Symbiotic fungal associations in 'lower' land plants

Symbiotic fungal associations inlsquolowerrsquo land plants

D J Read1 J G Duckett2 R Francis1 R Ligrone3 and A Russell2

1Department of Animal and Plant Sciences University of Shecurreneld Shecurreneld S10 2TN UK2School of Biological Sciences Queen Mary amp Westcenteld College Mile End Road London E1 4NS UK

3Facoltacopy di Scienze Ambientali Seconda Universitacopy di Napoli via Vivaldi 43 I- 81100 Caserta Italy

An analysis of the current state of knowledge of symbiotic fungal associations in `lowerrsquo plants is providedThree fungal phyla the Zygomycota Ascomycota and Basidiomycota are involved in forming theseassociations each producing a distinctive suite of structural features in well-decentned groups of `lowerrsquoplants Among the `lowerrsquo plants only mosses and Equisetum appear to lack one or other of these types ofassociation The salient features of the symbioses produced by each fungal group are described and therelationships between these associations and those formed by the same or related fungi in `higherrsquo plantsare discussed Particular consideration is given to the question of the extent to which root^fungus associa-tions in `lowerrsquo plants are analogous to `mycorrhizasrsquo of `higherrsquo plants and the need for analysis of thefunctional attributes of these symbioses is stressed

Zygomycetous fungi colonize a wide range of extant lower land plants (hornworts many hepatics lyco-pods Ophioglossales Psilotales and Gleicheniaceae) where they often produce structures analogous tothose seen in the vesicular-arbuscular (VA) mycorrhizas of higher plants which are formed by membersof the order Glomales A preponderance of associations of this kind is in accordance with palaeobotanicaland molecular evidence indicating that glomalean fungi produced the archetypal symbioses with the centrstplants to emerge on to land

It is shown probably for the centrst time that glomalean fungi forming typical VA mycorrhiza with ahigher plant (Plantago lanceolata) can colonize a thalloid liverwort (Pellia epiphylla) producing arbusculesand vesicles in the hepatic The extent to which these associations which are structurally analogous tomycorrhizas have similar functions remains to be evaluated

Ascomycetous associations are found in a relatively small number of families of leafy liverworts Thestructural features of the fungal colonization of rhizoids and underground axes of these plants are similarto those seen in mycorrhizal associations of ericaceous plants likeVaccinium Cross inoculation experimentshave concentrmed that a typical mycorrhizal endophyte of ericaceous plants Hymenoscyphus ericae will formassociations in liverworts which are structurally identical to those seen in nature Again the functionalsignicentcance of these associations remains to be examined

Some members of the Jungermanniales and Metzgeriales form associations with basidiomycetous fungiThese produce intracellular coils of hyphae which are similar to the pelotons seen in orchid mycorrhizaswhich also involve basidiomycetes The fungal associates of the autotrophic Aneura and of its heterotrophicrelative Cryptothallus mirabilis have been isolated In the latter case it has been shown that the fungal symbiontis an ectomycorrhizal associate of Betula suggesting that the apparently obligate nature of the associationbetween the hepatic and Betula in nature is based upon requirement for this particularheterotroph

Keywords bryophytes cross inoculation fungal symbioses pteridophytes ultrastructure

1 INTRODUCTION

It was evident to de Bary (1887) that intimate associationsbetween organisms of dissimilar genotype are widespreadin nature He used the term `symbiosisrsquo to decentne suchpartnerships but perceived that the symbiotic conditioncould embrace a very broad range of relationships someof which were of the antagonistic kind leading even todeath of a partner while in others both partners thrivedin a mutually benecentcial association de Baryrsquos view of theextent and nature of symbiosis has been supported bysubsequent research One of its main legacies isrecognition of the need to establish by experiment the

status of any apparently symbiotic association betweenorganisms However progress towards understanding offunction has generally lagged behind awareness of theextent of distribution of symbioses in biological systemsNowhere is this more true than in the case of thesymbiotic condition as demonstrated in `lowerrsquo landplants We have a broad base of knowledge of the distribu-tion of these relationships in those groups of plants whichare the descendants of the original colonists of the terres-trial environment but we know little of the status of thesesymbioses because experimental analyses have been scarce

The present paper has two objectives The centrst is todescribe the present state of knowledge of the taxonomic

Phil Trans R Soc Lond B (2000) 355 815^831 815 copy 2000 The Royal Society

and structural features of those associations betweensymbiotic fungi and lower plants which appear to beconsistently present in nature Second it is intended toaddress the important question of the functional basis ofthe associations described

The distinction between `lowerrsquo and `higherrsquo plants issomewhat arbitrary but for the present purpose is takento lie between the supposedly `primitiversquo land plants withlarge sometimes achlorophyllous gametophytes in theBryopsida Lycopsida Equisetopsida or Pteropsida (onlythe Psilotales and Ophioglossales) and all those pteropsidsgymnosperms and angiosperms in which the gam-etophyte is more diminutive

The roots of the majority of land plants are colonized bysymbiotic fungi to form dual organs called `mycorrhizasrsquo

the term being derived from the two Greek words `mykesrsquofungus and `rhizarsquo root Since in the strictest sense theunderground axes and rhizoids of lower plants are notroots any such fungal associations with them should notbe called mycorrhizas However recent decentnitions of thissymbiosis for example that of Trappe (1996) see it inbroader terms referring to mycorrhizas as `dual organs ofabsorption formed when symbiotic fungi inhabit healthytissues of most terrestrial plantsrsquo Under a decentnition of thiskind which does not specify roots any healthy fungus-containingabsorptive tissue of lower plants can legitimatelybe referred to as a mycorrhiza and more important ques-tions concerning the extent to which the relationship isfunctionally as well as structurally analagous with thesesymbioses in higher plants can be addressed

816 D J Read and others Symbiotic fungal associations in lowerrsquo land plants

PhilTrans R Soc Lond B (2000)

Table 1 The characteristics of the four most widespread mycorrhizal types found in `higherrsquo plants

kinds of mycorrhiza

VA ectomycorrhiza ericoid orchid

fungiseptate 7 + + +aseptate + 7 7 7

intracellular colonization + 7 + +fungal sheath 7 + 7 7Hartig net 7 + 7 7vesicles + or 7 7 7 7arbuscules + or 7 7 7 7achlorophylly 7( + ) 7 7 + a

fungal taxa zygomycetes basidio- and ascomycetes ascomycetes basidiomycetesplant taxa leptosporangiate ferns

gymnospermsangiosperms


Ericales Orchidaceae

a All orchids are achlorophyllous in the early seedling stagesThe structural characters given relate to the mature state not the developing or senescent states

Figure 1 Fossil evidence for the occurrenceof VA mycorrhizas in early land plants (a) Anarbuscule-like structure (a) in a cortical cell ofAglaophyton collected from Rhynie Chert rocksof Devonian age ca 400 Myr BP From Remyet al (1994) with permission CopyrightNational Academy of Sciences USA(b) Transverse section of a root of the cycad-related Antarcticycas obtained from rocks ofTriassic age ca 220 Myr BP in Antarctica Thecentral cortex contains areas in which the cellsare occupied by mycorrhizal fungi (arrowed)(cd ) Higher magnicentcation views of thesecortical cells of Antarcticycas showing coarselybranched arbuscules (a) and a vesicle (v)(b^d ) from Stubblecenteld et al (1987a) withpermission

The term mycorrhiza embraces four basic types of dualorgan each characterized by its own well-conservedstructural attributes which are a repoundection largely of theparticular fungi involved (table 1) Of the higher plantsexamined to date over 90 have been shown to formassociations of one or other of these types (Smith amp Read1997)

The type most widely distributed through the plantkingdom is that formed by zygomycetous fungi of theorder Glomales and referred to on the basis of two fungalstructures previously considered to be diagnostic for this

type of symbiosis namely `vesiclesrsquo and arbusculesrsquo asbeing vesicular-arbuscular (VA) or simply arbuscularrsquomycorrhiza (AM) It is however important to realizeparticularly when considering fungal associations of lowerplants that arbuscules and vesicles are not necessarilyproduced in all plants colonized by glomalean fungi Ithas been emphasized recently (Smith amp Read 1997Smith amp Smith 1997) that there are two basic classes ofVA mycorrhiza which are named after the `type speciesrsquoof plant in which they were originally described byGallaud (1905) These are the Arumrsquo and Parisrsquo types The

