12 Orchid Mycorrhizas: Molecular Ecology - ResearchGate

12 Orchid Mycorrhizas: Molecular Ecology, Physiology, Evolutionand Conservation Aspects

J.D.W. DEARNALEY1, F. MARTOS

2,M.-A. SELOSSE3

CONTENTS

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207II. New Techniques for Studying Orchid

Mycorrhizas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208A. Molecular Barcoding Approaches . . . . . . . . . 208B. Stable and Radioactive Isotopes . . . . . . . . . . . 210C. Other Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . 210

III. The Diversity of Orchid: FungusAssociations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212A. The Diversity of the ‘Rhizoctonias’ . . . . . . . . 212B. Fully Mycoheterotrophic Orchids and

Ectomycorrhizal Fungi . . . . . . . . . . . . . . . . . . . . . 213C. Fully Mycoheterotrophic Orchids and

Saprotrophic Agaricomycetes . . . . . . . . . . . . . . 214D. Mixotrophs: Green Orchids that Obtain

Carbon from Fungi . . . . . . . . . . . . . . . . . . . . . . . . . 215E. Epiphytic Orchids and ‘Rhizoctonias’. . . . . . 216

IV. Nutrient Exchanges BetweenOrchid and Mycobiont . . . . . . . . . . . . . . . . . . . . . . . 216

V. Fungal Specificity in Orchids . . . . . . . . . . . . . . . . 219A. Patterns and Evolutionary Significance . . . 219B. Adaptive Significance. . . . . . . . . . . . . . . . . . . . . . . 219C. The Impact of Mycorrhizal Specificity on

Orchid Speciation. . . . . . . . . . . . . . . . . . . . . . . . . . . 220VI. Orchid Mycorrhizas and Plant Conservation 220

A. Orchid Mycorrhizas and On-SiteManagement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221

B. Ex Situ Conservation and OrchidMycorrhizas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

C. Use of Mycorrhizal Fungi in RestorationProcedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

VII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

I. Introduction

Since the pioneering works by Noel Bernard(1874–1911; Boullard 1985), whose centenaryof death was celebrated in 2011, scientificinterest in orchid mycorrhizas has continuedto grow. This might seem somewhat surprisingas the association concerns a single angio-sperm family and is less widespread amongland plants than the ectomycorrhizal (ECM)or arbuscular mycorrhizal (AM) symbioses(Smith and Read 2008). Impetus for researchinto orchid mycorrhizas has been multifaceted.As the earth’s largest flowering plant familywith 27,135 accepted species (The Plant List2010), accumulating basic biological knowledgeof what represents approximately 10 % of thebotanical kingdom diversity is justified. For themycologist, these plants that shifted from anancestral AM symbiosis with Glomeromycetesto an original symbiosis with new fungal part-ners (Yukawa et al. 2009) offer a window onmycorrhizal abilities in numerous, and some-times unexpected, fungal lineages. Moreover,many species, such as aromatic Vanilla spp.or many ornamental species, are of economicvalue. Illuminating studies of orchid mycorrhi-zas have shown that some non-chlorophyllousplants live as parasites on ECM interactions orsaprotrophic fungi and that some green orchidsare partially heterotrophic as adults. Recentresearch has given insights into the nature ofthe mycorrhizal association of autotrophicorchids, suggesting that the association maybe mutualistic (Cameron et al. 2006, 2008).The mycorrhizas of orchids also offer generalperspectives on the evolution of specificity and

1Australian Centre for Sustainable Catchments and Faculty ofSciences, University of Southern Queensland, Toowoomba4350, Australia; e-mail: [email protected] of Life Sciences, University of KwaZulu-Natal, PrivateBag X01 Scottsville, Pietermaritzburg 3209, South Africa3Centre d’Ecologie Fonctionnelle et Evolutive (CNRS, UMR5157), 1919 Route de Mende, 34293 cedex 5 Montpellier, France

Fungal Associations, 2nd EditionThe Mycota IXB. Hock (Ed.)© Springer-Verlag Berlin Heidelberg 2012

mailto:[email protected]

mycorrhizal networks among plants. Finally, asorchids require the presence of suitable fungalpartners for seed germination and seedlingestablishment, a more complete understand-ing of the mycorrhizal biology of the manythreatened orchid species is required for con-servation action plans.

Orchids have historically been divided intothree main types on the basis of lifestyle, i.e.terrestrial (soil dwelling), epiphytic (plant sur-face dwelling) and lithophytic (rock surfacedwelling) species. Recent literature (e.g.Gebauer and Meyer 2003; Selosse et al. 2004;for reviews, see Merckx et al. 2009; Selosse andRoy 2009) has suggested a division of orchidsinto three physiological types based on carbonnutrition. Fully autotrophic species (the majorityof taxa) are those that are chlorophyllousand, as adults, obtain their carbon compoundsvia photosynthetic pathways. Fully mycoheter-otrophic (MH) species (approximately 200species worldwide; Leake 1994, 2004) aredependent on fungal carbon throughout theirlife cycle. A third type, the partial MH species ormixotrophs (Julou et al. 2005; Selosse and Roy2009) are intermediate, carrying out some pho-tosynthetic carbon fixation as well as receivingfungal carbon.

Regardless of their carbon nutrition at adultstage, all orchids produce minute, endosperm-lacking seeds and are dependent on fungal col-onization for germination and growth into anunderground heterotrophic, achlorophyllousstage called a protocorm (Rasmussen 1995;Smith and Read 2008). Environmental fungicolonize though embryo suspensor tissues orepidermal hairs and enter the cortical cells.Colonizing fungal hyphae do not breach thecortical cell membrane but ramify in the spacebetween cell wall and membrane, forming elab-orate coiled structures known as pelotons(Fig. 12.1) that collapse at later stages as a resultof plant digestion. Intact fungal coils and notcollapsed pelotons are likely the site of nutrientexchange between plant and fungus, as indi-cated by the fact that in vitro grown protocormsshow a growth response before peloton collapse(Hadley and Williamson 1971) and that thenutrient fluxes after labelling pulses occur toorapidly to be accounted for by hyphal digestion(Cameron et al. 2008).

This chapter will focus on current under-standing of the mycorrhizal associations ofthese three physiological orchid types. For amore comprehensive review of the colonizationprocess and anatomy of orchid mycorrhizasreaders are directed to Smith and Read (2008).Here it is intended to update the 10-year-oldexcellent reviews by Rasmussen (2002) andTaylor et al. (2002), to provide an overview ofthe contemporary approaches to studying theseinteractions, to elaborate on what has beengleaned from these studies with regards to theecology, physiology, evolution and conserva-tion aspects of orchid mycorrhizas and to high-light areas of the association that need furtherexploration.

II. New Techniques for StudyingOrchid Mycorrhizas

A. Molecular Barcoding Approaches

Historically, much knowledge about orchidmycorrhizas has been acquired from in vitroisolation of fungi. This has allowed basic fun-gal identification and simple in vitro seedgermination experiments conducted withsome root-isolated fungi (e.g. Warcup 1971;

H

S

C

O

Fig. 12.1. Light microscopy image showing healthy(H), slightly degraded (S) and collapsed (C) fungalpelotons of a Thelephora sp. in Cephalanthera long-ifolia roots (Ulle Puttsepp, unpublished micrograph;plant investigated in Abadie et al. (2006)). O Oxalatecrystal. Bar 50 mm

208 J.D.W. Dearnaley et al.

Clements 1988). Indeed, the orchid mycorrhi-zal association represents possibly one of theeasiest symbiotic systems to manipulate underlaboratory conditions as both partners can becultured axenically, at least in the case of theearly stages of the fully autotrophic orchids. Ahurdle in these types of investigations hasbeen an inability to accurately identify theisolated fungal partners and this has beenespecially critical to orchid conservation pro-cedures involving restorative work; moreover,isolation often provided mostly contaminantsor endophytes (i.e. fungi that for all or part oftheir life cycle inhabit living plant tissues butdo not form pelotons nor cause any obviousdisease symptoms; Wilson 1995).

