Wide geographical and ecological distribution of nitrogen and carbon gains from fungi in pyroloids and monotropoids (Ericaceae) and in orchids

* Stable isotope abundance analyses recently revealed that some European green orchids and pyroloids (Ericaceae) are partially myco-heterotrophic, exploiting mycorrhizal fungi for organic carbon and nitrogen. Here we investigate related species to assess their nutritional mode across various forest and climate types in Germany and California. * C- and N-isotope signatures of five green pyroloids, three green orchids and several obligate myco-heterotrophic species (including the putatively fully myco-heterotrophic Pyrola aphylla) were analysed to quantify the green plants' nutrient gain from their fungal partners and to investigate the constancy of enrichment in (13)C and (15)N of fully myco-heterotrophic plants from diverse taxa and locations relative to neighbouring autotrophic plants. * All green pyroloid and one orchid species showed significant (15)N enrichment, confirming incorporation of fungi-derived N compounds while heterotrophic C gain was detected only under low irradiance in Orthilia secunda. Pyrola aphylla had an isotope signature equivalent to those of fully myco-heterotrophic plants. * It is demonstrated that primarily N gain from mycorrhizal fungi occurred in all taxonomic groups investigated across a wide range of geographical and ecological contexts. The (13)C and (15)N enrichment of obligate myco-heterotrophic plants relative to accompanying autotrophic plants turned out as a fairly constant parameter.     

Further advances in orchid mycorrhizal research

Orchid mycorrhizas are mutualistic interactions between fungi and members of the Orchidaceae, the world’s largest plant family. The majority of the world’s orchids are photosynthetic, a small number of species are myco-heterotrophic throughout their lifetime, and recent research indicates a third mode (mixotrophy) whereby green orchids supplement their photosynthetically fixed carbon with carbon derived from their mycorrhizal fungus. Molecular identification studies of orchid-associated fungi indicate a wide range of fungi might be orchid mycobionts, show common fungal taxa across the globe and support the view that some orchids have specific fungal interactions. Confirmation of mycorrhizal status requires isolation of the fungi and restoration of functional mycorrhizas. New methods may now be used to store orchid-associated fungi and store and germinate seed, leading to more efficient culture of orchid species. However, many orchid mycorrhizas must be synthesised before conservation of these associations can be attempted in the field. Further gene expression studies of orchid mycorrhizas are needed to better understand the establishment and maintenance of the interaction. These data will add to efforts to conserve this diverse and valuable association.     

Evidence for mycorrhizal races in a cheating orchid

Disruptive selection on habitat or host-specificity has contributed to the diversification of several animal groups, especially plant-feeding insects. Photosynthetic plants typically associate with a broad range of mycorrhizal fungi, while non-photosynthetic plants that capture energy from mycorrhizal fungi ('mycoheterotrophs') are often specialized towards particular taxa. Sister myco-heterotroph species are often specialized towards different fungal taxa, suggesting rapid evolutionary shifts in specificity. Within-species variation in specificity has not been explored. Here, we tested whether genetic variation for mycorrhizal specificity occurs within the myco-heterotrophic orchid Corallorhiza maculata. Variation across three single-nucleotide polymorphisms revealed six multilocus genotypes across 122 orchids from 30 sites. These orchids were associated with 22 different fungal species distributed across the Russulaceae (ectomycorrhizal basidiomycetes) according to internal-transcribed-spacer sequence analysis. The fungi associated with four out of the six orchid genotypes fell predominantly within distinct subclades of the Russulaceae. This result was supported by Monte Carlo simulation and analyses of molecular variance of fungal sequence diversity. Different orchid genotypes were often found growing in close proximity, but maintained their distinct fungal associations. Similar patterns are characteristic of insect populations diversifying onto multiple hosts. We suggest that diversification and specialization of mycorrhizal associations have contributed to the rapid radiation of the Orchidaceae.     