Symbiotic fungal associations in lowerrsquo land plants D J Read and others 817

Phil Trans R Soc Lond B (2000)

origin of VAM fungi

land plants monocotsndashdicots

500 400 100200300 present

Cam

bria

n

Ord

ovic

ian

Sil

uria

n

Dev

onia

n

Car

boni

fero

us

Per

mia

n

Tri

assi

c

Jura

ssic

Cre

tace

ous

Tert

iary

time (Myr)

geologicalepoch

landmarks

estimated datesof divergence

eventsA B C D

Figure 2 Demonstrating thesynchronicity revealed bymolecular analysis of the originsof VA mycorrhizal fungi(VAM) and land plants in theBodovician^Silurian periodFrom Simon et al (1993)Reprinted with permissionCopyright of MacmillanMagazines Ltd

Table 2 The characteristics of fungal associations found in `lowerrsquo land plants

kinds of association

zygomycetous(cfVA mycorrhiza)

ascomycetous(cf ericoid mycorrhiza)

basidiomycetous(cf orchid orectomycorrhiza)

fungiseptate ^ + +aseptate + 7 7

intracellular colonization + or 7 a + +vesicles + or 7 7 7arbuscules + or 7 7 7achlorophylly ^ or + b 7 7 or + c

bacteria-like organisms + 7 7fungal taxa zygomycetes ascomycetes basidiomycetesplant taxa hornwortsd

Marchantialese

Metzgerialesk

Lycopodiumf

Botrychiumg

Psilotumh

Gleicheniaceaei

Jungermanniales j Jungermannialesk

Aneuraceael

aIn the protocormof Lycopodium cernuumbSubterranean gametophytesof Psilotales Ophioglossales some SchizaeaceaecCryptothallus onlydLigrone1988eLigrone amp Lopes1989 Ligrone amp Duckett1994fDuckett amp Ligrone1992 Schmid amp Oberwinkler19931995gSchmid amp Oberwinkler1994hPeterson et al1981iSchmid amp Oberwinkler1995jDuckett et al1991 Duckett amp Read1995kThis issue Pocock amp Duckett1984lLigrone et al1993

Arumrsquo type forms in roots with extensive cortical inter-cellular spaces through which fungal hyphae grow beforepenetrating into the cortical cells to produce arbusculesrsquoThe Parisrsquo type is found in species including as describedbelow many `lower plantsrsquo which lack well-developedsystems of intercellular spaces In this case after penetra-tion of the epidermis growth of the fungus occurs almostexclusively in the intracellular position where distinctcoils of hyphae are formed sometimes without any arbus-cules or vesicles

Smith amp Smith (1997) point out that because thedistinctive fungal structures of the two types resultprimarily from the anatomy of the absorbing organ thetype of VA mycorrhiza that develops must be controlledgenetically by the plant Evidence in support of this viewhas been provided in a number of studies of higherplants These show that a given species of glomaleanfungus can produce an Arumrsquo type mycorrhiza in onehost but a Parisrsquo type in another (Gerdemann 1965Jacquelinet-Jeanmougin amp Gianinazzi-Pearson 1983Daniels-Hetrick et al 1985)

Several lines of evidence suggest that these zygomy-cetous associations represent the archetypal mycorrhizaStructures reminiscent of the vesicles seen in modern AMassociations were seen and photographed by Kidston ampLang (1921) in underground axes of chert fossils fromDevonian times More convincing are the structures alsofrom Rhynie material and probably of Aglaophyton (centgure1a) which appear to be very similar to the intracellularàrbusculesrsquo formed by extant glomalean fungi (Remy etal 1994) Two further features strongly support the viewthat these structures represent ancient `mycorrhizasrsquo Thecentrst is that later fossils for example from gymnospermroots of the Carboniferous and cycad-like roots of theTriassic (centgure 1b^d) (Stubblecenteld et al 1987abc) suggesta continuous presence of these structures through thecourse of land plant evolution The second comes fromanalyses of substitutions in nucleic acid base sequences ofthe glomalean fungi forming this type of mycorrhiza inextant groups (Simon et al 1993) These suggest an originfor these fungi between 460 and 350 Myr BP over aperiod which according to the fossil record land plantsemerged (centgure 2) One centnal feature pointing to theantiquity of zygomycetous associations is that these are byfar the commonest type among lower land plants(table 2) In contrast the ascomycetous ericoid andbasidiomycetous orchid types are (i) far more restrictedin their distribution and (ii) concentned to supposedlyadvanced taxa in both jungermannialean and metzgeria-lean hepatics

A major factor selecting in favour of associationsbetween glomalean fungi and early land plants may havebeen the geometrical inadequacy of the undergroundaxes of autotrophs which were making the transitionfrom an aqueous to a soil-based system of nutrient supply(see Pirozynski amp Malloch 1975) In extant groups of`higherrsquo plants a relationship can be observed betweenresponsiveness to these fungi and the centbrosity of theirroot systems Thus species with coarse root systemssupporting few root hairs are usually more responsive tocolonization than those with centnely divided systems orhaving prolicentc root-hair development (Baylis 1972 1975)It has been hypothesized that the dependence of early

plants on colonization by organisms with more eiexclectiveabsorptive capabilities led to selective forces whichfavoured down-regulation of any mechanisms that wouldprovide resistance to pathogenic attack (Vanderplank1978) The observation that glomalean fungi penetrateand proliferate in the roots of so many species withoutapparently stimulating any of the physiological responsesnormally seen when plants are challenged by root patho-gens (Bonfante amp Perotto 1995 Gianinazzi-Pearson et al1996ab Kapulnik et al 1996) lends support to thishypothesis It suggests that compatibility was establishedbefore more advanced root systems were developed andthat lack of defence even in species with centbrous rootsystems is a genetically predetermined attribute in mostfamilies of land plants

The advantages to the fungus of an ability to down-regulate or bypass the defences of a wide range of auto-trophic species would be considerable The opportunitiesfor carbon acquisition particularly in species-richcommunities are greatly increased a feature thatprovides the resources required to generate a vigorousexternal mycelium This in turn enables the fungus tolocate and to colonize further root systems Herein liesthe challenge to those wishing to ascribe phylogenetic orfunctional signicentcance to associations between glomaleanfungi and their plant partners simply on the basis ofoccurrence of hyphae in the tissues The presence of colo-nization can be simply a manifestation of the almostuniversal ability of these heterotrophs to penetrate thebelow-ground structures of autotrophic land plants Ineach case the nature of the relationship which ensuesmust be ascertained by experiment

There are of course those families of higher plants inwhich root architecture is such that colonization by AMfungi appears to be essential for the acquisition of nutri-ents such as phosphorus which are poorly mobile in soil(Merryweather amp Fitter 1995 Smith amp Read 1997) Herethere is good reason to believe that coevolution of thepartners has been essential for this survival and that thesymbiosis is a mutualistic one Proven instances of inter-dependence of these kinds are likely to be of phylogeneticas well as functional signicentcance In other familieshowever particularly those consisting of plants withhighly centbrous root systems the colonization by the samefungi can have either neutral or antagonistic eiexclects(Francis amp Read 1995) The presence of the glomaleanfungi here may repoundect simply their ability to suppress hostdefence reactions and gain access to additional carbonsupplies It becomes obvious from these observations thatrecords of the presence of colonization by AM fungi arenot by themselves sucurrencient to enable either functionalor phylogenetic signicentcance to be inferred This caution isas much relevant to consideration of fungal associationsin lower plants as it is to the higher forms that have beenthe subject of more intensive study

The fossil evidence indicates that ectomycorrhizas are arelatively recent form of the symbiosis the centrst recordsbeing from Eocene rocks dated at ca 80 Myr BP (Le Pageet al 1997) This type is characterized by the formation ofa mantle or sheath of fungal mycelium over the outersurface of distal roots by the lack of intracellular penetra-tion of root cells and by the production of a very extensivenetwork of vegetative mycelia in the soil around the roots



The appearance of ectomycorrhizas in the fossil record isbroadly coincident with the assumed date of origin of thehomobasidiomycetes which are the most commonlyoccurring symbionts of roots in families such as the Pina-ceae Fagaceae and Betulaceae most members of whichform this type of association Because members of thesefamilies dominate the forest systems which cover a largeproportion of the Northern Hemisphere the ectomycor-rhizal symbiosis plays a central role in global nutrientcycling processes

