tolerate highlynegativewater potentials (=high tensions)withoutembolising varies greatly among species (cf Section 315) and isan important aspect of drought tolerance


Follow the above instructions for collecting for measurementof xylem conductivity

Measuring

Vulnerability to embolism is quantified by constructing axylem vulnerability curve This consists of plotting measuredvalues of xylem conductance on the y-axis against thevalues of stem water potential (Y) on the x-axis at whichthese conductance values (represented as a percentage of themaximum water-saturated conductance) were obtained Theshape of this curve is usually sigmoid To characterise thiscurve by one number the value of Y at its mid-point (50loss of conductance) is commonly used

Stem segments with differentY values can be obtained by anyof three possible methods

(1) Evaporative dehydration A large branch that includessome lateral secondary branches is cut from the plant andkept unwrapped during the day or longer for its xylem topartially dehydrate and develop tension as a result oftranspiration from its leaves Y is determined periodicallyby the pressure chamber method (cf Section 315) onsecondary branches that are removed at intervals for thispurposeWhen a targetY has been reached conductance of asegment of the main axis near the last-removed lateral ismeasuredWater is then flushed through this segment brieflyunder a pressure high enough to displace all air embolismsfrom it (see references cited below) and its conductance isre-measured to obtain the maximum conductance of thesegment This affords a value for the percentage loss ofconductance at that particular Y Separate branches aresimilarly tested to obtain conductance-loss values for othervalues of Y

(2) Centrifugation Attaching a short stem segmenthorizontally (and symmetrically) across the top of acentrifuge rotor and spinning it generates tension in thewater within the xylem conduits From the centrifugalforce that was applied a corresponding (negative) Y or Pcan be inferred Advantages of the method are that stemsegments can be brought to different effective waterpotentials very quickly However the technique is difficultfor stem segments longer than ~20 cm or ones that cannot bereliably attached to an available rotor and it cannot be usedfor segments longer than the width of the rotor chamberDetails needed for actually employing this method are givenin some of the cited references

(3) Air injection This simple method is based on the principleof substituting internal tension by external positive airpressure which is applied to a stem segment that islocated within a pressure chamber The procedure uses aspecial pressure chamber designed for a stem segment to passcompletely through it allowing a conductance measurementwhile the external pressure is applied This type of chamberis commercially available from the PMS Instrument Co


Albany Oregon USA (httppmsinstrumentcom accessed15 February 2013)

References on theory significance and large datasets Tyreeand Sperry (1989) Davis et al (2002) Brodribb et al (2003)Maherali et al (2004) Holbrook and Zwieniecki (2005) Choatet al (2007) Feild and Balun (2008) Sperry et al (2008a)

References on methods Cochard et al (1992) Sperry et al(1988 2008b) Alder et al (1997) Pammenter and VanderWilligen (1998)

5 Below-ground traits

Variation in root traits among species has large ramifications fortheir ecology Fine roots are the primary organs for water andnutrient acquisition and they are also responsible for transferringresources between below-ground and above-ground parts Fineroots can acquire resources directly or through symbionts Whenaccessing soil resources directly fine roots selectively acquiremineral nutrients from a complex range of soil solutions andsoil particles while modifying soil chemistry via exudation of arange of compounds Roots also must respond to heterogeneityof resource availability at multiple spatial and temporal scaleswhile resisting attacks from a wide range of organisms andenvironmental stresses

There is no single evolutionary solution to all the variablechallenges of acquiring soil resources in different ecosystemsMeasuring several independent root traits in combination may

help better understand the below-ground strategies of multiplespecies Here we describe three main sets of traits

51 Specific root length

Specific root length (SRL) the ratio of root length to dry mass offine roots is the below-ground analogue of SLA (see Section 31)providing a ratio of a standard unit of acquisition (root length) toresource investment (mass) Plants with high SRL build moreroot length for a given dry-mass investment and are generallyconsidered to have higher rates of nutrient and water uptake (perdry mass) shorter root lifespan and higher RGRs than for low-SRL plants Yet high SRL can result from having a low diameteror low tissue density each of which is independently associatedwith different traits For example thin roots exert less penetrativeforce on soil and transport less water whereas roots with lowtissue density have lower longevity but greater rates of uptakeunder high nutrient conditions As there is little operationaldifference in measuring just SRL or its two components werecommend that both metrics be measured when measuring thefunctional traits of roots


Roots are often measured in aggregate when comparing thelive fine roots of plants although individual fine roots or smallnumbers of fine roots can be enough to measure root diameter orbranching order Separating fine roots according to the timing

stem

debarkedend

(a)

stem segment

air under pressurewith syringe

rubber tubing

receptacle with water

(b)

stem

debarkedend

bubbles emerging

Fig 7 Procedure to determine the vessel length (a) A segment of approximately the probable length of the given species vessels is cut from the stem of interestandbark is removed from its basal end (b)A rubber tubingof a suitable tight-fittingdiameter is slippedover the segmentrsquos debarkedend anda relatively large (eg50mL) air-filled syringe is attached to the free end of this tubingWith the syringersquos plunger amild above-atmospheric air pressure is generatedwithin the tubingwhile holding the segmentrsquos free endunderwater If bubbles emerge from that end at least one vessel has been cut at both ends of the segment allowing air tomovefreely through the segmentrsquos entire length (if nobubblesemergeproceedasdescribedbelow)Repeat this test usingprogressively longer stemsegmentsuntil one isfound fromwhich bubbles donot emergeunder pressure From this segment cut off successively shorter slices and after each cut retest the segment until bubblesare first seen to emerge The segmentrsquos length at this point is just shorter than the length of its longest vessel If however no bubbles emerge from the first stemsegment that is tested the stemrsquos longest vessels are shorter than that segmentrsquos length so relatively long pieces must be cut successively from it until bubbles doemerge Then a segment slightly longer that this (but one that does not emit bubbleswhenfirst tested) can be used to come close to themaximumvessel length as atthe end of the foregoing procedure


or depth of sampling could be informative to answer particularquestions Roots measured in the field span a range of root agewhereas roots acquired from ingrowth cores or young plantswould constrain age The basis of comparison should be clear androot acquisition andpreparation consideredeach timeRoots fromthe top 20 cm are the standard basis of comparison however theactual depth sampled should be allowed to be as varied as theheight above ground from which to collect leaves

In mixed-species assemblages fine roots should be tracedback to shoots for positive identification This is not necessary inuniform stands and roots can often be distinguished amonga small number of species For small plants it is often mostfeasible to excavate the entire plant to be washed out lateraiding in identification A typical amount of root necessary formeasurement generally fits in the palm of your hand In general itis better to have a small amount of root that is better prepared thana larger amount of less well prepared root Preferably atypicallylarge or small individuals should be avoided


Unwashed roots can generally be stored under humid chilledconditions for a week with little degradation of structureWashing techniques should be gentle for species with low-density roots whereas more rigorous washing might be moresuitable for high-density roots in soils with heavy clays or coarseorganic matter that could compromise measurements Washingroots from a sandy soil can require as little as 30 s under a hosewhereas clearing roots of organic matter from a tundra soil mightrequire hours of painstaking plucking In general cleaningroots will require a combination of running water over a finemesh sieve (02ndash1mm) to remove fine heavy particles such assand rinsing in containers of water to remove coarser heavyparticles such as pebbles and plucking of debris with forceps toremove contaminants that are of a similar size and density as theroots of interest Often roots have to be finger-massaged andindividual roots separated to allow particles to be removed Ifsome fine particles such as clays are too difficult to remove rootscan be ashed at 650C later and ash mass subtracted from grossroot dry mass Washed roots can be stored in a 50 ethanolsolution for longer periods of time A useful rule of thumb is tostop washing roots when it appears that you are losing as much ofthe fine roots as you are removing soil or preferably slightlybefore

Measuring

If necessary under a dissecting microscope sort apparentlylive healthy roots from the recently washed sample Live rootsgenerally have a lighter fully turgid appearance compared withdead or dying roots of the same species which appear darker andfloppy or deflated however note that live and dead roots may notbedistinguishable bycolour Itwill help to observe a rangeof agesand colours of absorptive roots for each plant species beforemeasurement so as to properly identify healthy live roots Forwoody species roots are often divided by root (ramification)order to better standardise comparisons across species

Once roots have been obtained and prepared determiningSRL diameter and tissue density requires digitising the roots and

measuring their length and diameter Digitisation can be carriedout with almost any low-end flatbed scanner A scanner with aresolution of 1600 dpi provides a resolution of 15mm which isstill half the width of the finest roots of any plant Nevertheless ascanner with lower resolution may also work A scanner that hasa transparency adaptor that illuminates items on the scanner bedfrom above is recommended to provide crisp root images Rootsare best imaged while submerged in a small amount of waterwhich also aids in teasing individual roots apart A clear plastictray works well There should be no need to stain most roots toimage them After scanning scanned roots should be dried (48 hat 60C) andweighed These root samples can also be ground andanalysed for nutrient concentrations

When roots have been scanned units of root length need to betraced and their diameter determined For a small number ofroots this can be carried out with image-analysis software (seeSection 31 for free software) For a large number of samples orroot length the commercially available application WinRhizo(Reacutegent Instruments Quebec Canada) is recommended Thesoftware will automatically determine the length diameter androot volume distribution of a sample of root length enabling easycalculations of SRL average root diameter and root tissue density(root dry mass over volume the latter being derived from lengthand radius) Although the software is expensive for occasionaluse roots can be scanned independent of analysis software savedin the JPEG format and analysed later by someone who ownsthe software See under Special cases and extras in the presentSection for manual methods when none of the above facilities isavailable


(1) Root diameter and tissue density Not all roots of a givendiameter and tissue density have similar cellular structureRoots can vary in their relative proportions of cortex and stele(mainly phloem and xylem) as well as the construction ofeach For these reasons secondary to measuring grossmorphology of fine roots we also recommend cross-sectioning roots so as to determine their cellular structureFor this purposemultiple roots of each species are embeddedin a polymer cut on a microtome into 4-mm slices stainedwith toluidine blue which stains lignin bluendashgreen andcellulose purple or redndashviolet and then mounted on aglass slide Digital images are made for each species usinglight microscopy at 100 magnification and the cross-sectional areas of the root stele endodermis and largexylem elements are determined by tracing each portion ofthe root manually in image-analysis software With thesedata cell diameters and amounts of different tissues can becalculated relative to one another and to total cross-sectionalarea

References on theory significance and large datasetsEissenstat and Yanai (1997) Wahl and Ryser (2000) Steudle(2001) Pregitzer et al (2002) Roumet et al (2006) Craine(2009) Paula and Pausas (2011)

More on methods Newman (1966) Tennant (1975) Boumlhm(1979) Fitter (1996) Bouma et al (2000) Craine et al (2001)Craine (2009)


52 Root-system morphology

Characteristics of entire root systems can be independent ofindividual roots and need to be measured explicitly There arethree main traits of root systems that are best measured depthlateral extent and intensity of exploration For rooting depththe simplest metric to determine is maximum rooting depth(maximum soil depth from which resources can be acquiredranging from a few centimetres to tens of metres) The maximumlateral extent of roots defines the distance from the centre of theplant that roots can acquire resources from It also determines theability of plants to interact with spatial heterogeneity in soilresources The amount of fine-root biomass or root length perunit soil volume indicate the intensity of soil exploration and theability of a species to compete for soil nutrients

Thedepthdistributionof roots combinesdepth and intensity ofutilisation of soil Depth distributions are a better indicator of therelative relianceofplantsondifferent depths for soil resources anddefine their vertical distribution of influence on soil activity Ingeneral it is simpler to determine the root biomass with differentdepths whereas understanding root length with depth is likely tobe a better metric to understand the competitive ability of uptakecapacity for example In general biomass and length with depthwould be strongly correlated if there were no change in SRLwithdepth

Note that root tissue density and root diameter are positivelyrelated to longevity and negatively related to nutrient uptake Inaddition root tissue density is positively related to resistance topathogens and drought

Collection and analysis

Determining the maximum extent of roots depends on thespecies Excavating entire plants is reasonable for some shallow-rooted species For more deep-rooted species a pit must be dugand a cross-section of the soil from a pit face excavated In someextreme cases roots have to be accessed from caves or boreholes

Depth distributions can be determined by digging pits if aknown cross-sectional area can be excavated with depth Withpits a deep pit is dug and one pit face is smoothed vertically Thena cross-section is removedwith a flat shovel Root systems can beremoved in entirety or in sections In other cases an auger of5ndash10-cm diameter should be used to remove biomass with depthTypical depth distributions follow a somewhat exponentialrelationship A standard set of depths would be 5 10 20 4080 120 and 200 cm for root systems largely confined to the top2m of soil Incomplete root-depth distributions can be used toestimate maximum rooting depths however this will dependon the pattern of root biomass with depth For some speciesdepth distributions can be determined randomly relative to theindividual or at a point that represents the midpoint of its lateralextent whereas biomass will have to be determined directlybelow individuals for species with a tap root To determinelateral extent of root systems a horizontal strip of soil can beexcavated starting at the centre of the plant so as to trace rootsoutward In other caseswhere individuals are bunched the lateralextent of roots is likely to be equivalent to half the interplantdistance although this should be verified

Once soils have been excavated root storage andwashing usethe sameprotocols as described above Intact root systems arebest

laid out on a large mesh screen to be washed out with runningwater andor submerged in large tubs Confirming the identity ofspecies might require anatomical or molecular comparisons withother roots of that species however it ismost easily carried out bytracing roots back to their above-ground parts or sampling inconspecific stands If depthdistributions are tobedeterminedfine(lt2mm) and coarse roots should be separated Subsamples ofcleaned fine roots can then be scanned if desired for diameterlength and volume analyses Regardless root biomass should bedried and weighed for biomass distributions


(1) Large shrubs and trees When sampling larger shrubs andtrees the researcher will encounter thicker woody roots Thebest way to deal with this is to use a specialised wood-cuttingaugerWithin the coarse root fraction those root sections thatare obviously particularly important for mechanical supportor resource storage usually exceeding10mmindiameter arebest kept separate from the relatively thin sections They canstill be combined for certain analyses later on

(2) Clayey soil If the soil is particularly clayey aggregated orcontains calcium carbonate consider adding a dispersalagent (eg sodium hexametaphosphate) to the washingwater The best washing additive varies depending on theparticular condition of the soil

References on theory and significance Adiku et al (2000)Zwieniecki et al (2002) Hodge (2004) Schenk and Jackson(2002) Dunbabin et al (2004) Withington et al (2006) Craine(2009) Lambers et al (2011)

More on methods Boumlhm (1979) Caldwell and Virginia(1989) Jackson (1999) Linder et al (2000) Schenk andJackson (2002)

53 Nutrient-uptake strategy

The performance of different species in nutrient-limitedecosystems is undoubtedly affected by their inherentinterspecific differences in nutrient-uptake capacity One factoraffecting this is SRL (see Section 51) however quantitativedifferences in the affinities of the ion-transport carrier and in thecapacities of the roots among different species are superimposedon this Furthermore many plants exploit symbiotic associationswith bacteria or fungi to enhance their nutrient-competitiveability which is the subject of the present Section

Symbioses with N2-fixing bacteria or mycorrhizal fungi havebeen found in many plant species However literature is stronglybiased towards temperate species of Europe and North AmericaThere is still much to be learned about nutrient-uptake strategiesof species in less studied regions as exemplified by the recentdiscovery of specialised N-foraging snow roots in the CaucasusWe provide specific protocols in Material S2 that deal with thefollowing strategies

(1) N2-fixing bacteria ndash association with bacteria in nodules tofix atmospheric N2

(2) arbuscular mycorrhizae ndash symbiosis with arbuscularmycorrhizal fungi to aid in acquisition of nutrients andwater


(3) ecto-mycorrhizae ndash symbiosis with ecto-mycorrhizal fungito aid in uptake of inorganic nutrients and organic forms ofN

(4) ericoid-mycorrhizae ndash symbiosis with ericoid mycorrhizalfungi to aid in uptake of organic forms of N

(5) orchids ndash symbiosis with orchid mycorrhizal fungi foracquiring nutrients from litter

(6) rarer types of mycorrhizae eg arbutoid mycorrhizae(Arbutus Arctostaphylos) ecto-endo-mycorrhizae (certaingymnosperms) and pyroloid mycorrhizae (Pyrolaceae)

(7) myco-heterotrophic plants without chlorophyll that extractC and probablymost nutrients from dead organicmatter viamycorrhizal fungi

(8) root or stem-hemiparasitic green plants such as mistletoes(Loranthaceae) that extract nutrients such as N and P fromthe roots or stems of a host plant

(9) holoparasitic plants without chlorophyll that extract C andnutrients directly from a host plant

(10) carnivorous plants that capture organic forms of N and Pfrom animals

(11) hairy root clusters (proteoid roots) dauciform roots insedges and capillaroid roots in rushes that aid in P uptake

(12) other specialised strategies (mostly in epiphytes) including(a) tank plants (ponds) ndash nutrient and water capture and

storage(b) baskets ndash nutrient and water capture and storage(c) ant nests ndash nutrient uptake and storage(d) trichomes ndash nutrient and water capture through

bromeliad leaves and(e) root velamen radiculum ndash nutrient and water capture

and storage and

(13) none ndash no obvious specialised N- ort P-uptake mechanismuptake presumably directly through root hairs (or throughleaves eg in the case of certain fernswith very thin fronds)

Although most of the experimental work on the active uptakeof nutrient ions by roots has been carried out on agriculturalplants some information on that of wild plants is availablebut again relatively little for tropical and subtropical speciesThe radioisotope techniques for characterisation of ion-transportmechanisms are beyond the scope of the present handbookbut can be readily ascertained by consulting the ion-transportliterature

References on theory and significance see Material S2 inaddition for snow roots see Onipchenko et al (2009)

6 Regenerative traits

61 Dispersal syndrome

The mode of dispersal of the lsquodispersulersquo (or propagule = unit ofseed fruit or spore as it is dispersed)hasobvious consequences forthe distances it can cover the routes it can travel and its finaldestination

How to classify

This is a categorical trait Record all categories that areassumed to give significant potential dispersal (see Box 4) inorder of decreasing importance In the case of similar potentialcontributions prioritise the one with the presumed longer-distance dispersal eg wind dispersal takes priority over antdispersal

It is important to realise that dispersulesmay (occasionally) gettransported by one of the above modes even though they haveno obvious adaptation for it This is particularly true for endo-zoochory and exo-zoochory Note that there is ample literature(eg in Floras) for dispersal mode of many plant taxa

