DNA isolation and genome sequence of the 134-year-old holotype specimen of Boletus subvelutipes Peck.

Molecular characterization of type specimens is a powerful tool used in clarifying species identity/circumscription, as well as establishing the taxonomic and phylogenetic status of organisms in question. However, DNA sequencing of aged herbarium collections can be a challenge due to the quantity and quality of DNA still present in the specimens. Herein, we report a custom DNA isolation protocol suitable for processing minute quantities of old specimen tissue and its utilization via high-throughput sequencing technologies to obtain, for the first time, the genome assembly of the 134-year-old holotype of Boletus subvelutipes Peck, a North American fleshy pored mushroom of taxonomic and historical significance. A side-by-side evaluation of our DNA isolation method with that of a commercial "kit" by Qiagen is also presented. By relying on the type material, we have established the genetic identity of B. subvelutipes, as well as providing preliminary phylogenetic evidence for its generic affinities in Neoboletus within Boletaceae. The reference genome of the B. subvelutipes holotype provides a resource for future comparative genomic studies, taxonomic revisions in Boletaceae, and other evolutionary studies of fungi.

An Unexpectedly High Number of New Sutorius (Boletaceae) Species From Northern and Northeastern Thailand.

Sutorius is a poroid genus in Boletaceae that typically has chocolate brown to reddish brown or purplish brown basidiomata with a finely scaly stipe and produces a reddish brown spore deposit. During the survey on diversity of boletes in Northern and Northeastern Thailand, several Sutorius collections were obtained. Combined evidence from morphology and phylogenetic analyses of a combined three-gene data set (atp6, tef1 and rpb2) of the Sutorius collections along with selected Boletaceae in the Pulveroboletus group indicated that Thai collections represent seven new Sutorius species. The analyses also indicated that Tylopilus maculatoides belongs in Sutorius. Therefore, the transfer of T. maculatoides to Sutorius is proposed. Full descriptions and illustrations of the seven new species and S. maculatoides are presented in this study. With the seven new species and the new combination, eight of the eleven described Sutorius species are known to occur in Northern and Northeastern Thailand, whereas only one species is known from each of two continents, the Americas and Australia.

Squamanitaceae and three new species of Squamanita parasitic on Amanita basidiomes.

The systematic position of the enigmatically mycoparasitic genus Squamanita (Agaricales, Basidiomycota) together with Cystoderma, Phaeolepiota, Floccularia, and Leucopholiota is largely unknown. Recently they were recognized as Squamanitaceae, but previous studies used few DNA markers from a restricted sample of taxa from the family and lacked a formal taxonomic treatment. In this study, with newly generated sequences of the type of the genus Squamanita, S. schreieri, and several additional species of the family, the phylogeny is reinvestigated with a concatenated (18S-5.8S-nrLSU-RPB2-TEF1-α) dataset. This study reveals that Cystoderma, Phaeolepiota, Squamanita, Floccularia, and Leucopholiota are a monophyletic clade with strong statistical support in Bayesian analysis and form Squamanitaceae. Phaeolepiota nested within Cystoderma; Squamanita, Leucopholiota, and Floccularia clustered together as two monophyletic subclades; and Squamanita was present as a monophyletic clade with strong statistical support in both Maximum Likelihood and Bayesian analyses. The family name Squamanitaceae is formally emended and a detailed taxonomic treatment is presented to accommodate the five genera. Meanwhile, another concatenated (18S-ITS-nrLSU-RPB2-TEF1-α) dataset is used to investigate phylogenetic relationships and species delimitation in Squamanita. Our data indicates that "S. umbonata" from the Northern hemisphere forms two species complexes, one complex includes six specimens from North America, Europe, and East Asia, the other includes two specimens from Central America and North America respectively. Futhermore, species of Squamanita can parasitize species of Amanita, besides other fungal species. Squamanita mira parasitizes A. kitamagotake (A. sect. Caesareae), while S. orientalis and S. sororcula are parasites of species belonging to the A. sepiacea complex (A. sect. Validae). "Squamanita umbonata" from Italy occurs on A. excelsa (A. sect. Validae). Three new species of Squamanita from East Asia, viz. S. mira, S. orientalis and S. sororcula are documented with morphological, multi-gene phylogenetic, and ecological data, along with line drawings and photographs, and compared with similar species. A key for identification of the global Squamanita species is provided.

Two new Erythrophylloporus species (Boletaceae) from Thailand, with two new combinations of American species.

Erythrophylloporus is a lamellate genus in the family Boletaceae that has been recently described from China based on E.cinnabarinus, the only known species. Typical characters of Erythrophylloporus are reddish-orange to yellowish-red basidiomata, including lamellae, bright yellow basal mycelium and smooth, broadly ellipsoid, ellipsoid to nearly ovoid basidiospores. During our survey on diversity of Boletaceae in Thailand, several yellowish-orange to reddish- or brownish-orange lamellate boletes were collected. Based on both morphological evidence and molecular analyses of a four-gene dataset (atp6, tef1, rpb2 and cox3), they were recognised as belonging in Erythrophylloporus and different from the already known species. Two new species, E.paucicarpus and E.suthepensis are therefore introduced from Thailand with detailed descriptions and illustrations. Moreover, two previously described Phylloporus species, P.aurantiacus and P.fagicola, were also revised and recombined in Erythrophylloporus. A key to all known Erythrophylloporus species is provided.

