Mycorrhizal specificity differences in epiphytic habitat: three epiphytic orchids harbor distinct ecological and physiological specificity.

Orchidaceae has diversified in tree canopies and accounts for 68% of vascular epiphytes. Differences in mycorrhizal communities among epiphytic orchids can reduce species competition for mycorrhizal fungi and contribute to niche partitioning, which may be a crucial driver of the unusual species diversification among orchids. Mycorrhizal specificity-the range of fungi allowing mycorrhizal partnerships-was evaluated by assessment of mycorrhizal communities in the field (ecological specificity) and symbiotic cultures in the laboratory (physiological specificity) for three epiphytic orchids inhabiting Japan. Mycorrhizal communities were assessed with co-existing individuals growing within 10 cm of each other, revealing that ecological specificity varied widely among the three species, ranging from dominance by a single Ceratobasidiaceae fungus to diverse mycobionts across the Ceratobasidiaceae and Tulasnellaceae. In vitro seed germination tests revealed clear differences in physiological specificity among the three orchids, and that the primary mycorrhizal partners contributed to seed germination. In vitro compatibility ranges of three orchids strongly reflect the mycorrhizal community composition of wild populations. This suggests that differences in in situ mycorrhizal communities are not strongly driven by environmental factors, but are primarily due to physiological differences among orchid species. This study shows that the symbiotic strategy among the epiphytic orchid species varies from specialized to generalized association, which may contribute to biotic niche partitioning.

Evolutionary histories and mycorrhizal associations of mycoheterotrophic plants dependent on saprotrophic fungi.

Mycoheterotrophic plants (MHPs) are leafless, achlorophyllous, and completely dependent on mycorrhizal fungi for their carbon supply. Mycorrhizal symbiosis is a mutualistic association with fungi that is undertaken by the majority of land plants, but mycoheterotrophy represents a breakdown of this mutualism in that plants parasitize fungi. Most MHPs are associated with fungi that are mycorrhizal with autotrophic plants, such as arbuscular mycorrhizal (AM) or ectomycorrhizal (ECM) fungi. Although these MHPs gain carbon via the common mycorrhizal network that links the surrounding autotrophic plants, some mycoheterotrophic lineages are associated with saprotrophic (SAP) fungi, which are free-living and decompose leaf litter and wood materials. Such MHPs are dependent on the forest carbon cycle, which involves the decomposition of wood debris and leaf litter, and have a unique biology and evolutionary history. MHPs associated with SAP fungi (SAP-MHPs) have to date been found only in the Orchidaceae and likely evolved independently at least nine times within that family. Phylogenetically divergent SAP Basidiomycota, mostly Agaricales but also Hymenochaetales, Polyporales, and others, are involved in mycoheterotrophy. The fungal specificity of SAP-MHPs varies from a highly specific association with a single fungal species to a broad range of interactions with multiple fungal orders. Establishment of symbiotic culture systems is indispensable for understanding the mechanisms underlying plant-fungus interactions and the conservation of MHPs. Symbiotic culture systems have been established for many SAP-MHP species as a pure culture of free-living SAP fungi is easier than that of biotrophic AM or ECM fungi. Culturable SAP-MHPs are useful research materials and will contribute to the advancement of plant science.

The mycorrhizal community of the epiphytic orchid Thrixspermum japonicum is strongly biased toward a single Ceratobasidiaceae fungus, despite a wide range of fungal partners.

PREMISE: Orchids depend primarily on mycorrhizal fungi to obtain nutrients throughout their life cycle. Epiphytic orchids account for 69% of orchid diversity. The unstable availability of water and nutrients in their arboreal habitats often results in severe water and nutrient stresses. Consequently, mycorrhizal associations may be important for the survival of epiphytic orchids, but our understanding thereof remains limited. Here, we investigated the mycorrhizal community in a single epiphytic orchid species, using more samples than in any previous study. METHODS: We assessed the mycorrhizal communities of Thrixspermum japonicum, one of the most common epiphytic orchids in the temperate region of Japan. In total, 144 individuals were collected from 28 host tree species at 20 sites across 1300 km. The mycorrhizal fungi were identified based on nuclear ribosomal DNA internal transcribed spacer sequences and assigned operational taxonomic units (OTUs) based on 97% sequence similarity. RESULTS: We obtained 24 OTUs; 9 belonged to the Ceratobasidiaceae and 15 to the Tulasnellaceae. These OTUs are widely distributed throughout the phylogenetic trees of the two fungal families. However, a single Ceratobasidiaceae OTU accounted for 49.7% of all fungal sequences and was predominant in samples from 15 host tree species and 12 sites. CONCLUSIONS: Our results imply that despite having a broad range of mycorrhizal partners, T. japonicum was predominantly associated with a single fungal taxon at most of the sites among the host-tree species investigated. These findings contribute to elucidating mycorrhizal symbiosis in epiphytic habitats.

