Specificity and localised distribution of mycorrhizal fungi in the soil may contribute to co-existence of orchid species

Co-occurring orchid species tend to occupy different areas and associate with different mycorrhizal fungi, suggesting that orchid mycorrhizal (OrM) fungi may be unevenly distributed within the soil and, therefore, impact the aboveground spatial distribution of orchids. To test this hypothesis, we investigated spatial variations in the community of potential OrM associates within the roots of three co-habitating orchid species (Anacamptis morio, Gymnadenia conopsea, and Orchis mascula) and the surrounding soil in an orchid-rich calcareous grassland in Southern Belgium using 454 amplicon pyrosequencing. Putative OrM fungi were broadly distributed in the soil, although variations in community composition were strongly related to the proximal host plant. The diversity and frequency of sequences corresponding to OrM fungi in the soil declined with increasing distance from orchid plants, suggesting that the clustered distribution of orchid species may to some extent be explained by the localised distribution of species-specific mycorrhizal associates.     

Geographical range and host breadth of Sebacina orchid mycorrhizal fungi associating with Caladenia in south‐western Australia

Specialized mycorrhizal interactions have the potential to limit the geographical range of plant species and contribute to reproductive isolation. We investigated these predictions in Caladenia (Orchidaceae) from south‐western Australia, a group known to have specialized mycorrhizal associations with the genus Sebacina s.l. Sequencing of fungal isolates from 47 of the 136 species of Western Australian Caladenia was undertaken to resolve the geographical range and habitat preferences of mycorrhizal fungal operational taxonomic units (OTUs) and their host breadth in Caladenia. Eight different fungal OTUs were used by Caladenia, with the more frequently detected OTUs occurring in a wide range of habitats and geographical regions. Given the comparatively narrow geographical ranges of most Western Australian Caladenia taxa, this suggests that the geographical ranges of fungal OTUs are unlikely to limit the geographical range of Caladenia spp. Extensive sharing of fungal OTUs between closely related orchid species was detected, suggesting that in the main there is little potential for mycorrhizal fungi to contribute to reproductive isolation between Caladenia spp. Our data mostly support previous work suggesting high mycorrhizal specificity in Caladenia, but this may not be the case in all subgenera, highlighting that Caladenia may offer powerful opportunities for investigating the evolution of specialized mycorrhizal associations.     

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.     

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.     

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.     

Taxonomic and functional characterisation of fungi from the Sebacina vermifera complex from common and rare orchids in the genus Caladenia

The terrestrial orchid genus Caladenia contains many species which are threatened with extinction. They have highly specific associations with Sebacina vermifera and closely related fungi, and conservation of these terrestrial orchids, in part, relies on symbiotic propagation to produce plants for reintroduction and ex situ conservation collections. However, little is known of the diversity of mycorrhizal fungi associating with natural populations. Here, restriction fragment polymorphism analysis, internal transcribed spacer and nuclear large subunit sequencing and symbiotic seed germination were used to investigate the taxonomic and functional diversity of fungal isolates from single populations of six endangered Caladenia species and one common species across the same biogeographic range. Fifty-nine fungal isolates were collected for investigation including ten isolates from the six endangered species Caladenia audasii, Caladenia amoena, Caladenia sp. aff. fragrantissima (Central Victoria), Caladenia sp. aff. patersonii, Caladenia rosella and Caladenia orientalis and 49 isolates from six populations of the common species Caladenia tentaculata. While the common species associated with three distinct S. vermifera-like taxa, the six endangered species were restricted to one of these fungal taxa. No direct relationship between the taxonomic identity of the fungi and their ability to stimulate seed germination was observed; however, the majority of the fungi isolated from the Caladenia species were capable of germinating seed in vitro, indicating their mycorrhizal status and potential for symbiotic propagation in conservation programmes.     

药用植物绶草(兰科)的菌根真菌群落组成分析

兰科菌根真菌(OMF)被认为是影响兰科植物物种丰度和分布的一个重要因素.对广域分布兰科植物的菌根区系进行研究有助于人们更深入地了解兰科植物分布格局的形成机制.本研究以我国广域分布的兰科药用植物绶草Spiranthes sinensis为材料,采用Illumina Miseq高通量测序技术对北京、上海、江西、广西、云南、...     

