Mycorrhizal fungal community composition in seven orchid species inhabiting Song Mountain,Beijing, China

Mycorrhizal fungi play an important role in the germination and growth of orchids essentially influencing their survival,abundance, and spatial distribution. In this study, we investigated the composition of the mycorrhizal fungal community in seven terrestrial orchid species inhabiting Song Mountain, Beijing, China, using Illumina MiSeq high-throughput sequencing. The mycorrhizal communities in the seven orchids were mainly composed of members of the Ceratobasidiaceae, Sebacinales, and Tulasnellaceae, while a number of ectomycorrhizal fungi belonging to the Russulaceae, Tricholomataceae, Thelephoraceae, and Cortinariaceae were occasionally observed. However, the dominant fungal associates and mycorrhizal community differed significantly among the orchid species as well as subhabitats. These findings confirm the previous observation that sympatric orchid species show different preferences for mycorrhizal fungi, which may drive niche partitioning and contribute to their cooccurrence.     

Continental-scale distribution and diversity of Ceratobasidium orchid mycorrhizal fungi in Australia

Background and AimsMycorrhizal fungi are a critical component of the ecological niche of most plants and can potentially constrain their geographical range. Unlike other types of mycorrhizal fungi, the distributions of orchid mycorrhizal fungi (OMF) at large spatial scales are not well understood. Here, we investigate the distribution and diversity of Ceratobasidium OMF in orchids and soils across the Australian continent.MethodsWe sampled 217 Ceratobasidium isolates from 111 orchid species across southern Australia and combined these with 311 Ceratobasidium sequences from GenBank. To estimate the taxonomic diversity of Ceratobasidium associating with orchids, phylogenetic analysis of the ITS sequence locus was undertaken. Sequence data from the continent-wide Australian Microbiome Initiative were used to determine the geographical range of operational taxonomic units (OTUs) detected in orchids, with the distribution and climatic correlates of the two most frequently detected OTUs modelled using MaxEnt.Key ResultsWe identified 23 Ceratobasidium OTUs associating with Australian orchids, primarily from the orchid genera Pterostylis, Prasophyllum, Rhizanthella and Sarcochilus. OTUs isolated from orchids were closely related to, but distinct from, known pathogenic fungi. Data from soils and orchids revealed that ten of these OTUs occur on both east and west sides of the continent, while 13 OTUs were recorded at three locations or fewer. MaxEnt models suggested that the distributions of two widespread OTUs are correlated with temperature and soil moisture of the wettest quarter and far exceeded the distributions of their host orchid species.ConclusionsCeratobasidium OMF with cross-continental distributions are common in Australian soils and frequently have geographical ranges that exceed that of their host orchid species, suggesting these fungi are not limiting the distributions of their host orchids at large spatial scales. Most OTUs were distributed within southern Australia, although several OTUs had distributions extending into central and northern parts of the continent, illustrating their tolerance of an extraordinarily wide range of environmental conditions.     

High specificity of a rare terrestrial orchid toward a rare fungus within the North American tallgrass prairie

The Orchidaceae are globally distributed and represent a diverse lineage of obligate mycotrophic plants. Given their dependence on symbiotic fungi for germination and/or plant development, fungal community structure in substrates is expected to influence the distribution and persistence of orchid species. Yet, simultaneous characterization of orchid mycorrhizal fungal (OMF) communities in roots and in soil is rarely reported. To explain the co-distributions of OMF in roots, orchid-occupied, and bulk soil, we characterized mycorrhizal fungi associated with Platanthera praeclara over multiple years across its entire natural distribution within the North American tallgrass prairie. Root derived OMF communities included 24 Ceratobasidiaceae and 7 Tulasnellaceae operational taxonomic units (OTUs) though the orchid exhibited high spatio-temporal specificity toward a single Ceratobasidiaceae OTU, which was strongly stable across population sizes and phenological stages of the sampled individuals. The preferred OMF OTUs were primarily restricted to orchid-occupied locations while infrequent or absent in bulk soil. Variation in soil OMF assemblies was explained most by soil moisture, magnesium, manganese, and clay. In this first study of coupled root and soil OMF communities across a threatened grassland ecosystem, we report a strong relationship, further nuanced by soil chemistry, between a rare fungus and a rare orchid.     

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.     