The ectomycorrhizal specialist orchid Corallorhiza trifida is a partial myco-heterotroph

The leafless, circumboreal orchid Corallorhiza trifida is often assumed to be fully myco-heterotrophic despite contrary evidence concerning its ability to photosynthesize. Here, its level of myco-heterotrophy is assessed by analysing the natural abundance of the stable nitrogen and carbon isotopes (15)N and (13)C, respectively. The mycorrhizal associates and chlorophyll contents of C. trifida were investigated and the C and N isotope signatures of nine C. trifida individuals from Central Europe were compared with those of neighbouring obligate autotrophic and myco-heterotrophic reference plants. The results show that C. trifida only gains c. 52 +/- 5% of its total nitrogen and 77 +/- 10% of the carbon derived from fungi even though it has been shown to specialize on one specific complex of ectomycorrhizal fungi similar to fully myco-heterotrophic orchids. Concurrently, compared with other Corallorhiza species, C. trifida contains a remarkable amount of chlorophyll. Since C. trifida is able to supply significant proportions of its nitrogen and carbon demands through the same processes as autotrophic plants, this species should be referred to as partially myco-heterotrophic.     

Mixotrophy in orchids: insights from a comparative study of green individuals and nonphotosynthetic individuals of Cephalanthera damasonium

Some green orchids obtain carbon (C) from their mycorrhizal fungi and photosynthesis. This mixotrophy may represent an evolutionary step towards mycoheterotrophic plants fully feeding on fungal C. Here, we report on nonphotosynthetic individuals (albinos) of the green Cephalanthera damasonium that likely represent another evolutionary step. Albino and green individuals from a French population were compared for morphology and fertility, photosynthetic abilities, fungal partners (using microscopy and molecular tools), and nutrient sources (as characterized by 15N and 13C abundances). Albinos did not differ significantly from green individuals in morphology and fertility, but tended to be smaller. They harboured similar fungi, with Thelephoraceae and Cortinariaceae as mycorrhizal partners and few rhizoctonias. Albinos were nonphotosynthetic, fully mycoheterotrophic. Green individuals carried out photosynthesis at compensation point and received almost 50% of their C from fungi. Orchid fungi also colonized surrounding tree roots, likely to be the ultimate C source. Transition to mycoheterotrophy may require several simultaneous adaptations; albinos, by lacking some of them, may have reduced ecological success. This may limit the appearance of cheaters in mycorrhizal networks.     

Fungal‐host diversity among mycoheterotrophic plants increases proportionally to their fungal‐host overlap

The vast majority of plants obtain an important proportion of vital resources from soil through mycorrhizal fungi. Generally, this happens in exchange of photosynthetically fixed carbon, but occasionally the interaction is mycoheterotrophic, and plants obtain carbon from mycorrhizal fungi. This process results in an antagonistic interaction between mycoheterotrophic plants and their fungal hosts. Importantly, the fungal‐host diversity available for plants is restricted as mycoheterotrophic interactions often involve narrow lineages of fungal hosts. Unfortunately, little is known whether fungal‐host diversity may be additionally modulated by plant–plant interactions through shared hosts. Yet, this may have important implications for plant competition and coexistence. Here, we use DNA sequencing data to investigate the interaction patterns between mycoheterotrophic plants and arbuscular mycorrhizal fungi. We find no phylogenetic signal on the number of fungal hosts nor on the fungal hosts shared among mycoheterotrophic plants. However, we observe a potential trend toward increased phylogenetic diversity of fungal hosts among mycoheterotrophic plants with increasing overlap in their fungal hosts. While these patterns remain for groups of plants regardless of location, we do find higher levels of overlap and diversity among plants from the same location. These findings suggest that species coexistence cannot be fully understood without attention to the two sides of ecological interactions.     