The giant mycoheterotrophic orchid Erythrorchis altissima is associated mainly with a divergent set of wood‐decaying fungi

The climbing orchid Erythrorchis altissima is the largest mycoheterotroph in the world. Although previous in vitro work suggests that E. altissima has a unique symbiosis with wood‐decaying fungi, little is known about how this giant orchid meets its carbon and nutrient demands exclusively via mycorrhizal fungi. In this study, the mycorrhizal fungi of E. altissima were molecularly identified using root samples from 26 individuals. Furthermore, in vitro symbiotic germination with five fungi and stable isotope compositions in five E. altissima at one site were examined. In total, 37 fungal operational taxonomic units (OTUs) belonging to nine orders in Basidiomycota were identified from the orchid roots. Most of the fungal OTUs were wood‐decaying fungi, but underground roots had ectomycorrhizal Russula. Two fungal isolates from mycorrhizal roots induced seed germination and subsequent seedling development in vitro. Measurement of carbon and nitrogen stable isotope abundances revealed that E. altissima is a full mycoheterotroph whose carbon originates mainly from wood‐decaying fungi. All of the results show that E. altissima is associated with a wide range of wood‐ and soil‐inhabiting fungi, the majority of which are wood‐decaying taxa. This generalist association enables E. altissima to access a large carbon pool in woody debris and has been key to the evolution of such a large mycoheterotroph.     

Phenotypic selection on nectar amino acid composition in the Lepidoptera pollinated orchid species Gymnadenia conopsea

Plant–pollinator interactions are potential drivers of evolution in floral traits. Because nectar chemical composition is known to mediate both plant–pollinator interactions and plant reproductive success, it can be expected that chemical composition of nectar is subjected to strong pollinator‐mediated selective forces. However, the extent of natural selection on different nectar components has not been studied so far. Using the Lepidoptera pollinated fragrant orchid Gymnadenia conopsea as a model species, we used high‐performance anion‐exchange chromatography (HPAEC) to characterize the sugar and amino acid composition of floral nectar in three calcareous grassland populations of G. conopsea. We then measured phenotypic selection on nectar composition and on other plant and floral traits through applying both linear regression and structural equation modelling. We demonstrate phenotypic selection on plant height, inflorescence height and on specific nectar amino acids, whereas spur length, total sugar and amino acid concentration were not direct targets of selection. Chemical nectar composition is thus indeed under selective pressure but nectar amino acids are much more important to fitness of G. conopsea, as compared to nectar sugars. Furthermore, as we found no evidence of selection on the total amino acid concentration, it is unlikely that amino acids increase pollinator attraction because they are a pollinator nitrogen source. To further unravel the evolutionary ecology of floral nectar, behavioural experiments with pollinators exposed to different nectar components and studies experimentally identifying the selective agents are recommended.     

Low specificity and nested subset structure characterize mycorrhizal associations in five closely related species of the genus Orchis

Most orchid species rely on mycorrhizae to complete their life cycle. Despite a growing body of literature identifying orchid mycorrhizal associations, the nature and specificity of the association between orchid species and mycorrhizal fungi remains largely an open question. Nonetheless, better insights into these obligate plant–fungus associations are indispensable for understanding the biology and conservation of orchid populations. To investigate orchid mycorrhizal associations in five species of the genus Orchis (O. anthropophora, O. mascula, O. militaris, O. purpurea, and O. simia), we developed internal transcribed spacer‐based DNA arrays from extensive clone library sequence data sets, enabling rapid and simultaneous detection of a wide range of basidiomycetous mycorrhizal fungi. A low degree of specificity was observed, with two orchid species associating with nine different fungal partners. Phylogenetic analysis revealed that the majority of Orchis mycorrhizal fungi are members of the Tulasnellaceae, but in some plants, members of the Thelephoraceae, Cortinariaceae and Ceratobasidiaceae were also found. In all species except one (O. mascula), individual plants associated with more than one fungus simultaneously, and in some cases, associations with ≥3 mycorrhizal fungi at the same time were identified. Nestedness analysis showed that orchid mycorrhizal associations were significantly nested, suggesting asymmetric specialization and a dense core of interactions created by symmetric interactions between generalist species. Our results add support to the growing literature that multiple associations may be common among orchids. Low specificity or preference for a widespread fungal symbiont may partly explain the wide distribution of the investigated species.     