While AM and ECM symbioses are the most widelydistributed both through the plant kingdom and acrossthe terrestrial surface other forms of mycorrhiza becomeimportant in particular habitats Thus the ericoid typerestricted to members of the order Ericales is producedby ascomycetous fungi that invade the epidermal cells ofthe exceedingly centne `hair-rootsrsquo in major genera such asCalluna Erica Rhododendron and Vaccinium These plantscharacteristically dominate ecosystems at high latitudes oraltitudes where low temperature inhibits decompositionand the cycling of key nutrients such as nitrogen andphosphorus The fungi involved are known to play amajor role in mobilizing such nutrients from the recalci-trant organic residues in which they are deposited (Read1996)

The fourth distinctive mycorrhizal type is found exclu-sively in the largest of all terrestrial plant families theOrchidaceae All plants in this family are colonized byseptate basidiomycetous fungi which penetrate cells of theroots where coarse coils of hyphae called pelotons areproduced Most perhaps all of the chlorophyllousorchids are colonized by a distinctive group of basidiomy-cetous fungi in the form genus Rhizoctonia These can beisolated and sometimes induced to produce fruitingstructures enabling their full taxonomic identity to beestablished Among the genera identicented to date Ceratoba-sidium Thanatephorus and Sebacina are important In thoseorchids which retain the juvenile heterotrophic conditionthroughout the life cycle such as the achlorophyllousspecies Corallorhiza tricentda and Cephalanthera austinae newlyemerging evidence suggests that Rhizoctonia spp have beenreplaced as symbionts by fungi which at the same timeare forming typical ectomycorrhiza with autotrophic trees(Zelmer amp Currah 1995 Taylor amp Bruns 1997McKendrick et al 2000)

The fungi involved in the four basic types of mycor-rhiza seen in higher plants (table 1) are also known toform associations with distinctive groups of lower plants(table 2) In the remainder of this paper their particularrelationships with these groups will be described with aview to establishing the extents to which the associationsfound are structurally or functionally related to themycorrhizas seen in `higherrsquo plants

2 FUNGAL SYMBIOSES IN BRYOPSIDA

(a) Taxonomic aspectsWithin the Bryopsida interest lies chiepoundy in the hepa-

tics and hornworts in which the occurrence of fungalassociations possessing some of the structural attributes ofmycorrhizas has been recorded frequently It is a distin-guishing feature of mosses including Sphagnum that asso-ciations of these kinds have not been recorded Although

there are scattered publications dating from Peklo (1903)purporting to describe `symbioticrsquo fungi in various mosstissues both gametophytic and sporophytic (Mago et al1992) careful scrutiny of the data indicates that the fungiare concentned to dead or moribund host cells and are thusalmost certainly saprophytic or parasitic This absence offungi may in itself be a point of phylogenetic signicentcanceIt is certainly of physiological interest that mosses appearto resist colonization by mycorrhizal fungi so eiexclectivelyThis lack of susceptibility places them alongside a selectgroup of only a few higher plant families such as theCruciferae Caryophyllaceae and Polygonaceae whichare not normally colonized by these fungal symbionts

The occurrence of symbioses between hepatics andfungi has been known for over a century Schacht (1854)provided careful descriptions of fungi in the thalli of Pelliaand Preissia and observed fungal colonization of rhizoidsin Marchantia and Lunularia Later Janse (1897) providedillustrations of swollen rhizoid apices occupied by fungi inZoopsis a leafy jungermannialean hepatic Bernard (1909)used the widespread occurrence of fungus^hepatic asso-ciations as the basis for his theory that vascular crypto-gams were descended from mycotrophic bryophytes Aperiod of intensive light microscope (LM) analysis ofthese symbioses in Pellia (Ridler 1922 Magrou 1925)Marchantia (Burgeiexcl 1938) Lunularia (Ridler 1923) Aneura(Gavaudan 1930) Cryptothallus (Malmborgh 1935) andCalypogeia (Nemec 1904 Garjeanne 1903) concentrmed thatthese associations are a normal feature of hepatic biologyThis early work is reviewed by Stahl (1949) and Boullard(1988) It enabled two broad types of association to berecognized one involving aseptate fungi often witharbuscules and another formed by fungi with septatehyphae

More recently the use of combinations of histochem-istry LM and electron microscopy (EM) has providedfurther recentnement of our understanding These haveconcentrmed that zygomycetous infections of the AM kindoccur in Phaeoceros (Ligrone 1988) Conocephalum (Ligroneamp Lopes 1989) and Asterella (Ligrone amp Duckett 1994) aswell as in members of the Metzgeriales for example Pellia(Pocock amp Duckett 1984) In addition it has been possibleto discriminate between the kinds of colonization formedby septate fungi Thus a combination of ultrastructural(Duckett et al 1991) and cytochemical (Duckett amp Read1991) approaches has revealed that the rhizoid infectionsoccurring in leafy liverworts of the families LepidoziaceaeCalypogeiaceae Adelanthaceae Cephaloziaceae andCephaloziellaceae are caused by ascomycetous fungipossessing simple septa with Woronin bodies In contrastthe septate hyphae occurring as endophytes in a fewthalloid Metzgeriales such as Aneura and Cryptothallus(Ligrone et al 1993) and in a few jungermannialeanfamilies (Boullard 1988 this paper) have been shown bytheir possession of dolipore septa to be basidiomycetes

(b) Structural and functional aspects ofhepatic^fungus associations

(i) Zygomycetous associationsA prerequisite for the analysis of the functional basis of

any relationship between a micro-organism and its `hostrsquo isthat the putative partners be grown separately thenbrought together to determine whether the naturally



occurring symptoms and structures of the association arereproduced This approach to the study of inter-organisminteractions was established by Koch (1912) and hasbecome central to diagnosis of pathogenesis and theaetiology of disease Tests of what are now known as `Kochspostulatesrsquo have only rarely been applied to the study oflower plant symbioses They can be readily carried out incases where the supposed microbial associate is culturableas appears to be the case in many of the ascomycetous andbasidiomycetous associates of `lower plantsrsquo but are moredicurrencult to achieve in the case of zygomycetous infectionsbecause the glomalean fungi which are putatively the

causal organism cannot be grown free of their hostsSpores of these fungi can readily be obtained from soil or`pot culturesrsquo but they are not the propagules of choice instudies of infectivity because of the low vigour orìnoculum potentialrsquo sensu Garrett (1970) of the vegetativehyphae which they produce

Study of functional aspects of the relationship betweenPellia and its fungal endophytes was pioneered by Magrou(1925) He sowed spores on to soils bearing propagules ofAM fungi and produced young gametophytes that werecolonized at an early stage of development Magrou(1925) reported localized cell death and inhibitition of



Figure 3 Thalli of Pellia fabbroniana colonized by a VA fungus spreading from Plantago lanceolata (ab) 1 m m toluidine-blue-stainedsections showing the endophyte comprising trunk hyphae (arrows) and arbuscules (a) in the ventral thallus cells (a) pound 190(b) pound 500 (c) Scanning electron micrograph showing a trunk hypha and arbuscules pound 1900 (d) Transmission electronmicrographs showing trunk hypha (t) healthy (d) and collapsed (e) arbuscules surrounded by host cytoplasm (d) pound 7100(e) pound 3400

thallus development in colonized plants In naturallyoccurring plants he noticed the non-uniform nature ofinfections which were always absent from the thallus inthe vicinity of sex organs or developing sporophytes Onlyafter dehiscence of the capsule did extensive invasion ofmature tissues take place this being associated with theproduction of vesicles and arbuscules

An approach which mimics more eiexclectively the infec-tion process in nature was developed by Francis amp Read(1995) to investigate the impact of vigorous VA mycelialnetworks on the development of higher plants and toprovide distinction between true `hostsrsquo to these fungi and`non-hostsrsquo In this plants of species known to be `hostsrsquoare grown in a natural substrate with or without theirglomalean associates In the case of the pre-infectedmycorrhizal plants the fungus is allowed by growingthrough a mesh of centne pore size to colonize adjacentroot-free-soil The response of seedlings of test plantsintroduced to this soil and thus exposed to the fungus orwith `naturalrsquo inoculum potential is compared with that

obtained in the same substrate lacking the VA myceliumThis approach has been used to ask two basic questionsconcerning zygomycetous infections of thalloid hepaticsFirst do the coarse VA endophytes which are the normalcolonists of higher plant roots satisfy the requirements ofKochrsquos postulates by colonizing hepatics and reproducingtypical symptoms of infection Second does any infectionobserved stimulate growth or phosphorus content of thehepatic in a manner normally seen in higher plants when acompatible association forms Our experiments thus farindicate that VA fungi colonizing seedlings of Plantagowill spread into axenically grown thalli of Pellia (centgure 3)where they produce trunk hyphae arbuscles and vesiclesapparently similar to those seen in wild specimens Itremains to be demonstrated whether these infections arefunctionally similar to those in higher plants