Molecular taxonomy approaches haveenhanced fungal taxonomy, especially by iso-lating fungal DNA and sequencing the nuclearribosomal DNA (Seiffert 2009). The fungal part-ners of orchid mycorrhizas can be more accu-rately and routinely identified from culturedfungi or directly from orchid protocorms,roots, tubers and rhizomes (e.g. Bougoureet al. 2005; Martos et al. 2009; Swarts et al.2010). For mycobionts recalcitrant to axenicgrowth, PCR amplification of colonized orchidtissues using fungus-specific primers is com-monly used (Dearnaley and Le Brocque 2006;Dearnaley and Bougoure 2010).

Sequencing of the internal transcribed spacer (ITS) ofthe nuclear ribosomal DNA after PCR amplificationusing a variety of primer combinations (White et al.1990; Gardes and Bruns 1993) has been the method ofchoice for identifying orchid mycobionts over the pastdecade. One problem is the amplification recalcitranceof Tulasnellaceae, a frequent orchid mycorrhizal taxon(see Sect. III.A), to the ‘universal’ fungal ITS primers,because they have highly derived nuclear ribosomalDNA sequences. This entailed the need for additionalPCR amplifications using Tulasnellaceae-specific PCRprimers (Bidartondo et al. 2004; Selosse et al. 2004).Suarez et al. (2006) introduced the Tulasnellaceae-spe-cific primer 5.8S-Tul to amplify the 5’ part of 28S rDNA,which was used by Martos et al. (2012). This primerworks well on a wide range of clades of Tulasnellaceaeand is expected to be frequently used in future studiesof orchid mycorrhizal fungi because of the high hetero-geneity of the ITS alignment. Recently, some primerpairs specifically devoted to orchid mycorrhizal fungiwere described (Taylor and McCormick 2007), but theconstantly growing number of fungal taxa (see Sect. III)

questions their relevance in the new orchid lineages tobe explored. Sequencing of cloned ITS PCR products isoften carried out with orchids displaying low fungalspecificity (e.g. Selosse et al. 2002; Dearnaley 2006;Liebel et al. 2010; Martos et al. 2012). Sequencing ofthe large subunit (LSU) of the nuclear ribosomal DNAof the Sebacinales, common orchid mycobionts world-wide, is necessary for higher resolution separation ofgroups A and B, two major clades in this group (Weibet al. 2004, 2011; Selosse et al. 2009). Huynh et al. (2009)also recently showed that ITS sequencing may not suf-ficiently distinguish isolates of the ‘Sebacina vermifera’complex (Sebacinales group B), common mycobiontsof spider orchids in Australia. ITS sequencing andcloning may also reveal many endophytes (Bidartondoet al. 2004; Julou et al. 2005; Abadie et al. 2006; Roy et al.2009a). In Martos et al. (2012), a wide range of ascomy-cete and basidiomycete endophytes was identified, per-haps more than in any study of orchid associated fungito date.

Fungal endophytes are very frequentlyselected during in vitro isolation or PCRamplification from orchid tissues with fungus-specific primers: dissecting single fungalpelotons from roots tissues before in vitro iso-lations (Zhu et al. 2008) or PCR amplifications(Rasmussen 1995; Kristiansen et al. 2001) isthus strongly recommended in future work toavoid endophytes. The important diversity ofendophytic fungi, mainly from the Helotiales(e.g. Phialophora, Leptodontidium or Bisporellaspp.) or Xylariales, will not be discussed indetail here (for a review, see Bayman andOtero 2006), while their effect on orchid growth(potentially deleterious in some species;Bayman et al. 2002) and physiology deservesfurther study. Chaetotyriales are very commonorchid endophytes, at least in tropical areas.Capnodiales are also common in epiphytictaxa, but they might be involved in lichenicsymbioses. Many epiphytic orchids root inbryophytes or lichens.

Recently, Jacquemyn et al. (2010) andLievens et al. (2010) introducedDNA array tech-nologies for the identification of orchid fungalpartners: oligonucleotides were prepared froma preliminary exploration of fungal diversity ina limited number of individuals (Lievens et al.2010), and the array was successfully used toinvestigate the fungal partners of three closelyrelated Orchis species and their hybrids(Jacquemyn et al. 2011). This method allows

Orchid Mycorrhizas: Molecular Ecology, Physiology, Evolution and Conservation Aspects 209

fast and efficient handling of numerous sam-ples, especially compared to the cloning of PCRproducts. However, some fungal partners mayremain overlooked when using this procedurebecause preliminary exploration overlooks rarefungal taxa that may not be targeted duringfurther investigation (such as taxon 8 and 9from the Thelephoraceae and Cortinariaceae,respectively, in Lievens et al. 2010; Jacquemynet al. 2011).

B. Stable and Radioactive Isotopes

A common, but indirect approach to determinethe mode of nutrition of individual orchid taxaismass spectrometric analysis of natural C andN isotope abundances (Bidartondo et al. 2004;Julou et al. 2005; Abadie et al. 2006; Zimmeret al. 2007; Ogura-Tsujita et al. 2009; Martoset al. 2009). Fully MH species have been identi-fied to have 13C signatures similar to those oftheir mycorrhizal partners (Gebauer and Meyer2003; Trudell et al. 2003) and similar or higher15N abundance than their mycorrhizal fungi,suggesting a limited trend to 15N accumulationalong the food chain (Trudell et al. 2003). Asexpected, mixotrophs have stable isotope sig-natures intermediate between fully MH andautotrophic species (Julou et al. 2005; Abadieet al. 2006). Some fully autotrophic species suchas Goodyera spp. have even lower amounts ofthese natural isotopes as expected for plantsless reliant on nutrient acquisition from fungi(Gebauer and Meyer 2003; Bidartondo et al.2004). The strength of this method is thatabundances oversee the long-term metabolismof the plants, with little interference from theobserver.

On the fungal side, Latalova and Balaz(2010) showed that a Tulasnella species asso-ciated in vitro with the orchid Serapias stricti-flora was able to mix carbon from the orchid(a C3 plant) and dead maize roots (a C4 plantenriched in 13C). The fungus was able to growwith the orchid alone, with 13C abundance closeto its host, while addition of dead maize rootsresulted in an isotopic shift, so that the lattersource furnished ca. 30 % of the fungal biomass.

Experiments tracing the movement of iso-topically labelled compounds to orchid mycor-rhizas have been especially revealing. Althoughthey only provide snapshot views of the metab-olism at the time of pulse, they allow tracking ofexchanges between symbionts. McKendricket al. (2000) provided the first clear demon-stration of movement of 14C-labelled photo-synthates from tree species to the fully MHorchid Corallorhiza trifida via ECM fungi.Bougoure et al. (2010) recently demonstratedthe flow of 13C-labelled carbon from Melaleucascalena to the fully MH orchid Rhizanthellagardneri via an ECM fungal conduit. R. gardnerialso obtained nitrogen from its fungal partner asindicated by adding 13C+15N-labelled glycine tohyphae and surrounding soil. Labelling experi-ments also demonstrated that the fully autotro-phic orchid Goodyera repens acquires carbon,nitrogen and phosphorous from its fungal part-ner (Cameron et al. 2006, 2007, 2008). Notably,G. repens also transfers significant amounts ofphotosynthate (likely greater than 3 % of itsphotosynthetic carbon) back to its Ceratobasi-dium mycobiont – the first direct demonstra-tion of a net carbon flow from orchid to fungi(Cameron et al. 2006, 2008).

C. Other Approaches

In contrast to other mycorrhizal symbioses,such as ECM and AM associations, geneexpression studies in orchid mycorrhizas havelargely been neglected. Watkinson and Wel-baum (2003) analysed gene expression in themycorrhizal association of Cypripedium parvi-florum var. pubescens via differential mRNAdisplay. A trehalose phosphate phosphatasewas downregulated in the association, indicat-ing changes to orchid carbohydrate transport.Upregulation of a nucleotide binding proteinpossibly indicated increased cytokinesis duringorchid colonization. As indicated by Dearnaley(2007), modern gene expression techniquessuch as microarrays, RT-PCR and in situ hybri-dization may provide additional understandingof the molecular functioning of orchid mycor-rhizas. In particular, whole-genome sequencingand transcript profiling of orchid mycorrhizal


fungi, both free-living and in planta, may revealfungal genes that are upregulated in the symbi-osis (Martin et al. 2008).