References on theory significance and large datasets Howeand Smallwood (1982) Van der Pijl (1982) Bakker et al (1996)Howe andWestley (1997)Hulme (1998) Poschlod et al (2000)

Box 4 Dispersal syndromes

(1) Unassisted dispersal the seed or fruit has no obvious aids for longer-distance transport and merely falls passively from the plant

(2) Wind dispersal (anemochory) includes (A)minute dust-like seeds (eg Pyrola Orchidaceae) (B) seeds with pappus or other long hairs (eg willows(Salix) poplars (Populus)manyAsteraceae) lsquoballoonsrsquoor comas (trichomes at the endof a seed) (C)flattened fruits or seedswith large lsquowingsrsquo as seen inmany shrubs and trees (eg Acer birch (Betula) ash (Fraxinus) lime (Tilia) elm (Ulmus) pine (Pinus)) spores of ferns and related vascular cryptogams(Pteridophyta) and (D) lsquotumbleweedsrsquo where thewhole plant or infructescencewith ripe seeds is rolled over the groundbywind force therebydistributingthe seeds The latter strategy is known fromarid regions egBaptisia lanceolata in the south-easternUSAandAnastatica hierochuntica (rose-of-Jericho)in northern Africa and the Middle East(3) Internal animal transport (endo-zoochory) eg by birds mammals bats many fleshy often brightly coloured berries arillate seeds drupes and bigfruits (often brightly coloured) that are evidently eaten by vertebrates and pass through the gut before the seeds enter the soil elsewhere (eg holly (Ilex)apple (Malus))(4) External animal transport (exo-zoochory) fruits or seeds that become attached eg to animal hairs feathers legs bills aided by appendages such ashooks barbs awns burs or sticky substances (eg burdock (Arctium) many grasses)(5) Dispersal byhoarding brownor green seedsor nuts that arehoardedandburiedbymammals or birdsTough thick-walled indehiscent nuts tend tobehoarded by mammals (eg hazelnuts (Corylus) by squirrels) and rounded wingless seeds or nuts by birds (eg acorns (Quercus spp) by jays)(6) Ant dispersal (myrmecochory) dispersules with elaiosomes (specialised nutritious appendages) that make them attractive for capture transport anduse by ants or related insects(7) Dispersal by water (hydrochory) dispersules are adapted to prolonged floating on the water surface aided for instance by corky tissues and lowspecific gravity (eg coconut)(8) Dispersal by launching (ballistichory) restrained seeds that are launched away from the plant by lsquoexplosionrsquo as soon as the seed capsule opens (egImpatiens)(9) Bristle contraction hygroscopic bristles on the dispersule that promote movement with varying humidity


McIntyre and Lavorel (2001) Tackenberg et al (2003) Myerset al (2004)

More on methods Howe and Westley (1997) Forget andWenny (2005) Pons and Pausas (2007)

62 Dispersule size and shape

Of interest is the entire reproductive dispersule (= dispersalstructure or propagule) as it enters the soil The dispersule maycorrespondwith the seed however inmany species it constitutesthe seed plus surrounding structures eg the fruit Dispersule sizeis its oven-dry mass Dispersule shape is the variance of its threedimensions ie the length the width and the thickness (breadth)of thedispersule after eachof these valueshasbeendividedby thelargest of the three values Variances lie between 0 and 1 and areunitless Small dispersules with low shape values (relativelyspherical) tend to be buried deeper into the soil and live longerin the seed bank Seed size and shape are then fundamental forseed persistence in the soil (seed-bank persistence)


The same type of individuals as for leaf traits and plant heightshould be sampled Of interest is the unit that is likely to enter thesoil Therefore only parts that fall off easily (eg pappus) areremoved whereas parts such as eg wings and awns remainattached The flesh of fleshy fruits is removed too because theseeds are usually the units to get buried in this case (certainly ifthey have been through an animal gut system first) The seeds(or dispersules) should be mature and alive The dispersules caneither be picked off the plant or be collected from the soil surfaceIn some parts of the world eg in some tropical rain forest areasit may be efficient to pay local people specialised in tree climbing(and identification) to help with the collecting


Store the dispersules in sealed plastic bags and keep in a coolbox or fridge until measurement Process and measure as soonas possible For naturally dry dispersules air-dry storage is alsookay

Measuring

Remove any fruit flesh pappus or other loose parts (see abovein the present Section) For the remaining dispersule take thehighest standardised value for each dimension (length width andthickness) using callipers or a binocularmicroscope and calculatethe variance Then dry at 60C for at least 72 h (or else at 80C for48 h) and weigh (= dispersule size)


We recommend complementing this trait with other direct orindirect assessment of banks of seeds or seedlings for futureregeneration of a species For seed-bank assessment there aregood methods to follow (see More on methods below in thepresent Section) however (above-ground) canopy seeds banksof serotinous species of fire-prone ecosystems (eg Pinus andProteaceae such as Banksia Hakea and Protea) and long-livedseedling banks of woody species in the shaded understorey ofwoodlands and forests may also make important contributions

Viviparyas in somemangroves couldalsobepart included in suchassessments

References on theory significance and large datasets Hendryand Grime (1993) Thompson et al (1993 1997) Leishman andWestoby (1998) Funes et al (1999) Weiher et al (1999) Pecoet al (2003)

More onmethods Hendry andGrime (1993) Thompson et al(1993 1997) Weiher et al (1999) Pons and Pausas (2007)

63 Dispersal potential

Dispersal potential is defined as the proportion of dispersulesproduced by one individual that travelz a certain distance whichcan be chosen arbitrarily depending on the question Thedispersules may be seeds or fruits or vegetative propagules Incontrast to dispersal syndrome dispersal potential allows theassessment of dispersability of a seed in relation to distance Itvaries not only among species but also strongly among specieswith the same dispersal syndrome Therefore it is a crucialvariable when asking if dispersal is limiting the occurrence ofa species in suitable habitats or species richness of plantcommunities or if fragmentation is a threat to the survival ofspecies or populations The capacity to survive in disturbedhabitats or in fragmented landscapes is often correlated with ahigh dispersal potential Both seed production and also seedcharacters may be correlated with dispersal potential Themore seeds are produced the higher the probability that oneseed spans larger distances The seed characters such as egmassform and structure of seed surface responsible for a high dispersalpotential depend on the dispersal vector There may be a tradeoff between dispersal potential (in space) and maximum plantlifespan as well as seed-bank persistence (dispersal in time)Long-lived species often exhibit a low dispersal potential asdo species with a long-term persistent seed bank

How to record

Dispersal potential is a continuous variable and may berecorded either by direct measurements in the field or canbe identified by measurements of traits related to the dispersalpotential or by modelling approaches Wind-dispersal potentialis correlated with dispersule-releasing height and terminalvelocity dispersal potential by water to buoyancy of thedispersules and animal-dispersal potential to either attachmentpotential or survival after digestion Dispersal by humansmachines or vehicles is very complex Measuring dispersalpotential therefore requires studies adapted to the specificquestion

Measurements should be carried out on the intact dispersuleie seed or fruit with all the structures such as eg pappus andawns that are still attached when it is released Releasing heightshould bemeasuredduringdispersule release and is the differencebetween the highest elevation of the seed or fruit and the base ofthe plant Terminal velocity is measured on freshly collected air-dry dispersules and most simply by the actual rate of fall in stillair Floating capacity (proportion of dispersules floating after adefined time) is measured by putting dispersules in glass beakersthat are placed on a flask shaker moving with a frequency of100minndash1 Attachment capacity (proportion of dispersules stillattached after a defined time) is measured by putting seeds on


the respective animal fur which is then shaken by a shakingmachine Survival after digestion is measured either by digestionexperiments with the respective animals or by simulatingingestion by a standardised mechanical treatment and digestionby a standardised chemical treatment which have to be calibratedby digestion experiments

To assess animal-dispersal potential field studies shouldbe added where possible because the behaviour of animals(eg selection of species by grazing animals) stronglyinfluences dispersal potential Predicting animal-dispersalpotential requires process-based models with the ability topredict over a range of scenarios


(1) For water plants seed releasing height is the distancebetween the highest point of seeds or fruits andwater surface

(2) Secondary process eg dispersal by wind on the groundmay strongly affect dispersal potential Such processes areoften obvious only from field studies and may require theestablishment of additional new methods

References on theory and significance Bruun and Poschlod(2006) Poschlod et al (1998 2005) Tackenberg (2003)Tackenberg et al (2003) Schurr et al (2005) Will andTackenberg (2008) Cousens et al (2010)

More on methods Fischer et al (1996) Roumlmermann et al(2005a 2005b 2005c)

64 Seed mass

Seed mass also called seed size is the oven-dry mass of anaverage seed of a species expressed in mg Stored resources inlarge seeds tend to help the young seedling to survive andestablish in the face of environmental hazards (eg deepshade drought herbivory) Smaller seeds can be produced inlarger numbers with the same reproductive effort Smaller seedsalso tend to be buried deeper in the soil particularly if theirshape is close to spherical which aids their longevity in seedbanks Interspecific variation in seed mass also has an importanttaxonomic component more closely related taxa being morelikely to be similar in seed mass


The same type of individuals as for leaf traits and plant heightshould be sampled ie healthy adult plants that have their foliageexposed to full sunlight (or otherwise plants with the strongestlight exposure for that species) The seeds should be matureand alive If the shape of the dispersal unit (eg seed fruit) ismeasured too (see Section 62 above) do not remove any partsuntil dispersule measurement is finished We recommendcollecting at least 10 seeds from each of 10 plants of a speciesalthough more plants per species is preferred Depending on theaccuracy of the balance available 100 or even 1000 seeds perplant may be needed for species with tiny seeds (eg orchids)

In some parts of the world eg in some tropical rain forestareas it may be efficient to work in collaboration with localpeople specialised in tree climbing to help with collecting (andidentification)


If dispersule shape is also measured then store cool in sealedplastic bags whether or not wrapped in moist paper (see Section31) and process andmeasure as soon as possible Otherwise air-dry storage is also appropriate

Measuring

After measurements of dispersule shape (if applicable)remove any accessories (wings comas pappus elaiosomesfruit flesh) but make sure not to remove the testa in theprocess In other words first try to define clearly which partsbelong to the fruit as a whole and which belong strictly to theseed Only leave the fruit intact in cases where the testa and thesurrounding fruit structure are virtually inseparableDry the seeds(or achenes single-seeded fruits) at 80C for at least 48 h (or untilequilibriummass in very large or hard-skinned seeds) and weighBe aware that once taken from the oven the samples will take upmoisture from the air If they cannot beweighed immediately aftercooling down put them in the desiccator until weighing or elseback in the oven to dry off again Note that the average number ofseeds fromone plant (whether based onfive or 1000 seeds) countsas one statistical observation for calculations of mean standarddeviation and standard error


(1) Within individual variation Be aware that seed size mayvarymorewithin an individual than among individuals of thesame species Make sure to collect lsquoaverage-sizedrsquo seedsfromeach individual and not the exceptionally small or largeones

(2) Available databases Be aware that a considerable amountof published data are already available in the literature andsome of the large unpublished databases may be accessibleunder certain conditionsMany of these data can probably beadded to the database however make sure the methodologyused is compatible

(3) Seed volume There are also many large datasets forseed volume often measured as p6L1L2L3 (ieassuming an ellipsoidal shape) Most of these databasesactually include both seed mass and volume Using theappropriate calibration equations those data can be alsosuccessfully used

(4) Additional measurements For certain (eg allometric)questions additional measurements of the mass of thedispersule unit or the entire infructescence (reproductivestructure) may be of additional interest Both dry and freshmass may be useful in such cases

References on theory significance and large datasetsMazer (1989) Seiwa and Kikuzawa (1996) Reich et al(1998) Cornelissen (1999) Leishman et al (2000) Westobyet al (2002) Moles et al (2005) Moles and Westoby (2006)Wright et al (2007)

More onmethods Hendry andGrime (1993) Thompson et al(1997) Westoby (1998) Weiher et al (1999) Wright et al(2007)


65 Seedling functional morphology

Seedling functional types refer to morphology of seedlings inrelation to cotyledon function and position It is a categorical traitthat can be used to characterise plant regenerative strategiesThe distribution of seedling traits across families is still ratherpoorly known although the importance of seedling traits insystematics was recognised quite early This trait has beencreated on the basis of woody species (trees and shrubs) andhas been mainly used in tropical forests Garwood (1996)established the following five seedling categories based onthree cotyledon characters of presumed ecological significance(position texture and exposure) (Fig 8)

(1) cryptocotylar hypogeal with reserve storage cotyledons(CHR)

(2) cryptocotylar epigealwith reserve storage cotyledons (CER)(3) phanerocotylar epigeal with foliaceous cotyledons (PEF)(4) phanerocotylar epigeal with reserve storage cotyledons

(PER) and

(5) phanerocotylar hypogeal with reserve storage cotyledons(PHR)

These categories result from the combination of differentpossibilities in relation to cotyledon exposure (phanerocotylar orcryptocotylar) position (epigeal or hypogeal) and function(foliaceous or reserve storage see below within the presentProtocol) Although eight combinations are potentially possiblecryptocotylar foliaceous seedling types are biologically notpossible and phanerocotylar hypogeal foliaceous seedlings havenot yet been reported Seedling functional types are correlatedwith other plant traits such as seed size eg large seed sizes arerelated to reserve storage seedling types whereas small seed sizesare related to foliaceous and photosynthetic cotyledons Becausethe above-mentioned types have been particularly identified fortropical forests the occurrence and proportion of each type in otherecosystems should be tested


Because this is not a plastic trait we recommend germinatingsufficient number of seeds to obtain about five seedlings perspecies Seeds without evidence of for example pathogendamage and predation must be selected among the collectedones to run the experiments

Measuring

Seedling morphology (cotyledon exposure position andfunction) should be described when at least five individualshave developed at least three leaves each Species are thenassigned to the seedling morphological categories indicatedabove in the present Section (and in Fig 8)

cotyledon exposure ndashphanerocotylar if the seed coat opens andtwo cotyledons emerge from seed cryptocotylar if cotyledonsremain within the seed coatcotyledon position ndash epigeal when hypocotyl develops at least2 cmabove soil surface hypogealwhenhypocotyl developsonthe soil surface orcotyledon function ndash reserve cotyledon are fleshy foliaceouscotyledons (also called paracotyledons) are primarilyphotosynthetic


(1) Chlorophyll in fleshy cotyledons In many cases fleshycotyledons do contain chlorophyll however they are stillconsidered reserve organs (eg Aspidosperma spp)

References on theory significance and large datasets Ng(1978) De Vogel (1980) Hladik and Miquel (1990) Garwood(1996) Kitajima (1996) Wright et al (2000) Ibarra-Manriquezet al (2001) Zanne et al (2005) Leck et al (2008)

66 Resprouting capacity after major disturbance

The capacity of a plant species to resprout after destruction ofmost of its above-ground biomass is an important attribute forits persistence in ecosystems where recurrent major disturbancesare common Fire (natural or anthropogenic) hurricane-forcewind and logging are the most obvious and widespread majordisturbances however extreme drought or frost events severe

CERCHR

PEF

PERPHR

Fig 8 Seedling functional types Five seedling functional types asdescribed in Section 64 (leaves in white cotyledons in grey seeds inblack) Seedling functional morphology PHR=phanerocotylar hypogealwith storage cotyledons PER= phanerocotylar epigeal with storagecotyledons PEF= phanerocotylar epigeal with foliaceous cotyledonsCHR= cryptocotylar hypogeal with storage cotyledons and CER=cryptocotylar epigeal with storage cotyledons


grazing browsing or trashing by large herbivores landslidesflooding and other short-term large-scale erosion eventsalso qualify There appear to be ecological trade-offs betweensprouters and non-sprouter plants Comparedwith non-sprouterssprouters tend to show a larger allocation of carbohydrates tobelow-ground organs (or storage organs at soil-surface level)however their biomass growth tends to be slower and theirreproductive output lower The contribution of sprouters tospecies composition tends to be associated with the likelihoodof major biomass-destruction events as well as to the degree ofstress in terms of available resources

How to assess

Here we define resprouting capacity as the relative ability ofa plant species to form new shoots after destruction of most ofits above-ground biomass using reserves from basal or below-ground plant parts The following method is a clear compromisebetween general applicability and rapid assessment on the onehand and precision on the other It is particularly relevant forwoody plants and graminoids but may also be applied to forbsWithin the study site search for spots with clear symptoms of arecent major disturbance event In general this event should havebeen within the same year However if only woody species arebeing considered the assessment may be carried out up to 5 yearsafter thedisturbance (as longas shoots emerging fromnear the soilsurface can still be identified unambiguously as sprouts followingbiomass destruction) For each species try to find any number ofadult plants between 5 and 50 individuals (depending on timeavailable) fromwhich as much as possible but at least 75 of thelive above-ground biomass was destroyed including the entiregreen canopy This is to ensure that regrowth is only supported byreserves from basal or below-ground organs Note that in the caseof trunksandbranchesofwoodyplants old deadxylem(wood) isnot considered as part of the live biomass Thus if a tree is stillstanding after a fire but all its bark cambium and young xylemhave been killed it should be recorded as destruction of 100 ofthe above-ground biomass

Make sure that enough time has lapsed for possibleresprouting Estimate (crudely) the average percentage ofabove-ground biomass destroyed among these plants (ameasure of disturbance severity) by comparing against averageundamaged adult plants of the same species Multiply thispercentage by the percentage of the damaged plant populationthat has resprouted (ie formed new shoots emerging frombasal or below-ground parts) and divide by 100 to obtain thelsquoresprouting capacityrsquo (range 0ndash100 unitless) When data areavailable from more than one site take the highest value as thespecies value although this ignores the fact that great intraspecificvariability in sprouting capacity may occur In longer-termstudies resprouting may be investigated experimentally byclipping plants to simulate destruction of 75ndash100 of theabove-ground biomass (in which case the clipped parts can beused for other trait measurements as well) If fewer than fiveplants with lsquoappropriatersquo damage can be found give the speciesa default value of 50 if any resprouting is observed (50 beinghalfway between lsquomodestrsquo and lsquosubstantialrsquo resprouting seebelow) In species where no resprouting is observed merely

because no major biomass destruction can be found it isimportant to consider this a missing value (not a value of zero)

Broad interspecific comparisons have to take into account anintraspecific error of up to 25 units as a result of the dependence ofresprouting capacity on the severity of disturbance encounteredfor each species However within ecosystems where differentspecies suffer the same disturbance regime direct comparisonsshould be safe


