Mycorrhizal specificity in the fully mycoheterotrophic Hexalectris Raf. (Orchidaceae: Epidendroideae)

Mycoheterotrophic species have abandoned an autotrophic lifestyle and obtain carbon exclusively from mycorrhizal fungi. Although these species have evolved independently in many plant families, such events have occurred most often in the Orchidaceae, resulting in the highest concentration of these species in the tracheophytes. Studies of mycoheterotrophic species' mycobionts have generally revealed extreme levels of mycorrhizal specialization, suggesting that this system is ideal for studying the evolution of mycorrhizal associations. However, these studies have often investigated single or few, often unrelated, species without consideration of their phylogenetic relationships. Herein, we present the first investigation of the mycorrhizal associates of all species of a well-characterized orchid genus comprised exclusively of mycoheterotrophic species. With the employment of molecular phylogenetic methods, we identify the fungal associates of each of nine Hexalectris species from 134 individuals and 42 populations. We report that Hexalectris warnockii associates exclusively with members of the Thelephoraceae, H. brevicaulis and H. grandiflora associate with members of the Russulaceae and Sebacinaceae subgroup A, while each member of the H. spicata species complex associates primarily with unique sets of Sebacinaceae subgroup A clades. These results are consistent with other studies of mycorrhizal specificity within mycoheterotrophic plants in that they suggest strong selection within divergent lineages for unique associations with narrow clades of mycorrhizal fungi. Our results also suggest that mycorrhizal associations are a rapidly evolving characteristic in the H. spicata complex.     

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.     

Mycorrhizal diversity and specificity in Lecanorchis (Orchidaceae)

Lecanorchis is a nonphotosynthetic plant genus in Vanilloideae, Orchidaceae. Because of the distribution of many Lecanorchis taxa in various climate conditions, we hypothesized that mycorrhizal diversity and specificity are different among the different taxa of Lecanorchis. In the present study, identities of mycorrhizal fungi were examined for 90 individuals of 10 Lecanorchis taxa at 26 sites from Niigata to Okinawa Prefectures in Japan. Phylogenetic analyses of Lecanorchis taxa based on the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene (rDNA) divided the examined Lecanorchis taxa into three groups, groups A, B, and C. ITS rDNA sequences suggested that fungi associating with Lecanorchis were ectomycorrhiza-forming fungi in Lactarius, Russula, Atheliaceae, and Sebacina, with Lactarius and Russula dominant. Our results suggested some degree of mycorrhizal specialization among Lecanorchis taxa. Interestingly, the Lecanorchis group C had some specific relationships with Lactarius, whereas less specificity was found in the relationships with Russula. However, observed specificity results may be biased by geographic opportunity, and we suggest further research to assess whether Lecanorchis species are limited to the associations we observed.     

Mycorrhizal diversity in Apostasia (Orchidaceae) indicates the origin and evolution of orchid mycorrhiza

We demonstrated that "orchid mycorrhiza," a specialized mycorrhizal type, appeared in the common ancestor of the largest plant family Orchidaceae and that the fungal partner shifted from Glomeromycota to a particular clade of Basidiomycota in association with this character evolution. Several unique mycorrhizal characteristics may have contributed to the diversification of the family. However, the origin of orchid mycorrhiza and the diversity of mycobionts across orchid lineages still remain obscure. In this study, we investigated the mycorrhizae of five Apostasia taxa, members of the earliest-diverging clade of Orchidaceae. The results of molecular identification using nrDNA ITS and LSU regions showed that Apostasia mycorrhizal fungi belong to families Botryobasidiaceae and Ceratobasidiaceae, which fall within the order Cantharellales of Basidiomycota. Most major clades in Orchidaceae also form mycorrhizae with members of Cantharellales, while the sister group and other closely related groups to Orchidaceae (i.e., Asparagales except for orchids and the "commelinid" families) ubiquitously form symbioses with Glomeromycota to form arbuscular mycorrhizae. This pattern of symbiosis indicates that a major shift in fungal partner occurred in the common ancestor of the Orchidaceae.     