Fern gametophytes of Angiopteris lygodiifolia and Osmunda japonica harbor diverse Mucoromycotina fungi.

Mycorrhizal symbiosis between plants and fungi is ubiquitous, and has been played key roles in plant terrestrialization and diversification. Although arbuscular mycorrhizal (AM) symbioses with Glomeromycotina fungi have long been recognized as both ancient and widespread symbionts, recent studies showed that Mucoromycotina fungi were also ancestral symbionts and would thus be expected to co-exist with many land plants. To explore whether Mucoromycotina colonize fern gametophytes, we subjected fungal associations with gametophytes of two distantly related ferns, Angiopteris lygodiifolia (Marattiales) and Osmunda japonica (Osmundales), to molecular analysis. Direct PCR amplification from intracellular hyphal coils was also performed. We detected Mucoromycotina sequences in the gametophytes of A. lygodiifolia and O. japonica at rates of 41% (7/17) and 50% (49/98) of gametophytes, respectively, and assigned them to 10 operational taxonomic units of Endogonales lineages. In addition, we used AM fungal-specific primers and detected Glomeromycotina sequences in all individuals examined. The results suggest that Glomeromycotina and Mucoromycotina colonized fern gametophytes simultaneously. We found that Mucoromycotina were present in fern gametophytes of Marratiales and Osmundales, which implies that a variety of fern taxa have Mucoromycotina associations.

A leafless epiphytic orchid, Taeniophyllum glandulosum Blume (Orchidaceae), is specifically associated with the Ceratobasidiaceae family of basidiomycetous fungi.

Leafless epiphytes in the Orchidaceae undergo a morphological metamorphosis in which the root has chloroplast-containing cortical cells and is the sole photosynthetic organ for carbon gain. All orchids are entirely dependent on mycorrhizal fungi for their carbon supply during seed germination, and this mycorrhizal association generally persists in adult plants. However, our knowledge of the mycorrhizal association of leafless epiphytic orchids remains limited, and the contribution of the mycorrhizal association to nutrient acquisition in these orchid species is largely unknown. In this study, the mycorrhizal fungi of a leafless epiphytic orchid, Taeniophyllum glandulosum, were identified molecularly using 68 mature plants and 17 seedlings. In total, 187 fungal internal transcribed spacer sequences were obtained, of which 99% were identified as Ceratobasidiaceae. These sequences were classified into five operational taxonomic units (OTUs) based on 97% sequence similarity. The most frequent sequence was OTU1, which accounted for 91% of all Ceratobasidiaceae sequences, although other phylogenetically distinct Ceratobasidiaceae fungi were detected. These results show that T. glandulosum is specifically associated with a particular group of Ceratobasidiaceae. All mycorrhizal fungi found in T. glandulosum seedlings belonged to OTU1, which was also found in adult plants on the same host tree. The mycorrhizal fungi from 13 host tree species were compared, and T. glandulosum was preferentially associated with OTU1 on 11 tree species. In conclusion, T. glandulosum is specifically associated with Ceratobasidiaceae fungi and this specific association remains throughout the orchid life cycle and is found on divergent host tree species.

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

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

Fungal partner shifts during the evolution of mycoheterotrophy in Neottia.