(ii) Ascomycetous associations in hepaticsA range of leafy hepatics in the Cephaloziaceae and

related Jungermanniaceae develop swollen rhizoids



Figure 4 Swollen-tipped rhizoids of Cephalozia connivens after experimental inoculation with the ericoid mycorrhizal fungiHymenoscyphus ericae (ab) Light micrographs (a) pound 55 (b) pound 230 (c) Transmission electron micrograph showing numeroushyphae surrounded by host cytoplasm pound 4300 (d ) Detail of the fungus showing a simple septum with Woronin bodies pound 58 500

packed with fungal hyphae (Duckett et al 1991 Pocock ampDuckett 1985 Williams et al 1994) An EM study ofrhizoids in the Cephaloziaceae revealed the presence ofsimple septa and Woronin bodies in the hyphae charac-teristic of ascomycetous fungi (Duckett et al 1991) In

addition the hyphae showed high acurrennity for thepounduorescent dye 33rsquo-dihexyloxacarbocyanine iodide afurther distinguishing feature of the ascomycetes(Duckett amp Read 1991) Unlike VA fungi these can bereadily isolated and grown on water agar (Duckett amp



Figure 5 The basidiomycetous endophyte in the leafy hepatic Southbya tophacea (a) 1 m m toluidine-blue-stained transversesection showing fungus-containing cells in the centre of the stem pound 130 (bc) Scanning electron micrographs showing hyphalcoils in the inner stem cells (b) pound 280 (c) pound 5500 (d ) A mass of fungal hyphae within a host cell with healthy cytoplasmpound 9500 (e) Dolipore with imperforate parenthosomes pound 59 000



Figure 6 Cryptothallus mirabilis (a) Inner thallus cell showing numerous hyphal procentles pound 3200 (b) Dolipore with imperforateparenthosomes pound 44 000 (c^f ) Ectomycorrhiza in Betula formed by the Cryptothallus endophyte (c) 1 m m toluidine-blue-stainedsection showing a typical mantle and Hartig net pound 580 (d) Dolipore of the same with the same morphology as in Cryptothalluspound 46 000 (e f ) Details of the mantle (m) and Hartig net (h) In ( f ) note pseudoparenchymatous nature of the fungus a typicalfeature of Hartig nets (e) pound 2800 ( f ) pound 6400

Read 1995) In cross inoculation experiments the hepaticfungus isolated from Cephalozia and Kurzia producedtypical ericoid mycorrhizas following the introduction ofaxenically cultured seedlings of Calluna Erica and Vacci-nium While the identicentcation of the ascomycete isolatedfrom the liverworts is yet to be concentrmed several species

of leafy hepatics were readily infected by the ascomyceteHymenoscyphus ericae originally isolated from the roots ofCalluna (centgure 4) Not only do these experiments consider-ably extend the known host range of Hymenoscyphus ericaebut the sharing of a common endophyte may also haveconsiderable physiological and ecological impacts for



Figure 7 Aneura pinguis resynthesized fungal association (a) 1 m m toluidine-blue-stained section showing hyphal coils pound 470(bc) Scanning electron micrographs showing hyphal coils (b) pound 250 (c) pound 1300 (d) Hyphae with multilamellate wallsa characteristic feature of the Aneura endophyte pound 8500 (e) Transverse section of a dolipore pound 40 000

both the liverwort and ericaceous hosts We are currentlyinvestigating carbon exchange and nutrient relationshipsusing a similar approach to the studies with VA andbasidiomycete associations

(iii) Basidiomycetous associations in hepaticsIn contrast to the Cephaloziaceae other leafy hepatics

are colonized by endophytes inhabiting a discrete regionof the inner stem This type of association encompassesmembers of the Lophoziaceae Arnelliaceae and Scapa-niaceae (centgure 5) The presence of dolipores in EM

studies demonstrates we believe for the centrst time thatthe fungi involved are basidiomycetes The hyphal coilsseen within healthy host cytoplasm are reminiscent ofthose seen in orchid mycorrhizas

Light and ultrastructural analyses of the achloro-phyllous subterranean gametophytes of Cryptothallus andits closely allied photosynthetic relative Aneura haverevealed closely similar cytology of robust coiled intra-cellular hyphae with dolipore septa (centgures 6 and 7) Thisconcentrms the basidiomycete acurrennities of the endophytessuggested by Ligrone et al (1993)



Figure 8 (a) Transparent Plexiglas microcosm supporting Betula seedlings growing on sterilized Sphagnum peat with plants of theachlorophyllous hepatic Cryptothallus mirabilis (double arrows) Mycorrhizal fungal hyphae (single arrows) grow from C mirabilisto colonize the peat (b) A parallel microcosm with Betula grown on the same medium as in (a) but without Cryptothallus Noteabsence of fungal mycelia (c) Close-up view of C mirabilis thallus grown with B pendula as in (a) showing the conversion of rootsof Betula to ectomycorrhizas (single arrows) in the vicinity of the hepatic (d) Ectomycorrhizal laterals formed on monoxenicallygrown Betula seedlings inoculated with a pure culture of the mycorrhizal fungus of C mirabilis Sections of such roots (see centgure6c) reveal the typical ectomycorrhizal structures a Hartig net and mantle

Little is known about the functions of these basidio-mycete associations but our recent discovery that the fungifrom both Aneura and Cryptothallus can be grown axenicallyobviously opens the door to experimentation To this endwe have successfully reintroduced an Aneura fungal isolateof A pingus into thalli of this species grown axenically fromspores thus fulcentlling Kochrsquos postulates (centgure 7)

In view of its achlorophyllous nature it is logical tohypothesize that the carbon requirements of Cryptothallusare supplied by its fungal symbiont Until recently we hadno knowledge either of the source of any such carbon orits method of transfer That the plant has exacting habitatrequirements is however well documented It mostcommonly grows under Sphagnum lawns where there is anoverstory of Betula (Paton 1999) Under these circum-stances the source of carbon could therefore be eitherfrom the autotrophic associates or from the peat in whichthey are growing Field observation indicating that birchroots growing in the vicinity of C mirabilis plants wereheavily colonized by ectomycorrhizal fungi led us tohypothesize that the hepatic was part of a tripartite asso-ciation in which the fungus provided links to the treeThis hypothesis is being tested in a number of experi-ments Aseptically produced seedlings of Betula pendulahave been grown on thin layers of sterile Sphagnum peat ina series of transparent observation chambers After theBetula root system had begun to extend across the peatfreshly collected surface-washed thalli of C mirabilis wereplanted into half of the chambers (centgure 8a) Chamberswith and without thalli were incubated in a controlledenvironment growth cabinet with only the Betula shootsexposed to light While the birch plants without Crypto-thallus produced no ectomycorrhizas those in chamberscontaining the liverwort became heavily colonized by anectomycorrhizal fungus (centgure 8b) In a parallel experi-ment the basidiomycetous fungus was isolated fromCryptothallus and grown in association with asepticallygrown Betula seedlings using an agar-based Petri dishsystem developed by Brun et al (1995) After four weeksof incubation ectomycorrhizal roots were produced onthe seedlings (centgure 8c) concentrming that the endophyte ofC mirabilis is an ectomycorrhizal fungus

Sections of the birch roots from both experimentsexamined by LM and transmission electron microscopy(TEM) (centgure 6) concentrmed that the structures producedby the fungus including a mantle and Hartig net werethose of a typical ectomycorrhiza These observationsclearly demonstrate that Betula and Cryptothallus can beinterlinked structurally via a fungus but they do nothowever shed any light on the functional relationshipsbetween the partners

To investigate the potential of Betula seedlings to supplyCryptothallus with carbon a 14C-based labelling study wascarried out using tripartite systems set up in the transparentobservation chamberThe results to date indicate that thereis indeed a transfer of carbon from the autotrophic `higherrsquoplant to its heterotrophic `lowerrsquo plant neighbour

3 FUNGAL SYMBIOSES IN EXTANT LYCOPSIDA

The gametophytes of Lycopodium can be either sub-terranean and achlorophyllous or surface-living with

chlorophyll depending on the species It was establishedin classical early LM studies of their anatomy (Treub1884 1890ab Bruchmann 1885 1910 Lang 1899 1902Burgeiexcl 1938) that they were invariably invaded by fungiAccording to the results of these studies gametophytes ofalmost all species died at an early stage of development ifthey were not colonized by an appropriate fungus Morerecently there have been detailed ultrastructural analysesof a representative of the achlorophyllous types L clavatum(Schmid amp Oberwinkler 1993) and of a chlorophyll-bearing form L cernuum (Duckett amp Ligrone 1992)

