Takamichi Orihara

Mixotrophy of Platanthera minor, an orchid associated with ectomycorrhiza‐forming Ceratobasidiaceae fungi

• 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.

The sequestrate genus Rosbeeva T.Lebel & Orihara gen. nov. (Boletaceae) from Australasia and Japan: new species and new combinations

The sequestrate genus Chamonixia has been shown to have affinities to the Boletales, in particular the genus Leccinum. Australasian and Japanese species of Chamonixia were examined using morphological and molecular (ITS and nLSU rDNA) data and found to also have affinities with Leccinum and Leccinellum, however they form a distinct clade separate from the European type species C. caespitosa Rolland and North American species. A new genus, Rosbeeva T.Lebel & Orihara gen. nov., is proposed for the Australasian, Japanese and Chinese taxa. The species R. mucosa (Petri) T.Lebel comb. nov. is restricted in distribution to Singapore and Borneo, and R. pachyderma (Zeller & C.W. Dodge) T.Lebel comb. nov. to New Zealand, with Australian collections considered to belong to a revised R. vittatispora (G.W.Beaton, Pegler & T.W.K.Young) T.Lebel comb. nov. or a new species R. westraliensis T. Lebel sp. nov. The Chinese species R. bispora (B.C.Zhang & Y.N.Yu) T.Lebel & Orihara comb. nov is transferred to the new genus based upon morphological data. Two new species from Japan, Rosbeeva eucyanea Orihara and R. griseovelutina Orihara, are also described and illustrated. A key to all species of Rosbeeva is provided. Due to the highly modified gastroid sporocarp forms of both Chamonixia and Rosbeeva, many macroscopic characters of use in agaricoid taxonomy are difficult to interpret. However, color change and texture of sporocarps are of some use to distinguish genera and species. Microscopic characters such as spore shape, dimensions, and ornamentation, and pileipellis and hymenophoral trama structure, are essential for determining genera and species.

Taxonomic reconsideration of a sequestrate fungus, Octaviania columellifera, with the proposal of a new genus, Heliogaster, and its phylogenetic relationships in the Boletales

During taxonomic revision of genus Octaviania in Japan we examined herbarium and fresh specimens of O. columellifera and O. asterosperma sensu S. Yoshimi & Y. Doi with morphological and molecular techniques. These two species were identical in both macro- and micromorphological characters and were clearly different from the generally known O. asterosperma. The identity of the two species and their distinctness from O. asterosperma was further supported by both nuclear large subunit and ITS rDNA phylogeny. The molecular analyses also revealed that O. columellifera shares its lineage with the boletoid mushroom-forming Xerocomus chrysenteron complex and that it does not form a monophyletic clade with other Octaviania species. Our morphological reevaluation, including transmission electron microscopic observation of basidiospores, clarified the taxonomic boundary between O. columellifera and other Japanese Octaviania species. Accordingly we propose a new genus, Heliogaster, for O. columellifera with designation of the lectotype. We discuss phylogenetic relationships with Octaviania sensu stricto species and the closely related boletoid (pileate-stipitate) fungi, generic characters of Heliogaster and intraspecific phylogeny.

Diversity and systematics of the sequestrate genus Octaviania in Japan: two new subgenera and eleven new species

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.

Evolutionary history of the sequestrate genus Rossbeevera (Boletaceae) reveals a new genus Turmalinea and highlights the utility of ITS minisatellite-like insertions for molecular identification

The sequestrate (truffle-like) basidiomycete genera Rossbeevera, Chamonixia, and Octaviania are closely related to the epigeous mushroom genera Leccinum and Leccinellum. In order to elucidate the properties and placement of several undescribed sequestrate taxa in the group and to reveal the evolutionary history of Rossbeevera and its allies, we conducted phylogenetic analyses based on three nuclear (ITS, nLSU, EF-1α) and two mitochondrial DNA loci (ATP6 and mtSSU) as well as precise morphological observations. Phylogenetic analyses of three nuclear loci suggest a complex evolutionary history with sequestrate fruiting bodies present in several clades, including a previously unrecognized sister clade to Rossbeevera. Here we propose a new sequestrate genus, Turmalinea, with four new species and one new subspecies as well as two new species of Rossbeevera. The three-locus nuclear phylogeny resolves species-level divergence within the Rossbeevera-Turmalinea lineage, whereas a separate phylogeny based on two mitochondrial genes corresponds to geographic distance within each species-level lineage and suggests incomplete lineage sorting (ILS) and gene introgression within several intraspecific lineages of Rossbeevera. Furthermore, topological incongruence among the three nuclear single-locus phylogenies suggests that ancient speciation within Rossbeevera probably involved considerable ILS. We also found an unusually long, minisatellite-like insertion within the ITS2 in all Rossbeevera and Turmalinea species. A barcode gap analysis demonstrates that the insertion is more informative for discrimination at various taxonomic levels than the rest of the ITS region and could therefore serve as a unique molecular barcode for these genera.