PREMISE OF THE STUDY: Few previous studies have examined how mycobionts change during the evolution from autotrophy to mycoheterotrophy based on phylogenetic hypotheses. Neottia (Orchidaceae) comprises leafy species that are autotrophic and related leafless mycoheterotrophic species, and the phylogenetic relationships among them have been clarified. Accordingly, Neottia is a suitable taxon for investigating the question above. Here we clarified the diversity of mycobionts in Neottia plants and elucidated changes in the character of symbiotic associations during the evolution of mycoheterotrophy. METHODS: We sequenced the internal transcribed spacer (ITS) regions of nuclear ribosomal (nr) DNA for mycobionts of Neottia plants. Furthermore, we selected one representative DNA sample from each fungal operational taxonomic unit (OTU) and used it to amplify the large subunit (LSU) nrDNA sequences. Phylogenetic analyses of Sebacinales (basidiomycetes), the dominant mycobiont of Neottia, were conducted and sample-based rarefaction curves generated for the observed mycobiont richness on each OTU. KEY RESULTS: Leafy and leafless species in Neottia were associated with Sebacinales Group B and Sebacinales Group A, respectively. The composition and specificity level of fungal partners varied among Neottia species. CONCLUSIONS: Fungal partner composition and specificity level changed with speciation in both leafy and leafless Neottia species. In particular, mycorrhizal associations likely shifted from Sebacinales Group B to Group A during the evolution from autotrophy to mycoheterotrophy. Partner shifts to Sebacinales Group A have also been reported in the evolution of mycoheterotrophy of other plant groups, suggesting that convergence to this fungal group occurs in association with the evolution of mycoheterotrophy.

How do fungal partners affect the evolution and habitat preferences of mycoheterotrophic plants? A case study in Gastrodia.

PREMISE OF THE STUDY: Since mycoheterotrophic plants (MHPs) completely depend on their mycorrhizal fungi for carbon, selection of fungal partners has an important role in the speciation of MHPs. However, the causes and mechanisms of mycobiont changes during speciation are not clear. We tested fungal partner shifts and changes in mycorrhizal specificity during speciation of three closely related MHPs-Gastrodia confusa (Gc), G. pubilabiata (Gp), and G. nipponica (Gn) (Orchidaceae)-and correlations between these changes and the vegetation types where each species grows. METHODS: We investigated the diversity of mycobionts of the three species by sequencing nrDNA ITS, and the sequence data were subjected to test changes in fungal specificity and fungal partner shifts among the three species. Furthermore, we conducted multivariate analysis to test for differences in mycobiont communities of vegetation types where each species grows. KEY RESULTS: Two saprobic Basidiomycota, Marasmiaceae and Mycenaceae, were dominant fungal partners of the three species, and Gn was simultaneously associated with the ectomycorrhizal Russulaceae and Sebacinaceae. Although mycobiont composition differed among the three species, they also sometimes shared identical fungal species. Multivariate analysis revealed that mycobiont communities of the three species in bamboo thickets differed significantly from those in other vegetation types. CONCLUSIONS: Fungal partner shifts are not necessarily associated with the evolution of MHPs, and fungal specificity of Gc and Gp was significantly higher than that of Gn, implying that the specificity fluctuates during speciation. Further, Gc exclusively inhabits bamboo thickets, which suggests that adaptation to particular fungi specific to bamboo thickets triggered speciation of this species.

The tiny-leaved orchid Cephalanthera subaphylla obtains most of its carbon via mycoheterotrophy.

The evolution of mycoheterotrophy has been accompanied by extreme reductions in plant leaf size and photosynthetic capacity. Partially mycoheterotrophic plants, which obtain carbon from both photosynthesis and their mycorrhizal fungi, include species with leaves of normal size and others that are tiny-leaved. Thus, plant species may lose their leaves in a gradual process of size reduction rather than through a single step mutation. Little is known about how the degree of mycoheterotrophy changes during reductions in leaf size. We compared the degree of mycoheterotrophy among five Japanese Cephalanthera species, four with leaves of normal size (Cephalanthera falcata, Cephalanthera erecta, Cephalanthera longibracteata and Cephalanthera longifolia), one with tiny leaves (Cephalanthera subaphylla), and one albino form of C. falcata (as reference specimens for fully mycoheterotrophic plants). The levels of mycoheterotrophy were determined by stable isotope natural abundance analysis. All Cephalanthera species were relatively enriched in 13C and 15N in comparison with surrounding autotrophic plants. Cephalanthera subaphylla was strongly enriched in 13C and 15N to levels similar to the albinos. Species with leaves of normal size were significantly less enriched in 13C than C. subaphylla and the albinos. Thus, C. subaphylla was strongly mycoheterotrophic, obtaining most of its carbon from mycorrhizal fungi even though it has tiny leaves; species with leaves of normal size were partially mycoheterotrophic. Hence, during the evolutionary pathway to full mycoheterotrophy, some plant species appear to have gained strong mycoheterotrophic abilities before completely losing foliage leaves.

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.

First flowering hybrid between autotrophic and mycoheterotrophic plant species: breakthrough in molecular biology of mycoheterotrophy.

