Molecular identifications uncover diverse fungal symbionts of Pleione (Orchidaceae)

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

Culturable fungal endophytes in roots of Enkianthus campanulatus (Ericaceae)

Roots of plants in the genus Enkianthus, which belongs to the earliest diverging lineage in the Ericaceae, are commonly colonized by arbuscular mycorrhizal (AM) fungi. We documented the community of fungal root endophytes associated with Enkianthus species using a culture-based method for better understanding the members of root-colonizing fungi, except for AM fungi. Fungal isolates were successfully obtained from 610 out of 3,599 (16.9 %) root segments. Molecular analysis of fungal cultures based on ribosomal internal transcribed spacer (ITS) sequences yielded 63 operational taxonomical units (OTUs: 97 % sequence similarity cutoff) from 315 representative isolates. Further phylogenetic analysis showed that most (296 isolates) belonged to Ascomycota and were either members of Helotiales (Dermataceae, Hyaloscyphaceae, Phialocephala and Rhizoscyphus ericae aggregate), Oidiodendron, or other Pezizomycotina. Twenty-three out of 63 OTUs, which mainly consisted of Leotiomycetes, showed high similarities with reference sequences derived from roots of other ericaceous plants such as Rhododendron. The results indicated that Enkianthus houses variable root mycobionts including putative endophytic and mycorrhizal fungi in addition to AM fungi.     

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.     

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.     

Cryptostylis species (Orchidaceae) from a broad geographic and habitat range associate with a phylogenetically narrow lineage of Tulasnellaceae fungi

While many Australian terrestrial orchids have highly specialized mycorrhizal associations, we tested the hypothesis that the geographically widespread orchid genus Cryptostylis associates with a diversity of fungal species. Using fungal isolation and molecular approaches, we investigated the mycorrhizal associations of five Australian Cryptostylis species (27 sites sampled) and included limited sampling from three Asiatic Cryptostylis species (two sites). Like related orchid genera, Tulasnellaceae formed the main fungal associations of the Cryptostylis species we sampled, although some ectomycorrhizal, ericoid and saprotrophic fungi were detected infrequently. Each species of Australian Cryptostylis associated with three to seven Tulasnella Operational Taxonomic Units (OTUs), except for C. hunteriana where only one Tulasnella OTU was detected. In total, eleven Tulasnella OTUs associated with Australian Cryptostylis. The Asiatic Cryptostylis associated with four different Tulasnella OTUs belonging to the same lineage as the Australian species. While five Tulasnella OTUs (T. australiensis, T. prima, T. warcupii, T. densa, and T. punctata) were used by multiple species of Australian Cryptostylis, the most commonly used OTU differed between orchid species. The association with different Tulasnella fungi by Cryptostylis species co-occurring at the same site suggests that in any given environmental condition, Cryptostylis species may intrinsically favour different fungal OTUs.     

Many broadly-shared mycobionts characterize mycorrhizal interactions of two coexisting epiphytic orchids in a high elevation tropical forest

Interspecific interactions play an important role in community assembly. A basic ecological question is whether interactions are specialized (one to one) or generalized (many to many). Specialization of interactions should ideally be assessed across several populations because species could be specialists at a particular site but generalists when several sites are considered. Mycorrhizal interactions are fundamental for orchid life and distribution, but their level of specialization is still under debate. To understand the extent to which epiphytic orchids are specialists in their mycorrhizal interactions, we studied the richness and phylogenetic structure of mycobionts across different sites, and the similarity in the mycobiont composition between coexisting orchid species. We sequenced the nrDNA ITS2 region and explored the mycobiont communities associated with two epiphytic orchids, Epidendrum marsupiale and Cyrtochilum pardinum, at two elevations within two sites in Ecuador. We found 108 OTUs belonging to Serendipitaceae (66), Ceratobasidiaceae (22), Atractiellales (11) and Tulasnellaceae (9). Orchids at the highest elevations hosted the highest OTU richness. The two orchid species shared a high percentage of mycobionts between all sites. No phylogenetic structure within orchid mycorrhizal communities was found at any sites or elevations. Our results indicate that the studied orchids are generalists and share a broad group of mycobionts (16 OTUs) with no apparent niche segregation within or between sites.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Specificity and preference of mycorrhizal associations in two species of the genus Dendrobium (Orchidaceae)

Dendrobium is a large genus of tropical epiphytic orchids. Some members of this genus are in danger of extinction across China. To investigate orchid mycorrhizal associations of the genus Dendrobium, plants from two Dendrobium species (Dendrobium officinale and Dendrobium fimbriatum) were collected from two habitats in Guangxi Province, China, and clone libraries were constructed to identify the mycorrhizal fungi of individual plants. A low and high degree of specificity was observed in D. officinale and D. fimbriatum, respectively. Phylogenetic analysis revealed that the majority of Dendrobium mycorrhizal fungi are members of the Tulasnellaceae, but, in some plants, members of the Ceratobasidiaceae and Pluteaceae were also found. In D. officinale, individual plants associated with more than three fungi simultaneously, and, in some cases, associations with five fungi at the same time. One fungus was shared by individual plants of D. officinale collected from the two habitats. In D. fimbriatum, only one fungal partner was found in each population, and this fungus differed between populations. The two species of Dendrobium sampled from the same habitat did not share any fungal taxa. These results provide valuable information for conservation of these orchid species.     