Arbuscular mycorrhizal fungi of the Glomus-group A lineage (Glomerales; Glomeromycota) detected in myco-heterotrophic plants from tropical Africa

We amplified and sequenced partial 18S rDNA of fungi in the roots of 11 African myco-heterotrophic plants out of four angiosperm families (Burmanniaceae, Thismiaceae, Triuridaceae, and Gentianaceae). The sequences were cladistically analyzed with published sequences of arbuscular mycorrhizal fungi. We show that all investigated African myco-heterotrophic plants are associated with arbuscular mycorrhizal fungi within a clade of Glomus (Glomus-group A). We reveal a fine-level mycorrhizal specificity for a particular set of arbuscular mycorrhizal fungi within Glomus-group A by Afrothismia hydra (Thismiaceae). Furthermore, we show that the roots of two myco-heterotrophic plant individuals, besides being colonized by representatives of Glomus-group A, also contain DNA of Acaulospora sp. Consequently, Acaulospora is interpreted as a facultative mycorrhizal associate.     

Specialized cheating of the ectomycorrhizal symbiosis by an epiparasitic liverwort

Many non-photosynthetic vascular plants in 10 diverse families obtain all of their carbon from fungi, but in most cases the fungi and the ultimate sources of carbon are unknown. In a few cases, such plants have been shown to be epiparasitic because they obtain carbon from neighbouring green plants through shared mycorrhizal fungi. In all such cases, the epiparasitic plants have been found to specialize upon narrow lineages of ecto- or arbuscular mycorrhizal fungi. Here we show that a non-vascular plant, the non-photosynthetic liverwort Cryptothallus mirabilis, is epiparasitic and is specialized on Tulasnella species that form ectomycorrhizae with surrounding trees at four locations in England, France and Portugal. By using microcosm experiments we show that the interaction with Tulasnella is necessary for growth of Cryptothallus, and by using labelling experiments we show that (14)CO(2) provided to birch seedlings is transferred to Cryptothallus by Tulasnella. This is one of the first documented cases of epiparasitism by a non-vascular plant and of ectomycorrhizal formation by Tulasnella. These results broaden the emerging association between epiparasitism and mycorrhizal specialization into a new class of plants and a new order of fungi.     

'Prepackaged symbioses': propagules on roots of the myco-heterotrophic plant Arachnitis uniflora

Arachnitis uniflora, a myco-heterotrophic plant species, has fleshy tuberous roots colonized by the arbuscular mycorrhizal fungal genus Glomus (Phylum Glomeromycota). These roots produce apical and lateral propagules, both reported here for the first time. The objective of the study was to characterize the ontogeny and structure of the propagules, and to determine their function. Scanning electron microscopy, laser scanning confocal microscopy and light microscopy were used to study the ontogeny and structure of the propagules. Propagules developed either from cortical parenchyma cells or from cells immediately beneath the root cap; they developed a shoot meristem and cells in the basal region which were colonized by various fungal structures including hyphae and vesicles. These propagules may detach from the roots, establishing new plants.     

Grazing tolerance and mycorrhizal colonization: Effects of resource manipulation and plant size in biennial Gentianella campestris

As herbivory usually leads to loss of photosynthesizing biomass, its consequences for plants are often negative. However, in favorable conditions, effects of herbivory on plants may be neutral or even beneficial. According to the compensatory continuum hypothesis plants can tolerate herbivory best in resource-rich conditions. Besides herbivory, also primarily positive biotic interactions like mycorrhizal symbiosis, bear carbon costs. Tritrophic plant–fungus–herbivore interaction further complicates plant's cost-benefit balance, because herbivory of the host plant is expected to cause decline in mycorrhizal colonization under high availability of soil nutrients when benefits of symbiosis decline in relation to costs. To gain insight into above interactions we tested the effects of plant size and resource manipulation (simulated herbivory and fertilization) on both above-ground performance and on root fungal colonization of the biennial Gentianella campestris.Clipping caused allocation shift from height growth to branches in all groups except in large and fertilized plants. For large plants nutrient addition may have come too late, as the number of meristems was most likely determined already before the fertilization. Clipping decreased the amount of DSE (dark septate endophytic) fungi which generally are not considered to be mycorrhizal. The effect of clipping on total fungal colonization and colonization by arbuscular mycorrhizal (AM) fungal coils were found to depend on host size and resource level. Dissimilar mycorrhizal response to simulated herbivory in small vs. large plants could be due to more intensive light competition in case of small plants. Carbon limited small plants may not be able to maintain high mycorrhizal colonization, whereas large clipped plants allocate extra resources to roots and mycorrhizal fungi at the expense of above-ground parts. Our results suggest that herbivory may increase carbon limitation that leads re-growing shoots and fungal symbionts to function as competing sinks for the limited carbon reserves.     