The use of electron microscopy to investi-gate fungal symbionts in orchid mycorrhizashad somewhat of a rebirth in the past decade(e.g. Pereira et al. 2003; Selosse et al. 2004;Suarez et al. 2008; Martos et al. 2009; Kottkeet al. 2010; Schatz et al. 2010; Martos et al.2012). First, features of the fungal cell wall aswell as septal structure, e.g. dolipore and par-enthesomes, allow a distinction of the threemajor mycorrhizal taxa encompassed underthe name ‘rhizoctonia’ (see Sect. III.A; Moore1987). Moreover, it has been used to confirmhow some unexpected taxa do form pelotonsand thus are mycorrhizal. Kottke et al. (2010)has given support to molecular data suggestingthat Atractiellomycetes, members of the rustlineage (Pucciniomycotina), are mycorrhizalin some neotropical orchids. Selosse et al.(2004) corroborated molecular identificationof ascomyceteous Tuber spp. as the mainmycorrhizal partners in Epipactis microphyllaby using transmission electron microscopy tocheck for the presence of Woronin bodies inpelotons and immunogold reactions using anti-bodies specifically raised against a truffle phos-pholipase A2 (Fig. 12.2) – interestingly, in thisstudy, basidiomycetes that were found bymolecular means were never seen by micros-

copy. Immunolabelling transmission electronmicroscopy has been used to demonstrate pec-tin deposition in the interfacial matrix aroundCeratobasidium hyphae, but not Russulahyphae, in adjacent mycorrhizal root cells ofLimodorum abortivum, highlighting an orch-id’s exquisite capability to react distinctly todifferent fungal symbionts (Paduano et al.2011). Finally, Huynh et al. (2004) used scan-ning electron microscopy imaging of stems andprotocorms to determine the most effectivefungal isolates for conservation of thethreatened Caladenia formosa.

Other valuable new approaches include: (1) an orchidroot peloton isolation and culturing method that max-imizes the number of mycorrhizal fungi obtained butminimizes contamination from non-mycorrhizal fungiand bacteria (Zhu et al. 2008) and (2) a modification ofthe seed packet burial technique originally conceivedby Rasmussen and Whigham (1993) which involvesremoval of site soil and monitoring of symbiotic seedgermination under laboratory conditions (Brundrettet al. 2003). (3) Another method of orchid mycorrhizalfungal identification was proposed by Kristiansen et al.(2001), that is, PCR amplification from single pelotons.Now that large-scale environmental detection of fungiis possible through such approaches as t-RFLP (Dickieand FitzJohn 2007), DGGE (Bougoure and Cairney2005), pyrosequencing (Dumbrell et al. 2011) andDNA microarrays (Lievens et al. 2010), it will beintriguing to see how populations of orchid mycobiotachange with time, orchid life stage and environmentalconditions.

Fig. 12.2. TEM images of Epipactis microphylla cellscolonized by truffles. (A) Truffle septate hyphae (H)inside an orchid host cell, where the host plasma mem-brane (arrowheads) tightly surrounds the fungus (bar4 mm). (B) Gold granules are regularly distributed(arrows) on the longitudinal and septum wall of the

fungus after immunogold reaction with the anti-Tbsp1antibody specific for a truffle phospholipase. IM Inter-facial material, PL plasma membrane of the host cell,S septum, V vacuole, W Woronin bodies. Bar 0.6 mm(Modified from Selosse et al. (2004), reproduced withpermission of the publisher)


III. The Diversity of Orchid: FungusAssociations

A. The Diversity of the ‘Rhizoctonias’

For many years orchids were considered tointeract largely, if not only, with members ofthe ‘rhizoctonia’ complex. This assemblagecontains three now taxonomically disparateAgaricomycetes (¼Hymenomycetes) taxa:Sebacinales, Ceratobasidiaceae and Tulasnella-ceae (Table 12.1). None of them actually fit theexact definition of the asexual genus Rhizocto-nia by De Candolle (1815), i.e. the absence ofsporulation and formation of sclerotia, so thatthe name ‘rhizoctonia’ will be used here not in ataxonomic way, but only to convenientlyencompass the three above-mentioned taxa,which are common orchid partners. ‘Rhizocto-nias’ have also been divided into two asexualgenera, namely Ceratorhiza and Epulorhiza(Table 12.1), but this approach is uncomfortableto non-mycologists and, given the current trendto abandon asexual classification, we recom-mend no longer using these names.

Recent research has highlighted the diverseecology of these three ‘rhizoctonia’ taxa. Whilesome species are known to be parasitic, such asin the Ceratobasidiaceae, or are suspected to besaprotrophic, e.g. due to their cultivabilityin vitro on organic substrates, this classicalview (Smith and Read 2008) is now challengedat least for some species. Sebacinales encom-passes two major groups (Weib et al. 2011) thatboth occur as endophytes in the roots of manyplant species (Selosse et al. 2009): group Badditionally forms mycorrhizae with greenorchids and Ericaceae, while group A formsECM on trees and is also associated withsome MH orchids (see Sect. III.B; group A isusually not encompassed in ‘rhizoctonias’). Inan interesting example of convergent evolution,group B is involved in symbiotic germination ofPyrola spp. (Ericaceae), another taxon withdust-seeds and MH germination (Hashimotoet al. 2012). ECM clades may exist within theTulasnellaceae (Bidartondo et al. 2003) andCeratobasidiaceae (Yagame et al. 2008, 2012;

Collier and Bidartondo 2009), and noteworthyMH orchids were instrumental in establishingECM abilities in these taxa (see Sect. III.B).However, it is unlikely that ECM ‘rhizoctonias’are mycorrhizal in fully autotrophic orchids.We are far from a complete understanding ofthe diversity of nutritional strategies (out oforchid roots) for the Tulasnellaceae and Cera-tobasidiaceae: at least, we suspect that theirmain ecological niche exists out of orchidsroots.

Molecular taxonomic identification oforchid mycobionts has now revealed that thediversity of orchid associates is much morecomplex and that other basidiomycetes andeven ascomycetes can be involved in orchidmycorrhizas (Table 12.1). The recent overallpicture (discussed by Motomura et al. 2010) isthat autotrophic orchids largely associate with‘rhizoctonias’ worldwide. However, in tropicalregions, Atractiellomycetes (Pucciniomycotina)may be common mycorrhizal partners of someepiphytic and terrestrial autotrophic orchids, asshown in the neotropics (Kottke et al. 2010) andin the paleotropics (Martos et al. 2012). Thestudy of South African Diseae (Pterygodiumand Corycium spp.) revealed ECM Ascomycetessuch as Tricharina and Peziza (Waterman et al.2011), although no direct visualization wasobtained. One may expect this list of myco-bionts to enlarge in the future. Nevertheless,the study of the earliest-diverging orchidlineages and distribution of fungal associatesacross orchid phylogeny support that theancestral state is an association to the three‘rhizoctonia’ lineages (Yukawa et al. 2009).Interestingly, Tulasnellaceae turn out to be themost frequently found ‘rhizoctonias’, in bothtemperate and tropical regions (Rasmussen1995; Yuan et al. 2010): in a survey of 77 orchidspecies from La Reunion island (Indian Ocean),Martos et al. (2012) found them in 88 % of theinvestigated species (versus 42 % for Sebaci-nales and 18 % for the Ceratobasidiaceae). Bycontrast, mixotrophic or fully MH orchidsrevealed associations with more diverse fungallineages.