Effects of habitat fragmentation on the reproductive success of the nectar-producing orchid Gymnadenia conopsea and the nectarless Orchis mascula

Habitat fragmentation decreases plant population sizes and increases spatial isolation, which hampers the exchange of seeds and pollen between fragmented populations. This may result in decreased population viability. We compared the effects of population size and isolation on the reproductive success of two orchid species, Gymnadenia conopsea (nectar-producing) and Orchis mascula (nectarless) growing in highly fragmented calcareous grassland in southern Belgium. We expected that the nectar-producing species would be more susceptible to the negative reproductive consequences of habitat fragmentation compared to the nectarless species. Nectar production has been associated with increased geitonogamous pollination and, therefore, with lower seed viability. Our results show that seed viability increased with increasing population size in O. mascula, whereas it was always low in G. conopsea, even in large populations. In contrast, percentage fruit set was positively related to population size in the nectar-producing G. conopsea, but no such effect was observed in the nectarless O. mascula, where fruit set was low even in large populations. Population isolation was not related to reproductive success for either species. Our results suggest that even in large populations, where pollinators are expected to be more abundant, increased geitonogamous pollination reduces seed viability in the nectar-producing G. conopsea. In contrast, seed viability in O. mascula seems to benefit from increased pollinator availability in larger populations. For the latter species, however, fruit set remains low, even in large populations, compared to G. conopsea. This may be explained by the relatively low attractiveness of nectarless orchid species for pollinators. Our results indicate that small population size may negatively influence reproductive success in both nectarless and nectar-producing orchids by reducing seed viability and fruit set, respectively.     

Divergent selection on flowering phenology but not on floral morphology between two closely related orchids

Closely related species often differ in traits that influence reproductive success, suggesting that divergent selection on such traits contribute to the maintenance of species boundaries. Gymnadenia conopsea ss. and Gymnadenia densiflora are two closely related, perennial orchid species that differ in (a) floral traits important for pollination, including flowering phenology, floral display, and spur length, and (b) dominant pollinators. If plant–pollinator interactions contribute to the maintenance of trait differences between these two taxa, we expect current divergent selection on flowering phenology and floral morphology between the two species. We quantified phenotypic selection via female fitness in one year on flowering start, three floral display traits (plant height, number of flowers, and corolla size) and spur length, in six populations of G. conopsea s.s. and in four populations of G. densiflora. There was indication of divergent selection on flowering start in the expected direction, with selection for earlier flowering in two populations of the early‐flowering G. conopsea s.s. and for later flowering in one population of the late‐flowering G. densiflora. No divergent selection on floral morphology was detected, and there was no significant stabilizing selection on any trait in the two species. The results suggest ongoing adaptive differentiation of flowering phenology, strengthening this premating reproductive barrier between the two species. Synthesis: This study is among the first to test whether divergent selection on floral traits contribute to the maintenance of species differences between closely related plants. Phenological isolation confers a substantial potential for reproductive isolation, and divergent selection on flowering time can thus greatly influence reproductive isolation and adaptive differentiation.     

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.     

