Biocomplexity of Plant-Fungal Interactions (Southworth/Biocomplexity of Plant-Fungal Interactions)...

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BLBS099-c05 BLBS099-Southworth December 1, 2011 10:55 Trim: 244mm X 172mm

Chapter 5Biology of Mycoheterotrophic and

Mixotrophic PlantsHugues B. Massicotte,1 R. Larry Peterson,2 Lewis H. Melville,2

and Daniel L. Luoma3

1Ecosystem Science and Management Program, University of Northern British Columbia,Prince George, BC, Canada

2Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada3Department of Forest Ecosystems and Society, Oregon State University, Corvallis, OR, USA

The vast majority of plants on earth are autotrophic, using chlorophyll to convert solar energyinto metabolites for growth and reproduction. However, approximately 400 plant species in87 genera and 10 families (Furman and Trappe 1971; Leake 1994, 2004) have lost functioningchlorophyll. As a consequence, they have evolved different strategies to obtain the energythey need without photosynthesis. Species that receive all their carbon from autotrophic plantsvia fungal connections are referred to as mycoheterotrophs (Leake 1994). Achlorophyllousplants, due to their unusual and often ghostly appearance, fascinate people. How these plantsthrive, let alone survive, while exhibiting this apparent “carbon budget deficit” is a multifacetedtopic that leads to many questions. A mycoheterotrophic plant receives all of its carbon viathe fungus that serves as an intermediary between it and one or more associated autotrophicplants (Furman and Trappe 1971). The fungus does not receive obvious benefits from themycoheterotroph, and therefore, the mycoheterotroph can be considered a “cheater” (Bidartondoet al. 2003; Smith and Read 2008; Merckx et al. 2009). Brundrett (2004) has proposed thatthese plant–fungal relationships should be referred to as “exploitive mycorrhizae” since only thenongreen plant appears to gain from the association. Although these plants have been consideredto be saprotrophs, and are still referred to as such in some current literature, it is well establishedthat this is a fallacy (Leake 2005). Likewise, they are not direct parasites on autotrophic plantssince there are no connections such as root grafts or haustoria between the two plant species.

In addition to fully mycoheterotrophic species, there are approximately 20,000 species thatare partially mycoheterotrophic (Merckx et al. 2009). Most of these depend on fungi only duringearly stages of seedling establishment. However, several photosynthetic genera can, as juvenileor adult plants, obtain a portion of their carbon needs from neighboring photosynthetic plantsthrough fungal connections. The terms partial mycoheterotrophy or “mixotrophy” describe thisinteraction (Selosse et al. 2004; Smith and Read 2008). Plants that establish in light-deficientunderstory habitats may fit into this category; they may receive part of their carbon demandthrough fungal links to photosynthetic neighbors (see Chapter 4).

Biocomplexity of Plant–Fungal Interactions, First Edition. Edited by Darlene Southworth.C© 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

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110 Biocomplexity of Plant–Fungal Interactions

Morphological, anatomical, and cellular features of plant–fungal interactions have been usedto define mycorrhizal categories (Peterson and Massicotte 2004; Peterson et al. 2004; Imhof2009) and to aid in the clarification of the trophic nature of these associations. Informationobtained from microscopy has provided a valuable platform for investigations of the mecha-nisms involved in recognition between symbionts, in plant–fungal nutrient transfer and watermovement, in gene function, and in rhizosphere processes. The ability to contrast “normal”photosynthetic plants and their fungal associations with those of “abnormal” nonphotosyntheticplants may reveal insights into the functioning of mycorrhizal systems that occur in the majorityof autotrophic land plants.

Here, we consider categories of mycoheterotrophic and mixotrophic associations from theperspective of the fungal taxa involved, plant host–fungus specificity, seed germination and earlyseedling establishment, structural features of the interface between root cells and fungal hyphae,aspects of nutrient transfer, and the evolutionary and ecological aspects of these associations.

5.1. MYCOHETEROTROPHIC ASSOCIATIONS

5.1.1. Plants Associated with Arbuscular Mycorrhizal Fungi

Arbuscular mycorrhizae (AMs) involving fungi belonging to the Glomeromycota are formedin the majority of mycoheterotrophic plant species (Leake 1994, 2004). This is becoming moreapparent as increased numbers of plant species from the tropics and neotropics are examinedfor their symbiotic associations with fungi.

Seedless Vascular Plants

Several photosynthetic genera of seedless vascular plants, for example, Lycopodium, Huperzia(Lycopodiaceae), Ophioglossum, Botrychium (Ophioglossaceae), Psilotum, and Tmesipteris(Psilotaceae), form thick-walled spores that are shed and become buried in leaf litter andsoil. Spores of these genera require fungal symbionts for germination and development of theachlorophyllous gametophyte (Figure 5.1E). During these stages in their life cycle, these generaare mycoheterotrophic (Read et al. 2000). Experimental evidence is still lacking to confirm thetransfer of nutrients from the fungus into developing gametophytes.