B. Fully Mycoheterotrophic Orchids andEctomycorrhizal Fungi

MH orchids are achlorophyllous and receive alltheir carbon from their mycorrhizal fungi. Inthis way, they are paedomorphic, i.e. preservinga juvenile trait (heterotrophy, that is a featureof protocorms only in other orchid species)during the adult stage. Since the key studies ofCorallorhiza and Cephalanthera species by

Taylor and Bruns (1997) and McKendricket al. (2000), a large number of works indicatethat many other fully MH orchids receive car-bon from the ECM associations of autotrophicplants in temperate regions (e.g. Selosse et al.2002; Taylor et al. 2004; Dearnaley and Le Broc-que 2006; Roy et al. 2009a) and in some tropicalforests (Roy et al. 2009b). Two studies (Taylorand Bruns 1997; Selosse et al. 2002) providedevidence that the same fungal individual was

Table 12.1. Summary of the fungal genera forming orchid mycorrhizas. Examples of studies which have identifiedmycorrhizal genera are given. Taxa in bold are the three groups usually named ‘rhizoctonias’ in the orchidliterature (see text; including the asexual genera Ceratorhizab and Epulorhizaa)

Phylum BasidiomycotaSub phylum PucciniomycotinaClass Atractiellomycetes (e.g. Kottke et al. 2010)

Sub phylum AgaricomycotinaClass Agaricomycetes

Order AgaricalesArmillaria (e.g. Kikuchi et al. 2008)Campanella (e.g. Dearnaley and Bougoure 2010)Coprinus (e.g. Yagame et al. 2007)Gymnopus (e.g. Dearnaley 2006)Hymenogaster (e.g. Julou et al. 2005)Inocybe (e.g. Roy et al. 2009b)Marasmius (e.g. Burgeff 1959)Mycena (e.g. Ogura-Tsujita et al. 2009)Psathyrella (e.g. Yamato et al. 2005)

Order CantharellalesTulasnellaa (e.g. Jacquemyn et al. 2010)Clavulina (e.g. Selosse, unpublished data)Ceratobasidiumb (e.g. Otero et al. 2002)Thanatephorusb (e.g. Warcup 1991)

Order RussulalesGymnomyces (e.g. Dearnaley and Le Brocque 2006)Russula (e.g. Taylor et al. 2004)

Order HymenochaetalesErythromyces (e.g. Umata 1995)Resinicium (e.g. Martos et al. 2009)

Order SebacinalesSebacina group A (e.g. McKendrick et al. 2002)Sebacina group Ba (e.g. Bougoure et al. 2005)

Order ThelephoralesThelephora/Tomentella (e.g. Bidartondo et al. 2004)

Phylum AscomycotaSub phylum Pezizomycotina

Class PezizomycetesOrder PezizalesTuber (e.g. Selosse et al. 2004)Tricharina (e.g. Waterman et al. 2011)Peziza (e.g. Waterman et al. 2011)

aThe unrelated sexual genera Tulasnella and Sebacina encompass species from the asexual genus Epulorhiza.bThe sexual genera Ceratobasidium and Thanatephorus encompass species from the asexual genus Ceratorhiza.


present on MH orchids and surrounding ecto-mycorrhizal tree roots in situ: although thisrelied on the polymorphism of a single geneticmarker (nuclear ribosomal DNA), it supportsthat hyphal connection can transfer sufficientcarbon from surrounding trees to the MHplants to support their growth, as was morerecently supported by ex situ resynthesisexperiments (Bougoure et al. 2010). While theassociation is always specific in temperateregions, a recent study showed that, at least insome tropical areas, some Aphyllorchis MHspecies harboured several different ECM fungiin their roots (Roy et al. 2009b).

In most cases, the Russulaceae, Sebacinales and Thele-phoraceae are the most frequently involved taxa (Ken-nedy et al. 2011); Clavulinaceae may also occur in someGastrodia species (M.-A. Selosse, unpublished data) –interestingly, they also belong to the most frequent taxain ECM communities (Tedersoo and Nara 2010). Forthe less specific tropical orchids, lack of specificity mayensure the finding of suitable partners at most sites, andone can speculate that associating to less frequent part-ners may be evolutionarily risky. Nevertheless, someorchids do associate with rarer taxa, such as Inocybespp. (Roy et al. 2009a; Liebel and Gebauer 2011) or theECM Ceratobasidiaceae (Yagame et al. 2008, 2012;Bougoure et al. 2009, 2010): the latter are so rare inECM communities (Collier and Bidartondo 2009) thatMH orchids were instrumental in confirming their ECMstatus (Bougoure et al. 2010; see also Yagame et al.2012). The common features of ECM fungi supportingMH orchids are unclear, as they are dissimilar in phy-logenetic position, ecological preferences and mycelialmorphology (shape of mycorrhiza and soil explorationtype; R. Agerer, personal communication).

C. Fully Mycoheterotrophic Orchids andSaprotrophic Agaricomycetes

Shifts of fungal partners from non-ECM ‘rhi-zoctonia’ to various ECM fungi during MHorchid evolution are considered to give orchidsa more continuous carbon supply than thatprovided by the putatively saprotrophic ‘rhi-zoctonias’ (Taylor and Bruns 1997). However,some tropical MH orchids live in forests thatare devoid of ECM fungal communities (Smithand Read 2008). Other investigations usingmolecular fungal identification and stable iso-

tope analyses have now shown associations tonon-‘rhizoctonia’ saprotrophic partners.

In the fully MH orchid genus Gastrodia the main myco-bionts are related to Marasmius (Martos et al. 2009;Dearnaley and Bougoure 2010), Mycena (Martos et al.2009; Ogura-Tsujita et al. 2009), Resinicium (Martoset al. 2009), or Armillaria (Kikuchi et al. 2008), depend-ing on the species. Wood-decaying Erythromyces occurin Galeola species (Umata 1995) and litter-decayingMycena in Wullschlaegelia aphylla (Martos et al.2009). In both Epipogium roseum and Eulophiazollingeri, the mycobionts involved are saprotrophicCoprinaceae (Yamato et al. 2005; Yagame et al. 2007;Ogura-Tsujita and Yukawa 2008a). There are also a num-ber of pre-molecular, morphological studies identifyingdiverse saprobic fungal taxa in fully MH orchids (includ-ing Lycoperdon; for a review, see Ogura-Tsujita andYukawa (2008a) that need to be revisited by modernmolecular tools.

Although direct data (e.g. isotope tracerstudies) that tropical orchids receive carbonfrom decomposing plant matter via a hyphalconduit is still lacking (Selosse et al. 2010),fungal rhizomorphs linking dead organicmatter to the orchid mycorrhizal roots cansometimes be visualized (Kusano 1911; Martoset al. 2009; Fig. 12.3). Moreover, the stable iso-tope abundance signatures of these orchids aredistinctive: they often have slightly higher 13Cabundance but substantially lower 15N thanECM-associating plants (Ogura-Tsujita et al.2009), reflecting the higher 13C and lower 15Nabundance of saprotrophic fungi as comparedwith ECM fungi (Hobbie et al. 2001).

Typically fungal colonization is sparse infully MH orchids that rely on saprotrophicfungi than on ECM fungi (Dearnaley 2006;Dearnaley and Bougoure 2010), or even notcontinuous over the year for Wullschlaegeliaaphylla (Martos et al. 2009), suggesting thatthe mechanism of obtaining carbon is possiblymore efficient than with ECM fungi, but thisrequires further study. Additionally, hyphaecolonize some dead cortical root cells inWullschlaegelia aphylla (Martos et al. 2009),while a complicated pattern of colonizationexists in Gastrodia roots, with passage cellswhere hyphae enter the root, in host cellsthat are permanently colonized and in diges-tion cells where a carbon flux may occur


(Kusano 1911; Wang et al. 1997). Althoughtheir raison d’etre remains unclear, these pat-terns may be evolutionarily derived, empha-sizing the secondary evolution of this kind ofmycoheterotrophy. Martos et al. (2009) andSelosse et al. (2010) speculated that the shiftof fungal partners to various saprotrophicfungi during MH orchid evolution mighthave occurred in tropical and wet temperateregions, because these environmental condi-tions stimulate decomposing activity by fungiand might allow higher carbon gain for theplant. Indeed the need to support the largecarbon requirement of plants may explainwhy non-ECM ‘rhizoctonias’ are rarely foundin MH orchids, although they support MHgermination in many orchid species: theymay simply be too C-limited to fulfil theplant’s needs beyond the protocorm stage.