Among land plants, which generally exhibit autotrophy through photosynthesis, about 880 species are mycoheterotrophs, dependent on mycorrhizal fungi for their carbon supply. Shifts in nutritional mode from autotrophy to mycoheterotrophy are usually accompanied by evolution of various combinations of characters related to structure and physiology, e.g., loss of foliage leaves and roots, reduction in seed size, degradation of plastid genome, and changes in mycorrhizal association and pollination strategy. However, the patterns and processes involved in such alterations are generally unknown. Hybrids between autotrophic and mycoheterotrophic plants may provide a breakthrough in molecular studies on the evolution of mycoheterotrophy. We have produced the first hybrid between autotrophic and mycoheterotrophic plant species using the orchid group Cymbidium. The autotrophic Cymbidium ensifolium subsp. haematodes and mycoheterotrophic C. macrorhizon were artificially pollinated, and aseptic germination of the hybrid seeds obtained was promoted by sonication. In vitro flowering was observed five years after seed sowing. Development of foliage leaves, an important character for photosynthesis, segregated in the first generation; that is, some individuals only developed scale leaves on the rhizome and flowering stems. However, all of the flowering plants formed roots, which is identical to the maternal parent.

Significant difference in mycorrhizal specificity between an autotrophic and its sister mycoheterotrophic plant species of Petrosaviaceae.

Petrosaviaceae is a monocotyledonous plant family that comprises two genera: the autotrophic Japonolirion and the mycoheterotrophic Petrosavia. Accordingly, this plant family provides an excellent system to examine specificity differences in mycobionts between autotrophic and closely related mycoheterotrophic plant species. We investigated mycobionts of Japonolirion osense, the sole species of the monotypic genus, from all known habitats of this species by molecular identification and detected 22 arbuscular mycorrhizal (AM) fungal phylotypes in Archaesporales, Diversisporales, and Glomerales. In contrast, only one AM fungal phylotype in Glomerales was predominantly detected from the mycoheterotrophic Petrosavia sakuraii in a previous study. The high mycobiont diversity in J. osense and in an outgroup plant, Miscanthus sinensis (Poaceae), indicates that fungal specificity increased during the evolution of mycohetrotrophy in Petrosaviaceae. Furthermore, some AM fungal sequences of J. osense showed >99% sequence similarity to the dominant fungal phylotype of P. sakuraii, and one of them was nested within a clade of P. sakuraii mycobionts. These results indicate that fungal partners are not necessarily shifted, but rather selected for in the course of the evolution of mycoheterotrophy. We also confirmed the Paris-type mycorrhiza in J. osense.

Orchid Mycorrhizal Association of Cultivated Dendrobium Hybrid and Their Role in Seed Germination and Seedling Growth.

Orchids are crucial for the horticulture industry. Mycorrhizal fungi benefit crops by improving nutrition, plant growth, and disease resistance. However, the mycorrhizal association of horticultural hybrid orchids is poorly understood. To address this, we investigated mycorrhizal colonization in the entire root system and assessed the mycorrhizal community using a Dendrobium cultivar, D. Stardust 'Firebird', obtained from three nurseries. Additionally, we isolated and tested mycorrhizal fungi in symbiotic culture to assess their role in the seed germination and growth of Dendrobium species. All plants were colonized by mycorrhizal fungi, with a higher colonization rate in mature than in juvenile plants. Molecular identification of mycorrhizal fungi by Sanger and high-throughput sequencing revealed that the cultivar was associated with a phylogenetically diverse group of fungi, including mycorrhizal fungi from Tulasnellaceae, and several wood-decaying fungi. The Tulasnellaceae isolates significantly enhanced the seed germination of three Dendrobium species and increased the survival rate and growth of asymbiotic seedlings of D. moniliforme. This study is the first comprehensive examination of mycorrhizal associations in horticultural orchid hybrids, providing valuable insights for commercial production.

Arbuscular mycorrhiza formation in cordate gametophytes of two ferns, Angiopteris lygodiifolia and Osmunda japonica.