Based on structural features, the fungal symbionts in the gametophytes of seedless vascularplants appeared to be members of the Glomeromycota (Read et al. 2000; Leake et al. 2008);recent molecular studies have confirmed this for some species and have shown that the fungi as-sociated with Lycopodium clavatum and Huperzia spp. and with two species of the eusporangiatefern, Botrychium, belong to Glomus Group A (Winther and Friedman 2007a, 2007b).

Structurally, intracellular hyphal coils (Figure 5.1E), typical of Paris-type AM associationsreported in roots of many autotrophic angiosperm species, occur in gametophyte cells of severalgenera (Read et al. 2000; Winther and Friedman 2007a, 2007b). Vesicles (structures that storelipids and may act as fungal propagules) have also been reported. Gametophytes of the whiskfern, Psilotum nudum, contain dense coils of hyphae that lack cross-walls (septa) (Figure 5.2) andthat have infrequent terminal vesicles (Peterson et al. 1981). The association between fungi andgametophytes of L. clavatum has been described as the “lycopodioid mycothallus interaction”because of structural features that do not fit most AM associations (Schmid and Oberwinkler1993). Since typical arbuscules are absent, nutrient transfer might occur either via intact hyphalcoils or during breakdown of hyphae.

Monocotyledonous Angiosperms

Roots of several species in five monocot families (Burmanniaceae, Corsiaceae, Petrosaviaceae,Thismiaceae, and Triuridaceae) can be colonized by glomalean fungi; species in four of these

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Biology of Mycoheterotrophic and Mixotrophic Plants 111

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Figure 5.1. Life cycle of Lycopodium annotinum with fungal symbiont in gametophyte. (A) Sporo-phyte attached to subterranean gametophyte (arrow). (B) Strobilus with sporangia (arrowheads).(C) Sporangia with spores being released. (D) Early stage in spore germination. (E) Gameto-phyte showing archegonia (arrows), antheridia (arrowheads), zone of fungal colonization (doublearrowheads), and rhizoids (r).

families associate specifically with Glomus Group A (Bidartondo et al. 2002; Franke et al.2006). The very small seeds in these species contain embryos of only a few cells with limitedstorage compounds (Leake 1994). For germination to occur as well as subsequent seedlingestablishment, germinating seeds must be colonized by an appropriate fungus.

The AM association in most of these species is the Paris-type (Imhof 1998, 1999b, 1999c,2001, 2006; Imhof and Weber 1997, 2000). Intracellular hyphal coils (Figure 5.3) and, in somecases, vesicles develop within root cortical cells. Since arbuscules do not usually form, sugars

Figure 5.2. Psilotum nudum gametophyte (resin-embedded, acriflavin, blue light) with intracellu-lar coils (arrowheads). Bar, 10 �m. (For a color version of this figure, see the color plate section.)

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Figure 5.3. Afrothismia sp. root with intracellular fungal coils (arrowheads). Bar, 200 �m.(Courtesy of Stephan Imhof.)

may be transferred from the fungus to root cells either via fungal coils or when nutrients arereleased into root cells following degradation of fungal hyphae. Experimental evidence is lackingthat supports either mechanism.

The AM association with the mycoheterotroph, Arachnitis uniflora (Corsiaceae) (Figure 5.4), aspecies confined to a few locations in the southern hemisphere, is unique (Domınguez and Sersic

Figure 5.4. Two flowering stems of Arachnitis uniflora each with a single flower and fleshy roots.Bar, 1 mm. (Courtesy of Laura Domınguez.) (For a color version of this figure, see the color platesection.)

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Figure 5.5. Colonized Arachnitis uniflora root (resin-embedded, toluidine blue) with branchedhyphal structures (arrows) and intracellular hyphae (arrowheads). Bar, 50 �m.

2004). Although molecular studies have determined that A. uniflora associates with GlomusGroup A (Bidartondo et al. 2002), structural characteristics of the plant–fungus interaction isunlike that of other plant associations with Glomus spp. Unusual branched structures with inflatedends form in addition to hyphal coils in the cortical cells of the fleshy roots (Figures 5.5 and5.6). Arbuscules do not form and vesicles rarely occur. The function of the branched structuresis unknown but, along with the hyphal coils, they may be involved in the transfer of sugarsfrom fungus to root cells (Domınguez et al. 2009). A. uniflora also develops unusual asexualpropagules (Figures 5.7 and 5.8) on its fleshy roots; these propagules become colonized withfungi from the parent root before they detach (Domınguez et al. 2006). The propagules developa shoot apical meristem and adventitious roots and ultimately new plants that presumably linkto neighboring photosynthetic plants for their source of carbon.

Figure 5.6. Intracellular hyphal coil in a root cell of Arachnitis uniflora (confocal microscopy).Bar, 10 �m.