Widespread mycorrhizal specificity correlates to mycorrhizal function in the neotropical, epiphytic orchid Ionopsis utricularioides (Orchidaceae)

Tropical orchids constitute the greater part of orchid diversity, but little is known about their obligate mycorrhizal relationships. The specificity of these interactions and associated fungal distributions could influence orchid distributions and diversity. We investigated the mycorrhizal specificity of the tropical epiphytic orchid Ionopsis utricularioides across an extensive geographical range. DNA ITS sequence variation was surveyed in both plants and mycorrhizal fungi. Phylogeographic relationships were estimated for the mycorrhizal fungi. Orchid functional outcomes were determined through in vitro seed germination and seedling growth with a broad phylogenetic representation of fungi. Most fungal isolates derived from one clade of Ceratobasidium (anamorphs assignable to Ceratorhiza), with 78% within a narrower phylogenetic group, clade B. No correlation was found between the distributions of orchid and fungal genotypes. All fungal isolates significantly enhanced seed germination, while fungi in clade B significantly enhanced seedling growth. These results show that I. utricularioides associates with a phylogenetically narrow, effective fungal clade over a broad distribution. This preference for a widespread mycorrhizae may partly explain the ample distribution and abundance of I. utricularioides and contrasts with local mycorrhizal diversification seen in some nonphotosynthetic orchids. Enhanced orchid function with a particular fungal subclade suggests mycorrhizal specificity can increase orchid fitness.     

Molecular identification of mycorrhizal fungi in <Emphasis Type="Italic">Dactylorhiza sambucina</Emphasis> (Orchidaceae)

We amplified and sequenced internal transcribed spacer (ITS) region of the nuclear ribosomal repeat from fungi in roots of Dactylorhiza sambucina (Orchidaceae) and used the extended database to identify the mycorrhizal fungi. We molecularly identified three ITS recovered from D. sambucina roots, one belonging to Rhizoctonia group, and two to ascomycetes, for the first time in Orchidoidae. In many cases, two sequence types were found from the same orchid root, providing that two taxa may be involved in mycorrhizal formation (multiple mycobiont colonization). Moreover, we demonstrated that D. sambucina plants, irrespective of their colour polymorphism, possess roots containing several fungi belonging to both asco- and basidiomycetes.     

Identity and Specificity of Rhizoctonia-Like Fungi from Different Populations of Liparis japonica (Orchidaceae) in Northeast China

Mycorrhizal association is known to be important to orchid species, and a complete understanding of the fungi that form mycorrhizas is required for orchid ecology and conservation. Liparis japonica (Orchidaceae) is a widespread terrestrial photosynthetic orchid in Northeast China. Previously, we found the genetic diversity of this species has been reduced recent years due to habitat destruction and fragmentation, but little was known about the relationship between this orchid species and the mycorrhizal fungi. The Rhizoctonia-like fungi are the commonly accepted mycorrhizal fungi associated with orchids. In this study, the distribution, diversity and specificity of culturable Rhizoctonia-like fungi associated with L. japonica species were investigated from seven populations in Northeast China. Among the 201 endophytic fungal isolates obtained, 86 Rhizoctonia-like fungi were identified based on morphological characters and molecular methods, and the ITS sequences and phylogenetic analysis revealed that all these Rhizoctonia-like fungi fell in the same main clade and were closely related to those of Tulasnella calospora species group. These findings indicated the high mycorrhizal specificity existed in L. japonica species regardless of habitats at least in Northeast China. Our results also supported the wide distribution of this fungal partner, and implied that the decline of L. japonica in Northeast China did not result from high mycorrhizal specificity. Using culture-dependent technology, these mycorrhizal fungal isolates might be important sources for the further utilizing in orchids conservation.     

Assemblages of ericoid mycorrhizal and other root-associated fungi from Epacris pulchella (Ericaceae) as determined by culturing and direct DNA extraction from roots

Ericoid mycorrhizal fungal endophytes form mycorrhizal associations with Ericaceae plant taxa and are regarded as essential to the ecological fitness of the plants in extremely nutrient-poor soils worldwide. We isolated fungi from roots of Epacris pulchella (Ericaceae) in a south-eastern Australian sclerophyll forest and compared rDNA internal transcribed spacer (ITS) restriction fragment length polymorphisms (RFLPs) and sequences for the cultured isolate assemblage with fungi identified in DNA extracted directly from the same root systems by cloning or denaturing gradient gel electrophoresis (DGGE). The most abundant RFLP types in the cultured isolate assemblage were identified as putative ericoid mycorrhizal ascomycete endophytes, and these also represented the most abundant RFLP types in the cloned assemblage and the most intense bands in DGGE profiles. Each method identified unique taxa, notably putative basidiomycetes in the DNA extracted directly from E. pulchella roots. However, the relative abundance of these was low.     