D. Mixotrophs: Green Orchids that ObtainCarbon from Fungi

Stable isotope investigation of an increasinglylarge number of green orchids, e.g. in the generaCephalanthera, Epipactis or Cymbidium, hasrevealed natural abundances of 13C and 15Nhigher than surrounding autotrophic plants butless than that of fully MH orchids (Gebauer andMeyer 2003; Julou et al. 2005; Abadie et al. 2006).Such intermediate values suggest that these

orchids obtain part of their carbon via photo-synthesis and part through their mycorrhizalfungi – that is, these plants are mixotrophic(Julou et al. 2005). Mixotrophy is thought to bean intermediate step in the evolution of fullmycoheterotrophy (Bidartondo et al. 2004;Selosse et al. 2004; Abadie et al. 2006; Motomuraet al. 2010). Identifying photosynthesis ineffi-ciency in many chlorophyll-containing orchidsmay also uncover cryptic mixotrophic orchids(e.g. Girlanda et al. 2006).

These orchids are rarely specific in theirmycorrhizal associations and associate with sev-eral ECM fungi, with few exceptions, such asPlatanthera minor that is specific to an ECMCeratobasidium (Yagame et al. 2012). Epipactisspp. associate with truffles and related ECMPezizales, as one of the rare Ascomycete-associated orchid clades known so far(Fig. 12.2; Selosse et al. 2004; Bidartondo andRead 2008; Ogura-Tsujita and Yukawa 2008b;Shefferson et al. 2008). Tuber spp. also occur asrare mycobionts in the closely related mixo-trophic Limodorum abortivum (Girlanda et al.2006). Mycorrhizal associations with terrestrialorchid species were documented for 13 Tuberspecies that belong to five of the nine mainTuber clades (the Excavatum, Aestivum,Rufum, Maculatum and Puberulum clades;Bonito et al. 2010). In a thought-provokingpaper based on field data collection in Hungary,Ouanphanivanh et al. (2008) showed that Epi-

Fig. 12.3. Fungal rhizomorphs (arrows) of Mycenalinking dead leaves to mycorrhizal roots (arrowhead)of the fully MH orchid Wullschlaegelia aphylla, inwhich the fungus is mycorrhizal (bar 1 cm). Inset

Transverse section of a rhizomorph, with a centralhole, and hyphae with thicker, melanized walls at theexternal border (bar 100 mm). F. Martos, unpublishedmicrograph


pactis spp. co-occurred more often than at ran-dom with truffle stands (a similar situation wasshown for Cephalanthera and Hymenogaster)and could indicate truffle habitats. A noteworthyfeature is the presence of some ‘rhizoctonias’ inmixotrophic orchids, together with the domi-nant ECM fungi (Bidartondo et al. 2004; Julouet al. 2005; Abadie et al. 2006; Motomura et al.2010; Paduano et al. 2011). This feature alsosupports that these orchids are an intermediatestep in the evolution of full mycoheterotrophy,deriving from autotrophic orchid ancestorsassociated with ‘rhizoctonias’.

Some ECM fungi are found from time to time in auto-trophic orchids where ‘rhizoctonia’ are dominant: Rus-sulaceae occur in some Cypripedium spp. (Sheffersonet al. 2007) and Pterostylis nutans (Irwin et al. 2007),Thelephora and Cortinarius in Orchis spp. (Lievenset al. 2010) and putatively ECM Pezizomycetes andHelotiales in Gymnadenia conopsea (Stark et al. 2009).ECM Ascomycetes (Waterman et al. 2011) even domi-nate in some South African Diseae (Pterygodium andCorycium spp.). Although the exact interaction with theorchid is unknown, the detection of these fungi ishighly unexpected as they are not usually contaminantsof soil samples. We speculate that their presence in roottissues may give opportunity for their evolution intomycorrhizal partners, accompanying the emergence ofmixotrophy. The same may apply for evolution ofmycoheterotrophy based on saprotrophic fungi:Mycena-related fungi, that are sometimes associatedwith MH orchids as mentioned above (Sect. III.C), cansometime be found in roots of some green, ‘rhizocto-nia’-associated orchids (Fan et al. 1996; Guo et al. 1997).

E. Epiphytic Orchids and ‘Rhizoctonias’

Although epiphytic species represent the largestnumber of orchids worldwide (Jones 2006) theyare surprisingly less well studied with regards totheir mycorrhizal associations. Mycorrhizal fungisparsely colonize roots of epiphytic orchids incomparison with terrestrial orchids (Boddingtonand Dearnaley 2008; Smith and Read 2008; Gra-ham and Dearnaley 2012; Martos et al. 2012) butmolecular identification of the mycobionts pres-ent reveals them as the typical ‘rhizoctonias’ ofgreen orchids. This has included members of theCeratobasidiaceae (Otero et al. 2002, 2004, 2005,2007; Pereira et al. 2005; Gowland et al. 2007;

Graham and Dearnaley 2012; Martos et al.2012), the Tulasnellaceae (Pereira et al. 2003;Suarez et al. 2006; Kottke et al. 2008; Martoset al. 2012) and the Sebacinales (Suarez et al.2008; Martos et al. 2012).

In the largest survey of orchid mycorrhizas conductedfrom 77 orchid species from La Reunion island, Martoset al. (2012) found that communities of ‘rhizoctonias’significantly differed between epiphytic and terrestrialorchid communities in terms of OTUs, whereas thethree ‘rhizoctonia’ taxa did not differ in frequency.This may reflect that different fungal species are avail-able in soil and on tree bark, but we lack informationabout the diversity and ecology of ‘rhizoctonias’ in soilversus bark environments.

The dependency of epiphytic orchids onmycorrhizal fungi throughout the life cycle isnot surprising as the mycobionts, with theirincreased surface area, may improve access towater and minerals for plants which can be espe-cially limiting in the epiphytic state (Zotz andSchmidt 2006; Osorio-Gil et al. 2008). The habitatof many epiphytic species that live in the shade ofdense forest canopy is typified by low irradianceand it is possible that species will be soon identi-fied as mixotrophic with a dependence on exter-nal supplied carbon as well as photosynthesis.

IV. Nutrient Exchanges BetweenOrchid and Mycobiont

The minute seeds of orchids lack food reservesand colonization by a suitable fungus is neces-sary for further development under naturalconditions (Smith and Read 2008). Bothorganic and inorganic nutrients have beenshown to be transferred from mycorrhizalfungus to protocorms. Experiments usingsplit-plate systems and labelled glucose acces-sible only to the fungal partner have demon-strated carbon flow to orchid protocorms(Purves and Hadley 1975; Alexander and Had-ley 1985). A similar split-plate system indicatedthat phosphate (labelled by 32P) is passed fromfungus to protocorms of Dactylorhiza purpur-ella (Smith 1966).

Adult mycorrhizal orchids continue toreceive both organic and inorganic nutrients


from their fungal partners. When the Ceratoba-sidium partner of Goodyera repens is suppliedwith 14C-labelled glycine in the substrate,labelled carbon is passed to the orchid seed-lings (Cameron et al. 2006). The addition of13C/15N-labelled glycine to the fungal compart-ment also demonstrated a transfer of nitrogento orchid seedlings. Cameron et al. (2007) haveshown that adult Goodyera repens also receivephosphate from their fungal partner underexperimental conditions. Mycorrhizal fungimay be key to ensuring optimal water uptakefrom the environment: mycorrhizal Plan-tanthera integrilabia and Epidendrum conop-seum both had higher water content thanuncolonized controls (Yoder et al. 2000).

