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 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.

To determine the means and variations in CH4 uptake and N2O emission in the dominant soil and vegetation types to enable estimation of annual gases fluxes in the forest land of Japan, we measured monthly fluxes of both gases using a closed‐chamber technique at 26 sites throughout Japan over 2 years. No clear seasonal changes in CH4 uptake rates were observed at most sites. N2O emission was mostly low throughout the year, but was higher in summer at most sites. The annual mean rates of CH4 uptake and N2O emission (all sites combined) were 66 (2.9–175) µg CH4‐C m−2 h−1 and 1.88 (0.17–12.5) µg N2O‐N m−2 h−1, respectively. Annual changes in these fluxes over the 2 years were small. Significant differences in CH4 uptake were found among soil types (P < 0.05). The mean CH4 uptake rates (µg CH4‐C m−2 h−1) were as follows: Black soil (95 ± 39, mean ± standard deviation [SD]) > Brown forest soil (60 ± 27) ≥ other soils (20 ± 24). N2O emission rates differed significantly among vegetation types (P < 0.05). The mean N2O emission rates (µg N2O‐N m−2 h−1) were as follows: Japanese cedar (4.0 ± 2.3) ≥ Japanese cypress (2.6 ± 3.4) > hardwoods (0.8 ± 2.2) = other conifers (0.7 ± 1.4). The CH4 uptake rates in Japanese temperate forests were relatively higher than those in Europe and the USA (11–43 µg CH4‐C m−2 h−1), and the N2O emission rates in Japan were lower than those reported for temperate forests (0.23–252 µg N2O‐N m−2 h−1). Using land area data of vegetation cover and soil distribution, the amount of annual CH4 uptake and N2O emission in the Japanese forest land was estimated to be 124 Gg CH4‐C year−1 with 39% uncertainty and 3.3 Gg N2O‐N year−1 with 76% uncertainty, respectively.

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 specifi city 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 specifi city and fungal partner shifts among the three species. Furthermore, we conducted multivariate analysis to test for diff erences 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 diff ered among the three species, they also sometimes shared identical fungal species. Multivariate analysis revealed that mycobiont communities of the three species in bamboo thickets diff ered signifi cantly from those in other vegetation types.CONCLUSIONS: Fungal partner shifts are not necessarily associated with the evolution of MHPs, and fungal specifi city of Gc and Gp was signifi cantly higher than that of Gn , implying that the specifi city fl uctuates during speciation. Further, Gc exclusively inhabits bamboo thickets, which suggests that adaptation to particular fungi specifi c to bamboo thickets triggered speciation of this species.KEY WORDS ectomycorrhizal fungi; Gastrodia ; myceoheterotrophy; saprotrophic fungi; speciation; specifi city 208 • AMERICAN JOURNAL OF BOTANY Bruns, 2001 ), Corallorhiza of Orchidaceae ( Taylor and Bruns, 1999 ), Afrothismia of Th ismiaceae, Botrychium of Ophioglossaceae, Gymnosiphon of Burmanniaceae, Voyria of Gentianaceae, and Sciaphila of Triuridaceae ( Merckx et al., 2012 ). Th e fi rst two studies cited above revealed that fungal partner shift s occurred among closely related species within an MHP genus, whereas the third study confi rmed both patterns: partner shift s occurred in Gymnosiphon but not in the other four MHP genera examined. Th ese results indicate that partner shift s are not indispensable in the diversifi cation of MHPs. However, low sampling densities of plant species in some studies (e.g., Merckx et al., 2012 ) may have led to the diversity of mycobionts having been overlooked-and, thus, partner shift s may not be as common as currently thought. Meanwhile, changes in specifi city levels (i.e., phylogenetic breadth) of mycobionts among closely related MHP species have mostly been compared only for dominant fungal partners-for example, for the Corallorhiza striata complex ( Barrett ...

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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.