(1) Data from literature Useful and legitimate data may beobtained from the literature or by talking to local people(eg foresters farmers rangers) Make sure that the sameconditions of major destruction of above-ground biomasshave been met In such cases assign subjective numbers forresprouting capacity after major disturbance as follows 0never resprouting 20 very poor resprouting 40 moderateresprouting 60 substantial resprouting 80 abundantresprouting and 100 very abundant resprouting Thesame crude estimates may also be used for species forwhich the more quantitative assessment is not feasibleeg because the non-resprouting individuals are hard tofind after disturbance as is common in some herbs

(2) Strongly clonal plants In the case of strongly clonal plantsit is important to assesswhether damaged ramets can resproutfrom below-ground reserves and not from the foliage of aconnected ramet Therefore in such species resproutingshould be recorded only if most above-ground biomasshas been destroyed for all ramets in the vicinity

(3) Resprouting of young plants Additional recording ofresprouting ability of young plants may reveal importantinsights into population persistence although this could alsobe seen as a component of recruitment Thus data on the ageor size limits for resprouting ability may reveal importantinsights into population dynamics It is known that someresprouting species cannot resprout before a certain ageor size and others may lose their resprouting capacitywhen they attain a certain age or size

(4) Resprouting after smaller biomass destruction Additionalrecording of resprouting after less severe biomass destructionmay provide useful insights into plant response todisturbances For instance Quercus suber and manyEucalyptus spp can resprout from buds located in highpositions along the stem following a fire Be aware thatspecies highly adapted to fire (such as these examples) maygive the false impression that an area has not been exposed tosevere fires recently Other species in the same area or directfire observations should provide the evidence for that Theapproach of recording resprouting after less severe biomassdestruction could also be applied to the study of resproutingin the face of disturbances other thanfire such as herbivory ortrashing by vertebrates

References on theory significance and large datasets Nobleand Slatyer (1980) Everham and Brokaw (1996) Pausas (1997)Kammesheidt (1999) Bellingham and Sparrow (2000) Bondand Midgley (2001) Higgins et al (2000) Del Tredici (2001)


Burrows (2002) Vesk and Westoby (2004) Pausas andBradstock (2007) Poorter et al (2010)

Acknowledgements

This contribution was funded in part by the Inter-American Institute forGlobal Change Research (IAI) CRN 2015 and SGP-CRA2015 which weresupported by US National Science Foundation Grants GEO-0452325 andGEO-1138881 The work was also funded by FONCyT (PICT 20441 and365) CONICET (PIP 2006-2011 11220080101532) SECYT-UniversidadNacional de Coacuterdoba ndash Argentina and the US NSF (DEB-0620652) Theauthors particularly thank Phil Grime and the UCPEndashUniversity ofSheffield and Mark Westoby and the ARCndashNZ Network of VegetationFunctionndashMacquarie University for inspiring discussion and developmentof trait measurement The authors also thank Marcelo Cabido WilliamBond Paula Tecco Fernando Casanoves and Bryan Finegan for valuablediscussions The authors thank Diana Abal Solis and Juan Pablo Bellini forassistance with figures and diagrams and Valeria Falczuk for technicalassistance in the laboratory The authors appreciate helpful comments ofAdrienneNicotraHansLambers andone anonymous reviewerwhichgreatlyimproved the quality of themanuscriptMany users of the previous handbookgave useful feedback on trait measurements Finally we thank plant speciesaround the world for beginning to give away the secrets of their functioningand ecosystem-level impacts

References

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Ackerly DD Knight CA Weiss SB Barton K Starmer KP (2002) Leafsize specific leaf area and microhabitat distribution of chaparral woodyplants contrasting patterns in species level and community level analysesOecologia 130 449ndash457 doi101007s004420100805

Adair ECPartonWJDelGrossoSJ SilverWLHarmonMEHall SABurkeIC Hart SC (2008) Simple three-poolmodel accurately describes patternsof long-term litter decomposition in diverse climates Global ChangeBiology 14 2636ndash2660

Addington RN Donovan LA Mitchell RJ Vose JM Pecot SD Jack SBHacke UG Sperry JS Oren R (2006) Adjustments in hydraulicarchitecture of Pinus palustris maintain similar stomatal conductancein xeric and mesic habitats Plant Cell amp Environment 29 535ndash545doi101111j1365-3040200501430x

Adiku SGK Rose CW Braddock RD Ozier-Lafontaine H (2000) On thesimulation of root water extraction examination of a minimum energyhypothesis Soil Science 165 226ndash236doi10109700010694-200003000-00005

Aerts R (1995) The advantages of being evergreen Trends in Ecology ampEvolution 10 402ndash407 doi101016S0169-5347(00)89156-9

Aerts R (1996) Nutrient resorption from senescing leaves of perennials arethere general patterns Journal of Ecology 84 597ndash608doi1023072261481

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Albert CH Thuiller W Yoccoz NG Douzet R Aubert S Lavorel S (2010)Amulti-trait approach reveals the structure and the relative importance ofintra- vs interspecific variability in plant traits Functional Ecology 241192ndash1201 doi101111j1365-2435201001727x

Albert CH De Bello F Boulangeat I Pellet G Lavorel S Thuiller W (2012)On the importance of intraspecific variability for the quantification offunctional diversity Oikos 121 116ndash126doi101111j1600-0706201119672x

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Baraloto C Paine CET Patintildeo S Bonal D Heacuterault B Chave J (2010b)Functional trait variation and sampling strategies in species-rich plantcommunities Functional Ecology 24 208ndash216doi101111j1365-2435200901600x

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Bond WJ Midgley JJ (2001) Ecology of sprouting in woody plants thepersistence niche Trends in Ecology amp Evolution 16 45ndash51doi101016S0169-5347(00)02033-4

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Bouma TJ Nielsen KL Koutstaal B (2000) Sample preparation and scanningprotocol for computerised analysis of root length and diameter Plant andSoil 218 185ndash196 doi101023A1014905104017

Box EO (1996) Plant functional types and climate at the global scale Journalof Vegetation Science 7 309ndash320 doi1023073236274

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BrandoPMNepstadDCBalch JKBolkerBChristmanMCCoeMPutzFE(2012) Fire-induced treemortality in a neotropical forest the roles of barktraits tree size wood density and fire behavior Global Change Biology18 630ndash641 doi101111j1365-2486201102533x

Breckle SW (2002) Salinity halophytes and salt affected natural ecosystemsIn lsquoSalinity environmentndashplantsndashmoleculesrsquo (Eds A Laumluchli U Luumlttge)pp 53ndash77 (Kluwer Academic Publishers London)

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Brodribb TJ Feild TS (2010) Leaf hydraulic evolution led a surge in leafphotosynthetic capacity during early angiosperm diversification EcologyLetters 13 175ndash183 doi101111j1461-0248200901410x

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Buckley TN Roberts DW (2006) How should leaf area sapwood area andstomatal conductance vary with tree height to maximize growth TreePhysiology 26 145ndash157 doi101093treephys262145

Burd M (2007) Adaptive function of drip tips a test of the epiphyllhypothesis in Psychotria marginata and Faramea occidentalis(Rubiaceae) Journal of Tropical Ecology 23 449ndash455doi101017S0266467407004166

Burrows GE (2002) Epicormic strand structure in Angophora Eucalyptusand Lophostemon (Myrtaceae) ndash implications for fire resistance andrecovery New Phytologist 153 111ndash131doi101046j0028-646X200100299x

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Cain SA (1950) Life-forms and phytoclimate Botanical Review 16 1ndash32doi101007BF02879783

Caldwell MM Virginia RA (1989) Root systems In lsquoPlant physiogicalecology field methods and instrumentationrsquo (Ed RW Pearcy)pp 367ndash398 (Chapman and Hall London)

Cardinale BJ Duffy JE Gonzalez A Hooper DU Perrings C Venail PNarwani A Mace GM Tilman D Wardle DA Kinzig AP Daily GCLoreau M Grace JB Larigauderie A Srivastava DS Naeem S (2012)Biodiversity loss and its impact on humanity Nature 486 59ndash67doi101038nature11148

Casanoves F Pla L Di Rienzo JA Diacuteaz S (2011) FDiversity a softwarepackage for the integrated analysis of functional diversity Methods inEcology and Evolution 2 233ndash237doi101111j2041-210X201000082x

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Chapin FS III (1980) The mineral nutrition of wild plants Annual Review ofEcology and Systematics 11 233ndash260doi101146annureves11110180001313

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Chapin FS III Zaveleta ES Eviner VT Naylor RL Vitousek PM Lavorel SReynoldsHLHooperDUSalaOEHobbieSEMackMCDiaz S (2000)Consequences of changing biotic diversity Nature 405 234ndash242doi10103835012241

Chapin FS III Trainor SF Huntington O Lovecraft AL Zavaleta E NatcherDC McGuire D Nelson JL Ray L Calef M Fresco N Huntington HRupp TS DeWilde L Naylor RL (2008) Increasing wildfire in Alaskarsquosboreal forest pathways to potential solutions of a wicked problemBioscience 58 531ndash540 doi101641B580609

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Chen N Black TA (1992) Defining leaf area index for non-flat leaves PlantCell amp Environment 15 421ndash429doi101111j1365-30401992tb00992x

Cherubini P Gartner BL Tognetti R Braumlker OU SchochW Innes JL (2003)Identification measurement and interpretation of tree rings in woodyspecies from mediterranean climates Biological Reviews of theCambridge Philosophical Society 78 119ndash148doi101017S1464793102006000

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Choat B Sack L Holbrook NM (2007) Diversity of hydraulic traits innine Cordia species growing in tropical forests with contrastingprecipitation New Phytologist 175 686ndash698doi101111j1469-8137200702137x

Choong MF Lucas PW Ong JSY Pereira B Tan HTW Turner IM (1992)Leaf fracture toughness and sclerophylly their correlations and ecologicalimplications New Phytologist 121 597ndash610doi101111j1469-81371992tb01131x

Clements ES (1905) The relation of leaf structure to physical factorsTransactions of the American Microscopical Society 26 19ndash102doi1023073220956

Cochard H Cruizat P TyreeMT (1992) Use of positive pressures to establishvulnerability curves Plant Physiology 100 205ndash209doi101104pp1001205

Coley PD (1987) Patrones en las defensas de las plantas iquestpor queacute losherbiacutevoros prefieren ciertas especies Revista de Biologia Tropical 35151ndash164

Coley PD (1988) Effects of plant growth rate and leaf lifetime on the amountand type of anti-herbivore defense Oecologia 74 531ndash536doi101007BF00380050

Cooper SM Ginnett TF (1998) Spines protect plants against browsing bysmall climbing mammals Oecologia 113 219ndash221doi101007s004420050371

Cooper DJ DrsquoAmicoDR ScottML (2003) Physiological andmorphologicalresponse patterns of Populus deltoides to alluvial groundwater pumpingEnvironmental Management 31 215ndash226doi101007s00267-002-2808-2

Cornelissen JHC (1996) An experimental comparison of leaf decompositionrates in a wide range of temperate plant species and types Journal ofEcology 84 573ndash582 doi1023072261479

Cornelissen JHC (1999) A triangular relationship between leaf size and seedsize among woody species allometry ontogeny ecology and taxonomyOecologia 118 248ndash255 doi101007s004420050725

Cornelissen JHC Castro-Diacuteez P Hunt R (1996) Seedling growth allocationand leaf attributes in a wide range of woody plant species and typesJournal of Ecology 84 755ndash765 doi1023072261337

Cornelissen JHCWergerMJA Castro-Diacuteez P VanRheenen JWARowlandAP (1997)Foliar nutrients in relation to growth allocation and leaf traits inseedlings of a wide range of woody plant species and types Oecologia111 460ndash469 doi101007s004420050259

Cornelissen JHC Peacuterez-Harguindeguy N Diacuteaz S Grime JP Marzano BCabidoM Vendramini F Cerabolini B (1999) Leaf structure and defencecontrol litter decomposition rate across species and life forms in regionalfloras on two continents New Phytologist 143 191ndash200doi101046j1469-8137199900430x

Cornelissen JHC Lavorel S Garnier E Diacuteaz S Buchmann N Gurvich DEReich PB ter Steege H Morgan HD van der Heijden MGA Pausas JGPoorter H (2003) A handbook of protocols for standarised and easymeasurement of plant functional traits worldwide Australian Journalof Botany 51 335ndash380 doi101071BT02124

Cornelissen JHC Quested H Van Logtestijn RSP Peacuterez-Harguindeguy NGwynn-Jones D Diacuteaz S Callaghan TV Press MC Aerts R (2006) FoliarpH as a new plant trait can it explain variation in foliar chemistry andcarbon cycling processes among Subarctic plant species and typesOecologia 147 315ndash326 doi101007s00442-005-0269-z

Cornelissen JHC Van Bodegom PMAerts R Callaghan TV Van LogtestijnRSPAlatalo J StuartChapinFGerdolRGudmundsson JGwynn-JonesD Hartley AE Hik DS Hofgaard A Joacutensdoacutettir IS Karlsson S Klein JALaundre J Magnusson B Michelsen A Molau U Onipchenko VGQuested HM Sandvik SM Schmidt IK Shaver GR Solheim BSoudzilovskaia NA Stenstroumlm A Tolvanen A Totland Oslash Wada NWelker JM Zhao X MOL Team (2007) Global negative vegetationfeedback to climate warming responses of leaf litter decomposition ratesin cold biomes Ecology Letters 10 619ndash627doi101111j1461-0248200701051x

Cornelissen JHC Sibma F Van Logtestijn RSP Broekman R Thompson K(2011) Leaf pH as a plant trait species-driven rather than soil-drivenvariation Functional Ecology 25 449ndash455doi101111j1365-2435201001765x

CornwellWKBhaskarR SackLCordell S LunchCK(2007)Adjustment ofstructure and function of HawaiianMetrosideros polymorpha at high vslow precipitation Functional Ecology 21 1063ndash1071doi101111j1365-2435200701323x

Cornwell WK Cornelissen JHC Amatangelo K Dorrepaal E Eviner VTGodoy O Hobbie SE Hoorens B Kurokawa H Peacuterez-Harguindeguy NQuested HM Santiago LS Wardle DA Wright IJ Aerts R Allison SDVanBodegomPBrovkinVChatainACallaghanTVDiacuteaz SGarnier EGurvichDEKazakouEKlein JARead JReichPB SoudzilovskaiaNAVaieretti MVWestobyM (2008) Plant species traits are the predominantcontrol on litter decomposition rates within biomes worldwide EcologyLetters 11 1065ndash1071 doi101111j1461-0248200801219x

Cornwell WK Cornelissen JHC Allison SD Bauhus J Eggleton P PrestonCM Scarff F Weedon JT Wirth C Zanne AE (2009) Plant traits andwood fates across the globe rotted burned or consumedGlobal ChangeBiology 15 2431ndash2449 doi101111j1365-2486200901916x

Cousens RD Hill J FrenchK Bishop ID (2010) Towards better prediction ofseed dispersal by animals conceptual frameworks and process-basedmodels Functional Ecology 24 1163ndash1170doi101111j1365-2435201001747x

Craine JM (2009) lsquoResource strategies of wild plantsrsquo (Princeton UniversityPress Princeton NJ)


Craine JM Berin DM Reich PB Tilman D Knops J (1999) Measurementof leaf longevity of 14 species of grasses and forbs using a novelapproach New Phytologist 142 475ndash481doi101046j1469-8137199900411x

Craine JM Tilman DG Wedin DA Chapin S III (2001) The relationshipsamong root and leaf traits of 76 grassland species and relative abundancealong fertility and disturbance gradients Oikos 93 274ndash285doi101034j1600-07062001930210x

CramerW(1997)Usingplant functional types in a global vegetationmodel InlsquoPlant functional typesrsquo (Eds TM Smith HH Shugart FI Woodward)pp 271ndash288 (Cambridge University Press Cambridge UK)

CramerWBondeauAWoodwardFI Prentice ICBetts RABrovkinVCoxPMFisherVFoley JA FriendADKucharikCLomasMRRamankuttyNSitchSSmithBWhiteAYoung-MollingC(2001)Global responseofterrestrial ecosystem structure and function to CO2 and climate changeresults from six dynamic global vegetation models Global ChangeBiology 7 357ndash373 doi101046j1365-2486200100383x

Darvell BW Lee PKD Yuen TDB Lucas PW (1996) A portable fracturetoughness tester for biological materials Measurement Science ampTechnology 7 954ndash962 doi1010880957-023376016

David JS Valente F Gash JHC (2005) Evaporation of intercepted rainfallIn lsquoEncyclopedia of hydrological sciencesrsquo (Ed MG Anderson)pp 627ndash634 (John Wiley and Sons Chichester UK)

Davis SD Ewers FW Sperry JS Portwood KA Crocker MC Adams GC(2002) Shoot dieback during prolonged drought in Ceanothus(Rhamnaceae) chaparal of California a possible case of hydraulicfailure American Journal of Botany 89 820ndash828doi103732ajb895820

De Bello F Lavorel S Gerhold P Reier Uuml Paumlrtel M (2010) A biodiversitymonitoring framework for practical conservation of grasslands andshrublands Biological Conservation 143 9ndash17doi101016jbiocon200904022

De Bello F Lavorel S Albert CH ThuillerW Grigulis K Dolezal J JanecekS Lecircps J (2011) Quantifying the relevance of intraspecific trait variabilityfor functional diversity Methods in Ecology and Evolution 2 163ndash174doi101111j2041-210X201000071x

De Kroon H Van Groenendael JM (1997) lsquoThe ecology and evolution ofclonal plantsrsquo (Backhuys Publishers Leiden The Netherlands)

De Vogel EF (1980) lsquoSeedlings of dicotyledonsrsquo (Centre for AgriculturalPublishing and Documentation Wageningen The Netherlands)

De Witte LC Stoumlcklin J (2010) Longevity of clonal plants why it mattersand how to measure it Annals of Botany 106 859ndash870doi101093aobmcq191

Del Tredici P (2001) Sprouting in temperate trees a morphological andecological review Botanical Review 67 121ndash140doi101007BF02858075

Diacuteaz S Hodgson JG Thompson K Cabido M Cornelissen JHC Jalili AMontserrat-Martiacute G Grime JP Zarrinkamar F Asri Y Band SRBasconcelo S Castro-Diacuteez P Funes G Hamzehee B Khoshnevi MPeacuterez-Harguindeguy N Peacuterez-Rontomeacute MC Shirvany FA VendraminiF Yazdani S Abbas-Azimi R Bogaard A Boustani S Charles MDehghan M de Torres-Espuny L Falczuk V Guerrero-Campo JHynd A Jones G Kowsary E Kazemi-Saeed F Maestro-Martiacutenez MRomo-DiacuteezA ShawS SiavashBVillar-Salvador P ZakMR (2004)Theplant traits that drive ecosystems evidence from three continents Journalof Vegetation Science 15 295ndash304