The EM studies have concentrmed the earlier descriptionsof the fungus involved in the association as being aseptateand this together with the fact that the intracellularhyphae sometimes produce terminal vesicles albeit ofvery small size has led these symbioses to be placed inthe VA category (Harley amp Smith 1983 Harley amp Harley1987) It must be emphasized however that the taxo-nomic status of the fungi involved in lycopod gam-etophyte associations is not resolved Schmid ampOberwinkler (1993) contrast the apparent lack of arbus-cules which is evident from the earlier LM as well astheir own study with the persistent presence of dense andstrikingly regular coils of very centne hyphae havingdiameters in the range 08^18 m m Such coils againproduced by extremely narrow hyphae were also seen inL cernuum gametophytes by Duckett amp Ligrone (1992)The presence of coils rather than arbuscules is consistentwith the notion of a Paris-typersquo VA mycorrhiza as indi-cated above and one fungus Glomus tenuis whichproduces associations generally accepted to be of the VAtype in higher plants is characterized by having hyphaeand vesicles of the very centne dimensions seen in Lycopodiumprothalli (Hall 1977 Smith amp Read 1997)

A further feature suggesting that the fungus may haveglomalean acurrennities is that its intracellular hyphae inboth L clavatum and L cernuum are occupied by`bacterium-likersquo organelles (BLOs) which are indistin-guishable from those described in hyphae of coarser VAendophytes (MacDonald amp Chandler 1981 MacDonaldet al 1982) Apparently similar BLOs now thought on thebasis of molecular analysis to be of the Burkholdera type(Bianciotto et al 1996) have been observed in putative VAendophytes of the hornwort Phaeoceros laevis (Ligrone1988) and the thalloid hepatic Conocephalum conicum(Ligrone amp Lopes 1989) They have also been reported inthe Glomus-related zygomycete Endogone poundammicorona whichforms ectomycorrhiza (Bonfante-Fasolo amp Scannerini1977) and in some free-living fungi (Wilson amp Hanton1979)

There are some diiexclerences between the infectionsdescribed in L cernuum and L clavatum In the formervesicles were not seen and in addition to the pre-dominantly centne hyphae there were some of widerdiameter which were construed as being the trunks ofarbuscules Their presence led Duckett amp Ligrone (1992)to hypothesize that there may be two types of infection inthe same host genus though they acknowledged that ifarbuscules were formed they would be an unusual featurein Lycopodium

On the basis of the distinctive nature of the structuresseen in gametophytes of Lycopodiaceae Schmid ampOberwinkler (1993) suggested that the association be



described as a `lycopodioid mycothallus interactionrsquorather than as a mycorrhiza and this caution may wellbe appropriate

From the functional standpoint the tacit assumptioncan be made that Lycopodium gametophytes of the achloro-phyllous kind at least must be dependent on their fungusfor carbon Their failure to develop in the absence of colo-nization provides circumstantial evidence for this view Inthis case they can be regarded as `dual organs of absorp-tionrsquo and accurately described as being mycorrhizal

It is clear from the analyses of these symbioses made todate however that a disproportionate emphasis on struc-tural studies has left us without resolution of most of thekey questions concerning the homologies taxonomic andfunctional of fungus^lycopod associations A search ofthe literature suggests that in only one study has anattempt been made after appropriate surface steriliza-tion to isolate the fungus or fungi involved in the associa-tion in gametophytes and to reinoculate it to satisfyKochrsquos postulates In this (Freeberg 1962) a fungusremarkably reminiscent of a Rhizoctonia was isolatedProthalli grown with this fungus on a starch mediumwere found to gain weight more rapidly than those grownaxenically More studies of this kind are urgently neededIf the most important fungus turns out to have glomaleanacurrennities it is likely that it will not in fact be culturablebut even in this event molecular methods enabling char-acterization of these fungal taxa are now available andshould be applied

Since lycopod gametophytes can be readily obtainedfrom nature (Duckett amp Ligrone 1992) and grown in vitro(Whittier 1973 Whittier amp Webster 1986) there should beno impedance to progress in this important area ofresearch

Analyses of the mycorrhizal status of the sporophytegeneration of Lycopodium species have normally reportedthe presence of colonization by VA fungi (Boullard 1979Harley amp Harley 1987) This raises fascinating questionsconcerning the relationships taxonomic and functionalbetween the fungi colonizing the heterotrophic gameto-phyte and autotrophic sporophyte stages of the life cycleWe return to this issue later

4 FUNGAL SYMBIOSES IN EQUISETOPSIDA

There appears to be no record of any fungal associationin the autotrophic gametophyte generation of Equisetum

The question of the mycorrhizal status of the sporo-phyte generation has been the subject of some debate Thecentrst reported analyses of Equisetum roots (Holaquo veler 1892Stahl 1900) revealed no fungal colonization SubsequentlyBerch amp Kendrick (1982) examined the root systems ofnumerous Equisetum species and also reported them to belargely free of colonization On the basis of these resultsand an analysis of the early literature Berch amp Kendrickconcluded that the genus was non-mycorrhizal Theyventured also to suggest that the decline of the Equisetop-sida from the position of prominence which it occupied inCarboniferous and Jurassic forests was attributable to afailure to compete with mycotrophic neighbours Sub-sequently Koske et al (1985) have described what theycall `typicalrsquo VA mycorrhizas in several species ofEquisetum growing in sand-dune systems Conscious of the

possibility that the presence of VA fungi in their rootsmight repoundect simply the penetration of a `non-hostrsquoattempts were made to collect samples from plants thatwere growing in ìsolatedrsquo positions In fact a distance ofonly 05 m was achieved from a nearest non-Equisetumneighbour which in view of the extensive root systemstypical of mycotrophic plants particularly grasses indune systems is unlikely to have achieved the desiredeiexclect Their analyses led Koske et al (1985) to postulatethat the demise of Equisetales was attributable to a world-wide change from hydric to mesic conditions whichfavoured other groups and was not related to a geneticallydetermined inability to formVA mycorrhiza

This debate highlights the problems arising fromanalyses based purely on the presence or absence of fungalstructures Clearly Equisetum grows perfectly well in theabsence of mycorrhizal fungi under many circumstancesWhen associations between Equisetum roots and VA fungido occur the only way to establish the nature of the rela-tionship is by experiment In the absence to date of anyevidence for an absorptive role or of a contribution by thefungus to plant centtness there is no justicentcation for referringto these associations as being `mycorrhizalrsquo It follows thatthe phylogenetic signicentcance of the presence or absence ofcolonization in this group remains a matter of conjecture

5 FUNGAL SYMBIOSES IN PSILOTALES AND

OPHIOGLOSSALES

The gametophytes of all species in both of these ordersare achlorophyllous subterranean structures with endo-phytic fungal associations

In the Psilotales they have been most extensivelystudied in Psilotum nudum (Dangeard 1890 Darnell-Smith1917 Lawson 1918 Holloway 1939 Bierhost 1953 Boullard1957 1979 Peterson et al 1981 Whittier 1973) One typeof colonization produced by an aseptate fungus whichoccasionally bears vesicles is consistently present Thefungus involved was originally referred to the chytri-diaceous genus Cladochytrium and was considered to beparasitic These views of the taxonomic and functionalstatus of the fungus are no longer accepted Indeed sincecolonization by the same fungus seems to be a prerequi-site for healthy development of gametophytes there is aprima facie case for considering the association to be ofthe mycorrhizal kind The only published ultrastructuralaccount of Psilotum gametophytes describes cortical cellsoccupied by dense coils of aseptate hyphae some of whichbear terminal vesicles (Peterson et al 1981) A sequence ofhyphal development and degeneration was observed inthese cells but no arbuscules were seen Light and ultra-structural studies have revealed very similar endophyticfungal morphology and behaviour in the gametophytes ofBotrychium (Schmid amp Oberwinkler 1994 Nishida 1956)There is much to indicate therefore that these are zygo-mycetous fungi and it seems reasonable to hypothesizethat as in the case of lycopods Psilotum and Botrychiumgametophytes are supported by Paris-typersquo AM associa-tions The need to test this hypothesis by experiment isagain evident The only major structural distinctionbetween the Psilotum and Botrychium associations andthose described in Lycopodium (Duckett amp Ligrone 1992Schmid amp Oberwinkler 1994) is that the hyphae of the



