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.

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 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.

Evidence for novel and specialized mycorrhizal parasitism: the orchid Gastrodia confusa gains carbon from saprotrophic Mycena.

We investigated the physiological ecology of the Asian non-photosynthetic orchid Gastrodia confusa. We revealed its mycorrhizal partners by using molecular identification and identified its ultimate nutritional source by analysing carbon and nitrogen natural stable isotope abundances. Molecular identification using internal transcribed spacer and large subunit nrDNA sequences showed that G. confusa associates with several species of litter- and wood-decomposer Mycena fungi. The carbon and nitrogen isotope signatures of G. confusa were analysed together with photosynthetic plant reference samples and samples of the ectomycorrhizal epiparasite Monotropa uniflora. We found that G. confusa was highly enriched in (13)C but not greatly in (15)N, while M. uniflora was highly enriched in both (13)C and (15)N. The (13)C and (15)N signatures of G. confusa were the closest to those of the fruit bodies of saprotrophic fungi. Our results demonstrate for the first time using molecular and mass-spectrometric approaches that myco-heterotrophic plants gain carbon through parasitism of wood or litter decaying fungi. Furthermore, we demonstrate that, several otherwise free-living non-mycorrhizal, Mycena can be mycorrhizal partners of orchids.