Diacuteaz S Lavorel S De Bello F Queacutetier F Grigulis K Robson MT (2007)Incorporating plant functional diversity effects in ecosystem serviceassessments Proceedings of the National Academy of Sciences USA104 20684ndash20689 doi101073pnas0704716104

Diefendorf AFMueller KEWing SL Koch PL Freeman KH (2010) Globalpatterns in leaf 13C discrimination and implications for studies of past andfuture climate Proceedings of the National Academy of Sciences USA107 5738ndash5743 doi101073pnas0910513107

Diemer M (1998) Life span and dynamics of leaves of herbaceous perennialsin high-elevation environments lsquonews from the elephantrsquos legrsquoFunctional Ecology 12 413ndash425doi101046j1365-2435199800207x

Dilcher DL (1974) Approaches to the identification of angiosperm leafremains Botanical Review 40 1ndash157 doi101007BF02860067

Dimitrakopoulos AP Panov PI (2001) Pyric properties of some dominantMediterranean vegetation species International Journal ofWildlandFire10 23ndash27 doi101071WF01003

Doherty RM Sitch S Smith B Lewis SL Thornton PK (2010) Implicationsof future climate and atmospheric CO2 content for regionalbiogeochemistry biogeography and ecosystem services across EastAfrica Global Change Biology 16 617ndash640doi101111j1365-2486200901997x

Domingo F Saacutenchez G Moro MJ Brenner AJ Puigdefaacutebregas J (1998)Measurement and modelling of rainfall interception by three semi-aridcanopies Agricultural and Forest Meteorology 91 275ndash292doi101016S0168-1923(98)00068-9

Dunbabin V Rengel Z Diggle AJ (2004) Simulating form and function ofroot systems efficiency of nitrate uptake is dependent on root systemarchitecture and the spatial and temporal variability of nitrate supplyFunctional Ecology 18 204ndash211doi101111j0269-8463200400827x

Eamus D Prior L (2001) Ecophysiology of trees of seasonally dry tropicscomparisons among phenologies Advances in Ecological Research 32113ndash197 doi101016S0065-2504(01)32012-3

Earnshaw MJ Carver KA Gunn TC Kerenga K Harvey V Griffiths HBroadmeadow MSJ (1990) Photosynthetic pathway chilling toleranceand cell sap osmotic potential values of grasses along an altitudinalgradient in Papua New Guinea Oecologia 84 280ndash288

Edwards EJ Still CJ Donoghue MJ (2007) The relevance of phylogeny tostudies of global change Trends in Ecology amp Evolution 22 243ndash249doi101016jtree200702002

Ehleringer JR (1991) 13C12C fractionation and its utility in terrestrial plantstudies In lsquoCarbon isotopes techniquesrsquo (Eds DC Coleman B Fry)pp 187ndash200 (Academic Press London)

Ehleringer JR Osmond CB (2000) Stable isotopes In lsquoPlant physiologicalecology field methods and instrumentationrsquo (Eds RW Pearcy JEhleringer HA Mooney PW Rundel) pp 281ndash300 (Kluwer AcademicPublishers Dordrecht The Netherlands)

Ehleringer JR Cerling TE Helliker BR (1997) C4 photosynthesisatmospheric CO2 and climate Oecologia 112 285ndash299doi101007s004420050311

Eissenstat DM Yanai RD (1997) The ecology of root lifespan Advances inEcological Research 27 1ndash60 doi101016S0065-2504(08)60005-7

Elberse WTH Berendse F (1993) A comparative study of the growthand morphology of eight grass species from habitats with differentnutrient availabilities Functional Ecology 7 223ndash229doi1023072389891

Eliaacuteš P (1985) Leaf indices of woodland herbs as indicators of habitatconditions Ekologia 4 289ndash295

Ellenberg H Muumlller-Dombois D (1967) A key to Raunkiaer plant life formswith revised subdivisions Berichte des geobotanischen Institutes derETH Stiftung Ruumlbel 37 56ndash73

Ellsworth DS Reich PB (1992) Water relations and gas exchange of Acersaccharum seedlings in contrasting natural light and water regimes TreePhysiology 10 1ndash20 doi101093treephys1011

Enquist BJ (2002) Universal scaling in tree and vascular plant allometrytoward a general quantitative theory linking plant form and function fromcells to ecosystems Tree Physiology 22 1045ndash1064doi101093treephys2215-161045

Enriacutequez S Duarte CM Sand-Jensen K Nielsen SL (1996) Broad-scalecomparison of photosynthetic rates across phototrophic organismsOecologia 108 197ndash206


Etlinger MG Beall FC (2004) Development of a laboratory protocol for fireperformance of landscape plants International Journal of Wildland Fire13 479ndash488 doi101071WF04039

Evans GC (1972) lsquoThe quantitative analysis of plant growthrsquo (BlackwellScientific Publications Oxford UK)

Everham EM Brokaw NVL (1996) Forest damage and recovery fromcatastrophic wind Botanical Review 62 113ndash185doi101007BF02857920

Ewel JJ Bigelow SW (1996) Plant life forms and ecosystem functioningIn lsquoBiodiversity and ecosystem processes in tropical forestsrsquo (Eds GHOrians R Dirzo JH Cushman) pp 101ndash126 (Springer Berlin)

Faith DP Magalloacuten S Hendry AP Conti E Yahara T Donoghue MJ (2010)Evosystem services an evolutionary perspective on the links betweenbiodiversity and human well-being Current Opinion in EnvironmentalSustainability 2 66ndash74 doi101016jcosust201004002

FAO (1999) lsquoSoil salinity assessment Methods and interpretation ofelectrical conductivity measurementsrsquo FAO irrigation and drainagepaper 57 (FAO Rome)

Farquhar GD Ehleringer JR Hubick KT (1989) Carbon isotopediscrimination and photosynthesis Annual Review of Plant Physiologyand Plant Molecular Biology 40 503ndash537doi101146annurevpp40060189002443

Feild TS Balun L (2008) Xylem hydraulic and photosynthetic function ofGnetum (Gnetales) species from Papua New Guinea New Phytologist177 665ndash675 doi101111j1469-8137200702306x

FieldCMooneyHA(1986)Thephotosynthesisndashnitrogen relationship inwildplants In lsquoOn the economy of plant form and functionrsquo (Ed TJ Givnish)pp 25ndash55 (Cambridge University Press Cambridge UK)

Finzi AC Van Breemen N CanhamCD (1998) Canopy tree-soil interactionswithin temperate forests species effects on soil carbon and nitrogenEcological Applications 8 440ndash446

Fischer M Stoumlcklin J (1997) Local extinctions of plants in remnants ofextensively used calcareous grasslands 1950ndash85 Conservation Biology11 727ndash737 doi101046j1523-1739199796082x

Fischer S Poschlod P Beinlich B (1996) Experimental studies on thedispersal of plants and animals on sheep in calcareous grasslandsJournal of Applied Ecology 33 1206ndash1221 doi1023072404699

Fisher JB (1986) Branching patterns and angles in trees In lsquoOn the economyof plant form and functionrsquo (Ed TJ Givnish) pp 493ndash523 (CambridgeUniversity Press Cambridge UK)

Fitter A (1996) Characteristics and functions of root systems In lsquoPlant rootsthe hidden halfrsquo 2nd edn (Eds Y Waisel A Eshel U Kafkafi) pp 1ndash20(Marcel Dekker New York)

Flowers TJ Colmer TD (2008) Salinity tolerance in halophytes NewPhytologist 179 945ndash963 doi101111j1469-8137200802531x

Flowers TJ Troke PF Yeo AR (1977) The mechanism of salt tolerance inhalophytes Annual Review of Plant Physiology 28 89ndash121doi101146annurevpp28060177000513

FlowersTJHajibagheriMAClipsonNJW(1986)HalophytesTheQuarterlyReview of Biology 61 313ndash337 doi101086415032

Forget P Wenny D (2005) How to elucidate seed fate A review of methodsused to study seed removal and secondary seed dispersal In lsquoSeed fatepredation dispersal and seedling establishmentrsquo (Eds P Forget JLambert P Hulme S Vander Wal) pp 379ndash394 (CABI PublishingWallingford UK)

Fortunel C Garnier E Joffre J Kazakou E QuestedH Grigulis K Lavorel SAnsquerPCastroHCruzPDoležal J ErikssonOFreitasHGolodetsCJouany C Kigel J Kleyer M Lehsten V Lepš J Meier T Pakeman RPapadimitriou M Papanastasis VP Queacutetier F Robson M Sternberg MTheau J-P Theacutebault A Zarovali M (2009) Leaf traits capture the effectsof land use changes and climate on litter decomposability of grasslandsacross Europe Ecology 90 598ndash611 doi10189008-04181

Fortunel C Fine PVA Baraloto C (2012) Leaf stem and root tissue strategiesacross 758 Neotropical tree species Functional Ecology 26 1153ndash1161doi101111j1365-2435201202020x

FreschetGT Cornelissen JHCVanLogtestijnRSPAerts R (2010) Evidenceof the lsquoplant economics spectrumrsquo in a Subarctic flora Journal of Ecology98 362ndash373 doi101111j1365-2745200901615x

FreschetGTAertsRCornelissen JHC (2012)Aplant economics spectrumoflitter decomposability Functional Ecology 26 56ndash65doi101111j1365-2435201101913x

Funes G Basconcelo S Diacuteaz S Cabido M (1999) Seed bank dynamicsof Lachemilla pinnata (Rosaceae) in different plant communities ofmountain grassland in central Argentina Annales Botanici Fennici 36109ndash114

Gardner RO (1975) Overview of botanical clearing technique StainTechnology 50 99ndash105

Garnier E Laurent G (1994) Leaf anatomy specific mass and water contentin congeneric annual and perennial grass species New Phytologist 128725ndash736 doi101111j1469-81371994tb04036x

Garnier E Navas M-L (2012) A trait-based approach to comparativefunctional plant ecology concepts methods and applications foragroecology Agronomy for Sustainable Development 32 365ndash399doi101007s13593-011-0036-y

Garnier E Shipley B Roumet C Laurent G (2001a) A standardized protocolfor the determination of specific leaf area and leaf dry matter contentFunctional Ecology 15 688ndash695doi101046j0269-8463200100563x

Garnier E Laurent G Debain S Berthelier P Ducout B Roumet C NavasM-L (2001b) Consistency of species ranking based on functional leaftraits New Phytologist 152 69ndash83doi101046j0028-646x200100239x

Garnier E Cortez J Billegraves G Navas M-L Roumet C Debussche M LaurentG Blanchard A Aubry D Bellmann A Neill C Toussaint J-P (2004)Plant functional markers capture ecosystem properties during secondarysuccession Ecology 85 2630ndash2637 doi10189003-0799

Garnier E Lavorel S Ansquer P Castro H Cruz P Dolezal J Eriksson OFortunel C Freitas H Golodets C Grigulis K Jouany C Kazakou EKigel J KleyerM LehstenV Lepš JMeier T PakemanR PapadimitriouM Papanastasis VP Quested H Queacutetier F RobsonM Roumet C RuschG Skarpe C Sternberg M Theau J-P Vile D Zarovali MP (2007)Assessing the effects of land-use change on plant traits communitiesand ecosystem functioning in grasslands a standardized methodologyand lessons froman application to 11European sitesAnnals of Botany99967ndash985 doi101093aobmcl215

Gartner BL (1995) lsquoPlant stems physiology and functional morphologyrsquo(Academic Press San Diego CA)

Gartner BL Moore JC Gardiner BA (2004) Gas in stems abundanceand potential consequences for tree biomechanics Tree Physiology 241239ndash1250 doi101093treephys24111239

GarwoodNC(1996)Functionalmorphologyof tropical tree seedlings In lsquoTheecology of tropical forest tree seedlingsrsquo (Ed MD Swaine) pp 59ndash129(Parthenon New York)

Gatsuk LE Smirnova OV Vorontzova LI Zaugolnova LB Zhukova LA(1980) Age states of plants of various growth forms a review Journal ofEcology 68 675ndash696 doi1023072259429

Gaucherand S Lavorel S (2007) New method for rapid assessment ofthe functional composition of herbaceous plant communities AustralEcology 32 927ndash936 doi101111j1442-9993200701781x

Gaudet CL Keddy PA (1988) A comparative approach to predictingcompetitive ability from plant traits Nature 334 242ndash243doi101038334242a0

Gill AM Zylstra P (2005) Flammability of Australian forests AustralianForestry 68 88ndash94

GivnishTJ (1979)On the adaptive significanceof leaf form In lsquoTopics inplantpopulation biologyrsquo (Ed OT Solbrig S Jain GB Johnson PH Raven)pp 375ndash407 (Macmillan London)

Givnish TJ (1987) Comparative studies of leaf form assessing the relativeroles of selective pressures and phylogenetic constarintsNewPhytologist106 131ndash160 doi101111j1469-81371987tb04687x


Gowda JH Palo RT (2003) Age-related changes in defensive traits of Acaciatortilis Hayne African Journal of Ecology 41 218ndash223doi101046j1365-2028200300434x

Gowda J Raffaele E (2004) Spine production is induced by fire a naturalexperiment with three Berberis species Acta Oecologica 26 239ndash245doi101016jactao200408001

Graca MAS Baumlrlocher F Gessner MO (2005) lsquoMethods to study litterdecompositionrsquo (Springer Dordrecht The Netherlands)

Green DS Kruger EL (2001) Light-mediated constraints on leaf functioncorrelate with leaf structure among deciduous and evergreen tree speciesTree Physiology 21 1341ndash1346 doi101093treephys21181341

Grime JP (1979) Competition and the struggle for existence In lsquoPopulationdynamicsrsquo (Eds RM Anderson BD Turner LR Taylor) pp 123ndash139(Blackwell Scientific Publications Oxford UK)

Grime JP Hunt R (1975) Relative growth-rate its range and adaptivesignificance in a local flora Journal of Ecology 63 393ndash422doi1023072258728

Grime JPBlytheGMThornton JD (1970)Food selection by the snailCepaeanemoralis L In lsquoAnimal populations in relation to their food resourcesrsquo(Ed A Watson) pp 73ndash99 (Blackwell Scientific Publications OxfordUK)

Grime JP Cornelissen JHC Thompson K Hodgson JG (1996) Evidence of acausal connection between anti-herbivore defense and the decompositionrate of leaves Oikos 77 489ndash494 doi1023073545938

Grime JP ThompsonK Hunt R Hodgson JG Cornelissen JHC Rorison IHHendry GAF Ashenden TW Askew AP Band SR Booth RE BossardCC Campbell BD Cooper JEL Davison AW Gupta PL Hall W HandDW Hannah MA Hillier SH Hodkinson DJ Jalili A Liu Z MackeyJMLMatthewsNMowforthMANealAMReaderRJ ReilingKRoss-Fraser W Spencer RE Sutton F Tasker DE Thorpe PC Whitehouse J(1997) Integrated screening validates primary axes of specialisation inplants Oikos 79 259ndash281 doi1023073546011

GrubbPJ (1992)Apositive distrust in simplicityndash lessons fromplant defencesand from competition among plants and among animal Journal ofEcology 80 585ndash610 doi1023072260852

Gubsch M Buchmann N Schmid B Schulze ED Lipowsky A Roscher C(2011) Differential effects of plant diversity on functional trait variationof grass species Annals of Botany 107 157ndash169doi101093aobmcq220

GurvichDEDiacuteaz S FalczukV Peacuterez-HarguindeguyNCabidoMThorpeC(2002) Foliar resistence to simulated extreme temperature events incontrasting plant functional and chorological types Global ChangeBiology 8 1139ndash1145 doi101046j1365-2486200200540x

HanleyMELamontBB (2002)Relationships between physical and chemicalattributes of congeneric seedlings how important is seedling defenceFunctional Ecology 16 216ndash222doi101046j1365-2435200200612x

Harrison SP Marlon JR Bartlein PJ (2010) Fire in the Earth system InlsquoChanging climates earth systems and societyrsquo (Ed J Dodson) pp 21ndash48(Springer Dordrecht The Netherlands)

Hartley SE JonesCG (1997) Plant chemistry and herbivory or why theworldis green In lsquoPlant ecologyrsquo (Ed MJ Crawley) pp 284ndash324 (BlackwellScience Oxford UK)

Hattersley PWWatson L (1992) Diversification of photosynthesis In lsquoGrassevolution anddomesticationrsquo (EdGPChapman) pp 38ndash116 (CambridgeUniversity Press London)

Hendry GAF Grime JP (1993) lsquoMethods in comparative plant ecologyA laboratory manualrsquo (Chapman and Hall London)

Herwitz SR (1985) Interception storage capacities of tropical rainforestcanopy trees Journal of Hydrology 77 237ndash252doi1010160022-1694(85)90209-4

Hibberd JM QuickWP (2002) Characteristics of C4 photosynthesis in stemsand petioles of C3 flowering plants Nature 415 451ndash454doi101038415451a

Higgins SI Bond WJ Trolliope WSW (2000) Fire resprouting andvariability a recipe for grass-tree coexistence in savanna Journal ofEcology 88 213ndash229 doi101046j1365-2745200000435x

Hinckley TM Lassoie JP Running SW (1978) Temporal and spatialvariations in the water status of forest trees Forest Science 20a0001ndashz0001

Hirose T Werger MJA (1995) Canopy structure and shoton slux sartitioningsmong species in a herbaceous plant community Ecology 76 466ndash474doi1023071941205

Hladik A Miquel S (1990) Seedling types and plant establishment in anAfrican rain forest In lsquoReproductive ecology of tropical plantsrsquo (Eds KSBawa M Hadley) pp 261ndash282 (UNESCOParthenon Carnforth UK)

Hodge A (2004) The plastic plant root responses to heterogeneous suppliesof nutrients New Phytologist 162 9ndash24doi101111j1469-8137200401015x

Hodgson JG Wilson PJ Hunt R Grime JP Thompson K (1999) AllocatingC-S-R plant functional types a soft approach to a hard problemOikos 85282ndash294 doi1023073546494