Psilotum and Botrychium fungi are coarse Since the gam-etophytes of Psilotum Lycopodium and Ophioglossales cannow all be grown axenically (Renzaglia et al this issue)it should be possible to examine responses of the gam-etophyte to challenge by a range of glomalean fungi witha view to establishing the basis of any relationship whichthey may have with these plants

Peterson et al (1981) and Schmid amp Oberwinkler(1994) observed that many of the fungal hyphae andvesicle-like structures in Psilotum and Botrychium gam-etophytes stored lipids which appeared to be releasedinto the cortical cell cytoplasm on hyphal degradationThese authors speculated that the fungus may be able tosynthesize lipids from soil organic matter but there is noevidence for such a pathway or for metabolism of fungus-derived lipid by the plant

A relatively small number of observations on prothalliof Tmesipteris (Holloway 1917 Lawson 1918) suggest asimilar type of infection Spores of T elongata have alsobeen germinated axenically (Whittier amp Given 1987)

The sporophytes of Psilotales and Ophioglossales arenormally relatively massive autotrophic structures Whileit is recognized that rhizomes and roots respectively arenormally though not invariably colonized by mycorrhizalfungi it is necessary to emphasize at the outset that thereis no evidence that infection spreads from the gametophyteinto the developing sporophyte Boullard (1963) observedthat the rhizome of ophioglossaceous ferns was free ofcolonization and concluded that roots emerging from itwere infected `de novorsquo from soil Likewise Mesler (1976)saw no fungal colonization of the embryo The importanceof these observations which seem to parallel those in lyco-pods lies in the fact that the two stages of the life cyclemay be colonized by quite diiexclerent fungi This must betaken into account when considering carbon transfer intoand out of the respective generations (see below)

Janse (1897) pointed out that rhizomes of Psilotumpossessed hairs through which endophytic fungi passed toinfect cortical cells where they produced coils and vesi-cles The fungi are aseptate and in addition to vesiclesand coils arbuscules have also been seen (Burgeiexcl 1938)It would thus seem appropriate to regard this associationas being of the AM kind

A broadly similar picture emerges from studies ofOphioglossum sporophytes though here arbuscules of adistinctive structure appear to be the norm InO pendulum Burgeiexcl (1938) described single hyphaepenetrating the host cells and branching profusely toproduce many intracellular vesicles which he called `ster-narbuskelnrsquo In the ultrastructural analysis of Schmid ampOberwinkler (1996) these swellings were shown to ariseat the tips of what otherwise look like typical arbuscularbranches Some of these arbuscules arise directly fromhyphal coils in the manner described in Parisrsquo mycorrhizaby Gallaud (1905) A further feature suggestive of AMacurrennities is the presence of bacteria-like organisms inthe intracellular hyphae of O reticulatum (Schmid ampOberwinkler 1996)

Ascending centnally to `higherrsquo ferns here the greenphotosynthetic gametophytes are generally considered tobe fungus-free Light microscope studies do howeverdescribe the sporadic occurrence of endophytic fungi andsuggest they may be constantly present in basal families

like the Marattiaceae Osmundaceae Gleicheniaceae andSchizaeaceae (Boullard1979 Campbell 1908 Bower 1923)A recent LM and EM study (Schmid amp Oberwinkler1995) describing arbuscules and lipid-packed vesicles inhealthy gametophyte cells of Gleicheniaceae concludes thatthe association is closely similar to the infection inConocephalum and Phaeoceros As with every other pterido-phyte association investigationof function is now required

6 CONCLUSION

From the analyses presented above it becomes obviousthat a large discrepancy exists between knowledge of theoccurrence of fungal symbioses in `lowerrsquo plants and ourunderstanding of their functions Even in those cases suchas the achlorophyllous gametophytes of lower tracheo-phytes where it seems logical to hypothesize a functionbased on carbon supply by the fungus fundamental ques-tions remain What is the source of this carbon Are thesame fungal taxa colonizing the heterotrophic gameto-phyte and the autotrophic sporophyte If so how is theswitch in polarity of carbon movement achieved

Similar problems confront us when attempting tohypothesize functions for fungal symbioses in poikilo-hydric leafy hepatics Should we envisage that nutritionaladvantages of the kind known to accrue to homoiohydric`higherrsquo plants from mycorrhizal colonization will neces-sarily be observed in slow-growing poikilohydric liver-worts It is reasonable to hypothesize that thedevelopment of such plants is rarely if ever limited bynutrient availability This symbiosis could arise simplybecause selection has favoured loss of specicentcity in thesefungal biotrophs as it maximizes their access to carbon A`mycocentricrsquo view of this kind would place many of theassociations described above at the level of commensalrather than mutualistic symbiosis thus weakening anyargument in favour of them being similar to mycorrhizasAgain experiment alone will resolve these issues In viewof the consistency with which each of the described typesof colonization occurs in its respective group of `lowerrsquoplants it seems reasonable to hypothesize that physio-logical interactions of importance to both partners mustoccur There is an urgent need to test such hypotheses

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Baylis G T S 1975 The magnolioid mycorrhiza and myco-trophy in root systems derived from it In Endomycorrhizas (edF E Sanders B Mosse amp P B Tinker) pp 373^389 LondonAcademic Press

Berch S M amp Kendrick W B 1982 Vesicular-arbuscularmycorrhizae of southern Ontario ferns and fern-alliesMycologia 74 769^776

Bernard N 1909 Lrsquoevolution dans la symbiose Les orchidees etleur champignons commenseux Ann Sci Nat Paris 9 1^196

BianciottoV Bandi C Minerdi D Sironi M Tichy HV ampBonfante P 1996 An obligately endosymbiotic mycorrhizalfungus itself harbors obligately intracellular bacteria ApplEnviron Microbiol 62 3005^3010

Bierhorst D W 1953 Structure and development of the gam-etophyte of Psilotum nudum Am J Bot 40 649^658

Bonfante P amp Perotto S 1995 Strategies of arbuscular mycor-rhizal fungi when infectinghost plants New Phytol130 3^21



Bonfante-Fasolo P amp Scannerini S 1977 Cytological observa-tions on the mycorrhiza Endogone poundammicorona^Pinus strobusAllionia 22 23^34

Boullard B 1957 La mycotrophie chez les Pteridophytes Safrequence ses characterecopy res sa signicentcation Le Botaniste 415^185

Boullard B 1963 Le gametophyte des OphioglossaceesConsiderations biologiques Bull Soc Linn Norm 4 81^97

Boullard B 1979 Considerations sur la symbiose fongique chezles Pteridophytes Syllogeus No 19

Boullard B 1988 Observations on the coevolution of fungi withhepatics In Coevolution of fungi with plants and animals (edK A Pirozynk amp D L Hawkesworth) pp107^124 LondonAcademic Press

Bower F O 1923 The ferns vol 1 Cambridge University PressBruchmann H 1885 Das Prothallium von Lycopodium Bot

Centralb 21 23^28Bruchmann H 1910 Die Keimung der Sporen und die

Entwicklung der Prothallien von Lycopodium clavatumL annotinum und L selago Flora 101 220^267

Brun A Chalot M Finlay R D amp Solaquo derstrolaquo m B 1995Structure and function of the ectomycorrhizal associationbetween Paxillus involutus (Batsch) Fr and Betula pendulaRoth I Dynamics of mycorrhiza formation New Phytol 129487^493

Burgeiexcl H 1938 Mycorrhiza In Manual of pteridology vol 1 (edF Verdoorn) pp159^191 The Hague Martinus Nijhof

Campbell D H 1908 Symbiosis in fern prothallia Am Nat 42154^165

Dangeard P A 1890 Note sur les mycorhizes endotrophiquesLe Botaniste 2 223^230

Daniels-Hetrick B A Bloom J amp Feyerherm S M 1985Root colonization of Glomus epigaeum in nine host speciesMycologia 77 825^828

Darnell-Smith G P 1917 The gametophyte of Psilotum Trans RSoc Edinb 52 79^91

de Bary A 1887 Comparative morphology and biology of the fungimycetozoa and bacteria [English translation of 1884 edition]Oxford Clarendon Press

Duckett J G amp Ligrone R 1992 A light and electron micro-scope study of the fungal endophytes in the sporophyte andgametophyte of Lycopodium cernuum with observations onthe gametrophyte sporophyte junction Can J Bot 7058^72

Duckett J G amp Read D J 1991The use of the pounduorescent dye33rsquo-dihexyloxacarbocyanine iodide for selective staining ofascomycete fungi associated with liverwort rhizoids andericoid mycorrhizal roots New Phytol 118 259^272