Evolutionary histories and mycorrhizal associations of mycoheterotrophic plants dependent on saprotrophic fungi

Mycoheterotrophic plants (MHPs) are leafless, achlorophyllous, and completely dependent on mycorrhizal fungi for their carbon supply. Mycorrhizal symbiosis is a mutualistic association with fungi that is undertaken by the majority of land plants, but mycoheterotrophy represents a breakdown of this mutualism in that plants parasitize fungi. Most MHPs are associated with fungi that are mycorrhizal with autotrophic plants, such as arbuscular mycorrhizal (AM) or ectomycorrhizal (ECM) fungi. Although these MHPs gain carbon via the common mycorrhizal network that links the surrounding autotrophic plants, some mycoheterotrophic lineages are associated with saprotrophic (SAP) fungi, which are free-living and decompose leaf litter and wood materials. Such MHPs are dependent on the forest carbon cycle, which involves the decomposition of wood debris and leaf litter, and have a unique biology and evolutionary history. MHPs associated with SAP fungi (SAP-MHPs) have to date been found only in the Orchidaceae and likely evolved independently at least nine times within that family. Phylogenetically divergent SAP Basidiomycota, mostly Agaricales but also Hymenochaetales, Polyporales, and others, are involved in mycoheterotrophy. The fungal specificity of SAP-MHPs varies from a highly specific association with a single fungal species to a broad range of interactions with multiple fungal orders. Establishment of symbiotic culture systems is indispensable for understanding the mechanisms underlying plant–fungus interactions and the conservation of MHPs. Symbiotic culture systems have been established for many SAP-MHP species as a pure culture of free-living SAP fungi is easier than that of biotrophic AM or ECM fungi. Culturable SAP-MHPs are useful research materials and will contribute to the advancement of plant science.

     

Specialized mycorrhizal colonization pattern in achlorophyllous Epirixanthes spp. (Polygalaceae)

Roots of the achlorophyllous Epirixanthes papunana and E. elongata were sectioned in complete series in order to reconstruct the three-dimensional mycorrhizal colonization pattern within their tissues. Hyphal morphology, vesicles, as well as the exclusively intracellular mode of colonization indicate a PARIS-type of arbuscular mycorrhiza showing a hitherto unknown colonization pattern: (1) the outer cortex is colonized by persistent straight-growing hyphae which branch in a cascading manner, (2) a specific layer (called layer 2) is inhabited by persistent hyphal coils, (3) in the cells of the anatomically distinct inner cortex parenchyma layer (called layer 1) the hyphae immediately degenerate, and (4) the layer outside to layer 2 (called layer 3) is either transitional layer 2 when penetrated from the outer cortex or the fungal material degenerates when colonized from the layer 2. This complex colonization pattern is a reasonable adaptation to the particular demands of Epirixanthes as a myco-heterotrophic plant. It not only allows a sustained benefit from the fungal symbiont but also provides a two-level distribution system of hyphae within the roots. The outer cortex hyphae function as a permanent intraradical resource of living fungi providing connection to the external mycelium as well as a coarse distribution of hyphae within the root. Layer 2 represents the fine scale distribution of hyphae, having access to all potentially digesting cells of the layers 1 and 3. Common structural features of mycorrhizae in myco-heterotrophic plants are pointed out in order to find putative prerequisites for their heterotrophic mode of life.     