The importance of associations with saprotrophic non-Rhizoctonia fungi among fully mycoheterotrophic orchids is currently under-estimated: novel evidence from sub-tropical Asia

Background and Aims Most fully mycoheterotrophic (MH) orchids investigated to date are mycorrhizal with fungi that simultaneously form ectomycorrhizas with forest trees. Only a few MH orchids are currently known to be mycorrhizal with saprotrophic, mostly wood-decomposing, fungi instead of ectomycorrhizal fungi. This study provides evidence that the importance of associations between MH orchids and saprotrophic non-Rhizoctonia fungi is currently under-estimated.Methods Using microscopic techniques and molecular approaches, mycorrhizal fungi were localized and identified for seven MH orchid species from four genera and two subfamilies, Vanilloideae and Epidendroideae, growing in four humid and warm sub-tropical forests in Taiwan. Carbon and nitrogen stable isotope natural abundances of MH orchids and autotrophic reference plants were used in order to elucidate the nutritional resources utilized by the orchids.Key Results Six out of the seven MH orchid species were mycorrhizal with either wood- or litter-decaying saprotrophic fungi. Only one orchid species was associated with ectomycorrhizal fungi. Stable isotope abundance patterns showed significant distinctions between orchids mycorrhizal with the three groups of fungal hosts.Conclusions Mycoheterotrophic orchids utilizing saprotrophic non-Rhizoctonia fungi as a carbon and nutrient source are clearly more frequent than hitherto assumed. On the basis of this kind of nutrition, orchids can thrive in deeply shaded, light-limiting forest understoreys even without support from ectomycorrhizal fungi. Sub-tropical East Asia appears to be a hotspot for orchids mycorrhizal with saprotrophic non-Rhizoctonia fungi.     

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.     

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.     

Comparative study of nutritional mode and mycorrhizal fungi in green and albino variants of Goodyera velutina,an orchid mainly utilizing saprotrophic rhizoctonia

The majority of chlorophyllous orchids form mycorrhizal associations with so‐called rhizoctonia fungi, a phylogenetically heterogeneous assemblage of predominantly saprotrophic fungi in Ceratobasidiaceae, Tulasnellaceae, and Serendipitaceae. It is still a matter of debate whether adult orchids mainly associated with rhizoctonia species are partially mycoheterotrophic. Here, we investigated the nutritional modes of green and albino variants of Goodyera velutina, an orchid species considered to be mainly associated with Ceratobasidium spp., by measuring their ¹³C and ¹⁵N abundances, and by molecular barcoding of their mycorrhizal fungi. Molecular analysis revealed that both green and albino variants of G. velutina harbored a similar range of mycobionts, mainly saprotrophic Ceratobasidium spp., Tulasnella spp., and ectomycorrhizal Russula spp. In addition, stable isotope analysis revealed that albino variants were significantly enriched in ¹³C but not so greatly in ¹⁵N, suggesting that saprotrophic Ceratobasidium spp. and Tulasnella spp. are their main carbon source. However, in green variants, ¹³C levels were depleted and those of ¹⁵N were indistinguishable from the co‐occurring autotrophic plants. Therefore, we concluded that the albino G. velutina variants are fully mycoheterotrophic plants whose C derives mainly from saprotrophic rhizoctonia, while the green G. velutina variants are mainly autotrophic plants, at least at our study site, in spite of their additional associations with ectomycorrhizal fungi. This is the first report demonstrating that adult nonphotosynthetic albino variants can obtain their nutrition mainly from nonectomycorrhizal rhizoctonia.     

Arbuscular mycorrhizal colonization in field-collected terrestrial cordate gametophytes of pre-polypod leptosporangiate ferns (Osmundaceae,Gleicheniaceae, Plagiogyriaceae,Cyatheaceae)

To determine the mycorrhizal status of pteridophyte gametophytes in diverse taxa, the mycorrhizal colonization of wild gametophytes was investigated in terrestrial cordate gametophytes of pre-polypod leptosporangiate ferns, i.e., one species of Osmundaceae (Osmunda banksiifolia), two species of Gleicheniaceae (Diplopterygium glaucum, Dicranopteris linearis), and four species of Cyatheales including tree ferns (Plagiogyriaceae: Plagiogyria japonica, Plagiogyria euphlebia; Cyatheaceae: Cyathea podophylla, Cyathea lepifera). Microscopic observations revealed that 58 to 97 % of gametophytes in all species were colonized with arbuscular mycorrhizal (AM) fungi. Fungal colonization was limited to the multilayered midrib (cushion) tissue in all gametophytes examined. Molecular identification using fungal SSU rDNA sequences indicated that the AM fungi in gametophytes primarily belonged to the Glomeraceae, but also included the Claroideoglomeraceae, Gigasporaceae, Acaulosporaceae, and Archaeosporales. This study provides the first evidence for AM fungal colonization of wild gametophytes in the Plagiogyriaceae and Cyatheaceae. Taxonomically divergent photosynthetic gametophytes are similarly colonized by AM fungi, suggesting that mycorrhizal associations with AM fungi could widely occur in terrestrial pteridophyte gametophytes.