Molecular identification of mycorrhizal fungi in Neuwiedia veratrifolia (Orchidaceae)

We here apply a previously described method for identification of single peloton orchid mycorrhiza to a key orchid group and extend the usefulness in the heterobasidiomycetes of an existing fungal database for identification of mycorrhizal fungi. We amplified and sequenced mitochondrial ribosomal large subunit DNA from fungi in roots of Neuwiedia veratrifolia (Orchidaceae), a member of the small subfamily Apostasioideae that is sister to the remainder of Orchidaceae, and used the extended database to identify the mycorrhizal fungi. Sequences from fungi cultured from Neuwiedia roots and from direct peloton amplifications were analyzed cladistically with sequences determined from reference fungal collections and published sequences. The fungi from Neuwiedia are referred to the heterobasidiomycetous orders Tulasnellales and Ceratobasidiales, indicating that apostasioids utilize the same fungi as other photosynthetic orchids. The majority of Neuwiedia mycobionts came together in a clade with Tulasnella species, but some were most closely related to Thanatephorus. In some cases members of these two clades were isolated from the same orchid plant, providing another example of multiple mycobionts occurring in a single plant.     

Phylogenetic position of Apostasia and Neuwiedia (Orchidaceae)

JUDD, W. S., STERN, W. L. & CHEADLE, V. I. 1993. Phylogenetic position of Apostasia and Neuwiedia (Orchidaceae). Cladistic analyses of the phylogenetic relationships of selected orchid taxa were conducted in order to assess the phylogenetic position of Apostasia and Neuwiedia (Orchidaceae: Apostasioideae). These analyses employed newly available anatomical characters, along with several morphological features that had been used in recent phylogenetic analyses of Orchidaceae. Our analyses indicate that Apostasia is more closely related to Neuwiedia than it is to Cypripedioideae. The two genera comprise an apostasiad clade; this clade is the sister-group to a clade including Cypripedioideae and monandrous orchids. The apostasiad clade is diagnosed by the derived features of operculate pollen colpi, Apostasia-type seeds, and vessel members with simple perforation plates. Of these, the presence of simple perforation plates is considered to be the most significant phylogenetically. Therefore, the apostasiads should not be considered ancestral to the remaining orchid groups. Vessel members of the monandrous orchids, as well as the cypripediads, are multiperforate–the hypothesized ancestral state based on the condition in Hypoxidaceae.     

兰科菌根研究综述

兰科菌根是一种内生菌根,主要寄生于兰科（Orchidaceae）植物的种子及根系上。对兰科菌根真菌的分类及真菌资源多样性、兰科菌根的形态和菌根对兰科植物的效应等最新研究进展进行了综述。目前研究已知,感染兰科植物根部并能与之共生的真菌绝大多数属于担子菌门（Basidiomycota）和半知菌门（Deuteromycotha）,也有部分属于子囊菌门（Ascomycota）;兰科菌根的形成可分为两种情况：一是对兰科植物种子的侵染;二是对成长新根的侵染。菌根真菌对兰科植物的种子萌发及植株生长发育均有一定影响。     

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.     

Cultivable fungal community associated with the tropical orchid Dichaea andina

The orchid–fungus relationship has been studied since the discovery that the minute seeds of orchids depend on fungi to support the germination process. With the aim of describing the biodiversity of cultivable endophytic and mycorrhizal fungi from the orchid Dichaea andina, we isolated pure fungal cultures from its roots and identified them by sequencing the internal transcribed spacer. We recorded 22 fungal operational taxonomic units belonging to eight orders of Ascomycota: Eurotiales, Hypocreales, Xylariales, Helotiales, Boliniales, Chaetothyriales, Chaetosphaeriales and Pleosporales. The only Basidiomycota isolated belonged to the genus Ceratobasidium from the order Cantharellales, whose members are known as orchid mycorrhizal fungi. At the genus level, we identified 16 genera, the most common of which were Byssochlamys, Camarops, Trichoderma, Cladophialophora, Fusarium and Xylaria; some of them had been reported previously as orchid endophytes. The relevance of endophytic fungi to their hosts is still unclear, but this widely distributed interaction deserves further investigation.     