Orchid mycorrhizas have often been con-sidered to be atypical mycorrhizal associations,with the fungus deriving little benefit from theorchid host (Dearnaley 2007; Smith and Read2008). Key to this assumption was researchconducted by Hadley and Purves (1974) andAlexander and Hadley (1985) on mycorrhizalGoodyera repens. In their experiments, theorchid was exposed to 14CO2 and no subsequentpassage of labelled carbon to the fungal partnerwas detected. These experiments were morerecently repeated by Cameron et al. (2006,2008) using more naturally equivalent condi-tions (e.g. moderate temperature, lighting,humidity) with contrasting results. In the latterexperiments approximately 0.4–3.0 % of thecarbon label originally provided to the orchidwas passed to the fungal partner (Cameron et al.2006, 2008). Adult orchid mycorrhizas thuspotentially represent a truly mutualistic interac-tion similar to ECM and AM associations.

Rasmussen and Rasmussen (2007) and Hynson et al.(2009) rightly note that adult orchid mycorrhizal sys-tems other than Goodyera repens should be investigatedsimilarly, as well as under field conditions. The impor-tance of the carbon acquired from the orchids in thewhole nutritional budget of the fungus has also beenquestioned (Rasmussen and Rasmussen 2009). How-ever, using a clever experimental design where thecarbon source have different 13C abundances, Latalovaand Balaz (2010) showed that a Tulasnella speciesreceived 70 % of its carbon from its host, Serapiasstrictiflora and 30 % from dead maize roots added tothe system, but these experiments were again carriedout in vitro.

Most importantly, from an evolutionarypoint of view, the reciprocation in a mutualismcannot only be quantified in terms of nutrientflow, but should result in a fitness improve-ment. Fitness is notoriously difficult to measurein fungi (Pringle and Taylor 2002), and there iscurrently no evidence that fungi reproducebetter with the orchid than without.

Intriguing indirect evidence for mutualismarose recently from the analysis of the architec-ture of interaction networks between autotro-phic orchids and their ‘rhizoctonias’.These may vary according to the nature of theinteraction, especially when comparing mutu-alistic and trophic interactions (Thebault andFontaine 2010). Recent analyses (Jacquemynet al. 2010 and unpublished data) concludedthat orchid mycorrhizal interaction networksdisplayed a significantly nested structure, i.e.specialized (fungus-specific) orchid speciestended to associate with ‘rhizoctonia’species that themselves associated with moregeneralized (not fungus-specific) orchidspecies, and vice versa. Conversely, Martoset al. (2012) found that orchid–fungus networksdisplayed a highly modular structure in a trop-ical context, which could be interpreted as anecological divergence between epiphytic andterrestrial guilds of plant and fungal partnersin tropical communities, although the authorsalso confirmed the presence of some level ofnestedness in the epiphytic and terrestrialsub-networks (Fig. 12.4). The trend of nested-ness is a specific feature of mutualistic net-works, as opposed to parasitic or trophicnetworks that are more compartmentalized,and is viewed as a consequence of the recipro-cation process itself (Thebault and Fontaine2010). In other words, this may be an indirectindication that some reciprocation occurswith most ‘rhizoctonias’; thus, the investmentin protocorm development may be viewed as atransient cost to develop a host that willbe beneficial for the fungus later (Leakeet al. 2008).

Should we conclude that, conversely, mix-otrophic and MH orchids are fungal parasites?This is the tacit idea when calling these plantsepi-parasites, or cheaters on ECM symbioses(Merckx et al. 2009), but we still lack rigorous


evidence for this. Indeed, some vitamins, orsome protection at some time of the year mayenhance fungal fitness. Obviously, we needmore studies on the fungal side before anyconclusion can be made. Hopefully, the devel-

opment of models tractable in vitro for auto-trophic (Cameron et al. 2006, 2008) and MHorchids (Yagame et al. 2007; Bougoure et al.2010) will help in investigating these questions.

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Tulasnellaceae #01

Tulasnellaceae #04

Tulasnellaceae #09

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Sebacinales #04Sebacinales #07

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Ceratobasidiaceae #01Ceratobasidiaceae #03

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Tulasnellaceae #31

Tulasnellaceae #35

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Ceratobasidiaceae #07Ceratobasidiaceae #08


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Tulasnellaceae #29

Tulasnellaceae #38

Tulasnellaceae #43

Tulasnellaceae #48

Tulasnellaceae #54

Tulasnellaceae #02

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Tulasnellaceae #10Tulasnellaceae #16


Tulasnellaceae #18Tulasnellaceae #19

Tulasnellaceae #25

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Sebacinales #11


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Sebacinales #21

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Tulasnellaceae #32

Tulasnellaceae #37

Tulasnellaceae #42

Tulasnellaceae #44

Tulasnellaceae #52

Tulasnellaceae #55

Fig. 12.4. Nested architecture of an orchid–rhizoctonianetwork as formed by epiphytic orchids on La Reunionisland (from Martos et al. 2012). The left column showsorchid species with lines linking to various ‘rhizoctonia’

taxa (right column). A nested structure (i.e. lesscompartmentalized) is a specific feature of mutualisticnetworks


V. Fungal Specificity in Orchids

A. Patterns and Evolutionary Significance

Fungal specificity is the association of an orchidspecies with a small number of fungal partners(Irwin et al. 2007), and can be quantified as thephylogenetic breadth (¼ antiquity of the lastcommon ancestor) of its range of associates(Thompson 1994; Shefferson et al. 2010). Thiscan take the form of narrow specificity wherebyan orchid associates exclusively with a singlemycobiont across its range, such as the rareunderground orchid Rhizanthella gardneri(Bougoure et al. 2009). Typically, specificity isexpressed as an orchid species associating with alimited number of related fungal taxa, e.g. Cor-allorhiza maculata associating with Russulaceaespecies in the western United States (Taylor et al.2004), or Pterostylis nutans associating with twoCeratobasidium species in eastern Australia(Irwin et al. 2007). Both fully MH and autotro-phic species can display fungal specificity (e.g.McCormick et al. 2004; Yamato et al. 2005)although the phenomenon is more common tothe former orchid physiological type. A smallnumber of the investigated orchid species dis-play little fungal specificity. For example, thewidespread Australian grassland species Micro-tis intermedia associates with members of boththe Sebacinales and the Ceratobasidiaceae (Bon-nardeaux et al. 2007), while two fully MH Aphyl-lorchis species from Thailand associate with anarray of unrelated ECM fungi, including mem-bers of the Thelephoraceae, Russulaceae andSebacinales (Roy et al. 2009b).

The evolution of fungal specificity hasbeen recently evaluated by mapping the phylo-genetic breadth of mycorrhizal partners acrossorchid phylogenies. Shefferson et al. (2007,2010) analysed fungal specificity across twoorchid phylogenies of the genera Cypripediumand Goodyera and found that both wideningand broadening depended on orchid clades, sothat the level of fungal specificity was con-cluded to be an evolvable trait subjected toreversion in orchids. Considering the evolutionof fungal partners across orchid diversification,Waterman et al. (2011) showed that fungal

partners are conserved between closely relatedspecies of South African Coryciinae.

Martos et al. (2012) used both orchid and fungal phy-logenies to assess phylogenetic signal in the interactionnetwork of tropical angraecoid orchids on the island ofLa Reunion and found a stronger signal on the orchidside than on the fungal side: fungal partners that belongto the Tulasnellaceae, Sebacinales and Ceratobasidia-ceae are statistically more conserved between closelyrelated angraecoids than orchid partners of closelyrelated fungi are. Such an asymmetry of phylogeneticsignal may reveal different constraints for the partnersin orchid mycorrhiza, especially the lower dependenceof fungal partners on the symbiosis.