The evolutionary history of mycorrhizal specificity among lady's slipper orchids

Although coevolution is acknowledged to occur in nature, coevolutionary patterns in symbioses not involving species-to-species relationships are poorly understood. Mycorrhizal plants are thought to be too generalist to coevolve with their symbiotic fungi; yet some plants, including some orchids, exhibit strikingly narrow mycorrhizal specificity. Here, we assess the evolutionary history of mycorrhizal specificity in the lady's slipper orchid genus, Cypripedium. We sampled 90 populations of 15 taxa across three continents, using DNA methods to identify fungal symbionts and quantify mycorrhizal specificity. We assessed phylogenetic relationships among sampled Cypripedium taxa, onto which we mapped mycorrhizal specificity. Cypripedium taxa associated almost exclusively with fungi within family Tulasnellaceae. Ancestral specificity appears to have been narrow, followed by a broadening after the divergence of C. debile. Specificity then narrowed, resulting in strikingly narrow specificity in most of the taxa in this study, with no taxon rewidening to the same extant as basal members of the genus. Sympatric taxa generally associated with different sets of fungi, and most clades of Cypripedium-mycorrhizal fungi were found throughout much of the northern hemisphere, suggesting that these evolutionary patterns in specificity are not the result of biogeographic lack of opportunity to associate with potential partners. Mycorrhizal specificity in genus Cypripedium appears to be an evolvable trait, and associations with particular fungi are phylogenetically conserved.     

Relationships between orchid and fungal biodiversity: Mycorrhizal preferences in Mediterranean orchids

Orchidaceae is one of the most species-rich angiosperm families, and all orchids are fully dependent on fungi for their seed germination and their life cycle. The level of specificity of the association between orchid species and fungi can be related to the number of co-occurring orchid species. To investigate orchid mycorrhizal associations in adult-photosynthetic orchids, 16 Mediterranean orchid species belonging to 4 genera (Anacamptis, Ophrys, Orchis, and Serapias) at 11 different sites were subjected to DNA-based analysis. Eighteen operational taxonomic units representing two fungal families, Tulasnellaceae and Ceratobasidiaceae, were identified. All examined orchid species associated with different mycorrhizal fungi. Interestingly, there was a positive correlation between number of orchid species and number of mycorrhizal. Monospecific populations showed a lower number of fungi, while sympatric populations had a higher number of mycorrhizal fungi. Our results showed that Mediterranean orchid species associated with a higher number of mycorrhizal fungi confirming as photosynthetic orchids are typically generalists toward mycorrhizal fungi. Thus, photosynthetic orchids exhibit low specificity for fungal symbionts showing the potential for opportunistic associations with diverse fungi reducing competition for nutrient. We suggest that these characteristics could confer symbiotic assurance particularly in habitat with resource limitations or prone to stressful conditions.     

Diversity and host specificity of endophytic Rhizoctonia-like fungi from tropical orchids

All orchids have an obligate relationship with mycorrhizal symbionts. Most orchid mycorrhizal fungi are classified in the form-genus Rhizoctonia. This group includes anamorphs of Tulasnella, Ceratobasidium, and Thanatephorus. Rhizoctonia can be classified according to the number of nuclei in young cells (multi-, bi-, and uninucleate). From nine Puerto Rican orchids we isolated 108 Rhizoctonia-like fungi. Our isolates were either bi- or uninucleate, the first report of uninucleate Rhizoctonia-like fungi as orchid endophytes. We sequenced the internal transcribed spacer (ITS) region of nuclear ribosomal DNA from 26 isolates and identified four fungal lineages, all related to Ceratobasidium spp. from temperate regions. Most orchid species hosted more than one lineage, demonstrating considerable variation in mycorrhizal associations even among related orchid species. The uninucleate condition was not a good phylogenetic character in mycorrhizal fungi from Puerto Rico. All four lineages were represented by fungi from Tolumnia variegata, but only one lineage included fungi from Ionopsis utricularioides. Tropical epiphytic orchids appear to vary in degree of specificity in their mycorrhizal interactions more than previously thought.     