Duckett J G amp Read D J 1995 Ericoid mycorrhizas andrhizoidâscomycete associations in liverworts share the samemycobiont isolation of the partners and their resynthesis invitro New Phytol 129 439^447

Duckett J G Renzaglia K S amp Pell K 1991 A light andelectron microscope study of rhizoidâscomycete associationsand poundagelliform axes in British hepatics with observations onthe eiexclects of the fungi on host morphology New Phytol 118233^257

Francis R amp Read D J 1995 Mutualism and antagonism inthe mycorrhizal symbiosis with special reference to impactson plant community structure Can J Bot 73 1301^1309(suppl 1)

Freeberg J A 1962 Lycopodium prothalli and their endophyticfungi as studied in vitro Am J Bot 49 530^535

Gallaud I 1905 Etudes sur les mycorhizes endotrophes RevGen Bot 17 7^48 66^85123^136 223^239 313^325 423^433479^496

Garjeanne A J M 1903 Ulaquo ber die Mykorrhiza der LebermooseBeih Bot Zentralb 15 471^482

Garrett S D 1970 Pathogenic root-infecting fungi CambridgeUniversity Press

Gavaudan L 1930 Recherches sur la cellule des Hepatiques LeBotaniste 22 190^216

Gerdemann J W 1965 Vesicular-arbuscular mycorrhizasformed on maize and tulip tree by Endogone fasciculataMycologia 57 562^575

Gianinazzi-Pearson V Dumas-Gaudot E Gollottee ATahiri-Alaoui A amp Gianinazzi S 1996a Cellular and mole-cular defence-related root responses to invasion by arbuscularmycorrhizal fungi New Phytol 133 45^57

Gianinazzi-PearsonV GollotteA Cordier C amp Gianinazzi S1996b Root defence responses in relation to cell and tissueinvasion by symbiotic microorganisms cytological investiga-tions In Histology ultrastructure and molecular cytology of plant^microorganism interactions (ed M Nicole amp V Gianinazzi-Pearson) pp177^191 Dordrecht Kluwer

Hall I R 1977 Species and mycorrhizal infections of NewZealand EndogonaceaeTrans Br Mycol Soc 68 341^356

Harley J L amp Harley E L 1987 A check list of mycorrhiza inthe British poundora New Phytol 105(suppl 2) 1^102

Harley J L amp Smith S E 1983 Mycorrhizal symbioses LondonAcademic Press

Holloway J E 1917 The prothallus and young plant ofTmesipterisTrans R Soc NZ 50 1^44

Holloway J E 1939 The gametophyte embryo and youngrhizome of Psilotum triquetrum Sw Ann Bot Lond 3 313^336

Holaquo veler W 1892 Ulaquo ber die Verwerthung des Humus bei derErnahrung der chlorophyll fulaquo hrenden Ppoundangan Jahrbucherfur Wissenschaftliche Botanik 24 283^316

Jacquelinet-Jeanmougin S amp Gianinazzi-Pearson V 1983Endomycorrhizas in the Gentianaceae I The fungi asso-ciated with Gentiana lutea L New Phytol 95 663^666

Janse J M 1897 Les endophytes radicaux de quelques plantesjavanaises Ann Jard Bot Buitenz 14 53^212

Kapulnik Y Volpin H Itzhaki H Ganon D Elad YChet I amp Okon Y 1996 Suppression of defence response inmycorrhizal alfalfa and tobacco roots New Phytol 133 59^64

Kidston R amp Lang W H 1921 On old red sandstone plantsshowing structure from the Rhynie chert bed AberdeenshireTrans R Soc Edinb 52 855^901

Koch R 1912 Complete works vol I Leipzig George Thiemepp 650^660

Koske R E Friese C F Olexia P D amp Hauke R L 1985Vesicular-arbuscular mycorrhizas in Equistum Trans Br MycolSoc 85 350^353

Lang W H 1899 The prothallus of Lycopodium clavatum L AnnBot Lond 13 279^317

Lang W H 1902 On the prothalli of Ophioglossum pendulum andHelminthostachys zeylanica Ann Bot Lond 16 2^56

Lawson A A 1918 The gametophyte generation of thePsilotaceaeTrans R Soc Edinb 52 93^113

Le Page B A Currah R Stockey R amp Rothwell G W 1997Fossil ectomycorrhiza in Eocene Pinus roots Am J Bot84 410^412

Ligrone R 1988 Ultrastructure of a fungal endophyte inPhaeoceros laevis (L) Prosk (Anthoceratophyta) Bot Gaz 14992^100

Ligrone R amp Duckett J G 1994 Thallus diiexclerentiation in themarchantralean liverwort Asterella wilmsii (Steph) with parti-cular reference to longitudinal arrays of endoplasmicmicrotubules in the inner cells Ann Bot 73 577^586

Ligrone R amp Lopes C 1989 Cytology and development of amycorrhiza-like infection in the gametophyte of Conocephalumconicum (L) Dum (Marchantiales Hepatophyta) New Phytol111 423^433

Ligrone R Pocock K amp Duckett J G 1993 A comparativeultrastructural analysis of endophytic basidiomycetes in the



parasitic achlorophyllous hepatic Cryptothallus mirabilis and theclosely allied photosynthetic species Aneura pinguis(Metzgeriales) Can J Bot 71 666^679

MacDonald R M amp Chandler M R 1981 Bacterium-likeorganelles in the vesicular-arbuscular mycorrhizal fungusGlomus caledonius New Phytol 89 241^246

MacDonald R M Chandler M R amp Mosse B 1982 Theoccurrence of bacterium-like organelles in vesicular-arbuscular mycorrhizal fungi New Phytol 90 659^663

McKendrick S Leake J R Taylor D L amp Read D J 2000Symbiotic germination and development of mycohetero-trophic plants in nature ontogeny of Corallorhiza tricentda Chaordf teland characterisation of its mycorrhizal fungi New Phytol 145523^537

Mago P Agnes C A amp Mukerji K J 1992 VA mycorrhizalstatus of some Indian bryophytes Phytomorphology 42 231^239

Magrou J 1925 La symbiose chez les Hepatiques Le Pelliaepiphyllarsquo et son champignon commensal Ann Sci Nat Bot 7725^780

Malmborgh St V 1935 Cryptothallus ng Ein saprophytischesLebermoos Ann Bryol 6

Merryweather J amp Fitter A 1995 Arbuscular mycorrhiza andphosphorus as controlling factors in the life history of the obli-gately mycorrhizal Hyacinthoides non-scripta (L) Chouard exRothm New Phytol 129 629^636

Mesler M R 1976 Gametophytes and young sporophytes ofOphioglossum crotalophoroidesWalt Am J Bot 63 443^448

Nemec B 1904 Ulaquo ber die Mykorrhiza bei Calypogeia trichomanisBeih Bot Zentalb 16 253^268

Nishida M 1956 Studies on the systematic position and constitu-tion of Pteridophyta VI The gametophyte of Botrychiumvirginianum and its endogenous fungus Phytomorphology 6 67^73

Paton J A 1999 The liverwort poundora of the British Isles ColchesterHarley Books

Peklo J 1903 Kotazce mycorrhizy n muscinei Roz Abh Bolaquo timAkad Ztg 12 No 58

Peterson R L Howarth M J amp Whittier D P 1981Interactions between a fungal endophyte and gametophytecells in Psilotum nudum Can J Bot 59 711^720

Pirozynski K A amp Malloch D W 1975 The origin of landplants a matter of mycotrophism Biosystems 6 153^164

Pocock K amp Duckett J G 1984 A comparative ultra-structural analysis of the fungal endophyte in Cryptothallusmirabilis Malm and other British thalloid hepatics J Bryol13 227^233

Pocock K amp Duckett J G 1985 On the occurrence ofbranched and swollen rhizoids in British hepatics their rela-tionships with the substratum and associations with fungiNew Phytol 99 281^304

Read D J 1996 The structure and function of the ericoidmycorrhizal root Ann Bot 77 365^376

Remy W Taylor T N Hass H amp Kerp H 1994 Fourhundred-million-year-old vesicular arbuscular mycorrhizaeProc Natl Acad Sci USA 91 11841^11843

Ridler W F F 1922 The fungus present in Pellia epiphylla (L)Corda Ann Bot 36 193^207

Ridler W F F 1923 The fungus present in Lunularia cruciata (L)DumTrans Br Mycol Soc 9 82^92

Schacht H 1854 Pilzfaden im Innern der Zellen und derStarkmehlkolaquo rner vor Flora 1854 618^624