The magnitude and control of carbon transfer between plants linked by a common mycorrhizal network

Various claims have been made about the ecological significance of plant-to-plant carbon movement through common mycorrhizal networks (CMNs). Most suggest that resource competition among interconnected plants should be less important than previously thought. If true, that would profoundly alter our perception of how plants interact among themselves and with their environment. However, there are difficulties in quantifying the amounts of resource transferred via CMNs, ensuring that transfer is genuinely through hyphae, not soil, and understanding its control. Carbon movement has not been quantified in many of the published studies. Where it has, its likely functional role has not been clarified. Some recent, well-publicized research suggests that carbon transferred to trees via an ectomycorrhizal (EcM) network may be physiologically and ecologically important. Our view, however, is that the evidence for this remains equivocal. Appropriate controls for the possibility of carbon transfer via soil were not used under field conditions. In laboratory experiments, controls failed to clarify the role of EcM links in carbon transfer. To resolve some areas of uncertainty, abundances of ¹³C have been measured to estimate carbon transfers via an arbuscular mycorrhizal (AM) network connecting grasses and forbs of the same or different species. Permeable barriers to roots and hyphae allowed any direct carbon transfer via soil to be detected. Large amounts of carbon (typically 10% of that in roots) were transferred between linked plants via the CMN. Transferred carbon was never transported into shoots of 'receiver' plants. It remained in roots, probably inside fungal structures and, therefore, unavailable to the plants into which it was apparently transferred. Carbon transfer via an AM network does not allow 'resource sharing' among linked plants. It is probably irrelevant to the botanical components of a community, but it may be fundamental for fungal members. The 'mycocentric' view is that fungal structures within roots are parts of extended mycelia through which fungi move carbon according to their own carbon demands, not those of their autotrophic hosts.     

菌根真菌与植物共生营养交换机制研究进展

菌根是陆地生态系统普遍存在的、由土壤中的菌根真菌侵染宿主植物根系形成的联合共生体.菌根的建立是以共生体双方的营养交换为基础的:菌根真菌从土壤中吸收氮、磷等营养物质并转运给宿主植物,供其生长;作为交换,植物则以脂质或糖的形式向菌根真菌提供其生长所必需的碳水化合物.近年来,菌根真菌与宿主植物间的营养交换机制一直是研究的热点,国内外对菌根真菌介导的植物营养物质吸收和转运机制的研究也取得了巨大进展.本文综述了丛枝和外生两种菌根真菌与宿主植物间营养交换的最新研究进展,尤其是碳、氮、磷等几种重要营养物质的吸收与双向转运机制,以及营养交换在菌根形成中的潜在调控作用,并对目前存在的关键问题和未来研究方向进行了分析和展望,这对菌根模型的建立及菌根效益的优化具有重要意义.     

Mycorrhizal acquisition of inorganic phosphorus by the green-leaved terrestrial orchid Goodyera repens

BACKGROUND AND AIMS: Mycorrhizal fungi play a vital role in providing a carbon subsidy to support the germination and establishment of orchids from tiny seeds, but their roles in adult orchids have not been adequately characterized. Recent evidence that carbon is supplied by Goodyera repens to its fungal partner in return for nitrogen has established the mutualistic nature of the symbiosis in this orchid. In this paper the role of the fungus in the capture and transfer of inorganic phosphorus (P) to the orchid is unequivocally demonstrated for the first time. METHODS: Mycorrhiza-mediated uptake of phosphorus in G. repens was investigated using spatially separated, two-dimensional agar-based microcosms. RESULTS: External mycelium growing from this green orchid is shown to be effective in assimilating and transporting the radiotracer (33)P orthophosphate into the plant. After 7 d of exposure, over 10 % of the P supplied was transported over a diffusion barrier by the fungus and to the plants, more than half of this to the shoots. CONCLUSIONS: Goodyera repens can obtain significant amounts of P from its mycorrhizal partner. These results provide further support for the view that mycorrhizal associations in some adult green orchids are mutualistic.     