Phylogenetic constrains on mycorrhizal specificity in eight Dendrobium(Orchidaceae) species

Plant phylogeny constrains orchid mycorrhizal(OrM) fungal community composition in some orchids. Here, we investigated the structures of the OrM fungal communities of eight Dendrobium species in one niche to determine whether similarities in the OrM fungal communities correlated with the phylogeny of the host plants and whether the Dendrobium-OrM fungal interactions are phylogenetically conserved. A phylogeny based on DNA data was constructed for the eight coexisting Dendrobium species,and the OrM fungal communities were characterized by their roots. There were 31 different fungal lineages associated with the eight Dendrobium species. In total, 82.98% of the identified associations belonging to Tulasnellaceae, and a smaller proportion involved members of the unknown Basidiomycota(9.67%). Community analyses revealed that phylogenetically related Dendrobium tended to interact with a similar set of Tulasnellaceae fungi. The interactions between Dendrobium and Tulasnellaceae fungi were significantly influenced by the phylogenetic relationships among the Dendrobium species. Our results provide evidence that the mycorrhizal specificity in the eight coexisting Dendrobium species was phylogenetically conserved.     

Mycorrhizal specificity does not limit the distribution of an endangered orchid species

What factors determine the distribution of a species is a central question in ecology and conservation biology. In general, the distribution of plant species is assumed to be controlled by dispersal or environmentally controlled recruitment. For plant species which are critically dependent on mycorrhizal symbionts for germination and seedling establishment, specificity in mycorrhizal associations and availability of suitable mycorrhizal fungi can be expected to have a major impact on successful colonization and establishment and thus ultimately on a species distribution. We combined seed germination experiments with soil analyses and fungal assessments using 454 amplicon pyrosequencing to test the relative importance of dispersal limitation, mycorrhizal availability and local growth conditions on the distribution of the orchid species Liparis loeselii, which, despite being widely distributed, is rare and endangered in Europe. We compared local soil conditions, seed germination and mycorrhizal availability in the soil between locations in northern Belgium and France where L. loeselii occurs naturally and locations where conditions appear suitable, but where adults of the species are absent. Our results indicated that mycorrhizal communities associating with L. loeselii varied among sites and plant life cycle stages, but the observed variations did not affect seed germination, which occurred regardless of current L. loeselii presence and was significantly affected by soil moisture content. These results indicate that L. loeselii is a mycorrhizal generalist capable of opportunistically associating with a variety of fungal partners to induce seed germination. They also indicate that availability of fungal associates is not necessarily the determining factor driving the distribution of mycorrhizal plant species.     

Variation in Mycorrhizal Associations with Tulasnelloid Fungi among Populations of Five Dactylorhiza Species

Background

Orchid species rely on mycorrhizal symbioses with fungi to complete their life cycle. Although there is mounting evidence that orchids can associate with several fungi from different clades or families, less is known about the actual geographic distribution of these fungi and how they are distributed across different orchid species within a genus.

Methodology/Principal Findings

We investigated among-population variation in mycorrhizal associations in five species of the genus Dactylorhiza (D. fuchsii, D. incarnata, D. maculata, D. majalis and D. praetermissa) using culture-independent detection and identification techniques enabling simultaneous detection of multiple fungi in a single individual. Mycorrhizal specificity, determined as the number of fungal operational taxonomic units (OTUs), and phylogenetic diversity of fungi were compared between species, whereas discriminant analysis was used to compare mycorrhizal spectra across populations and species. Based on a 95% cut-off value in internal transcribed spacer (ITS) sequence similarity, a total of ten OTUs was identified belonging to three different clades within the Tulasnellaceae. Most OTUs were found in two or more Dactylorhiza species, and some of them were common and widespread, occurring in more than 50% of all sampled populations. Each orchid species associated with at least five different OTUs, whereas most individuals also associated with two or more fungal OTUs at the same time. Phylogenetic diversity, corrected for species richness, was not significantly different between species, confirming the generality of the observed orchid mycorrhizal associations.

Conclusions/Significance

We found that the investigated species of the genus Dactylorhiza associated with a wide range of fungal OTUs from the Tulasnellaceae, some of which were widespread and common. These findings challenge the idea that orchid rarity is related to mycorrhizal specificity and fungal distribution.     

Mycorrhizal preference promotes habitat invasion by a native Australian orchid: Microtis media

Background and Aims

Mycorrhizal specialization has been shown to limit recruitment capacity in orchids, but an increasing number of orchids are being documented as invasive or weed-like. The reasons for this proliferation were examined by investigating mycorrhizal fungi and edaphic correlates of Microtis media, an Australian terrestrial orchid that is an aggressive ecosystem and horticultural weed.

Methods

Molecular identification of fungi cultivated from M. media pelotons, symbiotic in vitro M. media seed germination assays, ex situ fungal baiting of M. media and co-occurring orchid taxa (Caladenia arenicola, Pterostylis sanguinea and Diuris magnifica) and soil physical and chemical analyses were undertaken.

