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.

Community of arbuscular mycorrhizal (AM) fungi in a coastal vegetation on Okinawa island in Japan was examined. A sampling plot was established in a colony of Ipomoea pes-caprae (Convolvulaceae) on the beach in Tamagusuku, Okinawa Pref, in which eight root samples of I. pes-caprae and three root samples each of Vigna marina (Leguminosae) and Paspalum distichum (Poaceae) were collected. Partial 18S rDNA of AM fungi was amplified from the root samples by polymerase chain reaction (PCR) with primers NS31 and AM1. Restriction fragment length polymorphism analysis with HinfI and RsaI for cloned PCR products revealed that two types of Glomus sp., type A and type B, were dominant in the colony. Among them, the fungi of type A were especially dominant near the edge of the colony facing the sea. A phylogenetic analysis showed that the AM fungi of type B are closely related to Glomus intraradices and those of type A are nearly related to type B. From the sequence data, it was also found that type A was further divided into two types, type A1 and A2. One representative strain each of the three types, type A1, A2, and B, propagated from single spore each, was examined for the growth of sorghum (Sorghum bicolor) at three different salinity levels, 0, 100, and 200 mM NaCl. At the non-salt-treated condition, the type B fungus was the most effective on shoot growth enhancement of the host plant, whereas at the salt-treated conditions, the type A2 fungus was the most effective. An efficient suppression of Na + translocation into the shoot by the examined AM fungi was found. These results suggested that the AM fungi dominant near the sea are adapted to salt-stressed environment to alleviate the salt stress of host plants.

The sequestrate fungi of Japan, including truffle and truffle-like fungi, have not been well characterized but are potentially diverse. We investigated the diversity and phylogeny of Japanese Octaviania specimens using a multifaceted approach including scanning and transmission electron microscopy as well as analysis of nuclear ribosomal DNA (ITS and LSU) and EF-1α (tef1) sequences. Phylogenetic analyses indicate that the genus Octaviania is divided into three major clades, and that there are at least 12 species-level lineages in Japan. Accordingly, we describe two new subgenera, Parcaea and Fulvoglobus, and eleven new species. Subgenus Parcaea accommodates four highly divergent, but macromorphologically almost indiscernible cryptic species. We discuss not only the diversity and species delimitation within the genus Octaviania but also the phylogeography of the Japanese taxa and their relatives.