Rossbeevera yunnanensis (Boletaceae, Boletales), a new sequestrate species from southern China

A new sequestrate (truffle-like) species, Rossbeevera yunnanensis, is described based on a collection from Chuxiong Prefecture, Yunnan Province, China. The species is morphologically characterized by its large, narrow, fusoid to fusiform basidiospores and remarkably thin peridium. Maximum likelihood and neighbor joining phylogenies of our nLSU rDNA dataset indicate that the species constitutes the earliest diverging lineage within the genus Rossbeevera and has a close phylogenetic relationship to species of Leccinellum. Morphological and phylogenetic relationships to the other Asian members of Rossbeevera are discussed.

Erratum to: The sequestrate genus Rossbeevera T.Lebel & Orihara gen. nov. (Boletaceae) from Australasia and Japan: new species and new combinations

We wish to correct a spelling error in the original publication for a newly described genus honouring the New Zealand mycologist Ross Beever. The correct spelling is Rossbeevera T. Lebel & Orihara gen. nov. A list of the species names follows.

Unique phylogenetic position of the African truffle-like fungus, Octaviania ivoryana (Boletaceae, Boletales), and the proposal of a new genus, Afrocastellanoa

ABSTRACT The sequestrate (truffle-like) basidiomycete Octaviania ivoryana was originally described based on collections from Zimbabwe, Kenya, Guinea, and Senegal. This species has basidiomes that stain blue-green and basidiospores with crowded spines that are characteristic of the genus Octaviania. However, O. ivoryana is the only Octaviania species described from sub-Saharan Africa, and the phylogenetic relationship of the species to other species of Octaviania sensu stricto has not been previously investigated. We examined the phylogenetic position of the isotype and paratype specimens of O. ivoryana based on two nuc rDNA loci—ITS1-5.8S-ITS2 (internal transcribed spacer [ITS]) and partial 28S—and the translation elongation factor 1-α gene. The resultant phylogenies indicate that O. ivoryana does not belong to Octaviania s. s. but instead forms a clade with the epigeous bolete genus, Porphyrellus sensu stricto (i.e., P. porphyrosporus and allies). The internal transcribed spacer phylogeny also recovers a monophyletic clade that includes sequences from O. ivoryana basidiomes as well as sequences from ectomycorrhizal root tips of Uapaca, Anthonotha, and assorted ectomycorrhizal Fabaceae species, suggesting that there is likely additional undescribed diversity within the lineage. We accordingly propose a new genus, Afrocastellanoa M.E. Sm. & Orihara, to accommodate the species O. ivoryana. Afrocastellanoa is morphologically distinct from Octaviania in the combination of a solid gleba, multilayered peridium, and the lack of distinct hymenium within the gleba. Our data suggest that the genus Afrocastellanoa is a unique sequestrate lineage with one described species and several undescribed species, all of which likely form ectomycorrhizas with African trees.

A molecular and morphological re-examination of the generic limits of truffles in the tarzetta-geopyxis lineage – Densocarpa, Hydnocystis, and Paurocotylis

Truffle species within the /tarzetta-geopyxis lineage share smooth, globose, hyaline spores, but differ in the amount of convolution of hymenia in ascomata. The relationships among truffle species in this lineage have historically been confused. Phylogenetic analyses of the ITS and 28S nuclear ribosomal DNA from recently collected members of the /tarzetta-geopyxis lineage from Asia, Austral Asia, North America, and South America prompted a reinvestigation of species and generic limits in the truffle genera Hydnocystis, Paurocotylis, and Stephensia. Our analyses support emendations of Hydnocystis and Paurocotylis, abandonment of Stephensia and the resurrection of the genus Densocarpa. Nomenclatural changes include the transfer of Stephensia bombycina to Hydnocystis, the transfer of Hydnocystis singeri and Stephensia bynumii to Paurocotylis, the reinstatement of Densocarpa for Stephensia shanori and transfer of Stephensia crocea to Densocarpa. This is the first detection of the genus Paurocotylis in the Americas. We describe three new species, Hydnocystis transitoria from North America, Paurocotylis patagonica from South America, and Paurocotylis watlingii from Australia. Our work highlights the unexplored diversity, morphological plasticity, and remaining taxonomic problems among truffles in the /tarzetta-geopyxis lineage.