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Figure 5.7. Propagule (arrowhead) on root of Arachnitis uniflora. Bar, 1 mm.

Dicotyledonous Angiosperms

A few mycoheterotrophic species in the families Gentianaceae and Polygalaceae associate withAM fungi (Imhof 1999a; Franke 2002; Franke et al. 2006; Imhof 2007). Molecular evidence hasshown that Sebaea oligantha (Gentianaceae) is associated with members of Glomus Group A(Franke et al. 2006). Based on the presence of auxiliary cells on external mycelium attached toroots of Voyria flavescens (Gentianaceae), the fungus is likely a member of the Gigasporaceae(Franke 2002). This needs to be confirmed using molecular methods. Structurally, host rootcells in species of both plant families are colonized by intracellular hyphal coils and fewvesicles without the formation of arbuscules (Figure 5.9); this is typical of a Paris-type AMassociation.

Figure 5.8. Section of Arachnitis uniflora root propagule with fungal colonization (arrows). Bar,100 �m.

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Figure 5.9. Epirixanthes papuana (Polygalaceae) root (transverse section) with intracellular hy-phal coils (arrows) and digested hyphae (arrowheads). Bar, 50 �m. (Courtesy of Stephan Imhof.)

5.1.2. Plants Associated with Ectomycorrhizal Fungi

A Nongreen Liverwort

The liverwort, Aneura mirabilis (synonym Cryptothallus mirabilis) (Aneuraceae, Metzgeriales),is the only nonvascular land plant with a dominant nonphotosynthetic haploid generation(Brundrett 2002). It is also the only known liverwort species that has become mycoheterotrophic(Bidartondo et al. 2003). This mycoheterotrophic thalloid, subterranean, liverwort species, alsoknown as ghostwort (Figure 5.10), has likely mutated from a photosynthetic Aneura relative(Wickett and Goffinet 2008). A. mirabilis has been found under leaf litter in birch, pine, willow,and oak forests in Europe. The fungal symbiont of A. mirabilis belongs to the genus Tulasnella,which is also the mycobiont of photosynthetic Aneura species (Kottke et al. 2003; Wickett andGoffinet 2008).

In microcosm experiments in which seedlings of Pinus muricata or Betula pendula wereinoculated with field-collected thalli of A. mirabilis, hyphae from the thalli contacted pine andbirch roots resulting in formation of ectomycorrhizae (Bidartondo et al. 2003). Carbon movedfrom photosynthetic B. pendula seedlings to thalli of A. mirabilis as long as hyphal connectionsremained intact.

Structural features of the fungal endophytes in A. mirabilis include coiled intracellular hyphaethat have dolipore septa and a surrounding plant host-cell plasma membrane, a characteristic ofmutualistic relationships (Pocock and Duckett 1984).

Monotropoid Ericaceae

Ten genera (Allotropa, Cheilotheca, Hemitomes, Monotropa, Monotropastrum, Monotropsis,Pityopus, Pleuricospora, Pterospora, Sarcodes) belonging to the Monotropoideae (Ericaceae)

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Figure 5.10. Cryptothallus (Aneura) mirabilis gametophytes (arrowheads) adjacent to Pinusmaritima roots from Portugal. Bar, 1 mm. (Courtesy of Martin Bidartondo.) (For a color version ofthis figure, see the color plate section.)

form monotropoid mycorrhizae with various fungal genera. Some of these plants have striking,colorful inflorescences that stand out among forest floor litter. For example, Sarcodes sanguinea(the snow plant) has a brilliant red inflorescence, whereas the inflorescence of Allotropa virgatais red and white striped giving it its common name, candystick.

Based on DNA sequences, many fungal species have been confirmed as major symbionts inmonotropoid mycorrhizae (Cullings et al. 1996; Kretzer et al. 2000; Bidartondo and Bruns 2001,2002), and the emerging pattern has furthered our understanding of fungal specificity phenomenaand the relationships between fungi and the plant species in the subfamily Monotropoideae(Bidartondo 2005; Smith and Read 2008).

The majority of fungi colonizing monotropoid roots are basidiomycetes, presumably withintermediate host range specificity, since these same fungi must also link with the autotrophicpartners, possibly involving multispecies mycorrhizal linkages in natural settings. S. sanguineaand Pterospora andromedea, species confined to western North America, associate almost ex-clusively with the truffle genus Rhizopogon (Rhizopogonaceae, Boletales), section Amylopogon.For Sarcodes, the confirmed fungal species are R. ellenae and R. subpurpurascens, whereas forPterospora, the fungal species include R. salebrosus, R. arctostaphyli, and an unknown taxon(Kretzer et al. 2000; Taylor et al. 2002; Bidartondo and Bruns 2001, 2002). Recent evidencefrom work on Pterospora in Yellowstone National Park (S. Miller, personal communication)suggests that R. ellenae should be added to the list of its fungal associates. Additionally, we haveobserved, as have others (Castellano and Trappe 1985), Pterospora with single-tip occurrencescolonized sporadically by Cenococcum in Oregon and in British Columbia.