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.     

Mycorrhizal fungi of Vanilla: diversity, specificity and effects on seed germination and plant growth

Mycorrhizal fungi are essential for the germination of orchid seeds. However, the specificity of orchids for their mycorrhizal fungi and the effects of the fungi on orchid growth are controversial. Mycorrhizal fungi have been studied in some temperate and tropical, epiphytic orchids, but the symbionts of tropical, terrestrial orchids are still unknown. Here we study diversity, specificity and function of mycorrhizal fungi in Vanilla, a pantropical genus that is both terrestrial and epiphytic. Mycorrhizal roots were collected from four Vanilla species in Puerto Rico, Costa Rica and Cuba. Cultured and uncultured mycorrhizal fungi were identified by sequencing the internal transcribed spacer region of nuclear rDNA (nrITS) and part of the mitochondrial ribosomal large subunit (mtLSU), and by counting number of nuclei in hyphae. Vanilla spp. were associated with a wide range of mycorrhizal fungi: Ceratobasidium, Thanatephorus and Tulasnella. Related fungi were found in different species of Vanilla, although at different relative frequencies. Ceratobasidium was more common in roots in soil and Tulasnella was more common in roots on tree bark, but several clades of fungi included strains from both substrates. Relative frequencies of genera of mycorrhizal fungi differed significantly between cultured fungi and those detected by direct amplification. Ceratobasidium and Tulasnella were tested for effects on seed germination of Vanilla and effects on growth of Vanilla and Dendrobium plants. We found significant differences among fungi in effects on seed germination and plant growth. Effects of mycorrhizal fungi on Vanilla and Dendrobium were similar: a clade of Ceratobasidium had a consistently positive effect on plant growth and seed germination. This clade has potential use in germination and propagation of orchids. Results confirmed that a single orchid species can be associated with several mycorrhizal fungi with different functional consequences for the plant.     

Studies of Mycorrhizal Fungi of Chinese Orchids and Their Role in Orchid Conservation in China—A Review

China has over 1,200 species of native orchids in nearly 173 genera. About one fourth of native species are of horticultural merit. Some species are of Chinese medicinal value. In fact, the demand on orchid species with high Chinese medicinal values such as Gastrodia elata, Dendrobium offcinale, along with demands on species of cultural importance, such as those in the genus of Cymbidium, is a major factor causing wild populations to diminish and in some cases, drive wild populations to the brink of extinction. These market demands have also driven studies on the role of mycorrhizal fungi in orchid seed germination, seedling and adult growth, and reproduction. Most of these mycorrhizal studies of Chinese orchids, however, are published in Chinese, some in medical journals, and thus overlooked by the mainstream orchid mycorrhizal publications. Yet some of these studies contained interesting discoveries on the nature of the mycorrhizal relationships between orchids and fungi. We present a review of some of these neglected publications. The most important discovery comes from the mycorrhizal studies on G. elata, in which the researchers concluded that those fungi species required to stimulate seed germination are different from those that facilitate the growth of G. elata beyond seedling stages. In addition, presence of the mycorrhizal fungi associated with vegetative growth of post-seedling G. elata hindered the germination of seeds. These phenomena were unreported prior to these studies. Furthermore, orchid mycorrhizal studies in China differ from the mainstream orchid studies in that many epiphytic species (in the genus of Dendrobium, as medicinal herbs) were investigated as well as terrestrial orchids (mostly in the genus Cymbidium, as traditional horticultural species). The different responses between epiphytic and terrestrial orchid seeds to fungi derived from roots suggest that epiphytic orchids may have a more general mycorrhizal relationship with fungi than do terrestrial orchid species during the seed germination stage. To date, orchid mycorrhizal research in China has had a strongly commercial purpose. We suggest that this continuing research on orchid mycorrhizal relationships are a solid foundation for further research that includes more rare and endangered taxa, and more in-situ studies to assist conservation and restoration of the endangered orchids. Knowledge on the identities and roles of mycorrhizal fungi of orchids holds one of the keys to successful restoration and sustainable use of Chinese orchids.     