Hodgson JG Montserrat-Martiacute G Charles M Jones G Wilson P Shipley BSharafi M Cerabolini BEL Cornelissen JHC Band SR Bogard ACastro-Diacuteez P Guerrero-Campo J Palmer C Peacuterez-Rontomeacute MCCarter G Hynd A Romo-Diacuteez A de Torres Espuny L Royo Pla F(2011) Is leaf dry matter content a better predictor of soil fertility thanspecific leaf area Annals of Botany 108 1337ndash1345doi101093aobmcr225

Holbrook NM Zwieniecki MA (2005) lsquoVascular transport in plantsrsquo(Elsevier Academic Press London)

Horn HS (1971) lsquoThe adaptive geometry of treesrsquo (Princeton UniversityPress Princeton NJ)

HorneckDAMillerRO (1998)Determination of total nitrogen in plant tissueIn lsquoHandbook and reference methods for plant analysisrsquo (Ed YP Kalra)pp 75ndash84 (CRC Press New York)

Howe HF Smallwood J (1982) Ecology of seed dispersal Annual Review ofEcology and Systematics 13 201ndash228doi101146annureves13110182001221

Howe HF Westley LC (1997) Ecology of pollination and seed dispersalIn lsquoPlant ecologyrsquo (Ed MJ Crawley) pp 262ndash283 (Blackwell OxfordUK)

Hulme PE (1998) Post-dispersal seed predation consequences for plantdemography and evolution Perspectives in Plant Ecology Evolutionand Systematics 1 32ndash46 doi1010781433-8319-00050

Hulshof CM Swenson NG (2010) Variation in leaf functional trait valueswithin and across individuals and species an example from aCosta Ricandry forest Functional Ecology 24 217ndash223doi101111j1365-2435200901614x

Hunt R (1982) lsquoPlant growth curves The functional approach to plant growthanalysisrsquo (Edward Arnold London)

Ibarra-Manriquez G Martiacutenez Ramos M Oyama K (2001) Seedlingfunctional types in a lowland rain forest in Meacutexico American Journalof Botany 88 1801ndash1812 doi1023073558356

Jackson RB (1999) The importance of root distributions for hydrologybiogeochemistry and ecosystem function In lsquoIntegrating hydrologyecosystem dynamics and biogeochemistry in complex landscapesrsquo(Eds JD Tenhunen P Kabat) pp 219ndash240 (Wiley Chichester UK)

JacksonJFAdamsDC JacksonUB(1999)Allometryof constitutivedefensea model and a comparative test with tree bark and fire regime AmericanNaturalist 153 614ndash632 doi101086303201

JacobsenALPrattRBDavisSDEwersFW(2008)Comparative communityphysiology nonconvergence in water relations among three semi-aridshrub communities New Phytologist 180 100ndash113doi101111j1469-8137200802554x

Jaureguiberry P Bertone GA Diacuteaz S (2011) Device for the standardmeasurement of shoot flammability in the field Austral Ecology 36821ndash829 doi101111j1442-9993201002222x


Jennings DH (1976) The effects of sodium chloride on higher plantsBiological Reviews of the Cambridge Philosophical Society 51 453ndash486doi101111j1469-185X1976tb01064x

Jones G Charles M Bogaard A Hodgson J (2010) Crops and weeds the roleof weed functional ecology in the identification of crop husbandrymethods Journal of Archaeological Science 37 70ndash77doi101016jjas200908017

Jow WM Bullock SH Kummerow J (1980) Leaf turnover rates ofAdenostoma fasciculatum (Rosaceae) American Journal of Botany 67256ndash261 doi1023072442650

Juneau KJ Tarasoff CS (2012) Leaf area and water content changes afterpermanent and temporary storage PLoS ONE 7 e42604doi101371journalpone0042604

Kammesheidt L (1999) Forest recovery by root suckers and abovegroundsprouts after slash-and-burn agriculture fire and logging in Paraguay andVenezuela Journal of Tropical Ecology 15 143ndash157doi101017S0266467499000723

Kattge J Diacuteaz S Lavorel S Prentice IC Leadley P Boumlnisch G Garnier EWestobyMReichPBWright IJCornelissen JHCViolleCHarrisonSPVan Bodegom PM Reichstein M Enquist BJ Soudzilovskaia NAAckerly DD Anand M Atkin O Bahn M Baker TR Baldocchi DBekker R Blanco CC Blonder B Bond WJ Bradstock R Bunker DECasanoves F Cavender-Bares J Chambers JQ Chapin III FS Chave JCoomes D Cornwell WK Craine JM Dobrin BH Duarte L Durka WElser J Esser G Estiarte M Fagan WF Fang J Fernaacutendez-Meacutendez FFidelisA FineganB FloresO FordH FrankD FreschetGT FyllasNMGallagherRVGreenWAGutierrezAGHickler THiggins SI HodgsonJG JaliliA JansenS JolyCAKerkhoffAJKirkupDKitajimaKKleyerMKlotzSKnopsJMHKramerKKuumlhnIKurokawaHLaughlinDLeeTD LeishmanM Lens F Lenz T Lewis SL Lloyd J Llusiagrave J Louault FMa SMahechaMDManning PMassad TMedlyn BEMessie JMolesATMuumlller SCNadrowskiKNaeemSNiinemetsUumlNoumlllert SNuumlskeAOgaya R Oleksyn J OnipchenkoVG OnodaY Ordontildeez J Overbeck GOzinga WA Patintildeo S Paula S Pausas JG Pentildeuelas J Phillips OL PillarV Poorter H Poorter L Poschlod P Prinzing A Proulx R Rammig AReinsch S Reu B Sack L Salgado-Negret B Sardans J Shiodera SShipley B Siefert A Sosinski E Soussana J-F Swaine E Swenson NThompson K Thornton P Waldram M Weiher E White M White SWright SJ Yguel B Zaehle S Zanne AEWirth C (2011) TRY ndash a globaldatabase of plant traits Global Change Biology 17 2905ndash2935doi101111j1365-2486201102451x

KazakouEViolleCRoumetC PintorCGimenezOGarnier E (2009)Litterquality and decomposability of species from a Mediterranean successiondepend on leaf traits but not on nitrogen supply Annals of Botany 1041151ndash1161 doi101093aobmcp202

King DA Davies SJ Noor NSM (2006) Growth and mortality arerelated to adult tree size in a Malaysian mixed dipterocarp forestForest Ecology and Management 223 152ndash158doi101016jforeco200510066

KitajimaK (1994) Relative importance of photosynthetic traits and allocationpatterns as correlates of seedling shade tolerance of 13 tropical treesOecologia 98 419ndash428 doi101007BF00324232

KitajimaK (1996)Cotyledon functionalmorphology patterns of seed reserveutilization and regeneration niches of tropical tree seedlings In lsquoTheecology of tropical forest tree seedlingsrsquo (Ed MD Swaine) pp 193ndash210(UNESCO Paris)

Kitajima K Poorter L (2010) Tissue-level leaf toughness but not laminathickness predicts sapling leaf lifespan and shade toleranceof tropical treespecies New Phytologist 186 708ndash721doi101111j1469-8137201003212x

KleyerMBekkerRMKnevel ICBakker JPThompsonKSonnenscheinMPoschlod PVanGroenendael JMKlimešLKlimešovaacute JKlotz S RuschGM Hermy M Adriaens D Boedeltje G Bossuyt B Dannemann AEndels P Goumltzenberger L Hodgson JG Jackel A-K Kuumlhn I Kunzmann

DOzingaWARoumlmermannC StadlerM Schlegelmilch J SteendamHJTackenbergOWilmannBCornelissen JHCErikssonOGarnierE PecoB (2008) The LEDA traitbase a database of life-history traits of thenorthwest European flora Journal of Ecology 96 1266ndash1274doi101111j1365-2745200801430x

Klimeš L Klimešovaacute J (2005) Clonal traits In lsquoThe LEDA traitbaseCollecting and measuring standards of life-history traits of thenorthwest European florarsquo (Eds IC Knevel RM Bekker D KunzmannMStadlerKThompson)pp 66ndash88 (University ofGroningenGroningenThe Netherlands)

Klimeš L Klimešovaacute J Hendriks RJJ Van Groenendael JM (1997)Clonal plant architecture a comparative analysis of form and functionIn lsquoThe ecology and evolution of clonal plantsrsquo (Eds H De KroonJM Van Groenendael) pp 1ndash29 (Backhuys Publishers Leiden TheNetherlands)

Klimešovaacute J Klimeš L (2007) Bud banks and their role in vegetativeregeneration ndash A literature review and proposal for simpleclassification and assessment Perspectives in Plant Ecology Evolutionand Systematics 8 115ndash129 doi101016jppees200610002

Knapp AK Carter GA (1998) Variability in leaf optical properties among 26species from a broad range of habitats American Journal of Botany 85940ndash946 doi1023072446360

Knevel IC Bekker RM Kleyer M (2003) Life-history traits of the northwestEuropean flora the LEDA database Journal of Vegetation Science 14611ndash614 doi101111j1654-11032003tb02188x

Kohyama T Suzuki E Partomihardjo T Yamada T Kubo T (2003) Treespecies differentiation in growth recruitment and allometry in relationto maximum height in a Bornean mixed dipterocarp forest Journal ofEcology 91 797ndash806 doi101046j1365-2745200300810x

Kocacinar F Sage RF (2003) Photosynthetic pathway alters xylem structureand hydraulic function in herbaceous plants Plant Cell amp Environment26 2015ndash2026 doi101111j1365-2478200301119x

Korning J Thomsen K (1994) A new method for measuring tree height intropical rain forest Journal of Vegetation Science 5 139ndash140doi1023073235647

LambersH PoorterH (1992) Inherent variation in growth rate between higherplants a search for physiological causes and ecological consequencesAdvances in Ecological Research 23 187ndash261doi101016S0065-2504(08)60148-8

Lambers H Finnegan PM Laliberteacute E Pearse SJ Ryan MH Shane MWVeneklaas EJ (2011) Phosphorus nutrition of proteaceae in severelyphosphorus-impoverished soils are there lessons to be learned forfuture crops Plant Physiology 156 1058ndash1066doi101104pp111174318

LarsonDW(2001)Theparadoxof great longevity in a short-lived tree speciesExperimental Gerontology 36 651ndash673doi101016S0531-5565(00)00233-3

Lavorel S Garnier E (2002) Predicting changes in community compositionand ecosystem functioning from plant traits revisiting the Holy GrailFunctional Ecology 16 545ndash556doi101046j1365-2435200200664x

Lavorel S Diacuteaz S Cornelissen JHC Garnier E Harrison SP McIntyre SPausas JG Peacuterez-Harguindeguy N Roumet C Urcelay C (2007) Plantfunctional types are we getting any closer to the holy grail In lsquoTerrestrialecosystems in a changing worldrsquo (Ed PD Canadell JG Pitelka L)pp 149ndash160 (Springer-Verlag Berlin)

Lavorel S Grigulis K McIntyre S Williams NSG Garden D Dorrough SBQueacutetier F Theacutebault A Bonis A (2008) Assessing functional diversity inthe field ndash methodology matters Functional Ecology 22 134ndash147

LeckMA Parker VT Simpson RL (2008) lsquoSeedling ecology and evolutionrsquo(Cambridge University Press Cambridge UK)

Leishman MR Westoby M (1998) Seed size and shape are not related topersistence in soil in Australia in the same way as in Britain FunctionalEcology 12 480ndash485 doi101046j1365-2435199800215x


LeishmanMWright IMolesAWestobyM(2000)Theevolutionary ecologyof seed size In lsquoThe ecology of regeneration in plant communitiesrsquo2nd edn (Ed M Fenner) pp 31ndash57 (CAB International London)

Lenz TIWright IJWestobyM (2006) Interrelations among pressure-volumecurve traits across species and water availability gradients PhysiologiaPlantarum 127 423ndash433 doi101111j1399-3054200600680x

Levitt J (1980) lsquoResponses of plants to environmental stressesrsquo (AcademicPress New York)

Linder CRMoore LA Jackson RB (2000) A universal molecular method foridentifying underground plant parts to species Molecular Ecology 91549ndash1559 doi101046j1365-294x200001034x

Litton CM Raich JW Ryan MG (2007) Carbon allocation in forestecosystems Global Change Biology 13 2089ndash2109doi101111j1365-2486200701420x

Loehle C (1988) Tree life histories the role of defenses Canadian Journalof Forest Research 18 209ndash222 doi101139x88-032

LucasPWChoongMFTanHTWTurner IMBerrickAJ (1991)The fracturetoughness of the leaf of the dicotyledon Calophyllum inophyllum L(Guttiferae) Philosophical Transactions of the Royal Society ofLondon Series B Biological Sciences 334 95ndash106doi101098rstb19910099

Lucas PWTurner IMDominyNJYamashitaN (2000)Mechanical defencesto herbivory Annals of Botany 86 913ndash920doi101006anbo20001261

Luumlttge (1997) lsquoPhysiolgical ecology of tropical plantsrsquo (Springer-VerlagBerlin)

MaSBaldocchiDDMambelli SDawsonTE (2011)Are temporal variationsof leaf traits responsible for seasonal and inter-annual variability inecosystem CO2 exchange Functional Ecology 25 258ndash270doi101111j1365-2435201001779x

Maas EV Hoffman GJ (1977) Crop salt tolerance ndash current assessmentJournal of the Irrigation and Drainage Division 103 115ndash134

MaceGM CramerWDiacuteaz S Faith DP Larigauderie A Le Prestre P PalmerM Perrings C Scholes RJ Walpole M Walther BA Watson JEMMooney HA (2010) Biodiversity targets after 2010 Current Opinion inEnvironmental Sustainability 2 3ndash8 doi101016jcosust201003003

Maherali H DeLucia EH (2001) Influence of climate-driven shifts in biomassallocation on water transport and storage in ponderosa pine Oecologia129 481ndash491

Maherali H Pockman WT Jackson RB (2004) Adaptive variation in thevulnerability of woody plants to xylem cavitation Ecology 852184ndash2199 doi10189002-0538

Maumlkelauml A Vanninen P (2001) Vertical structure of Scots pine crowns indifferent age and size classes Trees 15 385ndash392doi101007s004680100118

Marschner H (2012) lsquoMineral nutrition of higher plantsrsquo (Academic PressLondon)

Martorell C Ezcurra E (2007) The narrow-leaf syndrome a functional andevolutionary approach to the form of fog-harvesting rosette plantsOecologia 151 561ndash573 doi101007s00442-006-0614-x

Maseda PH Fernaacutendez RJ (2006) Staywet or else threeways inwhich plantscan adjust hydraulically to their environment Journal of ExperimentalBotany 57 3963ndash3977 doi101093jxberl127

Mazer J (1989)Ecological taxonomic and life history correlates of seedmassamong Indiana dune angiosperms Ecological Monographs 59 153ndash175doi1023072937284

McDowellNBarnardHBondBHinckleyTHubbardR IshiiHKoumlstnerBMagnani F Marshall J Meinzer F Phillips N Ryan M Whitehead D(2002) The relationship between tree height and leaf area sapwood arearatio Oecologia 132 12ndash20 doi101007s00442-002-0904-x

McGill BJ Enquist BJWeiher EWestobyM (2006) Rebuilding communityecology from functional traits Trends in Ecology amp Evolution 21178ndash185 doi101016jtree200602002

McIntyre S Lavorel S (2001) Livestock grazing in subtropical pasturessteps in the analysis of attribute response and plant functionaltypes Journal of Ecology 89 209ndash226doi101046j1365-2745200100535x

McIntyre S Lavorel S Landsberg J Forbes TDA (1999) Disturbanceresponse in vebetation ndash towards a global perspective on functionaltraits Journal of Vegetation Science 10 621ndash630 doi1023073237077

Medina E (1999) Tropical forests diversity and function of dominant life-forms In lsquoHandbook of plant functional ecologyrsquo (Eds FI PugnaireF Valladares) pp 407ndash448 (Marcel Dekker New York)

Meinzer FC Clearwater MJ Goldstein G (2001) Water transport in treescurrentperspectives new insights and somecontroversiesEnvironmentaland Experimental Botany 45 239ndash262doi101016S0098-8472(01)00074-0

Mencuccini M (2003) The ecological significance of long-distance watertransport short-term regulation long-term acclimation and the hydrauliccosts of stature across plant life forms Plant Cell amp Environment 26163ndash182 doi101046j1365-3040200300991x

Messier J McGill BJ Lechowicz MJ (2010) How do traits vary acrossecological scales A case for trait-based ecology Ecology Letters 13838ndash848 doi101111j1461-0248201001476x

Meyer LD (1994) Rainfall simulators for soil conservation research In lsquoSoilerosion researchmethodsrsquo (EdRLal) pp 83ndash104 (St Lucie Press DelrayBeach FL)

Milla R Reich PB (2007) The scaling of leaf area and mass the cost of lightinterception increases with leaf size Proceedings Biological Sciences274 2109ndash2115 doi101098rspb20070417

Milton SJ (1991) Plant spinescence in arid southern Africa ndash does moisturemediate selection by mammals Oecologia 87 279ndash287doi101007BF00325267

Mohr H Schopfer P (1995) lsquoPlant physiologyrsquo 4th edn (Springer Berlin)Moles A Westoby M (2006) Seed size and plant strategy across the

whole life cycle Oikos 113 91ndash105doi101111j0030-1299200614194x

Moles A Ackerly D Webb C Tweddle J Dickie J Pitman A Westoby M(2005) Factors that shape seed mass evolution Proceedings of theNational Academy of Sciences USA 102 10540ndash10544doi101073pnas0501473102

Moles AT Warton DI Warman L Swenson NG Laffan SW Zanne AEPitman A Hemmings FA Leishman MR (2009) Global patterns in plantheight Journal of Ecology 97 923ndash932doi101111j1365-2745200901526x

Moreira B Tavsanoglu C Pausas JG (2012) Local versus regionalintraspecific variability in regeneration traits Oecologia 168 671ndash677doi101007s00442-011-2127-5

Munns R Husain S Rivelli AR James RA Condon AG Lindsay MPLagudah ES Schachtman DP Hare RA (2002) Avenues for increasingsalt tolerance of crops and the role of physiologically based selectiontraits Plant and Soil 247 93ndash105 doi101023A1021119414799

Mutch RW (1970) Wildland fires and ecosystems ndash A hypothesis Ecology51 1046ndash1051 doi1023071933631

Myers J Vellend M Gardescu S Marks P (2004) Seed dispersal by white-tailed deer implications for long-distance dispersal invasion andmigration of plants in eastern North America Oecologia 139 35ndash44doi101007s00442-003-1474-2

Newman EI (1966) A method of estimating the total root length of a sampleJournal of Applied Ecology 3 139ndash145 doi1023072401670