The plant Pleuricospora fimbriolata grows only with the fungal species Gautieria monticola(Gomphaceae), another truffle-forming species (Bidartondo and Bruns 2001, 2002; Humpertet al. 2001). Monotropa uniflora (a Northern Hemisphere species) and Monotropastrum humile(an Asian species) have a strong affinity for fungi in the family Russulaceae, including manyspecies of Russula such as R. brevipes, R. decolorans, R. nitida, and Lactarius spp. (Young et al.2002; Bidartondo 2005; Bidartondo and Bruns 2005; Yang and Pfister 2006). Hemitomes con-gestum associates with Hydnellum spp. (Bankeraceae) (Bidartondo and Bruns 2001; Bidartondo2005).

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Monotropa hypopitys, Pityopus californicus, and A. virgata form mycorrhizae with fungalspecies in the Tricholomataceae. M. hypopitys associated with Tricholoma cingulatum whenSalix repens was the autotrophic host, and with Tricholoma terreum when Pinus sylvestris wasthe host (Leake et al. 2004). In P. californicus, Tricholoma myomyces is the fungal symbiont(Bidartondo and Bruns 2005) although a developmental study on young mycorrhizal embryosof P. californicus suggests that other fungi are present in earlier stages that are later replacedby T. myomyces (Massicotte et al. 2007). A. virgata, which associates exclusively with Tri-choloma magnivelare (Taylor et al. 2002), has one of the most specific host–fungal monotropoidsymbioses.

The fungi in these monotropoid associations form typical ectomycorrhizae with diverse au-totrophic tree species (Peterson et al. 2004). In the illustrated example (Figure 5.11A), thefungal symbiont links two plant species, the mycoheterotrophic Monotropa and the autotrophic(photosynthetic) pine. Structurally, monotropoid mycorrhizae resemble ectomycorrhizae, form-ing a mantle and a Hartig net that is confined to the epidermis (Figures 5.11B and 5.12).However, they possess a unique feature, the invasion of epidermal cells by fungal pegs, shorthyphae originating from the Hartig net or from the inner mantle (Figures 5.11C and 5.12; Lutzand Sjolund 1973; Duddridge and Read 1982; Robertson and Robertson 1982; Peterson andMassicotte 2004; Peterson et al. 2004). Fungal pegs form along the outer tangential wall ofepidermal cells in Monotropa (Figure 5.12) and Pityopus and at the base of the radial wall of

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Figure 5.11. (A) Diagram of autotrophic host (pine), the mycoheterotrophic Monotropa uniflora,and the bridging fungus. Fungal hyphae connect ectomycorrhizal roots (arrowheads) of pine witha root cluster (arrow) of Monotropa. (B) Diagram of transverse section of an ectomycorrhizal pineroot with mantle (arrows) and Hartig net (arrowheads). (C) Root epidermal cell of Monotropa withhyphal peg (arrowhead) and mantle (arrow).

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Figure 5.12. Monotropa uniflora root (toluidine blue) with multilayered mantle (*), Hartig net(arrow), and fungal peg (arrowhead) in epidermal cell. Bar, 10 �m.

epidermal cells in Pterospora and Sarcodes (Figure 5.13). Host cells respond by depositingadditional cell wall material, in finger-like projections, around each peg (Figure 5.14). It hasbeen hypothesized that fungal pegs, resembling “transfer cells” in other plant species, may beinvolved in nutrient transfer between the fungus and root cells (Massicotte et al. 2005). TheHartig net may also function in nutrient transfer but this needs to be confirmed.

The minute seeds produced by plants in the Monotropoideae have underdeveloped embryosand minimal nutritive tissue (Figure 5.15); they depend on a suitable fungus to provide sugars

Figure 5.13. Pterospora root (toluidine blue) showing peg apparatus in the radial wall (arrow).Bar, 10 �m.

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Figure 5.14. Ultrastructure of a fungal peg in root epidermal cell of Monotropa uniflora with wallingrowths (arrowheads). Bar, 5 �m.

and other nutrients needed for germination and seedling establishment (Bruns and Read 2000;Leake et al. 2004; Smith and Read 2008). In P. californicus, fungal hyphae become associatedwith germinating seeds and form a mantle as the embryo begins to elongate (Figure 5.16;Massicotte et al. 2007). Later, a mantle, a Hartig net, and fungal pegs form in the developingroot (Figure 5.17). Therefore, mycoheterotrophy is established very early in the life cycle of thisplant species.

