Running head: Yagame et al. Symbiosis between Psathyrellaceae and photosynthetic orchid IDENTIFICATION AND SYMBIOTIC ABILITY OF PSATHYRELLACEAE FUNGI ISOLATED FROM A PHOTOSYNTHETIC ORCHID, CREMASTRA APPENDICULATA (D. DON) MAKINO (ORCHIDACEAE)

Premise of the study: Photosynthetic orchids found in highly shaded forests are often mixotrophic, receiving part of their carbon energy via ectomycorrhizal fungi that had originally received carbohydrate from trees. A photosynthetic orchid, Cremastra appendiculata is also found under highly shaded forest, but our preliminary data suggested that its associated fungi were not ectomycorrhizal. We suspected this might be an unusual example of a mixotrophic orchid associating with saprobic fungi. Methods: For isolated mycobionts of C. appendiculata plants, two regions of nuclear ribosomal DNA, the internal transcribed spacer (ITS) and the large subunit (LSU), were sequenced and fruiting bodies of the one isolate, SI1-1 were induced. In addition, the two fungal isolates, SI1-1 and KI1-1 were grown in symbiotic cultures with C. appendiculata to verify their ability as mycobionts. Key results: In phylogenetic analyses, all isolates clustered with fungi belonging to Coprinellus in Psathyrellaceae of Agaricales. Phylogenetic analyses of these DNA sequences showed that five fungal isolates from C. appendiculata, including SI1-1 and two mycobionts isolated from the mycoheterotrophic orchid Epipogium roseum, have very similar ITS sequences. The fungal isolate, SI1-1 was identified as Coprinellus domesticus based on the morphological characteristics of the fruiting body. The two fungal isolates, SI1-1 and KI1-1 induced seed germination of C. appendiculata as mycobionts. Conclusions: This is the first report of relationships between fungi in Psathyrellaceae and photosynthetic orchids, in which new pathway to fully mycoheterotrophy could be revealed. The newly found mycorrhizal symbiosis could contribute to our understanding of evolution of mycoheterotrophy. Yagame et al. Page 4