天麻种子与小菇属真菌共生萌发的研究*

Four fungal species of Mycena were identificated from the strains isolated from the protocorm and roots of Orchidaceae species.M anoectochial was isolated from the Proocorm of G.elata, M orchidicola, M.dendrobii and M anoectochila respectively separated from the roots of Cymbidium sinense, Dendrobium candidum and Anoectochilus roxburghii. The symbiotic germination test beteen G. Elata seeds and the above fungal species demonstrated that these flingal species can stimulate the seed germination, Which means t…     

Monotropa uniflora plants of eastern Massachusetts form mycorrhizae with a diversity of russulacean fungi

Plant species in the subfamily Monotropoideae are mycoheterotrophs; they obtain fixed carbon from photosynthetic plants via a shared mycorrhizal network. Previous findings show mycoheterotrophic plants exhibit a high level of specificity to their mycorrhizal fungi. In this study we explore the association of mycorrhizal fungi and Monotropa uniflora (Monotropoideae: Ericaceae) in eastern North America. We collected M. uniflora roots and nearby basidiomycete sporocarps from four sites within a 100 km2 area in eastern Massachusetts. We analyzed DNA sequences of the internal transcribed spacer region (ITS) from the fungal nuclear ribosomal gene to assess the genetic diversity of fungi associating with M. uniflora roots. In this analysis we included 20 ITS sequences from Russula sporocarps collected nearby, 44 sequences of Russula or Lactarius species from GenBank and 12 GenBank sequences of fungi isolated from M. uniflora roots in previous studies. We found that all 56 sampled M. uniflora mycorrhizal fungi were members of the Russulaceae, confirming previous research. The analysis showed that most of the diversity of mycorrhizal fungi spreads across the genus Russula. ITS sequences of the mycorrhizal fungi consisted of 20 different phylotypes: 18 of the genus Russula and two of Lactarius, based on GenBank searches. Of the sampled plants, 57% associated with only three of the 20 mycorrhizal fungi detected in roots, and of the 25 sporocarp phylotypes collected three, were associated with M. uniflora. Furthermore the results indicate that the number of different fungal phylotypes associating with M. uniflora of eastern North America is higher than that of western North America but patterns of fungal species abundance might be similar between mycorrhizae from the two locations.     

Untangling above‐ and belowground mycorrhizal fungal networks in tropical orchids

Orchids typically depend on fungi for establishment from seeds, forming mycorrhizal associations with basidiomycete fungal partners in the polyphyletic group rhizoctonia from early stages of germination, sometimes with very high specificity. This has raised important questions about the roles of plant and fungal phylogenetics, and their habitat preferences, in controlling which fungi associate with which plants. In this issue of Molecular Ecology, Martos et al. (2012) report the largest network analysis to date for orchids and their mycorrhizal fungi, sampling a total of over 450 plants from nearly half the 150 tropical orchid species on Reunion Island, encompassing its main terrestrial and epiphytic orchid genera. The authors found a total of 95 operational taxonomic units of mycorrhizal fungi and investigated the architecture and nestedness of their bipartite networks with 73 orchid species. The most striking finding was a major ecological barrier between above‐ and belowground mycorrhizal fungal networks, despite both epiphytic and terrestrial orchids often associating with closely related taxa across all three major lineages of rhizoctonia fungi. The fungal partnerships of the epiphytes and terrestrial species involved a diversity of fungal taxa in a modular network architecture, with only about one in ten mycorrhizal fungi partnering orchids in both groups. In contrast, plant and fungal phylogenetics had weak or no effects on the network. This highlights the power of recently developed ecological network analyses to give new insights into controls on plant–fungal symbioses and raises exciting new hypotheses about the differences in properties and functioning of mycorrhiza in epiphytic and terrestrial orchids.     