Key Results

It was found that: (1) M. media associates with a broad taxonomic spectrum of mycobionts including Piriformospora indica, Sebacina vermifera, Tulasnella calospora and Ceratobasidium sp.; (2) germination efficacy of mycorrhizal isolates was greater for fungi isolated from plants in disturbed than in natural habitats; (3) a higher percentage of M. media seeds germinate than D. magnifica, P. sanguinea or C. arenicola seeds when incubated with soil from M. media roots; and (4) M. media–mycorrhizal fungal associations show an unusual breadth of habitat tolerance, especially for soil phosphorus (P) fertility.

Conclusions

The findings in M. media support the idea that invasive terrestrial orchids may associate with a diversity of fungi that are widespread and common, enhance seed germination in the host plant but not co-occurring orchid species and tolerate a range of habitats. These traits may provide the weedy orchid with a competitive advantage over co-occurring orchid species. If so, invasive orchids are likely to become more broadly distributed and increasingly colonize novel habitats.     

Can orchid mycorrhizal fungi be persistently harbored by the plant host?

The environmental distribution of non-obligate orchid mycorrhizal (OM) symbionts belonging to the ‘rhizoctonia’ complex remains elusive. Some of these fungi, indeed, are undetectable in soil outside the host rhizosphere. A manipulation experiment was performed to assess the importance of neighbouring non-orchid plants and soil as possible reservoirs of OM fungi for Spiranthes spiralis, a widespread photosynthetic European terrestrial orchid species. Fungi of S. spiralis roots were identified by DNA metabarcoding before and 4 months after the removal of the surrounding vegetation and soil. Although such a treatment significantly affected fungal colonization of newly-formed orchid roots, most OM fungi were consistently associated with the host roots. Frequency patterns in differently aged roots suggest that these fungi colonize new orchid roots from either older roots or other parts of the same plant, which may thus represent an environmental source for the subsequent establishment of the OM symbiosis.     

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.     

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.     

Continent-wide distribution in mycorrhizal fungi: implications for the biogeography of specialized orchids

Background and Aims Although mycorrhizal associations are predominantly generalist, specialized mycorrhizal interactions have repeatedly evolved in Orchidaceae, suggesting a potential role in limiting the geographical range of orchid species. In particular, the Australian orchid flora is characterized by high mycorrhizal specialization and short-range endemism. This study investigates the mycorrhizae used by Pheladenia deformis, one of the few orchid species to occur across the Australian continent. Specifically, it examines whether P. deformis is widely distributed through using multiple fungi or a single widespread fungus, and if the fungi used by Australian orchids are widespread at the continental scale.Methods Mycorrhizal fungi were isolated from P. deformis populations in eastern and western Australia. Germination trials using seed from western Australian populations were conducted to test if these fungi supported germination, regardless of the region in which they occurred. A phylogenetic analysis was undertaken using isolates from P. deformis and other Australian orchids that use the genus Sebacina to test for the occurrence of operational taxonomic units (OTUs) in eastern and western Australia.Key Results With the exception of one isolate, all fungi used by P. deformis belonged to a single fungal OTU of Sebacina. Fungal isolates from eastern and western Australia supported germination of P. deformis. A phylogenetic analysis of Australian Sebacina revealed that all of the OTUs that had been well sampled occurred on both sides of the continent.Conclusions The use of a widespread fungal OTU in P. deformis enables a broad distribution despite high mycorrhizal specificity. The Sebacina OTUs that are used by a range of Australian orchids occur on both sides of the continent, demonstrating that the short-range endemism prevalent in the orchids is not driven by fungal species with narrow distributions. Alternatively, a combination of specific edaphic requirements and a high incidence of pollination by sexual deception may explain biogeographic patterns in southern Australian orchids.     

兰科菌根的生态学研究进展

兰科植物(Orchidaceae)是典型的菌根植物,自然条件下其种子的成功萌发和生长的早期阶段对菌根真菌有绝对的依赖性,在有些成年兰科植物中菌根真菌仍起着重要的作用。目前大部分兰科植物已为濒危物种,鉴于兰科植物天然的菌根共生关系,开展兰科植物和菌根真菌互作的生态学研究不仅具有极高的科研价值,更有助于兰科植物的物种保护和野生种群的生态恢复。近年研究表明,兰科植物对真菌的选择和二者共生关系的建立与菌根真菌的空间分布和丰度密切相关,然而当前对自然环境中兰科菌根真菌的实际分布还了解甚少,因此文章从生态学角度系统分析兰科植物与菌根真菌的关系,探讨该领域的研究热点,旨在为兰科菌根的生态学研究提供参考。     

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、甘肃6个样地的绶草菌根区系进行了研究。一共检测到51个OMF分类单元,其中角担菌科Ceratobasidiaceae真菌是绶草菌根的主要类群,约有1/3的角担菌科种类存在于所有的样本中,说明该类真菌亦广域分布;胶膜菌科Tulasnellaceae、肉丝耳科Serendipitaceae、红菇科Russulaceae、革菌科Thelephoraceae、口蘑科Tricholomataceae等真菌亦有发现,只是其相对多度较低,且较多种类表现出明显的地域特异性;6个样地的绶草菌根区系组成存在显著性差异,且这种差异与地理距离之间并未表现出明显的相关性,暗示菌根区系组成更多受生境因素的影响。本研究结果可为进一步采用菌根技术实现该类兰科药用植物的种质保育及栽培生产提供理论参考。     

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.     