Ng FSP (1978) Strategies of establishment in Malayan forest trees InlsquoTropical trees as living systemsrsquo (Eds PB Tomlinson MHZimmerman)pp 129ndash162 (CambridgeUniversityPressCambridgeUK)

Niinemets Uuml (2001) Global-scale climatic controls of leaf dry mass per areadensity and thickness in trees and shrubs Ecology 82 453ndash469doi1018900012-9658(2001)082[0453GSCCOL]20CO2


Niinemets Uuml Portsmuth A Tena D Tobias M Matesanz S Valladares F(2007) Do we underestimate the importance of leaf size in planteconomics Disproportional scaling of support costs within thespectrum of leaf physiognomy Annals of Botany 100 283ndash303doi101093aobmcm107

Niklas KJ (1994) lsquoPlant allometry the scaling of form and processrsquo (TheUniversity of Chicago Press Chicago IL)

Niklas KJ Cobb ED Niinemets U Reich PB Sellin A Shipley B Wright IJ(2007) ldquoDiminishing returnsrdquo in the scaling of functional leaf traits acrossand within species groups Proceedings of the National Academy ofSciences USA 104 8891ndash8896 doi101073pnas0701135104

Noble IR Slatyer RO (1980) The use of vital attributes to predict successionalchanges in plant communities subject to recurrent disturbancesVegetatio43 5ndash21 doi101007BF00121013

North GB Noble PS (1992) Drought-induced changes in hydraulicconductivity and structure in roots of Ferocactus acanthodes andOpuntia ficus-indica New Phytologist 120 9ndash19doi101111j1469-81371992tb01053x

OrsquoLeary MH (1981) Carbon isotope fractionation in plants Phytochemistry20 553ndash567 doi1010160031-9422(81)85134-5

Olff H Vera FWM Bokdam J Bakker ES Gleichman JM de Maeyer KSmit R (1999) Shifting mosaics in grazed woodlands driven by thealternation of plant facilitation and competition Plant Biology 1127ndash137 doi101111j1438-86771999tb00236x

Ollinger SV Richardson ADMartin ME Hollinger DY Frolking SE ReichPB Plourde LC Katul GG Munger JW Oren R Smith M-L Pau U KTBolstad PV Cook BD Day MC Martin TA Monson RK Schmid HP(2008) Canopy nitrogen carbon assimilation and albedo in temperateand boreal forests functional relations and potential climate feedbacksProceedings of the National Academy of Sciences USA 10519 336ndash19 341 doi101073pnas0810021105

Onipchenko VG Makarov MI Van Logtestijn RSP Ivanov VBAkhmetzanova AA Teekev DK Ermak AA Salpargarova FSKozhevnikova AD Cornelissen JHC (2009) New nitrogen uptakestrategy specialized snow roots Ecology Letters 12 758ndash764doi101111j1461-0248200901331x

Onoda Y Schieving F Anten NPR (2008) Effects of light and nutrientavailability on leaf mechanical properties of Plantago major aconceptual approach Annals of Botany 101 727ndash736

Onoda Y Westoby M Adler PB Choong AMF Clissold FJ CornelissenJHCDiacuteaz S DominyNJ Elgart A Enrico L Fine PVAHoward JJ JaliliA Kitajima K Kurokawa H McArthur C Lucas PW Markesteijn LPeacuterez-HarguindeguyN Poorter L Richards L Santiago LS Sosinski EEVanBaelSAWartonDIWright IJ JosephWright SYamashitaN (2011)Global patterns of leaf mechanical properties Ecology Letters 14301ndash312 doi101111j1461-0248201001582x

Ordontildeez JC Van Bodegom PMWitte J-PM Bartholomeus RP Van Hal JRAerts R (2010) Plant strategies in relation to resource supply in mesic towet environments does theory mirror nature American Naturalist 175225ndash239 doi101086649582

Pakeman RJ Quested HM (2007) Sampling plant functional traits whatproportion of the species need to be measured Applied VegetationScience 10 91ndash96 doi101111j1654-109X2007tb00507x

Pammenter NW Van der Willigen C (1998) A mathematical and statisticalanalysis of the curves illustratingvulnerabilityof xylem to cavitationTreePhysiology 18 589ndash593 doi101093treephys188-9589

Papioacute C Trabaud L (1990) Structural characteristics of fuel components offiveMediterraean shrubsForest Ecology andManagement 35 249ndash259doi1010160378-1127(90)90006-W

Parkhurst DF (1994) Diffusion of CO2 and other gases inside leaves NewPhytologist 126 449ndash479 doi101111j1469-81371994tb04244x

Parkhurst DF LoucksOL (1972)Optimal leaf size in relation to environmentJournal of Ecology 60 505ndash537 doi1023072258359

Parton W Silver WL Burke IC Grassens L Harmon ME Currie WSKing JY Adair EC Brandt LA Hart SC Fasth B (2007) Global-scalesimilarities in nitrogen release patterns during long-term decompositionScience 315 361ndash364 doi101126science1134853

PatintildeoSLloydJPaivaRBakerTRQuesadaCAMercadoLMSchmerler JSchwarz M Santos AJB Aguilar A Czimczik CI Gallo J Horna VHoyosEJ JimenezEM PalominoW Peacock J Pentildea-CruzA SarmientoC Sota A Turriago JD Villanueva B Vitzthum P Alvarez E Arroyo LBaraloto C Bonal D Chave J Costa ACL Herrera R Higuchi N KilleenT Leal E Luizatildeo F Meir P Monteagudo A Neill D Nuacutentildeez-Vargas PPentildeuela MC Pitman N Priante Filho N Prieto A Panfil SN Rudas ASalomatildeo R Silva N Silveira M Soares de Almeida S Torres-Lezama AVaacutesquez-Martiacutenez RVieira IMalhiY PhillipsOL (2009)Branch xylemdensity variations across the Amazon BasinBiogeosciences 6 545ndash568doi105194bg-6-545-2009

Patintildeo S Fyllas NM Baker TR Paiva R Quesada CA Santos AJBSchwarz M ter Steege H Phillips OL Lloyd J (2012) Coordination ofphysiological and structural traits in Amazon forest trees Biogeosciences9 775ndash801 doi105194bg-9-775-2012

Paula S Pausas JG (2006) Leaf traits and resprouting ability in theMediterranean basin Functional Ecology 20 941ndash947doi101111j1365-2435200601185x

Paula S Pausas JG (2011) Root traits explain different foraging strategiesbetween resprouting life histories Oecologia 165 321ndash331doi101007s00442-010-1806-y

Paula S Arianoutsou M Kazanis Tavsanoglu Lloret F Buhk COjeda F Luna B Moreno JM Rodrigo A Espelta JM Palacio SFernandez-Santos B Fernandes PM Pausas JG (2009) Fire-relatedtraits for plant species of the Mediterranean Basin Ecology 90 1420doi10189008-13091

Pausas JG(1997)ResproutingofQuercus suber inNESpainafterfireJournalof Vegetation Science 8 703ndash706 doi1023073237375

Pausas JG Bradstock RA (2007) Fire persistence traits of plants along aproductivity and disturbance gradient in mediterranean shrublands ofsouth-east Australia Global Ecology and Biogeography 16 330ndash340doi101111j1466-8238200600283x

Pausas JG Alessio GA Moreira B Corcobado G (2012) Fires enhanceflammability in Ulex parviflorus New Phytologist 193 18ndash23doi101111j1469-8137201103945x

Peco B Traba J Levassor C Saacutenchez AM Azcaacuterate FM (2003) Seed sizeshape and persistence in dry Mediterranean grass and scrublands SeedScience Research 13 87ndash95 doi101079SSR2002127

Peacuterez-HarguindeguyNDiacuteazSVendraminiFCornelissen JHCGurvichDECabido M (2003) Leaf traits and herbivore selection in the field and incafeteria experiments Austral Ecology 28 642ndash650doi101046j1442-9993200301321x

Pickett STA Kempf JS (1980) Branching patterns in forest shrubs andunderstory trees in relation to habitat New Phytologist 86 219ndash228doi101111j1469-81371980tb03191x

Pierce S Winter K Griffiths H (2002) Carbon isotope ratio and the extent ofdaily CAM use by Bromeliaceae New Phytologist 156 75ndash83doi101046j1469-8137200200489x

Pisani JM Distel RA (1998) Inter- and intraspecific variations in productionof spines and phenols in Prosopis caldenia and Prosopis flexuosaJournal of Chemical Ecology 24 23ndash36 doi101023A1022380627261

Pons J Pausas JG (2007) Acorn dispersal estimated by radio-trackingOecologia 153 903ndash911 doi101007s00442-007-0788-x

Poorter L Bongers F (2006) Leaf traits are good predictors of plantperformance across 53 rain forest species Ecology 87 1733ndash1743doi1018900012-9658(2006)87[1733LTAGPO]20CO2

Poorter H Garnier E (1999) Ecological significance of relative growth rateand its components In lsquoHandbook of functional plant ecologyrsquo (Eds FIPugnaire F Valladares) pp 81ndash120 (Marcel Dekker New York)


PoorterHGarnierE (2007)The ecological significanceof variation in relativegrowth rate and its components In lsquoFunctional plant ecologyrsquo (Eds FIPugnaire F Valladares) pp 67ndash100 (CRC press Boca Raton FL)

Poorter H Lewis C (1986) Testing differences in relative growth ratea method avoiding curve fitting and pairing Physiologia Plantarum67 223ndash226 doi101111j1399-30541986tb02447x

Poorter H Nagel O (2000) The role of biomass allocation in the growthresponse of plants to different levels of light CO2 nutrients and water aquantitative review [published erratum appears in Australian Journal ofPlant Physiology 27 1191] Australian Journal of Plant Physiology 27595ndash607 doi101071PP99173_CO

Poorter H Rozendaal DMA (2008) Leaf size and leaf display of thirty-eighttropical tree species Oecologia 158 35ndash46doi101007s00442-008-1131-x

PoorterHWelschenRAM(1993)Analyzingvariation inRGRin termsof theunderlyingcarboneconomy In lsquoMethods in comparativeplant ecologyndashalaboratorymanualrsquo (EdsGAFHendry JPGrime) pp 107ndash110 (Chapmanand Hall London)

Poorter L Bongers L Bongers F (2006) Architecture of 54 moist-forest treespecies traits trade-oofs and functional groupsEcology 87 1289ndash1301doi1018900012-9658(2006)87[1289AOMTST]20CO2

Poorter L Wright SJ Paz H Ackerly DD Condit R Ibarra-Manriacutequez GHarmsKELicona JCMartiacutenez-RamosMMazerSJMuller-LandauHCPentildea-Claros M Webb CO Wright IJ (2008) Are functional traits goodpredictors of demographics rates Evidence from five neotropical forestsEcology 89 1908ndash1920 doi10189007-02071

Poorter H Niinemets Uuml Poorter L Wright IJ Villar R (2009) Causes andconsequences of variation in leaf mass per area (LMA) a meta-analysisNew Phytologist 182 565ndash588 doi101111j1469-8137200902830x

Poorter L Kitajima K Mercado P Chubintildea J Melgar I Prins HHT (2010)Resprouting as a persistence strategy of tropical forest trees its relationwith carbohydrate storage and shade tolerance Ecology 91 2613ndash2627doi10189009-08621

PoorterHNiklasKJReichPBOleksyn J PootPMommerL (2012)Tansleyreview Biomass allocation to leaves stems and roots meta-analyses ofinterspecific variation and environmental control New Phytologist 19330ndash50 doi101111j1469-8137201103952x

Poschlod P Kiefer S Traumlnkle U Bonn S (1998) Plant species richness incalcareous grasslands as affected by dispersability in space and timeApplied Vegetation Science 1 75ndash91 doi1023071479087

Poschlod PKleyerM TackenbergO (2000)Databases on life history traits asa tool for risk assessment in plant species Zeitschrift fuumlr Oumlkologie undNaturschutz 9 3ndash18

Poschlod P Tackenberg O Bonn S (2005) Plant dispersal potential and itsrelation to species frequency and coexistence In lsquoVegetation ecologyrsquo(Ed E Van der Maarel) pp 147ndash171 (Blackwell Oxford UK)

Pregitzer KS DeForest JL Burton AJ Allen MF Ruess RW Hendrick RL(2002) Fine root architecture of nine North American trees Ecology 72293ndash309

Preston KA Ackerly DD (2003) Hydraulic architecture and the evolution ofshoot allometry in contrasting climates American Journal of Botany 901502ndash1512 doi103732ajb90101502

Price CA Symonova O Mileyko Y Hilley T Weitz J (2011) Leaf extractionand analysis framework graphical user interface segmenting andanalyzing the structure of leaf veins and areoles Plant Physiology 155236ndash245 doi101104pp110162834

Puigdefaacutebregas J Pugnaire FI (1999) Plant survival in arid environments InlsquoHandbook of functional plant ecologyrsquo (Eds FI Pugnaire F Valladares)pp 381ndash405 (Marcel Dekker New York)

Putz FE Phyllis DC Lu K Montalvo A Aiello A (1983) Uprooting andsnapping of trees structural determinants and ecological consequencesCanadian Journal of Forest Research 13 1011ndash1020doi101139x83-133

Pyankov VI Gunin PD Tsoog S Black CC (2000) C-4 plants in thevegetation of Mongolia their natural occurrence and geographicaldistribution in relation to climate Oecologia 123 15ndash31doi101007s004420050985

RabotnovTA (1950) The life cycle of perennial herbaceous plants inmeadowcoenoses Trudy Botanicheskogo Instituta Akademii Nauk SSSR Seriia III6 7ndash204

Raunkiaer C (1934) lsquoThe life forms of plants and statistical plant geographyrsquo(Clarendon Press Oxford UK)

Read J Stokes A (2006) Plant biomechanics in an ecological contextAmerican Journal of Botany 93 1546ndash1565 doi103732ajb93101546

Rebollo S Milchunas DG Noy-Meir I Chapman PL (2002) The roleof a spiny plant refuge in structuring grazed shortgrass steppeplant communities Oikos 98 53ndash64doi101034j1600-07062002980106x

Rees M Osborne CP Woodward FI Hulme SP Turnbull LA Taylor SH(2010)Partitioning the components of relative growth rate how importantis plant size variation American Naturalist 176 E152ndashE161doi101086657037

Reich PB (2002) Root-shoot relations optimality in acclimation andadaptation or the lsquoEmperorrsquos new clothes In lsquoPlant roots the hiddenhalfrsquo (Eds Y Waisel A Eshel U Kafkafi) pp 314ndash338 (Marcel DekkerNew York)

Reich PB Uhl C Walters MB Ellsworth DS (1991a) Leaf lifespan as adeterminant of leaf structure and function among 23 Amazonian treespecies Oecologia 86 16ndash24 doi101007BF00317383

Reich PB Walters MB Ellsworth DS (1991b) Leaf age and season influencethe relationships between leaf nitrogen leaf mass per area andphotosynthesis in maple and oak trees Plant Cell amp Environment 14251ndash259 doi101111j1365-30401991tb01499x

Reich PBWaltersMB Ellsworth DS (1992) Leaf life-span in relation to leafplant and stand characteristics among diverse ecosystems EcologicalMonographs 62 365ndash392 doi1023072937116

Reich PB Walters MB Ellsworth DS (1997) From tropics to tundraglobal convergence in plant functioning Proceedings of the NationalAcademy of Sciences USA 94 13730ndash13734doi101073pnas942513730

Reich PB Tjoelker MG Walters MB Vanderklein DW Bushena C (1998)Close association ofRGR leaf and rootmorphology seedmass and shadetolerance in seedlings of nine boreal tree species grown in high and lowlight Functional Ecology 12 327ndash338doi101046j1365-2435199800208x

Reich PB Ellsworth DS Walters MB Vose JM Gresham C Volin JCBowmanWD (1999)Generality of leaf trait relationships a test across sixbiomes Ecology 80 1955ndash1969doi1018900012-9658(1999)080[1955GOLTRA]20CO2

Reich PB Tilman D Naeem S Ellsworth DS Knops J Craine J Wedin DTrost J (2004) Species and functional group diversity independentlyinfluence biomass accumulation and its response to CO2 and NProceedings of the National Academy of Sciences USA 10110 101ndash10 106 doi101073pnas0306602101

Reich PB Tjoelker MG Pregitzer KS Wright IJ Oleksyn J Machado JL(2008) Scaling of respiration to nitrogen in leaves stems and roots ofhigher land plants Ecology Letters 11 793ndash801doi101111j1461-0248200801185x

Reich PB Oleksyn J Wright IJ Niklas KJ Hedin L Elser J (2010) Evidenceof a general 23-power law of scaling leaf nitrogen to phosphorus amongmajor plant groups and biomes Proceedings Biological Sciences 277877ndash883 doi101098rspb20091818

Reyes G Brown S Chapman J Lugo AE (1992) Wood densities of tropicaltree species General technical report S0-88 US Department ofAgriculture Forest Service Southern Forest Experiment Station NewOrleans LA


RichterM (1992)Methods of interpreting climatological conditions based onphytomorphological characteristics in the cordilleras of the NeotropicsPlant Research and Development 36 89ndash114

Robertson GP Coleman DC Bledsoe CS Sollins P (1999) lsquoStandard soilmethods for long-term ecological researchrsquo (Oxford University PressOxford UK)

Rodriguez-Calcerrada J Atkin OK Robson TM Zaragoza-Castells J Gil LAranda I (2010) Thermal acclimation of leaf dark respiration of beechseedlingsexperiencingsummerdrought inhighand lowlightenvironmentsTree Physiology 30 214ndash224 doi101093treephystpp104

RoumlmermannCTackenbergO PoschlodP (2005a)How to predict attachmentpotential of seeds to sheep and cattle coat from simplemorphological seedtraits Oikos 110 219ndash230 doi101111j0030-1299200513911x

Roumlmermann C Tackenberg O Poschlod P (2005b) Buoyancy In lsquoTheLEDA traitbase collecting and measuring standardsrsquo (Eds IC KnevelRM Bekker D Kunzmann M Stadler K Thompson) pp 124ndash127(Groningen University Groningen The Netherlands)

Roumlmermann C Tackenberg O Poschlod P (2005c) External animal dispersal(epizoochory) In lsquoThe LEDA traitbase collecting and measuringstandardsrsquo (Eds IC Knevel RM Bekker D Kunzmann M Stadler KThompson) pp 127ndash129 (Groningen University Groningen TheNetherlands)

Roth-Nebelsick A Uhl D Mosbrugger V Kerp H (2001) Evolution andfunction of leaf venation architecture a review Annals of Botany 87553ndash566 doi101006anbo20011391