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Figure 5.15. Pityopus californicus seed (toluidine blue) with reduced embryo. Bar, 50 �m.Figure 5.16. Developing embryo of Pityopus californicus (toluidine blue) with multilayered mantle(arrowheads). Bar, 100 �m.Figure 5.17. Pityopus californicus root (toluidine blue) with multilayered mantle (*), Hartig net(arrowheads), and fungal peg (arrow). Bar, 10 �m.

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Pyrola aphylla

Most species in the Pyroleae (Ericaceae) are either autotrophic, capable of providing all theircarbon needs through photosynthesis, or mixotrophic, gaining some carbon through fungal linkswith autotrophic plants. Pyrola picta exhibits a leafless form (f. aphylla), but reduced leaveshave been reported on some specimens. The trophic status of P. picta f. aphylla remaineduncertain until the stable isotope ratios of carbon and nitrogen in P. picta f. aphylla were shownto be similar to those of fully mycoheterotrophic species associated with ectomycorrhizal fungi(Hynson et al. 2009). However, unlike other mycoheterotrophic species in the Ericaceae, P. pictaf. aphylla does not show specificity for the fungi with which it associates (Hynson and Bruns2009). Instead, a suite of ectomycorrhizal and other fungi have been identified by molecularmethods.

5.1.3. Orchids

The Orchidaceae has the largest number of mycoheterotrophic genera of any plant family (Smithand Read 2008), accounting for 35% of the 400 fully mycoheterotrophic angiosperm species(Leake 1994). In nature, all orchid species require a suitable fungal partner so that their “dustseeds” will germinate and for subsequent development of the protocorm, a structure formed byan increase in cell number of the embryo followed by the organization of a shoot apical meristemand adventitious root primordia (Peterson et al. 1998, 2004; Smith and Read 2008). The fungalsymbiont invades the embryo through the suspensor end, through the micropyle, or throughepidermal hairs and forms intracellular hyphal coils referred to as pelotons (Figure 5.18). Allorchid species can be considered mycoheterotrophic during this early stage of their life cycle(Leake 2004). The Basidiomycota genera most frequently reported to be the fungal symbionts atthis stage are the asexual anamorphs, Ceratorhiza, Epulorhiza, and Moniliopsis. These generaare able to enzymatically digest complex carbohydrates to simple sugars that are used for fungalgrowth and that are transferred to developing protocorms to support seedling establishment(Figure 5.18).

To determine the effects of different autotrophic hosts as well as the influence that proximityto mature orchids has on germination and seedling development, seeds of Corallorhiza trifidawere enclosed in nylon mesh packets and placed in soil under either a Salix repens community

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Figure 5.18. (A) Orchid embryo development with intracellular fungal hyphae (pelotons) (arrow).(B) Protocormwith pelotons (arrow), developing shoot (arrowhead), and adventitious root (doublearrowhead).

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or a Betula–Alnus community, at various distances from adult orchid plants (McKendricket al. 2000b). Highest germination occurred under S. repens, at considerable distances fromadult orchid plants. Based on molecular analyses, the fungal symbionts were in the familyThelephoraceae. In a microcosm experiment, C. trifida seedlings that had been generated inthe field were placed in proximity to S. repens, B. pendula, or P. sylvestris roots; typicalectomycorrhizae formed with Salix and Betula but not with Pinus (McKendrick et al. 2000a).Pelotons were present in the orchid seedlings, and radioactive tracer experiments confirmed thetranslocation of carbon from S. repens and B. pendula to orchid seedlings as long as hyphallinks were present.

In a field study to determine the effect of distance from host trees on germination and proto-corm development, seeds of Neottia nidus-avis were enclosed in packets and placed either neartrees of Fagus sylvatica or at some distance from them (McKendrick et al. 2002). Germinationwas low when packets were 5 m or more from mature plants. Although various fungi wereassociated with the seeds, DNA analyses of fungi in protocorms showed that Sebacina(anamorph, Epulorhiza) was the symbiont most likely involved in the stimulation of seedgermination.

Although the majority of orchid species develop photosynthetic adult plants, several generaremain dependent on mycorrhizal fungi for carbon compounds throughout their life cycle andcontinue to be mycoheterotrophs. In these cases, developing seedlings link to photosyntheticplant species via fungal mycelia, most belonging to the Basidiomycota (Taylor and Bruns 1997).A basidiomycete fungal species forming pelotons in rhizomes of C. trifida (Figure 5.19) wasisolated and used as inoculum with Pinus contorta seedlings. It induced root dichotomy andtypical ectomycorrhizae (Figures 5.19 and 5.20) (Zelmer and Currah 1995). More recently,Zimmer et al. (2008) used molecular methods to show that the fungus associated with C. trifidabelongs to the basidiomycete genus Tomentella.