Experimental evidence of ericoid mycorrhizal potential within Serendipitaceae (Sebacinales)

The Sebacinales are a monophyletic group of ubiquitous hymenomycetous mycobionts which form ericoid and orchid mycorrhizae, ecto- and ectendomycorrhizae, and nonspecific root endophytic associations with a wide spectrum of plants. However, due to the complete lack of fungal isolates derived from Ericaceae roots, the Sebacinales ericoid mycorrhizal (ErM) potential has not yet been tested experimentally. Here, we report for the first time isolation of a serendipitoid (formerly Sebacinales Group B) mycobiont from Ericaceae which survived in pure culture for several years. This allowed us to test its ability to form ericoid mycorrhizae with an Ericaceae host in vitro, to describe its development and colonization pattern in host roots over time, and to compare its performance with typical ErM fungi and other serendipitoids derived from non-Ericaceae hosts. Out of ten serendipitoid isolates tested, eight intracellularly colonized Vaccinium hair roots, but only the Ericaceae-derived isolate repeatedly formed typical ericoid mycorrhiza morphologically identical to ericoid mycorrhiza commonly found in naturally colonized Ericaceae, but yet different from ericoid mycorrhiza formed in vitro by the prominent ascomycetous ErM fungus Rhizoscyphus ericae. One Orchidaceae-derived isolate repeatedly formed abundant hyaline intracellular microsclerotia morphologically identical to those occasionally found in naturally colonized Ericaceae, and an isolate of Serendipita (= Piriformospora) indica produced abundant intracellular chlamydospores typical of this species. Our results confirm for the first time experimentally that some Sebacinales can form ericoid mycorrhiza, point to their broad endophytic potential in Ericaceae hosts, and suggest possible ericoid mycorrhizal specificity in Serendipitaceae.     

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.     

Distribution of different mycorrhizal classes on Mount Koma, northern Japan

To investigate the role of mycorrhizae in nutrient-poor primary successional volcanic ecosystems, we surveyed mycorrhizal frequencies on the volcano Mount Koma (42°04N, 140°42E, 1,140 m elevation) in northern Japan. After the 1929 eruptions, plant community development started at the base of the volcano. Ammonia and nitrate levels, along with plant cover, decreased with increasing elevation, whereas phosphorus did not. In total, 305 individuals of 56 seed plant species were investigated in three elevational zones (550–600 m, 650–700 m, and 750–800 m). Five mycorrhizal classes were classified based on morphological traits: ecto- (ECM), arbuscular (AM), arbutoid, ericoid, and orchid mycorrhiza. All plant species were mycorrhizal to at least some extent, with most widespread tree species being heavily ectomycorrhizal. In addition, of 16 tree species collected in all three zones, 6 differed in the frequencies of ECM on roots between elevational zones, and 3 of these 6 species increased in frequency with increasing elevation. These results suggest that ECM colonization in some tree species is related to establishment in nutrient-poor habitats. All species of Ericaceae and Pyrolaceae had ericoid mycorrhizae, and an Orchidaceae species had orchid mycorrhizae. Herbaceous species, except for the low mycorrhizal frequency of Carex oxyandra and two Polygonaceae species, and ericoid and orchid mycorrhizal species, were generally AM. Of herbaceous species, Anaphalis margaritacea var. angustior increased AM frequency and decreased ECM frequency with increasing elevation, and Hieracium umbellatum increased ECM frequency. In total, the establishment of herbaceous species was not sufficiently explained by AM colonization on roots. Tree individuals developed 2–3 classes of mycorrhizae more than herbs at each elevational zone. We conclude that the symbiosis between seed plants and mycorrhizae, ECM in particular, greatly influences plant community structures on Mount Koma. Not only a single mycorrhizal class, but combinations of mycorrhizal classes should be studied to clarify effects on plant community dynamics.     

Phylogeny of the glomeromycota (arbuscular mycorrhizal fungi): recent developments and new gene markers

The fungal symbionts of arbuscular mycorrhiza form a monophyletic group in the true Fungi, the phylum Glomeromycota. Fewer than 200 described species currently are included in this group. The only member of this clade known to form a different type of symbiosis is Geosiphon pyriformis, which associates with cyanobacteria. Because none of these fungi has been cultivated without their plant hosts or cyanobacterial partners, progress in obtaining multigene phylogenies has been slow and the nuclear-encoded ribosomal RNA genes have remained the only widely accessible molecular markers. rDNA phylogenies have revealed considerable polyphyly of some glomeromycotan genera that has been used to reassess taxonomic concepts. Environmental studies using phylogenetic methods for molecular identification have recovered an amazing diversity of unknown phylotypes, suggesting considerable cryptic species diversity. Protein gene sequences that have become available recently have challenged the rDNA-supported sister group relationship of the Glomeromycota with Asco/Basidiomycota. However the number of taxa analyzed with these new markers is still too small to provide a comprehensive picture of intraphylum relationships. We use nuclear-encoded rDNA and rpb1 protein gene sequences to reassess the phylogeny of the Glomeromycota and discuss possible implications.     