Comparison of green and albino individuals of the partially mycoheterotrophic orchid Epipactis helleborine on molecular identities of mycorrhizal fungi,nutritional modes and gene expression in mycorrhizal roots

Some green orchids obtain carbon from their mycorrhizal fungi, as well as from photosynthesis. These partially mycoheterotrophic orchids sometimes produce fully achlorophyllous, leaf‐bearing (albino) variants. Comparing green and albino individuals of these orchids will help to uncover the molecular mechanisms associated with mycoheterotrophy. We compared green and albino Epipactis helleborine by molecular barcoding of mycorrhizal fungi, nutrient sources based on ¹⁵N and ¹³C abundances and gene expression in their mycorrhizae by RNA‐seq and cDNA de novo assembly. Molecular identification of mycorrhizal fungi showed that green and albino E. helleborine harboured similar mycobionts, mainly Wilcoxina. Stable isotope analyses indicated that albino E. helleborine plants were fully mycoheterotrophic, whereas green individuals were partially mycoheterotrophic. Gene expression analyses showed that genes involved in antioxidant metabolism were upregulated in the albino variants, which indicates that these plants experience greater oxidative stress than the green variants, possibly due to a more frequent lysis of intracellular pelotons. It was also found that some genes involved in the transport of some metabolites, including carbon sources from plant to fungus, are higher in albino than in green variants. This result may indicate a bidirectional carbon flow even in the mycoheterotrophic symbiosis. The genes related to mycorrhizal symbiosis in autotrophic orchids and arbuscular mycorrhizal plants were also upregulated in the albino variants, indicating the existence of common molecular mechanisms among the different mycorrhizal types.     

High mycorrhizal specificity in a widespread mycoheterotrophic plant, Eulophia zollingeri (Orchidaceae)

Because mycoheterotrophic plants fully depend on their mycorrhizal partner for their carbon supply, the major limiting factor for the geographic distribution of these plants may be the presence of their mycorrhizal partner. Although this factor may seem to be a disadvantage for increasing geographic distribution, widespread mycoheterotrophic species nonetheless exist. The mechanism causing the wide distribution of some mycoheterotrophic species is, however, seldom discussed. We identified the mycorrhizal partner of a widespread mycoheterotrophic orchid, Eulophia zollingeri, using 12 individuals from seven populations in Japan, Myanmar, and Taiwan by DNA-based methods. All fungal ITS sequences from the roots closely related to those of Psathyrella candolleana (Coprinaceae) from GenBank accessions and herbarium specimens. These results indicate that E. zollingeri is exclusively associated with the P. candolleana species group. Further, the molecular data support the wide distribution and wide-ranging habitat of this fungal partner. Our data provide evidence that a mycoheterotrophic plant can achieve a wide distribution, even though it has a high mycorrhizal specificity, if its fungal partner is widely distributed.     

Saprotrophic fungal mycorrhizal symbionts in achlorophyllous orchids: Finding treasures among the ‘molecular scraps’?