Using molecular techniques, identification of orchid fungal symbionts has resulted in therealization that there is some specificity with respect to the fungal associates of several orchidgenera. For example, adult plants of the mycoheterotroph Neottia nidus-avis associate primar-ily with the Sebacinaceae (Selosse et al. 2002), whereas Cephalanthera austinae associateswith members of the Thelephoraceae (Taylor and Bruns 1997). The mycoheterotrophic orchidEulophia zollingeri shows extreme specificity with the fungal symbiont, Psathyrella candolleana

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Figure 5.19. Corallorhiza trifida root cell (arrow) showing intracellular hyphal coils (arrowheads)(chlorazol black E). Bar, 10 �m. (Courtesy of Carla Zelmer and Randy Currah.)Figure 5.20. The same fungus isolated from Corallorhiza trifida root cells and inoculated onPinus contorta roots forming ectomycorrhizae with a mantle (arrow) and Hartig net (arrowheads).Bar, 25 �m. (Courtesy of Carla Zelmer and Randy Currah.)

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(Psathyrellaceae, Agaricales) (Ogura-Tsujita and Yukawa 2008). In contrast, mycoheterotrophicspecies of Epipactis associate not only with members of the Basidiomycota but also withmembers of the Ascomycota, including Tuber species (Selosse et al. 2004).

5.2. MIXOTROPHIC ASSOCIATIONS

Several species in the Orchidaceae and Ericaceae bridge autotrophy and mycoheterotrophy(Selosse and Roy 2009). Although these species are photosynthetic, they often occur in theforest understory where light is more limited and, consequently, they acquire part of their carbonneeds via fungal links to other neighboring autotrophic plants (Abadie et al. 2006; Tedersooet al. 2007; Smith and Read 2008). The term mixotrophy or partial mycoheterotrophy describesthis mode of carbon acquisition (Selosse et al. 2004).

The orchid, Cephalanthera longifolia, has two growth forms, photosynthetic and albino. Greenindividuals receive a portion of carbon from fungal symbionts and are, therefore, mixotrophs(Julou et al. 2005; Abadie et al. 2006). However, albino individuals are entirely myco-heterotrophic receiving all of their carbon via fungal symbionts. Fungi in the Thelephoraceaewere most frequently associated with both phenotypes. Fungal pelotons with hyphal clamp con-nections formed within root cortical cells. Albino forms had higher root colonization levels thangreen forms. Studies of another orchid provide more insights on mixotrophy. In populations ofCephalanthera damasonium that exhibited variable levels of chlorophyll, Stockel et al. (2011)found that leaf chlorophyll concentrations were linearly related to the proportional reliance onfungi as a C source. They also found that the level of dependence on mycoheterotrophy affectedleaf total C and N concentrations. Partial mycoheterotrophy in C. damasonium was found to bea malleable response related to leaf chlorophyll concentrations.

The orchid genus Limodorum consists of fully mycoheterotrophic species as well as onespecies, L. abortivum, which is described as being capable of some “inefficient photosynthesis”while relying on associated fungi for a portion of its carbon requirements (Girlanda et al.2006). The dominant fungal symbionts for this orchid genus were identified as Russula spp.(Russulaceae), and ectomycorrhizal roots belonging to surrounding tree species harbored similarfungi. Intracellular pelotons formed within cortical cells of L. abortivum.

C. trifida (Orchidaceae) associates with ectomycorrhizal fungi that link to neighbor treespecies such as P. contorta (Zelmer and Currah 1995). Stable isotope ratios of nitrogen andcarbon show that this orchid is partially mycoheterotrophic (Zimmer et al. 2008), and becauseonly a limited amount of carbon is gained through photosynthesis, C. trifida may be closer tobeing mycoheterotrophic than mixotrophic (Cameron et al. 2009).

To summarize the concept of mixotrophy, a Pyrola species capable of some photosynthesisbut relying on an autotrophic plant (in this case, an angiosperm tree species) for a portion ofits required carbon is illustrated in Figure 5.21. As in mycoheterotrophic relationships, fungalmycelia link the roots of participating plant species.

In the Pyroleae (Ericaceae), species in Pyrola, Orthilia, and Chimaphila obtain 10–68% oftheir carbon via association with both ascomycete and basidiomycete fungal symbionts (Tedersooet al. 2007); they can, therefore, be considered mixotrophic. However, only one species (Orthiliasecunda) gained carbon from its fungal symbiont, whereas other pyroloid species gained fungal-derived nitrogen compounds (Zimmer et al. 2007; Hynson et al. 2009). In contrast to thefully mycoheterotrophic P. picta f. aphylla that has stable isotope signatures indicating fullmycoheterotrophy, two closely related autotrophic species, P. picta and Chimaphila umbellata,showed nitrogen signatures resembling mycoheterotrophic species but carbon signatures typicalof autotrophic species.