Rothermel RC (1972) A mathematical model for predicting fire spread inwildland fuels USDAForest Service Research Paper INT (USDAForestService Washington DC)

Roumet C Urcelay C Diacuteaz S (2006) Suites of root traits differ betweenannual and perennial species growing in the field New Phytologist 170357ndash368 doi101111j1469-8137200601667x

Royer DL McElwain JC Adams JM Wilf P (2008) Sensitivity of leaf sizeand shape to climate within Acer rubrum and Quercus kelloggii NewPhytologist 179 808ndash817 doi101111j1469-8137200802496x

Rozema J Bijwaard P Prast G Broekman R (1985) Ecophysiologicaladaptations of coastal halophytes from foredunes and salt marshesVegetatio 62 499ndash521 doi101007BF00044777

Rozema JWeijers SBroekmanRBlokker PBuizerBWerlemanCYaqineHEHoogedoornHMayoral FuertesM Cooper E (2009) Annual growthof Cassiope tetragona as a proxy for Arctic climate developingcorrelative and experimental transfer functions to reconstruct pastsummer temperature on a millennial time scale Global ChangeBiology 15 1703ndash1715 doi101111j1365-2486200901858x

Rundel PW (1991) Shrub life-forms In lsquoResponse of plants to multiplestressesrsquo (EdsHAMooneyWEWinner EJ Pell) pp 345ndash370 (AcademicPress San Diego CA)

Ryser P Bernardi J Merla A (2008) Determination of leaf fresh mass afterstorage between moist paper towels constraints and reliability of themethod Journal of Experimental Botany 59 2461ndash2467doi101093jxbern120

SackLFroleK (2006)Leaf structural diversity is related to hydraulic capacityin tropical rain forest trees Ecology 87 483ndash491 doi10189005-0710

Sack L Holbrook NM (2006) Leaf hydraulics Annual Review of PlantBiology 57 361ndash381 doi101146annurevarplant56032604144141

Sack L Grubb PJ Marantildeoacuten T (2003) The functional morphology of juvenileplants tolerant of strong summer drought in shaded forest understories insouthern Spain Plant Ecology 168 139ndash163doi101023A1024423820136

SackLCornwellWKSantiagoLSBarbourMMChoatBEvans JRMunnsR Nicotra A (2010) A unique web resource for physiology ecology andthe environmental sciences PrometheusWiki Functional Plant Biology37 687ndash693 doi101071FP10097

Sage RF (2001) Environmental and evolutionary preconditions for the originand diversification of the C4 photosynthetic syndrome Plant Biology 3202ndash213 doi101055s-2001-15206

Santiago LS Goldstein G Meinzer FC Fisher JB Machado K Woodruff DJones T (2004) Leaf photosynthetic traits scale with hydraulicconductivity and wood density in Panamanian forest canopy treesOecologia 140 543ndash550 doi101007s00442-004-1624-1

Scarff FR Westoby M (2006) Leaf litter flammability in some semi-aridAustralian woodlands Functional Ecology 20 745ndash752doi101111j1365-2435200601174x

SchenkHJ Jackson RB (2002) The global biogeography of rootsEcologicalMonographs 72 311ndash328doi1018900012-9615(2002)072[0311TGBOR]20CO2

Scholander PF (1966) The role of solvent pressure in osmotic systemsProceedings of the National Academy of Sciences USA 55 1407ndash1414doi101073pnas5561407

Schurr FM Bond WJ Midgley GF Higgins SI (2005) A mechanistic modelfor secondary seed dispersal by wind and its experimental validationJournal of Ecology 93 1017ndash1028doi101111j1365-2745200501018x

Schweingruber FH (1996) lsquoTree rings and environment Dendroecologyrsquo(Haupt-Verlag Bern Switzerland)

Schweingruber FH Poschlod P (2005) Growth rings in herbs and shrubs lifespan age determination and stem anatomy Forest Snow and LandscapeResearch 79 195ndash415

Schwilk DW Ackerly DD (2001) Flammability and serotiny as strategiescorrelated evolution in pines Oikos 94 326ndash336doi101034j1600-07062001940213x

Seibt URajabiAGriffithsHBerry JA (2008)Carbon isotopes andwater useefficiency sense and sensitivity Oecologia 155 441ndash454doi101007s00442-007-0932-7

Seiwa K Kikuzawa K (1996) Importance of seed size for the establishmentof seedlings of five deciduous broad-leaved tree species Vegetatio 12351ndash64 doi101007BF00044887

Shipley B (1995) Structured interspecific determinants of specific leaf area in34 species of herbaceous angiosperms Functional Ecology 9 312ndash319doi1023072390579

Shipley B Vu TT (2002) Dry matter content as a measure of dry matterconcentration in plants and their parts New Phytologist 153 359ndash364doi101046j0028-646X200100320x

SingerMC(2000)Reducingambiguity indescribingplant-insect interactionslsquopreferencersquo lsquoacceptabilityrsquo and lsquoelectivityrsquo Ecology Letters 3 159ndash162doi101046j1461-0248200000136x

Skinner FKRotenbergYNeumannAW(1989)Contact anglemeasurementsfrom the contact diameter of sessile drops by means of a modifiedaxisymmetric drop shape analysis Journal of Colloid and InterfaceScience 130 25ndash34 doi1010160021-9797(89)90074-X

Smith WK Bell DT Shepherd KA (1998) Associations between leafstructure orientation and sunlight exposure in five Western Australiancommunities American Journal of Botany 85 56ndash63doi1023072446554

SouthwoodTRE(1977)Habitat the templet for ecological strategies Journalof Animal Ecology 43 337ndash365

Southwood TRE Brown VK Reader PM (1986) Leaf palatability lifeexpectancy and herbivore damage Oecologia 70 544ndash548doi101007BF00379901

Sperry JS (2003) Evolution of water transport and xylem structureInternational Journal of Plant Sciences 164 S115ndashS127doi101086368398

Sperry JS Donnelly JR TyreeMT (1988) Amethod for measuring hydraulicconductivity and embolism in xylem Plant Cell amp Environment 1135ndash40 doi101111j1365-30401988tb01774x


Sperry JSMeinzer FCMcCullohKA (2008a) Safety and efficiency conflictsin hydraulic architecture scaling from tissues to trees Plant Cell ampEnvironment 31 632ndash645 doi101111j1365-3040200701765x

Sperry JS Taneda H Bush SE Hacke UG (2008b) Evaluation of centrifugalmethods for measuring xylem cavitation in conifers diffuse and ring-porous angiosperms New Phytologist 177 558ndash568

Srivastava DS Cadotte MW MacDonald AAM Marushia RG MirotchnickN (2012) Phylogenetic diversity and the functioning of ecosystemsEcology Letters 15 637ndash648 doi101111j1461-0248201201795x

StaverACBondWJ February EC (2011)Historymatters tree establishmentvariability and species turnover in anAfrican savannaEcosphere 2 art49doi101890ES11-000291

Stephens SL Gordon DA Martin RE (1994) Combustibility of selecteddomestic vegetation subjected to desiccation In lsquo12th conference on fireand forest meteorologyrsquo Jekyll Island GA Available from httpwwwcnrberkeleyedustephens-labPublicationsStephensetal_93_20Comb_Vegpdf [Verified 22 February 2013]

Steudle E (2001) Water uptake by plant roots an integration of viewsIn lsquoRecent advances of plant root structure and functionrsquo (Eds OGašpariacutekovaacute M Ciamporovaacute I Mistriacutek F Baluška) pp 71ndash82 (KluwerAcademic Publishers Dordrecht The Netherlands)

Stich B PiephoH-P Schulz BMelchinger AE (2008)Multi-trait associationmapping in sugar beet (Beta vulgaris L) Theoretical and AppliedGenetics 117 947ndash954 doi101007s00122-008-0834-z

Strauss SY Agrawal AA (1999) The ecology and evolution of plant toleranceto herbivory Trends in Ecology amp Evolution 14 179ndash185doi101016S0169-5347(98)01576-6

Swenson NG Enquist BJ (2008) The relationship between stem andbranch specific gravity and the ability of each measure to predictleaf area American Journal of Botany 95 516ndash519doi103732ajb954516

Tackenberg O (2003) Modeling long distance dispersal of plant diaspores bywind Ecological Monographs 73 173ndash189doi1018900012-9615(2003)073[0173MLDOPD]20CO2

Tackenberg O Poschlod P Bonn S (2003) Assessment of wind dispersalpotential in plant species Ecological Monographs 73 191ndash205doi1018900012-9615(2003)073[0191AOWDPI]20CO2

Taiz L Zeiger E (2010) lsquoPlant physiologyrsquo (The BenjaminCummingsPublishing Redwood City CA)

Tamm CO (1972) Survival and flowering of some perennial herbs II Thebehaviour of some orchids on permanent plots Oikos 23 23ndash28doi1023073543923

Taylor B Parkinson D (1988) A new microcosm approach to litterdecomposition studies Canadian Journal of Botany 66 1933ndash1939

Temminghoff EEJM Houba VJG (2004) lsquoPlant analyses proceduresrsquo(Kluwer Academic Publishers Dodrecht The Netherlands)

Tennant D (1975) A test of amodified line intersect method of estimating rootlength Journal of Ecology 63 995ndash1001 doi1023072258617

Thomas SC (1996) Asymptotic height as a predictor of growth and allometriccharacteristics inMalaysian rain forest trees American Journal of Botany83 556ndash566 doi1023072445913

ThompsonK Band SR Hodgson JG (1993) Seed size and shape predict seedpersistence in the soil Functional Ecology 7 236ndash241doi1023072389893

Thompson K Bakker JP Bekker RM (1997) lsquoThe soil seed bank of NorthWest Europe methodology density and longevityrsquo (CambridgeUniversity Press Cambridge UK)

Tjoelker MG Oleksyn J Reich PB (2001) Modelling respiration ofvegetation evidence for a general temperature-dependent Q10 GlobalChange Biology 7 223ndash230 doi101046j1365-2486200100397x

Turner NC (1988) Measurement of plant water status by the pressurechamber technique Irrigation Science 9 289ndash308doi101007BF00296704

Turner IM (1994) Sclerophylly primarily protective Functional Ecology 8669ndash675 doi1023072390225

Tyree MT Sperry JS (1989) Vulnerability of xylem to cavitation andembolism Annual Review of Plant Physiology and Plant MolecularBiology 40 19ndash36 doi101146annurevpp40060189000315

TyreeMT ZimmermannMH (2002) lsquoXylem structure and the ascent of saprsquo(Springer-Verlag Berlin)

Uhl D Mosbrugger V (1999) Leaf venation density as a climate andenvironmental proxy a critical review and new dataPalaeogeography Palaeoclimatology Palaeoecology 149 15ndash26doi101016S0031-0182(98)00189-8

Vaieretti MV Diacuteaz S Vile D Garnier E (2007) Two measurementmethods of leaf dry matter content produce similar results in a broadrange of species Annals of Botany 99 955ndash958doi101093aobmcm022

Valette J-C (1997) Inflammabilities of Mediterranean species In lsquoForest firerisk and management EUR 16719 ENrsquo (Ed P Balabanis F EftichidisR Fantechi) pp 51ndash64 (European Commission Environment andQualityof Life Brussels)

Van Altena C Van Logtestijn RSP Cornwell WK Cornelissen JHC (2012)Species composition and fire non-additivemixture effects on ground fuelflammability Frontiers in Plant Science 3 63doi103389fpls201200063

Van der Pijl L (1982) lsquoPrinciples of dispersal in higher plantsrsquo (Springer-Verlag Berlin)

VanGelderHA Poorter L Sterck FJ (2006)Woodmechanics allometry andlife-history variation in a tropical rain forest tree community NewPhytologist 171 367ndash378 doi101111j1469-8137200601757x

Van Groenendael JM Klimeš L Klimešova J Hendriks RJJ (1997)Comparative ecology of clonal plants In lsquoPlant life historiesrsquo (Eds JLHarper J Silvertown M Franco) pp 191ndash209 (Cambridge UniversityPress Cambridge UK)

Vendramini F Diacuteaz S Gurvich DE Wilson PJ Thompson K Hodgson JG(2002) Leaf traits as indicators of resource-use strategy in floras withsucculent species New Phytologist 154 147ndash157doi101046j1469-8137200200357x

Veneklaas EJ Poorter L (1998) Growth and carbon partitioning of tropicaltree seedlings in contrasting light environments In lsquoInherent variationin plant growthrsquo (Eds H Lambers H Poorter MMI Van Vuuren)pp 337ndash361 (Backhuys Publishers Leiden The Netherlands)

Veneklaas EJ Zagt RJ Van Leerdam A Van Ek R Broekhoven AJ VanGenderen M (1990) Hydrological properties of the epiphyte mass of amontane tropical rain forest Colombia Vegetatio 89 183ndash192doi101007BF00032170

VeskPAWestobyM(2004)Sproutingability acrossdiversedisturbances andvegetation types worldwide Journal of Ecology 92 310ndash320doi101111j0022-0477200400871x

Vile D Garnier E Shipley B Laurent G Navas M-L Roumet C Lavorel SDiacuteaz S Hodgson JG Lloret F Midgley GF Poorter H Rutherford MCWilson PJ Wright IJ (2005) Specific leaf area and dry matter contentestimate thickness in laminar leaves Annals of Botany 96 1129ndash1136doi101093aobmci264

Violle C Navas M-L Vile D Kazakou E Fortunel C Hummel I Garnier E(2007) Let the concept of trait be functional Oikos 116 882ndash892doi101111j0030-1299200715559x

Wahl S Ryser P (2000) Root tissue structure is linked to ecological strategiesof grasses New Phytologist 148 459ndash471doi101046j1469-8137200000775x

Walters MB Reich PB (1999) Low-light carbon balance and shade tolerancein the seedlings of woody plants do winter deciduous and broad-leavedevergreen species differ New Phytologist 143 143ndash154doi101046j1469-8137199900425x

Wand SJE Midgley GG Jones MH Curtis PS (1999) Responses of wildC4 and C3 grasses (Poaceae) species to elevated atmospheric CO2

concentration a meta-analytical test of current theories andperceptions Global Change Biology 5 723ndash741doi101046j1365-2486199900265x


Weiher E Clarke GDP Keddy PA (1998) Community assembly rulesmorphological dispersion and the coexistence of plant species Oikos81 309ndash322 doi1023073547051

Weiher E Van derWerf A ThompsonK RoderickM Garnier E ErikssonO(1999) Challenging Theophrastus a common core list of plant traits forfunctional ecology Journal of Vegetation Science 10 609ndash620doi1023073237076

Westoby M (1998) A leaf-height-seed (LHS) plant ecology strategy schemePlant and Soil 199 213ndash227 doi101023A1004327224729

Westoby M Cunningham SA Fonseca C Overton J Wright IJ (1998)Phylogeny and variation in light capture area deployed per unitinvestment in leaves designs for selecting study species with a view togeneralizing In lsquoVariation in growth rate and productivity of higherplantsrsquo (Eds H Lambers H Poorter H MMI Van Vuuren) pp 539ndash566(Backhuys Publishers Leiden The Netherlands)

Westoby M Warton D Reich PB (2000) The time value of leaf areaAmerican Naturalist 155 649ndash656 doi101086303346

Westoby M Falster D Moles A Vesk P Wright I (2002) Plant ecologicalstrategies some leading dimensions of variation between speciesAnnualReview of Ecology and Systematics 33 125ndash159doi101146annurevecolsys33010802150452

Whittaker RH (1975) lsquoCommunities and ecosystemsrsquo 2nd edn (MacmillanPublishing New York)

Will H Tackenberg O (2008) A mechanistic simulation model of seeddispersal by animals Journal of Ecology 96 1011ndash1022doi101111j1365-2745200701341x

Williamson GB Wiemann MC (2010) Measuring wood specificgravity Correctly American Journal of Botany 97 519ndash524doi103732ajb0900243

Wilson PJ Thompson K Hodgson JG (1999) Specific leaf area and leaf drymatter content asalternativepredictorsofplant strategiesNewPhytologist143 155ndash162 doi101046j1469-8137199900427x

Withington JM Reich B Oleksyn J Eissenstat DM (2006) Comparisons ofstructure and life span in roots and leaves among temperate treesEcological Monographs 76 381ndash397doi1018900012-9615(2006)076[0381COSALS]20CO2

WitkowskiETFLamontBB(1991)Leaf specificmass confounds leaf densityand thickness Oecologia 88 486ndash493

Wong S-C Cowan IR Farquhar GD (1979) Stomatal conductance correlateswith photosynthetic capacity Nature 282 424ndash426doi101038282424a0

Wong S-C Cowan IR Farquhar GD (1985a) Leaf conductance in relationto rate of CO2 assimilation I Influence of nitrogen nutrition phosphorusnutrition photon flux density and ambient partial pressure of CO2

during ontogeny Plant Physiology 78 821ndash825doi101104pp784821

Wong S-C Cowan IR Farquhar GD (1985b) Leaf conductance in relation torate of CO2 assimilation II Effects of short-term exposures to differentphoton flux densities Plant Physiology 78 826ndash829doi101104pp784826

Wong S-C Cowan IR Farquhar GD (1985c) Leaf conductance in relationto rate of CO2 assimilation III Influences of water stress andphotoinhibitionPlant Physiology 78 830ndash834 doi101104pp784830

Wright IJCannonK (2001)Relationships between leaf lifespanand structuraldefences in a low-nutrient sclerophyll flora Functional Ecology 15351ndash359 doi101046j1365-2435200100522x

WrightW Illius AW (1995) A comparative study of the fracture properties of5 grasses Functional Ecology 9 269ndash278 doi1023072390573

Wright W Vincent JFK (1996) Herbivory and the mechanics of fracture inplants Biological Reviews of the Cambridge Philosophical Society 71401ndash413 doi101111j1469-185X1996tb01280x

Wright IJWestobyM (2002) Leaves at low versus high rainfall coordinationof structure lifespan and physiology New Phytologist 155 403ndash416doi101046j1469-8137200200479x

Wright IJ Clifford HT Kidson R Reed ML Rice BL Westoby M (2000)A survey of seed and seedling characters in 1744 Australian dicotyledonspecies cross-species trait correlations and correlated trait-shifts withinevolutionary lineagesBiological Journal of the Linnean Society LinneanSociety of London 69 521ndash547doi101111j1095-83122000tb01222x

Wright IJ Westoby M Reich PB (2002) Convergence towards higher leafmass per area in dry and nutrient-poor habitats has different consequencesfor leaf life span Journal of Ecology 90 534ndash543doi101046j1365-2745200200689x