Sudden partner shifts have also occurredduring the evolution of the MH genus Hexalec-tris (Kennedy et al. 2011), or in the genusEpipogium, where E. aphyllum associateswith ECM Inocybe spp. (Roy et al. 2009b)while E. roseum associates with saprotrophicPsathyrella-related partners (Yamato et al.2005). Partner shift can thus rapidly evolve,although how the shift occurs remains unclear.As mentioned below (Sect. V.B), the observa-tion that some unexpected fungi are sometimesdetected in roots, in addition to the majormycorrhizal fungi, may be relevant as a startingpoint in the transition – this led to the sugges-tion that ‘molecular scraps’ (unexpected fungiconsidered as contaminant or marginal in themycobiont spectrum) obtained in symbionttyping should always be reported (Selosseet al. 2010). A particularly interesting stage forthe transition may be the germination step: insome orchid species at least, the fungi enhancingthe first stage of germination are more diversethan the fungi allowing further development(Vujanovic et al. 2000; Bidartondo and Read2008). From this situation, where early embryoscontact diverse fungi, a mutant for specificitymay survive.

B. Adaptive Significance

The adaptive significance of fungal specificityin orchid mycorrhizas is a source of some con-jecture. Specific fungal partners may lead to


enhanced seed germination rates (Otero et al.2004; Bonnardeaux et al. 2007) and thusincreased fitness. The efficiency of nutrientexchange between partners may be heightenedwith specific plant–fungus combinations (Bon-nardeaux et al. 2007) and this may be critical forcarbon uptake for mixotrophic and fully MHorchids in low-light habitats where there is ahigher dependency on fungal carbon (e.g.Girlanda et al. 2006). More efficient nutrientexchange as a driver for fungal specificity inorchids can be suggested by examples of partnerswitching in adult orchids. For example Good-yera pubescens switched from one Tulasnellaspecies to another when plants were drought-stressed (McCormick et al. 2006). Several studieshave suggested that autotrophic orchids associ-ate with different clades of ‘rhizoctonias’depending on the environment, e.g. when com-paring terrestrial and epiphytic orchid commu-nities in tropical forests (Martos et al. 2012), orEuropean terrestrial orchids in dry and wet habi-tats (where different Tulasnellaceae subcladesdominate; Illyes et al. 2009). However, it remainsunknown whether this results from choosingoptimal fungal partners or simply from differentavailability of fungal taxa.

C. The Impact of Mycorrhizal Specificity onOrchid Speciation

Fungal specificity was recently linked to specia-tion in the Orchidaceae by a number of authors(Otero and Flanagan 2006; Shefferson et al. 2007;Waterman and Bidartondo 2008; Waterman et al.2011). Distribution of fungi in soils is highlyheterogeneous (Richard et al. 2004; Pickleset al. 2010) and this, combined with narrowfungal specificity, may determine the small,over-dispersed populations of many orchid spe-cies (Otero and Flanagan 2006). The resultingpatchiness of orchid distribution may limitgene flow between isolated populations and thenumber of reproducing individuals, leading inturn to genetic drift and allopatric speciation(Tremblay et al. 2005; Waterman and Bidartondo2008). Support for this process comes from theobservation that different populations of theHex-alectris spicata complex display distinct mycor-

rhizal fungi (Taylor et al. 2003). Natural selectionmay also act on small, isolated populations oforchids, as highlighted in the study of Oteroet al. (2005) that showed varying levels of germi-nation rates (or fitness) after reproducing in vitroassociations between Tolumnia variegata and dif-ferent ‘rhizoctonia’ fungi.

In contrast, Roche et al. (2010) showed thatmultiple species of Chiloglottis associated witha narrow group of Tulasnellaceae fungi acrosseastern Australia. The fact that each of thesespecies associates with a distinct wasp pollinatorsuggests that pollination systems and notfungal specificity is driving speciation in theorchid genus. A similar interpretation wasmade by Waterman et al. (2011) when studyingshifts of pollination modes and mycorrhizalpartners across the phylogeny of South AfricanCoryciinae orchids. Roche et al. (2010)suggested that a common mycorrhizal fungusin Chiloglottis spp. has enabled rapidpollination-mediated speciation via co-occurrence of multiple potential species types.

Mycorrhizal associations during species hybridization,a potential source of speciation in the Orchidaceae,have been examined by some researchers. In crossesbetween Caladenia spp., Hollick et al. (2005) showedthat hybrids have genetically similar fungi to one of thetwo parents. The hybrid formed between crosses ofOrchis simia and Orchis anthropophora also had similarTulasnellaceae fungi to its parents (Schatz et al. 2010).Interestingly, hybrid Orchis plants had higher levels ofmycorrhizal colonization than the parents but this waspossibly related to the inability to attract pollinatorsand to produce seeds, therefore providing more carbonfor the colonizing fungus. Jacquemyn et al. (2010) alsoinvestigated the mycorrhizal associations of Orchishybrids and concluded from common mycobionts inprotocorms and adults that mycorrhizal fungi play asmall role in reproductive isolation. One generalizingspeculation that can be derived from these studies isthat mycorrhizal symbiosis acts in a permissive way, i.e.that, for successful hybridization to occur, the parent’sfungi need to be related or identical.

VI. Orchid Mycorrhizas and PlantConservation

A dependence on narrowly specific interactionswith fungi and pollinators may predisposemany orchids to become rare (Bonnardeaux


et al. 2007; Dearnaley 2007; Swarts et al. 2010).However, Phillips et al. (2011) have recentlyshown that fungal specificity has not led torarity in West Australian Drakaea spp. asthe associated Tulasnella fungus is widelydistributed in the environment. Nevertheless,as humankind continues to have negativeimpacts on natural ecosystems through suchperturbations as vegetation clearing, alteredfire regimes, weed and feral animal introductionand climate change, populations of many rareorchid taxa are further declining (Brundrett2007). Conservation approaches for such orch-ids include on site protection of existing popu-lations, ex situ storage of tissues and restorationprocedures (Swarts and Dixon 2009). All ofthese approaches require an understanding ofthe mycorrhizal biology of the species in ques-tion, since fungi are vital for orchid seed germi-nation and adult vegetative life.

A. Orchid Mycorrhizas and On-SiteManagement

Molecular identification of the mycobionts ofmany orchid species has given an insight intothe ecological position of fungal species. Thishas highlighted management procedures thatare needed to protect existing populations. Theconservation of fully MH and mixotrophicorchids dependent on ECM associations suchas Hexalectris, Epipactis, Dipodium and Rhi-zanthella (Taylor et al. 2003; Selosse et al.2004; Bougoure and Dearnaley 2005; Bougoureet al. 2010) clearly need maintenance of standsof suitable host trees. Fully MH species such asGastrodia, Epipogium and Erythrorchis, whichare nutritionally dependent on wood-rottingfungi (Yamato et al. 2005; Dearnaley 2006; Mar-tos et al. 2009; Dearnaley and Bougoure 2010),will need the retention of a suitable decompos-able substrate. For the majority of (autotro-phic) orchids, preservation of the uppermostorganic layer of soils is essential, as this loca-tion is the key habitat of their ‘rhizoctonias’associates (Brundrett et al. 2003). As this layeris particularly susceptible to frequent burning

(Brundrett 2007), careful monitoring of fireregimes is a necessary conservation measure.

For all orchids with partial or full mycoheterotrophy,the fungus cannot be separated from its own carbonsource and, if such occurs during relocation, both thefungus and the plant may die. This was shown in anoverlooked book by Sadovsky (1965) dealing with thecultivation of ‘orchids in your own garden’: amongother studies, Sadovsky trialled the relocation of anumber of orchid species at a time where protectionlaws were more flexible in Europe, and the resulting listshowed that mixotrophic and MH species could not betransplanted. Thus, there may be problems savingpopulations of such orchids by transferral to anothersite in the case of major disturbance. However, theeffective glasshouse relocation of Rhizanthella slateri(with its ECM partner and photosynthetic host)threatened by a major road development in easternAustralia (M. Clements, personal communication) pro-vides an exemplar of success.