Minor structural differences occur among mycorrhizae of pyroloid species although alldevelop a mantle, Hartig net, and intracellular hyphal complexes (Massicotte et al. 2008).The mantle can vary in thickness and, depending on the fungal symbiont, hyphae may haveprominent clamp connections (Figure 5.22) or none. The Hartig net and intracellular hyphae are

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Autotrophic host

Pyrola

Figure 5.21. (A) Diagram of autotrophic angiosperm host, with Pyrola the mixotrophic plant, andthe bridging fungus. Fungal hyphae connect ectomycorrhizal roots (arrowheads) of the host treeand roots (arrow) of Pyrola. (B) Angiosperm host root (transverse section) with mantle (arrows)and Hartig net (arrowheads) confined to the epidermis. (C) Intracellular hyphae (*) in epidermalcells, Hartig net (arrowheads), and hyphae linking the Hartig net to intracellular hyphae along theinner epidermal tangential wall (double arrowhead) of a Pyrola root.

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Figure 5.22. Mantle hyphae with clamp connections (arrows) over the root surface of Pyrola sp.(scanning electron microscopy). Bar, 5 �m.Figure 5.23. Pyrola asarifolia root (toluidine blue) with Hartig net (arrowheads), mantle (doublearrowhead), and intracellular coils (arrow). Bar, 50 �m.Figure 5.24. Pyrola asarifolia epidermal cell with Hartig net (arrowheads), mantle (arrow), andintracellular hyphae (*). Bar, 25 �m.

confined to the enlarged epidermal cells (Figure 5.23); the Hartig net is usually uniseriate andthe intracellular hyphae highly branched (Figure 5.24).

5.3. ECOLOGICAL CONSIDERATIONS

Increasing interest concerning the ecological significance of mycoheterotrophic and mixotrophicsystems reflects the realization that these life strategies occur worldwide and include speciesfrom liverworts to eudicots. The repeated evolution of the mycoheterotrophic habit, presumablyevolving independently over 40 times from autotrophic relatives, argues against the view thatmycoheterotrophy is rare and anomalous (Taylor et al. 2002; Merckx et al. 2009).

The documentation of mixotrophy (partial mycoheterotrophy) among species in the Orchi-daceae and Ericaceae (e.g., Abadie et al. 2006; Bidartondo et al. 2004; Tedersoo et al. 2007;Zimmer et al. 2007) provides the impetus for further related research; this, in part, is due to thepotential importance that mixotrophy might have on the survival of these plants in light-limitedenvironments where photosynthesis provides only a fraction of their carbon, the remaining com-ing from symbiotic fungi through their connections to other autotrophic plants. Mixotrophicassociations may also be significant for Pyrola species that often form a major component of theunderstory flora of boreal and temperate forests in the Northern Hemisphere and that contributeto the ecosystem functioning (Tedersoo et al. 2007). Similarly, Simard et al. (1997) and Simardand Durall (2004) suggest that, during seedling establishment of both conifers and hardwoodsunder situations where light is limited due to canopy closure, sugars may be obtained fromadjacent mature plants by “tapping” into mycorrhizal networks via source-sink mechanisms.

The use of stable isotope ratios to monitor carbon and nitrogen has facilitated progress indetermining the source of nutrients in mycoheterotrophic and mixotrophic species (Gebauerand Meyer 2003; Tedersoo et al. 2007; Zimmer et al. 2007, 2008; Hynson et al. 2009; Selosseand Roy 2009). This technique has confirmed that the fungal symbiont provides the source ofcarbon for mycoheterotrophic species as well as a portion of the nitrogen. Although nutrientadvantages to nongreen plants are clear in both mycoheterotrophy and mixotrophy systems,what the associated fungi receive remains uncertain. Presumably, the benefits outweigh the costsfor all members in the tripartite arrangement, but this has been difficult to assess.

Fungal–host specificity is often so extreme for many mycoheterotrophic plants that seedsmay not germinate and seedlings may not develop in the absence of their preferred fungalsymbionts (Bruns and Read 2000). Such cases present a challenge for conservation efforts. Forexample, seed germination may be stimulated by a “close relative” of the preferred fungus,

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but the seedlings may not survive past early developmental stages. Based on morphologicalcharacteristics, early postgermination stages of Pityopus seeds with developing embryos clearlyshowed an association with a Cenococcum-like fungus (Massicotte et al. 2007); however, onlyTricholoma was detected on roots of mature plants (Bidartondo and Bruns 2005). The specializedbiology of mycoheterotrophic species and the relative rarity of some species have raised concernsabout how best to meet their conservation needs (e.g., Lichthardt 1995; WNHP 2005). Inmanaged forest settings, silvicultural methods that are designed to maintain high levels ofbiodiversity are relatively new practices (Halpern et al. 2005). Through green-tree retention,ectomycorrhizal fungal diversity is maintained at higher levels than in clearcuts, and retainedtrees provide some legacy to ectomycorrhizal fungi for development of the next stand (Luomaet al. 2006). The practice of green-tree retention shows potential to benefit mycoheterotrophicspecies, but the effectiveness of various implementation scenarios remains to be tested.