Schmid E amp Oberwinkler F 1993 Mycorrhiza-like interactionbetween the achlorophyllous gametophyte of Lycopodiumclavatum L and its fungal endophyte studied by light and elec-tron microscopy New Phytol 124 69^81

Schmid E amp Oberwinkler F 1994 Light and electronmicroscopy of the host^fungus interaction in the achloro-phyllous gametophyte of Botrychium lunaria Can J Bot 72182^188

Schmid E amp Oberwinkler F 1995 A light- and electron-microscope study on a vesicular-arbuscular host^fungusinteraction in gametophytes and young sporophytes of theGleicheniaceae (Filicales) New Phytol 129 317^324

Schmid E amp Oberwinkler F 1996 Light and electronmicroscopy of a distinctive VA mycorrhiza in maturesporophytes of Ophioglossum reticulatum Mycol Res 100843^849

Simon L Bousquet J Levesque R C amp Lalonde M 1993Origin and diversicentcation of endomycorrhizal fungi and co-incidence with vascular land plants Nature 363 67^69

Smith S E amp Read D J 1997 Mycorrhizal symbiosis San DiegoAcademic Press

Smith S E amp Smith S E 1997 Structural diversity in(vesicular)-arbuscular mycorrhizal symbioses New Phytol 137373^388

Stahl E 1900 Der Sinn der Mycorhizenbildung JahrbWiss Bot34 539^668

Stahl M 1949 Die Mycorrhiza der Lebermoose mit besondererBerucksichtigung der thallosen Formen Planta 37 103^148

Stubblecenteld S P Taylor T N amp Seymour R L 1987a Apossible endogonaceous fungus from the Triassic ofAntarctica Mycologia 79 905^906

Stubblecenteld S P Taylor T N amp Trappe J M 1987b Fossilmycorrhizae a case for symbiosis Science 237 59^60

Stubblecenteld S P Taylor T N amp Trappe J M 1987c AntarcticVAM fossils Am J Bot 74 1904^1911

Taylor D L amp Bruns T D 1997 Independent specialized inva-sions of ectomycorrhizal mutualism by two nonphotosyntheticorchids Proc Natl Acad Sci USA 94 4510^4515

Trappe J M 1996 What is a mycorrhiza Proceedings of the 4thEuropean Symposium on Mycorrhizae Granada Spain EC ReportEUR 16728 pp 3^9

Treub M 1884 Etude sur les Lycopodiacees I Le prothalle duLycopodium cernuum L Ann Jard Bot Buitenz 4 107^138

Treub M 1890a Etude sur les Lycopodiacees VI Lrsquoembryon etle plantule du Lycopodium cernuum Ann Jard Bot Buitenz 81^15

Treub M 1890b Etude sur les Lycopodiacees VIII Les tuber-cles radicaux du Lycopodium cernuum L Ann Jard Bot Buitenz8 15^23

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DiscussionA E Newton (Department of Botany Natural HistoryMuseum London UK) Following up on the statement thatmosses lack fungal associations while many other lowerplants (hepatics anthocerotes early fossils etc) havebeen found to have several diiexclerent types of symbioticassociation (a) Which taxa of mosses have beenstudied and (b) Why do you think mosses lack theseassociations



J G Duckett I have examined over 200 taxa throughoutthe mosses

D J Read Response to (a) above Mosses may not havebeen systematically searched for the presence of fungalsymbionts However the view that they lack fungal asso-ciations is based on extensive observations carried outover many years by bryologists on the one hand and thoseinterested in mycorrhizas on the other

D J Read Response to (b) above In the absence ofexperimental analysis of the responses of mosses to chal-lenge by fungal symbionts it is possible to speculate thatsome feature of the wall structure of moss cells rendersthem impenetrable Mosses are hosts to remarkably fewfungal pathogens and are also generally unpalatable toherbivores both features being indications of eiexclective`defencersquoagainst intrusion

J G Duckett Response to (b) above Mosses possessextensive protonemalrhizoidal systems that may mimic thefungal systems making the fungal association unnecessary

This is turn raised the question `What is the situation inAndreaea and Andraeaobryum taxa that lack rhizoidalsystemsrsquo

D J Read We have looked at both Andreaea andAndraeaobryumoumlthey are never infected However Iwould not use the `rhizoidrsquo-based response of Jeiexcl Bothliverworts and mosses have rhizoids Indeed in liverwortsit is the rhizoids that provide the channels for penetrationand in many cases they harbour the fungal colonies

P Kenrick (Department of Palaeontology Natural HistoryMuseum London UK) You have discussed fungus associa-tions of terrestrial plants rooted in soil and shown ussome striking examples of how the absence of a fungusunder experimental conditions aiexclects the vigour of theplant Do epiphytic and epilithic plants also have fungalassociations

D J Read The patterns of fungal symbiont colonizationseen in hepatics of normal terrestrial habitats arerepeated in epiphytic and epilithic situations



and structural features of those associations betweensymbiotic fungi and lower plants which appear to beconsistently present in nature Second it is intended toaddress the important question of the functional basis ofthe associations described

The distinction between `lowerrsquo and `higherrsquo plants issomewhat arbitrary but for the present purpose is takento lie between the supposedly `primitiversquo land plants withlarge sometimes achlorophyllous gametophytes in theBryopsida Lycopsida Equisetopsida or Pteropsida (onlythe Psilotales and Ophioglossales) and all those pteropsidsgymnosperms and angiosperms in which the gam-etophyte is more diminutive

The roots of the majority of land plants are colonized bysymbiotic fungi to form dual organs called `mycorrhizasrsquo

the term being derived from the two Greek words `mykesrsquofungus and `rhizarsquo root Since in the strictest sense theunderground axes and rhizoids of lower plants are notroots any such fungal associations with them should notbe called mycorrhizas However recent decentnitions of thissymbiosis for example that of Trappe (1996) see it inbroader terms referring to mycorrhizas as `dual organs ofabsorption formed when symbiotic fungi inhabit healthytissues of most terrestrial plantsrsquo Under a decentnition of thiskind which does not specify roots any healthy fungus-containingabsorptive tissue of lower plants can legitimatelybe referred to as a mycorrhiza and more important ques-tions concerning the extent to which the relationship isfunctionally as well as structurally analagous with thesesymbioses in higher plants can be addressed



Table 1 The characteristics of the four most widespread mycorrhizal types found in `higherrsquo plants

kinds of mycorrhiza

VA ectomycorrhiza ericoid orchid

fungiseptate 7 + + +aseptate + 7 7 7

intracellular colonization + 7 + +fungal sheath 7 + 7 7Hartig net 7 + 7 7vesicles + or 7 7 7 7arbuscules + or 7 7 7 7achlorophylly 7( + ) 7 7 + a

fungal taxa zygomycetes basidio- and ascomycetes ascomycetes basidiomycetesplant taxa leptosporangiate ferns



Ericales Orchidaceae

a All orchids are achlorophyllous in the early seedling stagesThe structural characters given relate to the mature state not the developing or senescent states

Figure 1 Fossil evidence for the occurrenceof VA mycorrhizas in early land plants (a) Anarbuscule-like structure (a) in a cortical cell ofAglaophyton collected from Rhynie Chert rocksof Devonian age ca 400 Myr BP From Remyet al (1994) with permission CopyrightNational Academy of Sciences USA(b) Transverse section of a root of the cycad-related Antarcticycas obtained from rocks ofTriassic age ca 220 Myr BP in Antarctica Thecentral cortex contains areas in which the cellsare occupied by mycorrhizal fungi (arrowed)(cd ) Higher magnicentcation views of thesecortical cells of Antarcticycas showing coarselybranched arbuscules (a) and a vesicle (v)(b^d ) from Stubblecenteld et al (1987a) withpermission






origin of VAM fungi


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6 CONCLUSION



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origin of VAM fungi


500 400 100200300 present

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bria

n

Ord

ovic

ian

Sil

uria

n

Dev

onia

n

Car

boni

fero

us

Per

mia

n

Tri

assi

c

Jura

ssic

Cre

tace

ous

Tert

iary

time (Myr)

geologicalepoch

landmarks


eventsA B C D










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Metzgerialesk

Lycopodiumf

Botrychiumg

Psilotumh

Gleicheniaceaei


Aneuraceael






















































































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6 CONCLUSION



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6 CONCLUSION



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6 CONCLUSION



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6 CONCLUSION



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6 CONCLUSION



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6 CONCLUSION



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Documents

Symbiotic fungal associations in 'lower' land plants