Mycorrhizal symbiosis is common among land plants including pteridophytes (monilophytes and lycophytes). In pteridophytes with diplohaplontic life cycle, mycorrhizal formations were mostly reported for sporophytes, but very few for gametophytes. To clarify the mycorrhizal association of photosynthetic gametophytes, field-collected gametophytes of Angiopteris lygodiifolia (Marattiaceae, n = 52) and Osmunda japonica (Osmundaceae, n = 45) were examined using microscopic and molecular techniques. Collected gametophytes were mostly cut into two pieces. One piece was used for light and scanning microscopic observations, and the other for molecular identification of plant species (chloroplast rbcL sequences) and mycorrhizal fungi (small subunit rDNA sequences). Microscopic observations showed that 96 % (50/52) of Angiopteris and 95 % (41/43) of Osmunda gametophytes contained intracellular hyphae with arbuscules and/or vesicles and fungal colonization was limited to the inner tissue of the thick midribs (cushion). Fungal DNA analyses showed that 92 % (48/52) of Angiopteris and 92 % (35/38) of Osmunda have sequences of arbuscular mycorrhizal fungi, which were highly divergent but all belonged to Glomus group A. These results suggest that A. lygodiifolia and O. japonica gametophytes consistently form arbuscular mycorrhizae. Mycorrhizal formation in wild fern gametophytes, based on large-scale sampling with molecular identification of host plant species, was demonstrated for the first time.

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.

Shifts in mycorrhizal fungi during the evolution of autotrophy to mycoheterotrophy in Cymbidium (Orchidaceae).

PREMISE OF THE STUDY: Mycoheterotrophic plants, which completely depend upon mycorrhizal fungi for their nutrient supply, have unusual associations with fungal partners. The processes involved in shifts in fungal associations during cladogenesis of plant partners from autotrophy to mycoheterotrophy have not been demonstrated using a robust phylogenetic framework. METHODS: Consequences of a mycorrhizal shift were examined in Cymbidium (Orchidaceae) using achlorophyllous and sister chlorophyllous species. Fungal associates of the two achlorophyllous mycoheterotrophs (C. macrorhizon and C. aberrans), their close relatives, the chlorophyllous mixotrophs (C. goeringii and C. lancifolium) and an outgroup, the chlorophyllous autotroph C. dayanum, were identified by internal transcribed spacers of the nuclear ribosomal DNA sequences. KEY RESULTS: Molecular identification of mycorrhizal fungi revealed: (1) the outgroup autotroph is predominantly dependent on saprobic Tulasnellaceae, (2) the mixotrophs associate with the Tulasnellaceae and ectomycorrhizal groups including the Sebacinales, Russulaceae, Thelephoraceae and Clavulinaceae, and (3) the two mycoheterotrophs are mostly specialized with ectomycorrhizal Sebacinales. CONCLUSION: Fungal partners in Cymbidium have shifted from saprobic to ectomycorrhizal fungi via a phase of coexistence of both nutritional types of fungi. These three phases correspond to the evolution from autotrophy to mycoheterotrophy via mixotrophy in Cymbidium. We demonstrate that shifts in mycorrhizal fungi correlate with the evolution of nutritional modes in plants. Furthermore, gradual shifts in fungal partners through a phase of coexistence of different types of mycobionts may play a crucial role in the evolution of mycoheterotrophic plants.

In-vitro symbiotic germination of seeds of five mycoheterotrophic Gastrodia orchids with Mycena and Marasmiaceae fungi.

We performed in-vitro germination tests on seeds from five Gastrodia orchids (G. confusa, G. elata var. elata, G. elata var. pallens, G. nipponica, and G. pubilabiata) using one Marasmiaceae and two Mycena isolates. Mycena sp. 1 promoted germination of all five Gastrodia orchids, with root and/or tuber formation observed in G. confusa, G. nipponica, and G. pubilabiata. No additional growth was observed in the other two orchids. Mycena sp. 2 induced G. confusa, G. elata var. elata, and G. nipponica germination, whereas Marasmiaceae sp. 1 induced G. nipponica and G. pubilabiata germination. Phylogenetic analyses indicated that the two Mycena isolates represent distinct lineages within the Mycenaceae. Mycena sp. 1 and Marasmiaceae sp. 1 are closely related to Mycena abramsii and Marasmiellus rhizomorphogenus, respectively. Our results imply that Mycena and marasmioid fungi play important roles in early development in Gastrodia species, and that Mycena fungi in particular may be common mycobionts of Gastrodia species. Root and/or tuber development was observed with four plant-fungus combinations, implying that these associations persist throughout the life cycle, whereas G. elata var. elata may require different associates over time. Our findings will contribute to elucidating the mycorrhizal associations of mycoheterotrophic orchids throughout their life cycle.

Specific arbuscular mycorrhizal fungi associated with non-photosynthetic Petrosavia sakuraii (Petrosaviaceae).