5.4. EVOLUTIONARY CONSIDERATIONS

Mycoheterotrophy has evolved separately in several plant lineages; these include one nonvascu-lar plant, gametophytes of seedless vascular plants, and both monocotylenous and eudicotyle-nous angiosperms (Brundrett 2002). Depending on the plant group, the ancestral photosyntheticspecies were associated with AM fungi, ectomycorrhizal fungi, or orchid fungi (Brundrett 2004).These ancestral species likely shared at least one fungal symbiont with adjacent photosyntheticspecies (Bidartondo 2005)—a situation that would help facilitate the evolution of mycoheterotro-phy. In the angiosperms, convergent evolution has taken place in mycoheterotrophic species: allshow extremely reduced seed size, a reduction in shoot and root systems, and a loss of photo-synthesis (Leake 1994; Brundrett 2004). All species in the Orchidaceae have evolved a closeassociation with mycorrhizal fungi, requiring specific fungi for seed germination, the formationof the protocorm, and subsequent seedling establishment (Peterson et al. 2004). In this family,known for rapid evolution, mycoheterotrophy has evolved at least 20 times (Molvray et al. 2000).The evolutionary advantage of mycoheterotrophy remains unclear and will not be resolved untilthe fitness of both symbionts in these associations is determined (Taylor 2004).

5.5. FUTURE RESEARCH

Interest in mycoheterotrophic and mixotrophic plant species has increased rapidly, and valuableinformation has been obtained concerning the relationship between these species and their fungalsymbionts. Although the taxonomic resolution has improved for many of the fungal symbiontsinvolved in different groups of mycoheterotrophs, questions remain. Some mycoheterotrophicplants appear to be specific with regard to the fungi with which they associate (e.g., Bidartondoand Bruns 2001, 2005; Bidartondo et al. 2002), while others associate with diverse fungi (Hynsonand Bruns 2009). Study of additional mycoheterotrophic plants may determine if the majorityof species show a high degree of specificity concerning fungal associates. For these “tripartite”complex interactions to persist, all species involved must adapt to landscape disturbances,anthropogenic events, and to climate change. A critical question concerns whether plant hostsand their associated fungi will be able to relocate together in response to climate change. Forinstance, British Columbia, Canada, has seen unprecedented destruction of mature lodgepolepine stands in the last decade due to the increase in the overwinter survival of mountain pinebeetle, a consequence of warming brought about by climate change. Some of these pine forestsharbored large populations of P. andromedea, a mycoheterotrophic species associated withRhizopogon spp. (Bidartondo and Bruns 2002). Will populations of P. andromedea survive theloss of mature pine stands and subsequent changes created by salvage-harvest, replanting, orspecies succession?

The early stages in seed germination and seedling establishment are unknown for manymycoheterotrophic species (Leake 1994). Physiological and structural studies using either seed

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packet systems in field situations or microcosms could advance this research (McKendrick et al.2000a, 2000b; Bidartondo and Bruns 2002). Processes that depend on associated fungi involvesignaling and recognition between the symbiont partners. Seeds of P. andromedea have beenstimulated to germinate by Rhizopogon species even when seeds and fungal inoculum wereseparated by cellophane; a diffusible or volatile substance, as yet unidentified, must stimulategermination of Pterospora seeds (Bruns and Read 2000). The timing of early recognition eventsand the establishment of fungal bridges between compatible neighboring photosynthetic plantsis challenging to study in field situations. For example, some monotropoid plants depend on alimited number of specific fungal-root associates out of hundreds of available ectomycorrhizalfungi. Although the functional implications of this are not well understood, certain fungi maybring benefits to particular host plants that others cannot.

Research is ongoing to document the mycoheterotrophic status of poorly known plant taxa.For instance, one species in the Iridaceae, Geosiris aphylla, restricted to Madagascar andother islands in the Indian Ocean, is nonphotosynthetic and presumably gains carbon fromphotosynthetic plants via AM fungi, although this remains to be confirmed.

A suspected mycoheterotroph, the only known nongreen gymnosperm, Parasitaxus ustus(= usta), has been reported to receive carbon via fungal hyphae but in a manner unlike an-giosperm mycoheterotrophs or holoparasites (Feild and Brodribb 2005). This bizarre conifer,restricted to New Caledonia, grows attached to Falcatifolium taxoides, another conifer fromwhich the linking fungus presumably receives carbon. Molecular evidence has confirmed thatP. ustus is a distinct species (Sinclair et al. 2002). Structural information suggests that P. ustusis a holoparasite, but detailed anatomical studies and stable isotope ratios are needed to confirmits status.

ACKNOWLEDGMENTS

We are grateful to Linda Tackaberry for editorial assistance and to the Natural Sciences andEngineering Research Council of Canada for financial support to H.B.M and R.L.P. We thankour colleagues who have generously provided some of the images.

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