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。     

Guilds of mycorrhizal fungi and their relation to trees,ericads, orchids and liverworts in a neotropical mountain rain forest

Mycorrhizas of vascular plants and mycorrhiza-like associations of liverworts and hornworts are integral parts of terrestrial ecosystems, but have rarely been studied in tropical mountain rain forests. The tropical mountain rain forest area of the Reserva Biológica San Francisco in South Ecuador situated on the eastern slope of the Cordillera El Consuelo is exceptionally rich in tree species, ericads and orchids, but also in liverworts. Previous light and electron microscopical studies revealed that tree roots are well colonized by structurally diverse Glomeromycota, and that epiphytic, pleurothallid orchids form mycorrhizas with members of the Tulasnellales and the Sebacinales (Basidiomycota). Sebacinales also occurred in mycorrhizas of hemiepiphytic ericads and Tulasnellales were found in liverworts belonging to the Aneuraceae. On the basis of these findings, we hypothesized that symbiotic fungi with a broad host range created shared guilds or even fungal networks between different plant species and plant families. To test this hypothesis, molecular phylogenetic studies of the fungi associated with roots and thalli were carried out using sequences of the nuclear rDNA coding for the small subunit rRNA (nucSSU) of Glomeromycota and the large subunit rRNA (nucLSU) of Basidiomycota. Sequence analyses showed that Sebacinales and Tulasnellales were only shared within but not between ericads and orchids or between liverworts and orchids, respectively. Regarding arbuscular-mycorrhiza-forming trees, however, 18 out of 33 Glomus sequence types were shared by two to four tree species belonging to distinct families. Nearly all investigated trees shared one sequence type with another tree individual. Host range and potential shared guilds appeared to be restricted to the plant family level for Basidiomycota, but were covering diverse plant families in case of Glomeromycota. Given that the sequence types as defined here correspond to fungal species, our findings indicate potential fungal networks between trees.     

A narrow group of monophyletic Tulasnella (Tulasnellaceae) symbiont lineages are associated with multiple species of Chiloglottis (Orchidaceae): Implications for orchid diversity

? Premise of the study: The Orchidaceae is characterized by exceptional species diversity. Obligate orchid mycorrhizae are predicted to determine orchid distributions, and highly specific relationships between orchids and fungi may drive orchid diversification. In this study, mycorrhizal diversity was examined in the terrestrial, photosynthetic orchid genus Chiloglottis to test the hypothesis of mycorrhizal-mediated diversification in the genus Chiloglottis. This orchid genus secures pollination by sexual deception, an obligate and highly specific pollination strategy. Here we asked whether the obligate orchid-fungal interactions are also specific. ? Methods: Two sequenced loci, the internal transcribed spacer region (ITS) and mitochondrial large subunit (mtLSU), were used to identify fungal isolates and assess fungal species diversity. Symbiotic germination of two species Chiloglottis aff. jeanesii and C. valida were used to assess germination potential of isolates and confirm mycorrhizal association. ? Key results: Phylogenetic analyses revealed that six representative Chiloglottis species spanning a broad survey of the genus were all associated with a narrow group of monophyletic Tulasnella fungal lineages. ? Conclusions: The Chiloglottis-Tulasnella interaction appears to be the first known case of such a narrow symbiont association across a broadly surveyed orchid genus. It appears that the specific pollination system of Chiloglottis, rather than specific orchid-fungal interactions has been the key driving force in the diversification of the genus. These findings also indicate that plant groups with highly specific mycorrhizal partners can have a widespread distribution.     

Ultrastructural studies and molecular characterization of root-associated fungi of $$\textit{}$$ (D. Don) Szlach.: a threatened and medicinally important taxon

Crepidium acuminatum (Orchidaceae) is a threatened medicinal orchid that grows under shady and moist forest floor where light remains for a very short period of time. Mycorrhizal association is known to be essential for seed germination and seedling establishment in a majority of orchids. Identification of fungi that form mycorrhizae with orchids is of crucial importance for orchid conservation. We used both morphological as well as molecular approaches to study this plant–fungal interaction. Scanning electron microscopy showed that fungi grow and proliferate in the middle layers of the cortex. Also, spiral-root hairs were found along with root hairs, which is an unusual observation. Spiral-root hairs provide more surface area for fluid absorption and entrance of colonizers. Further, total root genomic DNA was isolated and fungal internal-transcribed spacer (ITS) regions were polymerase chain reaction (PCR)-amplified using specific primer combinations ITS1F/ITS4 and ITS1/ITS4tul. ITS sequences were obtained and analysed to know the closest sequence matche in the GenBank using BLASTn hosted by NLM-NCBI. Subject sequences were identified to be belonging to three main genera, namely, Tulasnella, Aspergillus and Penicillium. Results indicate that mycorrhizal association is necessary for the growth and development of the plant. In addition, this symbiosis influences the distribution and rarity of this medicinally valuable taxon. Specific fungal partners may lead to an enhanced seed germination rate and increased efficiency of nutrient exchange between both the partners. Hence, knowledge of mycorrhizal fungi is essential for future in vitro germination and seedling establishment programmes, because they rely on fungi for germination. Identification of mycorrhizal fungi can be used for orchid propagation and conservation programmes.     

