Microscopic investigations revealed that thick rhizomes were densely colonized by fungi bearing clamp-connections and dolipores, i.e. basidiomycetes. Molecular analysis identified Inocybe species as exclusive symbionts of 75 % of the plants investigated and, more rarely, other basidiomycetes (Hebeloma, Xerocomus, Lactarius, Thelephora species). Additionally, ascomycetes, probably endophytes or parasites, were sometimes present. Although E. aphyllum associates with diverse species from Inocybe subgenera Mallocybe and Inocybe sensu stricto, no evidence for cryptic speciation in E. aphyllum was found. Since basidiomycetes colonizing the orchid are ectomycorrhizal, surrounding trees are probably the ultimate carbon source. Accordingly, in one population, ectomycorrhizae sampled around an individual orchid revealed the same fungus on 11.2 % of tree roots investigated. Conversely, long, thin stolons bearing bulbils indicated active asexual multiplication, but these propagules were not colonized by fungi. These findings are discussed in the framework of ecology and evolution of mycoheterotrophy.

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C, d15 N. Summary• We investigated the fungal symbionts and carbon nutrition of a Japanese forest photosynthetic orchid, Platanthera minor, whose ecology suggests a mixotrophic syndrome, that is, a mycorrhizal association with ectomycorrhiza (ECM)-forming fungi and partial exploitation of fungal carbon.• We performed molecular identification of symbionts by PCR amplifications of the fungal ribosomal DNA on hyphal coils extracted from P. minor roots. We tested for a 13 C and 15 N enrichment characteristic of mixotrophic plants. We also tested the ectomycorrhizal abilities of orchid symbionts using a new protocol of direct inoculation of hyphal coils onto roots of Pinus densiflora seedlings.• In phylogenetic analyses, most isolated fungi were close to ECM-forming Ceratobasidiaceae clades previously detected from a few fully heterotrophic orchids or environmental ectomycorrhiza surveys. The direct inoculation of fungal coils of these fungi resulted in ectomycorrhiza formation on P. densiflora seedlings. Stable isotope analyses indicated mixotrophic nutrition of P. minor, with fungal carbon contributing from 50% to 65%.• This is the first evidence of photosynthetic orchids associated with ectomycorrhizal Ceratobasidiaceae taxa, confirming the evolution of mixotrophy in the Orchideae orchid tribe, and of ectomycorrhizal abilities in the Ceratobasidiaceae. Our new ectomycorrhiza formation technique may enhance the study of unculturable orchid mycorrhizal fungi.

Mycorrhizal fungi were isolated from the nonphotosynthetic orchid Chamaegastrodia sikokiana and identified as members of Ceratobasidiaceae by phylogenetic analysis of the internal transcribed spacer (ITS) region of ribosomal deoxyribonucleic acid. The ITS sequences were similar among geographically separated samples obtained from Mt. Kiyosumi in Chiba Prefecture and Mt. Yokokura in Kochi Prefecture. One of the isolated fungi, KI1-2, formed ectomycorrhiza on seedlings of Abies firma in pot culture, suggesting that tripartite symbiosis exists among C. sikokiana, mycorrhizal fungi, and A. firma in nature, and carbon compounds are supplied from A. firma to C. sikokiana through the hyphae of the mycorrhizal fungi. To our knowledge, this is the second study to suggest the involvement of Ceratobasidiaceae fungi in tripartite symbiosis with achlorophyllous orchids and photosynthetic host plants.

This report is the first of a mycorrhizal symbiosis between a fungus in Psathyrellaceae and a photosynthetic orchid, revealing a new pathway to full mycoheterotrophy and contributing to our understanding of the evolution of mycoheterotrophy.