Clonal spore populations in sporocarps of arbuscular mycorrhizal fungi.

Some arbuscular mycorrhizal (AM) fungal species known to form sporocarps (i.e., aggregations of spores) are polyphyletic in two orders, Glomerales and Diversisporales. Spore clusters (sporocarp-like structures) often formed in pot cultures or in vitro conditions are supposed to be clonal populations, while sporocarps in natural habitats with a fungal peridium are morphologically similar to those of epigeous sexual (zygosporic) sporocarps of Endogone species. Thus, in this study, we explored the genetics of sporocarpic spores of two AM fungi with a view to possibilities of clonal or sexual reproduction during sporocarps formation. To examine these possibilities, we investigated single-nucleotide polymorphisms (SNPs) in reduced genomic libraries of spores isolated from sporocarps molecularly identified as Rhizophagus irregularis and Diversispora epigaea. In addition, partial sequences of the MAT locus HD2 gene of R. irregularis were phylogenetically analyzed to determine the nuclear status of the spores. We found that most SNPs were shared among the spores isolated from each sporocarp in both species. Furthermore, all HD2 sequences from spores isolated from three R. irregularis sporocarps were identical. These results indicate that those sporocarps comprise clonal spores. Therefore, sporocarps with clonal spores may have different functions than sexual reproduction, such as massive spore production or spore dispersal via mycophagy.

Multilocus phylogenies reveal three new truffle-like taxa and the traces of interspecific hybridization in Octaviania (Boletaceae, Boletales).

Among many convergently evolved sequestrate fungal genera in Boletaceae (Boletales, Basidiomycota), the genus Octaviania is the most diverse. We recently collected many specimens of Octaviania subg. Octaviania, including several undescribed taxa, from Japan and the Americas. Here we describe two new species in subgenus Octaviania, O. tenuipes and O. tomentosa, from temperate to subtropical evergreen Fagaceae forests in Japan based on morphological observation and robust multilocus phylogenetic analyses (nrDNA ITS and partial large subunit [LSU], translation elongation factor 1-α gene [TEF1] and the largest subunit of RNA polymerase II gene [RPB1]). Based on specimens from the Americas as well as studies of the holotype, we also taxonomically re-evaluate O. asterosperma var. potteri. Our analysis suggests that O. asterosperma var. potteri is a distinct taxon within the subgenus Octaviania so we recognize this as O. potteri stat. nov. We unexpectedly collected O. potteri specimens from geographically widespread sites in the USA, Japan and Colombia. This is the first verified report of Octaviania from the South American continent. Our molecular analyses also revealed that the RPB1 sequence of one O. tenuipes specimen was identical to that of a closely related species, O. japonimontana, and that one O. potteri specimen from Minnesota had an RPB1 sequence of an unknown species of O. subg. Octaviania. Additionally, one O. japonimontana specimen had an unusually divergent TEF1 sequence. Gene-tree comparison and phylogenetic network analysis of the multilocus dataset suggest that these heterogenous sequences are most likely the result of previous inter- and intra-specific hybridization. We hypothesize that frequent hybridization events in Octaviania may have promoted the high genetic and species diversity found within the genus.

Tuber iryudaense and T. tomentosum: Two new truffles encased in tomentose mycelium from Japan.

We describe two new Japanese truffle species, Tuber iryudaense and Tuber tomentosum, based on molecular and morphological analyses. Both species are clearly distinguishable from other Tuber species by the ocher tomentose mycelium covering the ascoma surfaces. Tuber iryudaense has one-spored asci that each contain a large (68-97 × 51-80 µm), reddish-brown ascospore; these microscopic characters are similar to those of closely related Chinese species, T. calosporum, T. gigantosporum, T. glabrum, T. monosporum, and T. sinomonosporum. Tuber tomentosum forms one to four ascospores per ascus with a reddish-brown color similar to that of ascospores of T. macrosporum and T. canaliculatum, although their spores are much larger than those of T. tomentosum (27‒64 × 26‒55 µm). Molecular phylogenetic analyses based on ribosomal internal transcribed spacer and partial 28S nuc rDNA sequences support that both species are distinct within the Macrosporum group.

Longistriata flava (Boletaceae, Basidiomycota) - a new monotypic sequestrate genus and species from Brazilian Atlantic Forest.

A new monotypic sequestrate genus, Longistriata is described based on collections from the Neotropical forest of Atlantic forest in Paraíba, Northeast Brazil - an area known for its high degree of endemism. The striking features of this new fungus are the hypogeous habit, the vivid yellow peridium in mature basidiomes, broadly ellipsoid basidiospores with a distinct wall that is ornamented with longitudinal striations and lageniform cystidia with rounded apices. Phylogenetic analysis, based on LSU and tef-1α regions, showed that the type species, Longistriata flava, is phylogenetically sister to the monotypic sequestrate African genus Mackintoshia in Boletaceae. Together these two species formed the earliest diverging lineage in the subfamily Zangioideae. Longistriata flava is found in nutrient-poor white sand habitats where plants in the genera Coccoloba (Polygonaceae) and Guapira (Nyctaginaceae) are the only potential ectomycorrhizal host symbionts.

Type studies of Rossbeeverabispora, and a new species of Rossbeevera from south China.

The type of Rossbeeverabispora and additional collections from the type location and adjacent areas were studied. Molecular data for R.bispora derived from the new collections are provided. In addition, R.griseobrunnea, a new species of Rossbeevera, is described from Xiangtoushan National Nature Reserve, Guangdong Province of China. The new species is characterized by its globose to subglobose sequestrate basidiomata with grayish white to grayish brown pileus, pale bluish discoloration in some parts of the hymenophore when injured becoming rusty brown to dark brown after being exposed to the air, fusoid (star-shaped in cross section) basidiospores 17-20 × 9-12 µm, and subcutis elements in the pileus. Based on multi-locus (ITS+nrLSU+tef1-α+rpb2) molecular phylogenetic analyses, both species appear as sister to R.paracyanea. We present color photos, macro- and micro-description, SEM basidiospores, molecular affinities of the species and compare them with morphologically similar taxa within the genus. A key to the species known from northern and southern hemispheres is provided.

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

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.

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.

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.