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.     

Variation in mycorrhizal performance in the epiphytic orchid <Emphasis Type="Italic">Tolumnia variegata in vitro</Emphasis>: the potential for natural selection

Symbiotic seed germination is a critical stage in orchid life histories. Natural selection may act to favor plants that efficiently use mycorrhizal fungi. However, the necessary conditions for natural selection – variation, heritability, and differences in fitness – have not been demonstrated for either orchid or fungus. With the epiphytic orchid Tolumnia variegata as a model system, we ask the following questions: (1) Do seeds from different individuals in a population differ in germination and seedling development in the presence of the same fungi? (2) Do different mycorrhizal fungi (Ceratobasidium spp.) differ in ability to stimulate seed germination and growth in T. variegata? And (3) are the Ceratobasidium isolates that best induce seed germination and seedling development more closely related to each other than to isolates that are less effective? We performed symbiotic seed germination experiments in vitro. The experiments were done using mycorrhizal fungi isolated from T. variegata; relationships among the fungi were inferred from nuclear ribosomal ITS sequences. We found significant variation for both symbiotic germination and seedling growth among biparental seed crops obtained from a population of T. variegata plants. Differences among Ceratobasidium fungi in seed germination were significant. The fungi that induced highest seed germination and seedling development belonged to two of four clades of Ceratobasidium. The two experiments show that there is potential for natural selection to act on orchid–fungus relationships. Given that orchids vary in performance, and that mycorrhizal fungi are not geographically distributed homogeneously, mycorrhizae may affect population size, distribution and evolution of orchids.     

Influence of mycorrhizal fungi on seed germination and growth in terrestrial and epiphytic orchids

Epiphytes constitute over 70% of orchid diversity, but little is known about the functioning of their mycorrhizal associations. Terrestrial orchid seeds germinate symbiotically in soil and leaf litter, whereas epiphytic orchids may be exposed to relatively high light levels from an early stage of development and often produce green seeds. This suggests that seedlings of the two groups of orchids may differ in their responses to light and requirements for mycorrhiza-supplied carbon. The interactive effects of light, exogenous carbon and mycorrhizal status on germination and growth were investigated in vitro using axenic agar microcosms for one tropical epiphyte and three geophytic orchid species. The geophytic species strongly depended on their mycorrhiza for growth and this could not be substituted by exogenous sucrose, whereas the epiphytic species achieved 95% of the mycorrhizal seedling volume when supplied with exogenous sucrose in the dark. Mycorrhiza status strongly interacted with light exposure, enabling germination. Light inhibited or severely reduced growth, especially for the terrestrial orchids in the absence of mycorrhiza. For the first time, this study showed the parallel ecological importance of mycorrhizal fungi in overcoming light inhibition of seed germination and growth in both terrestrial and epiphytic orchids.     

Fungi from the roots of the common terrestrial orchid Gymnadenia conopsea

The fungal community associated with the terrestrial photosynthetic orchid Gymnadenia conopsea was characterized through PCR-amplification directly from root extracted DNA and cloning of the PCR products. Six populations in two geographically distinct regions in Germany were investigated. New ITS-primers amplifying a wide taxonomic range including Basidiomycetes and Ascomycetes revealed a high taxonomic and ecological diversity of fungal associates, including typical orchid mycorrhizas of the Tulasnellaceae and Ceratobasidiaceae as well as several ectomycorrhizal taxa of the Pezizales. The wide spectrum of potential mycorrhizal partners may contribute to this orchid's ability to colonize different habitat types with their characteristic microbial communities. The fungal community of G. conopsea showed a clear spatial structure. With 43 % shared taxa the species composition of the two regions showed only little overlap. Regardless of regions, populations were highly variable concerning taxon richness, varying between 5 and 14 taxa per population. The spatial structure and the continuous presence of mycorrhizal taxa on the one hand and the low specificity towards certain fungal taxa on the other hand suggest that the fungal community associated with G. conopsea is determined by multiple factors. In this context, germination as well as pronounced morphological and genetic differentiation within G. conopsea deserve attention as potential factors affecting the composition of the fungal community.