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.     

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.     

Atractiellomycetes belonging to the ‘rust’ lineage (Pucciniomycotina) form mycorrhizae with terrestrial and epiphytic neotropical orchids

Distinctive groups of fungi are involved in the diverse mycorrhizal associations of land plants. All previously known mycorrhiza-forming Basidiomycota associated with trees, ericads, liverworts or orchids are hosted in Agaricomycetes, Agaricomycotina. Here we demonstrate for the first time that Atractiellomycetes, members of the ‘rust’ lineage (Pucciniomycotina), are mycobionts of orchids. The mycobionts of 103 terrestrial and epiphytic orchid individuals, sampled in the tropical mountain rainforest of Southern Ecuador, were identified by sequencing the whole ITS1-5.8S-ITS2 region and part of 28S rDNA. Mycorrhizae of 13 orchid individuals were investigated by transmission electron microscopy. Simple septal pores and symplechosomes in the hyphal coils of mycorrhizae from four orchid individuals indicated members of Atractiellomycetes. Molecular phylogeny of sequences from mycobionts of 32 orchid individuals out of 103 samples confirmed Atractiellomycetes and the placement in Pucciniomycotina, previously known to comprise only parasitic and saprophytic fungi. Thus, our finding reveals these fungi, frequently associated to neotropical orchids, as the most basal living basidiomycetes involved in mycorrhizal associations of land plants.     

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.     

Identification of Picea-ectomycorrhizae by comparing DNA-sequences

Identification of the fungi forming ectomycorrhiza is still a great challenge. Ectomycorrhizae of Picea abies, collected in southwest Germany during several years and described as morphotypes, were identified using LSU and ITS sequences. To this the fungal sequences amplified from the mycorrhizae were compared with sequences from identified sporocarps. The fungal partner of Piceirhiza gelatinosa was identified as Hygrophorus olivaceoalbus, the fungal partner of Piceirhiza rosa-nigrescens was identified as Dermocybe cf. semisanguinea, and the fungal partner of a white mycorrhiza, described here for the first time, belongs to the Hebeloma velutipes group (Hebeloma crustuliniforme complex). Identification to genus level was possible for Piceirhiza lanuginosa where a Cortinarius-species is the fungal partner. A Tomentella-species forms a newly described light-brown mycorrhiza. Identification to family and to order-level was achieved for a milky-dull silvery mycorrhiza (Cortinariaceae), and Piceirhiza globulifera (Aphyllophorales), respectively. Ten samples of white, slightly bent mycorrhizae were formed by 8 different Cortinarius-species, including among others Cortinarius traganus, C. delibutus, and C. brunneus. The fungal partners of several brown, smooth mycorrhizae displaying only a Hartig net are Ascomycetes, among these are Wilcoxina cf. mikolae and Hymenoscyphus cf. ericae.     

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.     

The rare terrestrial orchid Nervilia nipponica consistently associates with a single group of novel mycobionts

Nervilia nipponica is a tuberous terrestrial orchid that has a highly restricted distribution within common secondary evergreen forest communities in central and western Japan. Such a limited occurrence could be attributable to a requirement for a specific mycorrhizal fungus. As part of a broader examination of this hypothesis, we sought to elucidate the mycorrhizal associations of N. nipponica. Seventy-five samples of mycorrhizae from forty individuals were collected at ten populations throughout the orchid’s range in Japan. The identity of mycorrhizal fungi was investigated by sequencing two genetic markers (nrDNA ITS and nrDNA 28S LSU) and their relationships were assessed via phylogenetic analyses. The most frequently encountered mycorrhizal fungi consisted of four closely related Agaricomycetes that infected an average of 78.7 % of individuals per population. All four formed a discrete, monophyletic clade with low sequence homology to other fungi registered in GenBank, indicating that they belong to a novel, unnamed family. Two additional fungal groups, belonging to Ceratobasidiaceae and “Group B” Sebacinales, were found in 22.0 and 21.5 % of individuals per population, respectively. The orchid probably uses these two groups opportunistically, because they were found at lower densities and always in combination with the unidentified Agaricomycete. These findings suggest that a group of novel Agaricomycete fungi constitutes the dominant mycobiont of N. nipponica.