Wright IJ Reich PB Westoby M Ackerly DD Baruch Z Bongers FCavender-Bares J Chapin T Cornelissen JHC Diemer M Flexas JGarnier E Groom PK Gulias J Hikosaka K Lamont BB Lee T LeeWLuskCMidgley JJNavasMLNiinemetsUOleksyn JOsadaNPoorterH Poot P Prior L Pyankov VI Roumet C Thomas SC Tjoelker MGVeneklaas EJ Villar R (2004) The worldwide leaf economics spectrumNature 428 821ndash827 doi101038nature02403

Wright IJ Falster DS PickupMWestobyM (2006) Cross-species patterns inthe coordination between leaf and stem traits and their implications forplant hydraulics Physiologia Plantarum 127 445ndash456doi101111j1399-3054200600699x

Wright IJ Ackerly D Bongers F Harms KE Ibarra-Manriacutequez G Martiacutenez-RamosMMazer SJMuller-Landau HC Paz H PitmanNCA Poorter LSilman MR Vriesendorp CF Webb CO Westoby M Wright SJ (2007)Relationships among ecologically important dimensions of plant traitvariation in seven neotropical forests Annals of Botany 99 1003ndash1015doi101093aobmcl066

Yeo AR (1983) Salinity resistance physiologies and prices PhysiologiaPlantarum 58 214ndash222 doi101111j1399-30541983tb04172x

Zanne AE Chapman CA Kitajima K (2005) Evolutionary and ecologicalcorrelates of early seedling morphology in east African trees and shrubsAmerican Journal of Botany 92 972ndash978 doi103732ajb926972

Zanne AE Westoby M Falster DS Ackerly DD Loarie SR Arnold SEJCoomesDA (2010)Angiospermwood structure global patterns in vesselanatomy and their relation to wood density and potential conductivityAmerican Journal of Botany 97 207ndash215 doi103732ajb0900178

Zhu JK (2001) Plant salt tolerance Trends in Plant Science 6 66ndash71doi101016S1360-1385(00)01838-0

Zimmermann MH Jeje AA (1981) Vessel-length distribution in stems ofsome American woody plants Canadian Journal of Botany 591882ndash1892 doi101139b81-248

Zinke PJ (1962) The pattern of influence of individual forest trees on soilproperties Ecology 43 130ndash133 doi1023071932049

Zotz G Ziegler H (1997) The occurrence of crassulacean metabolism amongvascular epiphytes from central Panama New Phytologist 137 223ndash229doi101046j1469-8137199700800x

ZwienieckiMAMelcherPJHolbrookNM(2001)Hydrogel control of xylemhydraulic resistance in plants Science 291 1059ndash1062doi101126science1057175

Zwieniecki MA Melcher PJ Boyce CK Sack L Holbrook NM (2002)Hydraulic architecture of leaf venation in Laurus nobilis L Plant Cell ampEnvironment 25 1445ndash1450 doi101046j1365-3040200200922x


Appendix 1 Summary of plant traitsSummary of plant traits included in the handbook

The range of values corresponds to those generally reported forfield-grownplants Ranges of values are based on the literature and the authorsrsquo datasets and do notalways necessarily correspond to the widest ranges that exist in nature or are theoretically possible Recommended sample size indicates the minimum andpreferred number of individuals to be sampled so as to obtain an appropriate indication of the values for the trait of interest when only one value is given itcorresponds to the number of individuals (=replicates) when two values are given thefirst one corresponds to the number of individuals and the second one to thenumber of organs to bemeasured per individual Note that one replicate can be compounded from several individuals (for smaller species)whereas one individualcannotbeused for different replicatesThe expectedcoefficient of variation (CV) rangegives the20th and the80thpercentile of theCV(=sd scaled to themean) asobserved in a number of datasets obtained for a range offield plants for different biomesNumbering of plant traits correspondswith the numbering of the chapters

in the handbook

Plant trait Preferred unit Range of values Recommended noof replicates

CVrange

Minimum Preferred ()

2 Whole-plant traits21 Life history Categorical ndash 3 5 ndash

22 Life form Categorical ndash 3 5 ndash

23 Growth form Categorical ndash 3 5 ndash

24 Plant height m lt001ndash140 10 25 17ndash3625 Clonality Categorical ndash 5 10 ndash

26 Spinescence 5 10 ndash

Spine length mm 05ndash300Spine width mm 05ndash30Spine length leaf length Unitless 0ndash30

27 Branching architecture No of ramificationsper branch

0 ndash gt100 5 10 ndash

28 Leaf to sapwood area Unitless 100ndash103 5 10 ndash

29 Root-mass fraction Unitless 015ndash040(for seedlingsdown to 010)

5 10 ndash

210 Salt-tolerance traits 5 10Selective root cation uptake Unitless ndash ndash

Salt excretion and compartmentalisation Categorical ndash ndash

211 Relative growth rate mg gndash1 dayndash1 2ndash300 10 20212 Shoot flammability Unitless 0 ndash ~3 5 10213 Water-flux traits 10 20Gap fraction Unitless 0ndash1Stem flow 0ndash50Water retention on plant surface gmndash2 0ndash500Leaf wettability degrees (contact angle) 0ndash180Droplet retention ability degrees (slope angle) 0ndash90

3 Leaf traits31 Specific leaf area m2 kg1 (mm2mg1)A lt1ndash300 5 5 10 4 8ndash1632 Area of a leaf mm2 1 ndash gt206 5 5 10 4 17ndash3633 Leaf dry-matter content mg g1 50ndash700 5 5 10 4 4ndash1034 Leaf thickness mm lt01ndash5B 5 1035 pH of green leaves or leaf litter Unitless 35ndash65 5 5 10 4 1ndash636 Leaf nitrogen and phosphorus concentrations(a) Leaf nitrogen concentration mg g1 5ndash70 5 5 10 4 8ndash19(b) Leaf phosphorus concentration mg g1 02ndash5 5 5 10 4 10ndash28

37 Physical strength of leaves 5 5 10 4 14ndash29Force to tear Nmm1 017ndash40Work to shear Jmndash1 002ndash05Force to punch Nmmndash1 003ndash16

38 Leaf lifespan and duration of green foliage(a) Leaf lifespan Month 05ndash200 5 40 10 160 11ndash39(b) Duration of green foliage Month 1ndash12 5 10 ndash

39 Photosynthetic pathway Categorical ndash 3 3 ndash

310 Vein density mmmmndash2 05ndash25 5 10311 Light-saturated photosynthetic rate mmolmndash2 sndash1 2ndash30 5 10312 Leaf dark respiration mmolmndash2 sndash1 04ndash4 5 10313 Electrolyte leakage 2ndash100 5 5 10 4 9ndash26

(Continued )


Appendix 1 (continued )


CVrange


314 Leaf water potential MPa 70 5 5 5 10 11ndash33315 Leaf palatability Leaf area consumed 0ndash100 10 20316 Litter decomposabilityC Mass loss 0ndash100 10 20 7ndash14

4 Stem traits41 Stem-specific density mgmm3 (kg l1)A 01ndash13 5 10 5ndash942 Twig dry-matter content mg g1 150ndash850 5 10 2ndash843 Bark thickness mm 01 ndash gt30 5 1044 Xylem conductivity 5 10 21ndash63Stem-specific xylem hydraulic

conductivity (KS)kgmndash1 sndash1MPandash1 1 (gymnosperms)

to 200 (tropical lianas)Leaf-area-specific xylem hydraulic

conductivity (KL)kgmndash1 sndash1MPandash1 6 10ndash5

(gymnosperms)to 1 10ndash2

(tropical lianas)45 Vulnerability to embolism (Y50) MPa ndash025ndash14 5 10 20ndash45

5 Below-ground traits51 Specific root length m gndash1 3ndash350 5 10 10 10 15ndash2452 Root-system morphology 5 10Depth m 005ndash70Lateral extent m 005ndash40Density of exploration mmmmndash3 10ndash4ndash1

53 Nutrient uptake strategy Categorical ndash 5 10 ndash

6 Regenerative traits61 Dispersal mode Categorical ndash 3 6 ndash

62 Dispersule size and shapeSize (mass) mg (g)A 5 10Shape Unitless 0ndash1 3 6 ndash

63 Dispersal potential Dispersulesdisperseddispersulesproduced

ndash 10 20 ndash

64 Seed mass mg 103ndash107 5 10 14ndash2765 Seedling morphology Categorical ndash 3 6 ndash

66 Resprouting capacity Unitless 0ndash100 5 10 ndash

AAlternative preferred units in parenthesesBConsidering only photosynthetic tissue total leaf thickness can be gt40mm in some succulent plantsCReplicate numbers correspond to the number of individual plants (replicates) fromwhich to collect leaf litter number of leaves in each samplewill depend onits weight and the size of the litterbag


wwwpublishcsiroaujournalsajb

Abstract Plant functional traits are the features (morphological physiological phenological) that represent ecologicalstrategies and determine how plants respond to environmental factors affect other trophic levels and influence ecosystemproperties Variation in plant functional traits and trait syndromes has proven useful for tacklingmany important ecologicalquestions at a range of scales giving rise to a demand for standardised ways to measure ecologically meaningful plant traitsThis line of research has been among the most fruitful avenues for understanding ecological and evolutionary patterns andprocesses It also has the potential both to build a predictive set of local regional and global relationships between plants andenvironment and to quantify a wide range of natural and human-driven processes including changes in biodiversity theimpacts of species invasions alterations in biogeochemical processes and vegetationndashatmosphere interactions Theimportance of these topics dictates the urgent need for more and better data and increases the value of standardisedprotocols for quantifying trait variation of different species in particular for traitswith power to predict plant- and ecosystem-level processes and for traits that can be measured relatively easily Updated and expanded from the widely used previousversion this handbook retains the focus on clearly presentedwidely applicable step-by-step recipeswith aminimumof texton theory and not only includes updated methods for the traits previously covered but also introduces many new protocolsfor further traits This new handbook has a better balance between whole-plant traits leaf traits root and stem traits andregenerative traits and puts particular emphasis on traits important for predicting speciesrsquo effects on key ecosystempropertiesWehope this newhandbookbecomes a standard companion in local andglobal efforts to learn about the responsesand impacts of different plant species with respect to environmental changes in the present past and future

Additional keywords biodiversity ecophysiology ecosystem dynamics ecosystem functions environmental changeplant morphology

Received 23 November 2011 accepted 29 January 2013 published online 26 April 2013

Contents

Introduction and discussion 1691 Selection of species and individuals 17011 Selection of species17012 Selection of individuals within a species17113 Replicate measurements1722 Whole-plant traits17221 Life history and maximum plant lifespan 17222 Life form 17323 Growth form 17324 Plant height 17525 Clonality bud banks and below-ground storage

organs17626 Spinescence17727 Branching architecture 17828 Leaf area sapwood area ratio 17829 Root-mass fraction 179210 Salt resistance179211 Relative growth rate and its components181212 Plant flammability182213 Water-flux traits 1843 Leaf traits 18631 Specific leaf area 18632 Area of a leaf 18933 Leaf dry-matter content19034 Leaf thickness 19035 pH of green leaves or leaf litter 19136 Leaf nitrogen (N) concentration and leaf phosphorous

(P) concentration19237 Physical strength of leaves19338 Leaf lifespan and duration of green foliage 195

39 Vein density 197310 Light-saturated photosynthetic rate 198311 Leaf dark respiration 198312 Photosynthetic pathway 199313 C-isotope composition as a measure of intrinsic

water-use efficiency 200314 Electrolyte leakage as an indicator of frost

sensitivity 201315 Leaf water potential as a measure of water

status 202316 Leaf palatability as indicated by preference by

model herbivores203317 Litter decomposability 2054 Stem traits 20741 Stem-specific density 20742 Twig dry-matter content and twig drying time20943 Bark thickness (and bark quality) 20944 Xylem conductivity21045 Vulnerability to embolism 2115 Below-ground traits 21251 Specific root length 21252 Root-system morphology21453 Nutrient-uptake strategy2146 Regenerative traits 21561 Dispersal syndrome21562 Dispersule size and shape 21663 Dispersal potential 21664 Seed mass21765 Seedling functional morphology21866 Resprouting capacity after major disturbance218Acknowledgements220References220







































How to assess

(A) Life history























22 Life form


23 Growth form


How to record






































24 Plant height























Categories are then


























26 Spinescence




How to measure







(1) No spines











How to measure









2

1

3 4

5 6

branch length

startingpoint

trunk



Measuring
















210 Salt resistance






























































(m)

(e)

(g)

(h)(i )

( j ) (k )

( l )

(a)

(d )

(b )

(c )

(f )





























How to assess





















3 Leaf traits












Measuring

























32 Area of a leaf





Measuring

















Measuring






34 Leaf thickness






Measuring




















Measuring












Measuring




ndash NO3ndash and













Measuring


















(a) (b)

(c) (d )





(3) Punch tests






























39 Vein density




Measuring





































Measuring



























ndash8permil)



Measuring
















Measurement



Calculations


























Measuring










Pressuregauge

Chamber

Lid







Measuring








1 2 3 4 5 6

313029282726

51 52 53 54 55 56

818079787776

101 102 103 104 105 106

131130129128

(a)

(b)

(c)

127126










Measuring









(a) (b)


(c)











4 Stem traits






Measuring






























Measuring













How to measure














Measuring

















Measuring


















stem

debarkedend

(a)

stem segment


rubber tubing


(b)

stem

debarkedend

bubbles emerging







Measuring










































and storage and







How to classify
















Measuring









How to record











64 Seed mass







Measuring














(PER) and





Measuring








CERCHR

PEF

PERPHR




How to assess













Acknowledgements


References



































































































































































































































































































































































































































































































in the handbook


CVrange












5 10 ndash









(Continued )




CVrange














ndash 10 20 ndash











































How to assess

(A) Life history























22 Life form


23 Growth form


How to record






































24 Plant height























Categories are then


























26 Spinescence




How to measure







(1) No spines











How to measure









2

1

3 4

5 6

branch length

startingpoint

trunk



Measuring
















210 Salt resistance






























































(m)

(e)

(g)

(h)(i )

( j ) (k )

( l )

(a)

(d )

(b )

(c )

(f )





























How to assess





















3 Leaf traits












Measuring

























32 Area of a leaf





Measuring

















Measuring






34 Leaf thickness






Measuring




















Measuring












Measuring




ndash NO3ndash and













Measuring


















(a) (b)

(c) (d )





(3) Punch tests






























39 Vein density




Measuring





































Measuring



























ndash8permil)



Measuring
















Measurement



Calculations


























Measuring










Pressuregauge

Chamber

Lid







Measuring








1 2 3 4 5 6

313029282726

51 52 53 54 55 56

818079787776

101 102 103 104 105 106

131130129128

(a)

(b)

(c)

127126










Measuring









(a) (b)


(c)











4 Stem traits






Measuring






























Measuring













How to measure














Measuring

















Measuring


















stem

debarkedend

(a)

stem segment


rubber tubing


(b)

stem

debarkedend

bubbles emerging







Measuring










































and storage and







How to classify
















Measuring









How to record











64 Seed mass







Measuring














(PER) and





Measuring








CERCHR

PEF

PERPHR




How to assess













Acknowledgements


References



































































































































































































































































































































































































































































































in the handbook


CVrange












5 10 ndash









(Continued )




CVrange














ndash 10 20 ndash


































How to assess

(A) Life history























22 Life form


23 Growth form


How to record






































24 Plant height























Categories are then


























26 Spinescence




How to measure







(1) No spines











How to measure









2

1

3 4

5 6

branch length

startingpoint

trunk



Measuring
















210 Salt resistance






























































(m)

(e)

(g)

(h)(i )

( j ) (k )

( l )

(a)

(d )

(b )

(c )

(f )





























How to assess





















3 Leaf traits












Measuring

























32 Area of a leaf





Measuring

















Measuring






34 Leaf thickness






Measuring




















Measuring












Measuring




ndash NO3ndash and













Measuring


















(a) (b)

(c) (d )





(3) Punch tests






























39 Vein density




Measuring





































Measuring



























ndash8permil)



Measuring
















Measurement



Calculations


























Measuring










Pressuregauge

Chamber

Lid







Measuring








1 2 3 4 5 6

313029282726

51 52 53 54 55 56

818079787776

101 102 103 104 105 106

131130129128

(a)

(b)

(c)

127126










Measuring









(a) (b)


(c)











4 Stem traits






Measuring






























Measuring













How to measure














Measuring

















Measuring


















stem

debarkedend

(a)

stem segment


rubber tubing


(b)

stem

debarkedend

bubbles emerging







Measuring










































and storage and







How to classify
















Measuring









How to record











64 Seed mass







Measuring














(PER) and





Measuring








CERCHR

PEF

PERPHR




How to assess













Acknowledgements


References



































































































































































































































































































































































































































































































in the handbook


CVrange












5 10 ndash









(Continued )




CVrange














ndash 10 20 ndash
























How to assess

(A) Life history























22 Life form


23 Growth form


How to record






































24 Plant height























Categories are then


























26 Spinescence




How to measure







(1) No spines











How to measure









2

1

3 4

5 6

branch length

startingpoint

trunk



Measuring
















210 Salt resistance






























































(m)

(e)

(g)

(h)(i )

( j ) (k )

( l )

(a)

(d )

(b )

(c )

(f )





























How to assess





















3 Leaf traits












Measuring

























32 Area of a leaf





Measuring

















Measuring






34 Leaf thickness






Measuring




















Measuring












Measuring




ndash NO3ndash and













Measuring


















(a) (b)

(c) (d )





(3) Punch tests






























39 Vein density




Measuring





































Measuring



























ndash8permil)



Measuring
















Measurement



Calculations


























Measuring










Pressuregauge

Chamber

Lid







Measuring








1 2 3 4 5 6

313029282726

51 52 53 54 55 56

818079787776

101 102 103 104 105 106

131130129128

(a)

(b)

(c)

127126










Measuring









(a) (b)


(c)











4 Stem traits






Measuring






























Measuring













How to measure














Measuring

















Measuring


















stem

debarkedend

(a)

stem segment


rubber tubing


(b)

stem

debarkedend

bubbles emerging







Measuring










































and storage and







How to classify
















Measuring









How to record











64 Seed mass







Measuring














(PER) and





Measuring








CERCHR

PEF

PERPHR




How to assess













Acknowledgements


References



































































































































































































































































































































































































































































































in the handbook


CVrange












5 10 ndash









(Continued )




CVrange














ndash 10 20 ndash






Documents

New handbook for standardised measurement of plant