Regular monitoring for the continued pres-ence of orchid-associated fungi is a necessarymanagement procedure. This can be done sim-ply by seasonal observations of macrofungalfruiting bodies for some associated orchid spe-cies. For microfungi and rarely sporulatingfungi, such a most ‘rhizoctonias’ (e.g. clade BSebacinales that do not fruit; Weib et al. 2004),seed baiting procedures carried out both in situand ex situ, are cost-effective (Brundrett et al.2003). Molecular detection of orchid-associatedfungal DNA using specific or general fungal pri-mers will also ensure that sites continue to har-bour the appropriate mycobionts. The best wayto preserve orchids is therefore to preserve theirfungi and, from there, given the uncertainties onthe ecology of fungi, the whole environment.This is indeed good news for mycologists sinceorchid protection therefore protects fungi – notonly those taxa involved in mycorrhizal associa-tions, but the surrounding ones as well.

Some fully MH orchids rely on ECM fungithat have fruiting bodies that are consumed bynative mammals (Bidartondo et al. 2004; Selosseet al. 2004; Dearnaley and Le Brocque 2006). InAustralia, members of the Russulaceae are con-sumed by marsupials such as Bettongs andPotoroos (Claridge and May 1994). To ensurecontinued cycling of fungal propagules through


ecosystems, protection of these spore-dispersinganimals should be a long-term priority.

Two recent works suggest that the presence of fungimay not be the sole limiting factor for orchid settle-ment: in experimental seed sowing at different siteswhere the focal orchid species does not grow, there isevidence that early development into a protocorm canoccur in Cephalanthera spp. (Bidartondo and Read2008) and Epipactis spp. (Tesitelov et al. 2012), withsuccessful access to appropriate fungal partners.Although these plants are mixotrophic and may notrepresent general models, limitations to orchid devel-opment may thus be more than fungal – as the authorsdiscuss, a limitation on seed dispersal or abiotic factorsmay also be involved. Thus, the fungal symbiosis,although crucial to the orchids, may not be seen asthe sole factor explaining why orchids develop andwhy a given site is suitable for orchid growth.

B. Ex Situ Conservation and OrchidMycorrhizas

Ex situ symbiotic germination of seed is a com-mon approach in conservation procedures forthreatened orchids (Batty et al. 2006b; Stewartand Kane 2007; Zettler et al. 2007). Mycorrhizalfungi can be obtained from adult plants in situor via buried seed germination packets (Battyet al. 2001; Dearnaley et al. 2009). Damage toadult threatened orchids can be minimized bytaking a small sliver of colonized stem (Wrightet al. 2009; Smith et al. 2010) and the best plantsto isolate fungi from are leafing to floweringstages (Huynh et al. 2004). Surface sterilizationof isolated orchid tissues reduces the amount ofcontamination from bacteria and faster-growing ascomycetes (Huynh et al. 2009).Once pelotons are separated from the host tis-sue, the best ‘rhizoctonias’ to choose for symbi-otic autotrophic orchid seed germination arethose with fine loose hyphae and monilioidcells (Huynh et al. 2004). Pure fungal inoculumand surface-sterilized orchid seed are tradition-ally co-cultured on oatmeal-based agar medium(Clements et al. 1986). The growth of orchidsdependent on ECM fungi and photosynthetichosts requires special culturing set-ups suchas that developed by Bougoure et al. (2010) forRhizanthella gardneri (Fig. 12.5). Fully MH

orchids reliant on saprotrophic Agaricomy-cetes can be grown in seed packets with amedium of sawdust and fungal inoculum(Yagame et al. 2007). Ex vitro approacheswhereby seed is sown in pot soil inoculatedwith the appropriate mycorrhizal fungus hasan additional advantage in that seedlings mayform associations with other micro-organismspresent in the medium (Wright et al. 2009).Procedures for maintaining orchid mycorrhizalfungi in the long term include immersing theinoculum in liquid nitrogen (Batty et al. 2001)or via encapsulation of both seed and fungi inalginate beads, with low-temperature storage(Sommerville et al. 2008). It is important thata range of fungal taxa and isolates are preservedin orchid conservation work as multiple fungimight co-exist in plants (Irwin et al. 2007;Wright et al. 2010) or orchids may switchfungi as they mature (Xu and Guo 2000) oreven as adults (McCormick et al. 2004; Dearna-ley 2006). Furthermore, the fungal isolate that isbest at germinating seed does not necessarilyensure the best long-term survival of orchidspecies (Wright 2007; Huynh et al. 2009).

C. Use of Mycorrhizal Fungi in RestorationProcedures

Symbiotically grown orchid seedlings can betransferred directly to the natural state butplant persistence is enhanced by growth in pot-ting media for several seasons (Swarts 2007).Moving plants from Petri dish to soil can be asignificant hurdle (Wright et al. 2009) but anintermediate deflasking procedure involvingcarefully aerated sand–agar containers hasbeen shown to effectively prepare agar-grownsymbiotic seedlings for transfer to soil in pots(Batty et al. 2006a). There appears to be nobenefit to inoculating the pot soil with compat-ible fungi, with the original colonizing fungusproving sufficient for nutrient uptake for theseedlings (Batty et al. 2006a). For establishingorchid populations in the natural state, seed-lings and tubers appear to be better than seedsowing, although the latter is more cost- andtime-effective (Batty et al. 2006b; Wright et al.


2009). Both in situ (Rasmussen and Whigham1993) and ex situ (Brundrett et al. 2003) seedbaiting can be used to confirm the presenceof fungi at introduction sites. Translocationsuccess can be enhanced by a combination ofadding fungal inoculum and loosening soil atsites; the latter potentially enhances the activityof the fungi at the location (Smith et al. 2009).Fungal inoculum can be introduced to new siteswithout orchids and can persist in soils forseveral seasons in preparation for restorationprocedures (Hollick et al. 2007).

VII. Conclusions

Orchid mycorrhizas are predominantly repre-sented by associations between photosynthetic

plants and ‘rhizoctonia’ fungi. These associa-tions, which likely represent the plesiomorphiccondition for orchids, gave rise through repeatedevolutionary shifts to interactions with otherdiverse fungal lineages and diversification oforchid metabolism. How orchids recruit andallow new fungi (even some ‘naıve’ fungi fromnon-mycorrhizal clades) to enter the dual mor-phogenesis of mycorrhizas remains unclear.However, orchid mycorrhizas are excellentmodels to reveal the general properties of mycor-rhizal systems as well as providing insights intothe fungal world via specificity aspects, ecologicalnetworks and evolution of the mycorrhizal state.

Although considerable advances have beenmade in understanding the ecology and evolutionof orchid mycorrhizas in recent years, substantialknowledge gaps still exist. In particular, many

Fig. 12.5. Rhizanthella gardneri growth pot arrange-ment (from Bougoure et al. 2010; used with the permis-sion of author and publisher). The orchid (shown in C)is grown in the inner of three pots while the ECM

Ceratobasidium partner, inoculated into the middlepot, passes photosynthate from the autotrophic Mela-leuca scalena (outer pot, also seen in A and B) to theorchid via small holes in each pot


aspects of orchid mycorrhizal physiology stillrequire investigation, for example the ubiquityof plant to fungus carbon transfer in green orch-ids, the metabolism of fungi involved in the pro-cess and the expression of genes throughout thesymbiosis. Moreover, research is often orchid-focussed, so that a lot of questions remain onthe fungal side which is probably less easy toinvestigate. The exact nutrition, diversity, benefitsfrom the association (if any) and repartition insoil of many mycobionts, such as the Tulasnella-ceae, are often ignored and with some exceptions(e.g. Selosse et al. 2002; McCormick et al. 2009),the fungus is rarely investigated out of the orchidroots. It is hoped that these and other areas willcontinue to contribute to understanding thesefascinating mycorrhizal interactions, with moreemphasis on the involved fungal taxa.

Acknowledgements The authors would like to thankthe large number of students and colleagues who havesupported their research on orchid mycorrhizas inrecent years. J.D. thanks the Australian Orchid Foun-dation for support of his research. M.-A.S. and F.M.thank the Societe Francaise d’Orchidophile and itsmembers for continuous support and participation intheir research programmes.

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