A narrowly endemic photosynthetic orchid is non‐specific in its mycorrhizal associations

Mycorrhizal association is a common characteristic in a majority of land plants, and the survival and distribution of a species can depend on the distribution of suitable fungi in its habitat. Orchidaceae is one of the most species‐rich angiosperm families, and all orchids are fully dependent on fungi for their seed germination and some also for subsequent growth and survival. Given this obligate dependence, at least in the early growth stages, elucidating the patterns of orchid–mycorrhizal relationships is critical to orchid biology, ecology and conservation. To assess whether rarity of an orchid is determined by its specificity towards its fungal hosts, we studied the spatial and temporal variability in the host fungi associated with one of the rarest North American terrestrial orchids, Piperia yadonii. The fungal internal transcribed spacer region was amplified and sequenced by sampling roots from eight populations of P. yadonii distributed across two habitats, Pinus radiata forest and maritime chaparral, in California. Across populations and sampling years, 26 operational taxonomic units representing three fungal families, the Ceratobasidiaceae, Sebacinaceae and Tulasnellaceae, were identified. Fungi belonging to the Sebacinaceae were documented in orchid roots only at P. radiata forest sites, while those from the Ceratobasidiaceae and Tulasnellaceae occurred in both habitats. Our results indicate that orchid rarity can be unrelated to the breadth of mycorrhizal associations. Our data also show that the dominance of various fungal families in mycorrhizal plants can be influenced by habitat preferences of mycorrhizal partners.     

Molecular identifications uncover diverse fungal symbionts of Pleione (Orchidaceae)

Fungal mutualisms are essential for the evolution and diversification of Orchidaceae, yet the fungal symbionts of Pleione orchids are poorly understood because molecular data are unavailable for this genus. Based on ITS-rDNA sequencing for mycobionts of 15 Pleione species (both wild and cultivated plants were included), we conducted phylogenetic analyses for the most dominant mycobionts, and compared the operational taxonomic units (OTUs) of mycorrhizal fungi among species within Pleione. Tulasnellaceae, Ceratobasidiaceae, Serendipitaceae (Sebacinales), Atractiellales, and Auriculariales were reported as putative mycobionts of Pleione. In particular, the mycorrhizal associations between subtropical orchids and Atractiellales have not been observed before. For the dominant mycobionts in the roots of Pleione and its related genera, Bletilla and Coelogyne, we detected no fungal OTU that was shared. Within Pleione, species with a sympatric distribution showed preferences for different fungi. Epiphytic and lithophytic individuals of Pleione albiflora shared OTUs of Tulasnellaceae but harbored different OTUs of Sebacinales, indicating some degree of fungal specificity toward certain habitats. These findings provide new insights into the ecological adaptation and evolution of orchids, and will contribute to the conservation and utilization of species resources.     

High specificity generally characterizes mycorrhizal association in rare lady's slipper orchids, genus Cypripedium

Lady's slipper orchids (Cypripedium spp.) are rare terrestrial plants that grow throughout the temperate Northern Hemisphere. Like all orchids, they require mycorrhizal fungi for germination and seedling nutrition. The nutritional relationships of adult Cypripedium mycorrhizae are unclear; however, Cypripedium distribution may be limited by mycorrhizal specificity, whether this specificity occurs only during the seedling stage or carries on into adulthood. We attempted to identify the primary mycorrhizal symbionts for 100 Cypripedium plants, and successfully did so with two Cypripedium calceolus, 10 Cypripedium californicum, six Cypripedium candidum, 16 Cypripedium fasciculatum, two Cypripedium guttatum, 12 Cypripedium montanum, and 11 Cypripedium parviflorum plants from a total of 44 populations in Europe and North America, yielding fungal nuclear large subunit and mitochondrial large subunit sequence and RFLP (restriction fragment length polymorphism) data for 59 plants. Because orchid mycorrhizal fungi are typically observed without fruiting structures, we assessed fungal identity through direct PCR (polymerase chain reaction) amplification of fungal genes from mycorrhizally colonized root tissue. Phylogenetic analysis revealed that the great majority of Cypripedium mycorrhizal fungi are members of narrow clades within the fungal family Tulasnellaceae. Rarely occurring root endophytes include members of the Sebacinaceae, Ceratobasidiaceae, and the ascomycetous genus, Phialophora. C. californicum was the only orchid species with apparently low specificity, as it associated with tulasnelloid, ceratobasidioid, and sebacinoid fungi in roughly equal proportion. Our results add support to the growing literature showing that high specificity is not limited to nonphotosynthetic plants, but also occurs in photosynthetic ones.