Mycorrhizal fungi in roots of the achlorophyllous Petrosavia sakuraii (Petrosaviaceae) were identified by molecular methods. Habitats examined were plantations of the Japanese cypress Chamaecyparis obtusa in Honshu, an evergreen broad-leaved forest in Amami Island in Japan and a mixed deciduous and evergreen forest in China. Aseptate hyphal coils were observed in root cortical cells of P. sakuraii, suggesting Paris-type arbuscular mycorrhiza (AM). Furthermore, hyphal coils that had degenerated to amorphous clumps were found in various layers of the root cortex. Despite extensive sampling of P. sakuraii from various sites in Japan and China, most of the obtained AM fungal sequences of the nuclear small subunit ribosomal RNA gene were nearly identical and phylogenetic analysis revealed that they formed a single clade in the Glomus group A lineage. This suggests that the symbiotic relationship is highly specific. AM fungi of P. sakuraii were phylogenetically different from those previously detected in the roots of some mycoheterotrophic plants. In a habitat in C. obtusa plantation, approximately half of the AM fungi detected in roots of C. obtusa surrounding P. sakuraii belonged to the same clade as that of P. sakuraii. This indicates that particular AM fungi are selected by P. sakuraii from diverse indigenous AM fungi. The same AM fungi can colonize both plant species, and photosynthates of C. obtusa may be supplied to P. sakuraii through a shared AM fungal mycelial network. Although C. obtusa plantations are widely distributed throughout Japan, P. petrosavia is a rare plant species, probably because of its high specificity towards particular AM fungi.

Strong primer bias for Tulasnellaceae fungi in metabarcoding: Specific primers improve the characterization of the mycorrhizal communities of epiphytic orchids.

Primer bias toward Tulasnellaceae fungi during PCR is a known issue with metabarcoding analyses for the assessment of orchid mycorrhizal communities. However, this bias had not been evaluated for the fungal communities of epiphytic orchids, which account for 69% of all orchid species diversity. We compared the mycorrhizal communities detected using two primer pairs, a fungal universal primer pair (ITS86F/ITS4) and Tulasnella-specific primer pair (5.8STulngs/ITS4-Tul2), using a mock community of fungal isolates from epiphytic orchids and also environmental samples, including orchid roots and a tree bark tip from the host tree of an epiphytic orchid collected. The detected mycorrhizal communities differed widely depending on the primer pairs used. The fungal universal primer pair successfully identified Ceratobasidiaceae and Serendipitaceae fungi but did not reflect Tulasnellaceae diversity. Tulasnellaceae fungi were mainly detected using the Tulasnella-specific primer pair. These tendencies were observed in both the mock community and environmental samples. These results strongly suggest that the use of a Tulasnella-specific primer in combination with a fungal universal primer is essential for assessing the mycorrhizal communities of orchids through metabarcoding analysis, especially in epiphytic orchids. Our study contributes to further understanding of the diversity of mycorrhizal fungi in orchids.

Evolution of host breadth in broad interactions: mycorrhizal specificity in East Asian and North American rattlesnake plantains (Goodyera spp.) and their fungal hosts.

Host breadth is often assumed to have no evolutionary significance in broad interactions because of the lack of cophylogenetic patterns between interacting species. Nonetheless, the breadth and suite of hosts utilized by one species may have adaptive value, particularly if it underlies a common ecological niche among hosts. Here, we present a preliminary assessment of the evolution of mycorrhizal specificity in 12 closely related orchid species (genera Goodyera and Hetaeria) using DNA-based methods. We mapped specificity onto a plant phylogeny that we estimated to infer the evolutionary history of the mycorrhiza from the plant perspective, and hypothesized that phylogeny would explain a significant portion of the variance in specificity of plants on their host fungi. Sampled plants overwhelmingly associated with genus Ceratobasidium, but also occasionally with some ascomycetes. Ancestral mycorrhizal specificity was narrow in the orchids, and broadened rarely as Goodyera speciated. Statistical tests of phylogenetic inertia suggested some support for specificity varying with increasing phylogenetic distance, though only when the phylogenetic distance between suites of fungi interacting with each plant taxon were taken into account. These patterns suggest a role for phylogenetic conservatism in maintaining suits of fungal hosts among plants. We stress the evolutionary importance of host breadth in these organisms, and suggest that even generalists are likely to be constrained evolutionarily to maintaining associations with their symbionts.