Ecological specialization in mycorrhizal symbiosis leads to rarity in an endangered orchid

Terrestrial orchid germination, growth and development are closely linked to the establishment and maintenance of a relationship with a mycorrhizal fungus. Mycorrhizal dependency and specificity varies considerably between orchid taxa but the degree to which this underpins rarity in orchids is unknown. In the context of examining orchid rarity, large scale in vitro and in situ germination trials complemented by DNA sequencing were used to investigate ecological specialization in the mycorrhizal interaction of the rare terrestrial orchid Caladenia huegelii. Common and widespread sympatric orchid congeners were used for comparative purposes. Germination trials revealed an absolute requirement for mycorrhisation with compatibility barriers to germination limiting C. huegelii to a highly specific and range limited, efficacious mycorrhizal fungus. DNA sequencing confirmed fidelity between orchid and fungus across the distribution range of C. huegelii and at key life history stages within its life cycle. It was also revealed that common congeners could swap or share fungal partners including the fungus associated with the rare orchid but not vice versa. Data from this study provides evidence for orchid rarity as a cause and consequence of high mycorrhizal specialization. This interaction must be taken into account in efforts to mitigate the significant extinction risk for this species from anthropogenically induced habitat change and illustrates the importance of understanding fungal specificity in orchid ecology and conservation.     

Addition of fungal inoculum increases germination of orchid seeds in restored grasslands

Grasslands restored on arable land often retain high residual nutrients, modified soil biota, and lower plant species diversity. Establishment of rare plant species with complex multitrophic interactions, typical of undisturbed nutrient-poor environments, may be hindered by the absence of interacting organisms. We hypothesised that the addition of a mycorrhizal symbiont improves the seed germination of orchids that crucially depend on fungi. We focused on grasslands restored on arable land 1–15 years ago featuring residual mineral nutrients and low organic matter contents compared to semi-natural grasslands and on four orchid species differing in the level of mycorrhizal specificity: high – Anacamptis pyramidalis and Orchis mascula – and low – Platanthera bifolia and Gymnadenia conopsea. Five fungal isolates obtained from non-green underground mycorrhizal orchid seedlings (protocorms) or adults' roots were tested for orchid-fungus compatibility under conditions in vitro. Orchid seeds inserted in retrievable seed packets were subsequently co-introduced with selected fungal isolates grown either on agar or sterilized hay into the soil of nine restored grasslands and incubated for twelve months. The identity of mycorrhizal fungi in retrieved protocorms was verified by molecular methods. The isolates that supported protocorm establishment in vitro enabled also protocorm formation in situ, but success rates differed among orchid species. While mycorrhizal specialists produced most protocorms after inoculation, the mycorrhizal generalists took advantage of naturally occurring fungi and produced some protocorms both in inoculated and uninoculated treatments. We showed that the addition of mycorrhizal fungi enhanced protocorm formation regardless of the modified soil environment, especially in mycorrhizal specialist orchids. This method may help to restore populations of native orchid species in their former distribution ranges, including farming-altered habitats.     

Genetic diversity in the rare orchid Gymnadenia odoratissima and a comparison with the more common congener, G. conopsea

Patterns and levels of genetic diversity mayhave significant influence on the long termpersistence of local populations and revealingsuch information is important in protectingrare species. In this study we investigated thegenetic pattern in five microsatellite lociwithin five Swedish populations of the rareorchid species Gymnadenia odoratissima. Thegeographic distribution of G. odoratissima isrestricted to Europe and in Scandinavia it isonly found in three provinces in southernSweden; Östergötland,Västergötland and on the island ofGotland.Compared with the more widespread congener G.conopsea our results indicate lower levels ofgenetic variation within and higher degrees ofgenetic differentiation among populations ofG. odoratissima (H_EL = 0.6–0.8 in G. conopseaand 0.3–0.7 in G. odoratissima; F_ST over allpopulations = 0.06 in G. conopsea and 0.19 inG. odoratissima). Also, we found a cleardistinction among mainland and islandpopulations of G. odoratissima wherepopulations on the island of Gotland seem toexhibit higher levels of gene flow andintragenetic variation, probably as a result ofa larger number of existing populations.Future conservation of this species shouldfocus on facilitation on colonisation events,especially on the mainland, and preservation ofthe genetically more variable Gotlandpopulations.     