Mycoheterotrophic plants are achlorophyllous plants that obtain carbon from their mycorrhizal fungi. They are usually considered to associate with fungi that are (1) specific of each mycoheterotrophic species and (2) mycorrhizal on surrounding green plants, which are the ultimate carbon source of the entire system. Here we review recent works revealing that some mycoheterotrophic plants are not fungal-specific, and that some mycoheterotrophic orchids associate with saprophytic fungi. A re-examination of earlier data suggests that lower specificity may be less rare than supposed in mycoheterotrophic plants. Association between mycoheterotrophic orchids and saprophytic fungi arose several times in the evolution of the two partners. We speculate that this indirectly illustrates why transition from saprotrophy to mycorrhizal status is common in fungal evolution. Moreover, some unexpected fungi occasionally encountered in plant roots should not be discounted as ‘molecular scraps’, since these facultatively biotrophic encounters may evolve into mycorrhizal symbionts in some other plants.Key words: endophytic fungi, evolution of mycorrhizae, mycoheterophy, mycorrhizae, saprophytic fungi, specificityConsiderable advances were recently made in the ecology of achlorophyllous, heterotrophic plants that obtain carbon from their mycorrhizal fungi (Fig. 1). Most plants have contact with soil through mycorrhizal symbioses, in which roots associate with a suitable fungal partner. Fungi utilize soil mineral nutrients, and while sharing them with host plants, they generally receive carbon as a reward. In contrast, some achlorophyllous plants living in the shaded forest understorey have reversed the process. They receive carbon from their mycorrhizal fungi exclusively, hence the designation ‘mycoheterotrophic’ (MH) plants.¹ Mycoheterotrophy has appeared several times during the evolution of land plants, and more than 20 times among orchids that encompass half of all MH species.² In the last decade, the development of molecular tools has enabled researchers to identify many fungal symbionts, which are often uncultivable. The fungi occurring in the densely colonized roots of MH species often produce a stronger PCR signal than any fungal contaminant, making molecular tools very effective for this field of study.Open in a separate windowFigure 1Wullschlaegelia aphylla, a mycoheterotrophic orchid unspecifically associated with saprotrophic Mycena and Gymnopus species. (A) Whole plant at flowering time, with reduced, tuberoid root system at that period. (B) Section of mycorrhizal root showing intracellular hyphal pelotons at early stage (p), or late stage (undergoing lysis, lp); among orchids, the colonization of dead cortical cell (cc) is a unique feature to some saprotrophic fungi (picture by A. Faccio, University of Torino).     

Myco-heterotrophy: when fungi host plants

Background

Myco-heterotrophic plants are partly or entirely non-photosynthetic plants that obtain energy and nutrients from fungi. These plants form a symbiosis with arbuscular mycorrhizal, ectomycorrhizal or saprotrophic fungi to meet their nutrient demands.

Scope

This Botanical Briefing summarizes current knowledge about myco-heterotrophy, discusses its controversial aspects and highlights future directions for research.

Conclusions

Considerable recent progress has been made in terms of understanding the evolutionary history, germination and nutrition of myco-heterotrophic plants. Myco-heterotrophic plants: (1) are diverse and often ancient lineages that have coevolved with fungi, (2) often demonstrate unusually high specificity towards fungi during germination and maturity, and (3) can either cheat common mycorrhizal networks supported by neighbouring photosynthetic plants to satisfy all or part of their energetic and nutritional needs, or recruit free-living saprotrophic fungi into novel mycorrhizal symbioses. However, several fundamental aspects of myco-heterotrophy remain controversial or unknown, such as symbiotic costs and physiology.Key words: Cheater, common mycorrhizal network, mutualism, myco-heterotrophy, non-photosynthetic, symbiosis     

Symbiotic germination and development of myco-heterotrophic plants in nature: ontogeny of Corallorhiza trifida and characterization of its mycorrhizal fungi

The processes of symbiotic germination and seedling development were analysed in the myco-heterotrophic orchid Corallorhiza trifida , seeds of which were buried in 'packets' either adjacent to or at varying distances from adult plants in defined communities of ectomycorrhizal tree species. Germination occurred within eight months of burial under Betula – Alnus and within seven months under Salix repens . It was always associated with penetration of the suspensor by a clamp-forming mycorrhizal fungus. Four distinct developmental stages were defined and the rates of transition through these stages were plotted. There was no evidence of a relationship between extent of germination or rate of development and the presence of naturally distributed plants of C. trifida at the spatial scale of 1 m. The best germination and the most rapid rate of development of C. trifida seedlings occurred in a Salix repens community located at a considerable distance from any extant C. trifida population. Determination of internal transcribed spacer (ITS) RFLPs and of gene sequences of the fungi involved in symbiotic germination and growth of C. trifida , revealed them to belong exclusively to the Thelephora – Tomentella complex of the Thelephoraceae. These fungi are known also to be ectomycorrhizal associates of trees. It is hypothesized that the rate of growth of the C. trifida seedlings is determined by the ability of the fungal symbionts to transfer carbon from their ectomycorrhizal co-associates.