Mycorrhizal diversity,seed germination and long‐term changes in population size across nine populations of the terrestrial orchid Neottia ovata

In plant species that rely on mycorrhizal symbioses for germination and seedling establishment, seedling recruitment and temporal changes in abundance can be expected to depend on fungal community composition and local environmental conditions. However, disentangling the precise factors that determine recruitment success in species that critically rely on mycorrhizal fungi represents a major challenge. In this study, we used seed germination experiments, 454 amplicon pyrosequencing and assessment of soil conditions to investigate the factors driving changes in local abundance in 28 populations of the orchid Neottia ovata. Comparison of population sizes measured in 2003 and 2013 showed that nearly 60% of the studied populations had declined in size (average growth rate across all populations: ?0.01). Investigation of the mycorrhizal fungi in both the roots and soil revealed a total of 68 species of putatively mycorrhizal fungi, 21 of which occurred exclusively in roots, 25 that occurred solely in soil and 22 that were observed in both the soil and roots. Seed germination was limited and significantly and positively related to soil moisture content and soil pH, but not to fungal community composition. Large populations or populations with high population growth rates showed significantly higher germination than small populations or populations declining in size, but no significant relationships were found between population size or growth and mycorrhizal diversity. Overall, these results indicate that temporal changes in abundance were related to the ability of seeds to germinate, but at the same time they provided limited evidence that variation in fungal communities played an important role in determining population dynamics.     

Colonization patterns of mycorrhizal fungi associated with two rare orchids, <Emphasis Type="Italic">Cephalanthera</Emphasis> <Emphasis Type="Italic">falcata</Emphasis> and <Emphasis Type="Italic">C. erecta</Emphasis>

We investigated the spatial distribution and taxonomic identity of mycorrhizal fungi colonizing the root systems of two threatened Cephalanthera species, C. falcata and C. erecta, in naturally regenerated forests. Peloton formation was observed in both plant species, confirming the existence of orchid mycorrhizas. For C. falcata, mycorrhization was significantly different among individuals, ranging from 14 to 63%, and no significant difference among C. erecta individuals was detected (57–68%). Mycorrhization among three growth directions of roots and between orchid species was not significantly different. The spatial distribution of mycorrhizas in both orchids showed significant differences, being most frequent at an apical position. Based on DNA sequencing and phylogenetic analyses, we inferred that the families Thelephoraceae and Sebacinaceae were mycobionts for C. falcata and Thelephoraceae for C. erecta. Our findings indicated that mycorrhizal colonization occurs at a distal position from the base of these orchid root systems and that mycorrhizal fungi are restricted to few ectomycorrhizal fungal families.     

Highly diverse and spatially heterogeneous mycorrhizal symbiosis in a rare epiphyte is unrelated to broad biogeographic or environmental features

Symbiotic interactions are common in nature. In dynamic or degraded environments, the ability to associate with multiple partners (i.e. broad specificity) may enable species to persist through fluctuations in the availability of any particular partner. Understanding how species interactions vary across landscapes is necessary to anticipate direct and indirect consequences of environmental degradation on species conservation. We asked whether mycorrhizal symbiosis by populations of a rare epiphytic orchid (Epidendrum firmum) is related to geographic or environmental heterogeneity. The latter would suggest that interactions are governed by environmental conditions rather than historic isolation of populations and/or mycorrhizal fungi. We used DNA-based methods to identify mycorrhizal fungi from eleven E. firmum populations in Costa Rica. We used molecular and phylogenetic analyses to compare associations. Epidendrum firmum exhibited broad specificity, associating with diverse mycorrhizal fungi, including six Tulasnellaceae molecular operational taxonomic units (MOTUs), five Sebacinales MOTUs and others. Notably, diverse mycorrhizal symbioses formed in disturbed pasture and roadside habitats. Mycorrhizal fungi exhibited significant similarity within populations (spatial and phylogenetic autocorrelation) and significant differences among populations (phylogenetic community dissimilarity). However, mycorrhizal symbioses were not significantly associated with biogeographic or environmental features. Such unexpected heterogeneity among populations may result from complex combinations of fine-scale environmental factors and macro-evolutionary patterns of change in mycorrhizal specificity. Thus, E. firmum exhibits broad specificity and the potential for opportunistic associations with diverse fungi. We suggest that these characteristics could confer symbiotic assurance when mycorrhizal fungi are stochastically available, which may be crucial in dynamic or disturbed habitats such as tropical forest canopies.