Phylogeny, biogeography and taxonomic re-assessment of Multifurca (Russulaceae, Russulales) using three-locus data.

Multifurca is a small genus newly established to accommodate lactarioid and russuloid species with some characters reminiscent of corticoid members of Russulaceae. It shows an amphi-pacific distribution with strong preference for the tropical zone of the Northern Hemisphere and thus has particular significance for biogeographical study. Using worldwide samples and three loci (ITS, 28S rDNA and rpb2), we demonstrated that Multifurca is split into two highly supported major clades that are here recognized at the subgeneric level: subg. Furcata subg. nov. exclusively includes lactarioid species, while subg. Multifurca includes species with a russuloid habit. Using phylogenetic species recognition and comparison of genetic distances we recognize five new and six previously described species, almost double the known number of species before this study. Molecular dating using a Bayesian method suggested that Multifurca originated in early Paleocene and diversified in the Eocene. The most recent interspecific divergences occurred both in Asia and America, roughly at the same time around the Pliocene. Ancestral area reconstruction and comparisons of genetic distances and morphology suggested an early divergence within Australasia or tropical Asia. From the early Miocene to Pliocene, multiple dispersals/migrations to Australasia and North America by island hopping or land bridge likely happened. Vicariance at the late Tertiary might be the most likely mechanism accounting for the eastern Asia-southeastern North America and Australasia-tropical Asia disjunct distributions. The shared polymorphisms in the ITS alignment, numerous degenerated base pairs in the rpb2 sequences and weak conflict between the ITS and LSU genealogies of M. subg. Furcata suggest recent speciation. Host specificity of Multifurca species or species pairs is relatively low. Host shifts are believed to have aided establishment in new territories during the dispersals and migrations.

A global view of Gyroporus: molecular phylogenetics, diversity patterns, and new species.

Gyroporus (Gyroporaceae, Boletales) is a highly diverse genus of poroid ectomycorrhizal mushrooms with a nearly worldwide distribution. Previous attempts to unravel the diversity within this genus proved difficult due to the presence of semicryptic species and ambiguous results from analysis of ribosomal RNA markers. In this study, we employ a combined morphotaxonomic and phylogenetic approach to delimit species and elucidate geographic and evolutionary patterns in Gyroporus. For phylogenetic analyses, the protein-coding genes atp6 (mitochondrial adenosine triphosphate [ATP] synthase subunit 6) and rpb2 (nuclear second largest subunit of RNA polymerase II) were selected based on their utility in studies of Boletales. We infer several distinct clades, most notably one corresponding to G. castaneus as a speciose Northern Hemisphere group, another unifying G. cyanescens and like entities, and a third group unifying G. longicystidiatus and a New World sister species. Also notable is the recovery of a sister relationship between the cyanescens and longicystidiatus clades. We formally describe five new species of Gyroporus, outline a number of provisional species, and briefly discuss distributional patterns. This study provides an important scaffold for future work on this well-known but poorly understood genus of fungi.

The Macrofungi Collection Consortium.

PREMISE OF THE STUDY: The Macrofungi Collection Consortium (MaCC) is a digitization project funded by the National Science Foundation's Advancing Digitization of Biodiversity Collections program. The main scientific objective of the MaCC project was to provide baseline data for determining the extent and distribution of macrofungal diversity. METHODS AND RESULTS: Between 2012 and 2017, 39 participating institutions digitized approximately 1,250,000 specimens of macrofungi from U.S. herbaria. These newly digitized data, combined with existing data and contributions from the Microfungi Collections Consortium, have created a database of approximately 3.4 million specimen records that are shared online through MyCoPortal, a Symbiota-based data portal. In addition to the digitized herbarium specimen data, MyCoPortal also contains descriptions, illustrations, and observational records. DISCUSSION: The database of digitized specimen data created through this project is a resource for both amateur and professional mycologists. The data provided through MyCoPortal will provide a foundation for a comprehensive Mycoflora of North America. Such a project is now under development as a collaboration between the professional and amateur mycological communities, with the goal of documenting the macrofungi of North America with gene sequences as well as phenotypic descriptions and images.

New Porcini (Boletus sect. Boletus) from Australia and Thailand.

Boletus albobrunnescens and B. austroedulis are described as new species in section Boletus from Thailand and Australia respectively. The former is easily characterized by the pure white basidiomata that stain brown. Boletus austroedulis has a gray-brown, slightly rugulose pileus with hymeniform pileipellis producing pileocystidia, and the stipe is only apically reticulate if at all. These new species represent ancient lineages inferred from prior molecular phylogenetic analyses.

Phylogenetic overview of the Boletineae.

The generic and sub-generic relationships in the Boletineae (Boletales) were studied using nuclear large subunit (nuc-lsu), translation elongation factor 1-alpha (tef1), and DNA directed RNA polymerase largest subunit (RPB1). The Boletineae, with the exclusion of Hydnomerulius pinastri, was strongly supported and the status of the families Boletaceae and Paxillaceae is discussed. Members of the genus Boletus are found throughout the phylogeny, with the majority not closely related to the type species, Boletus edulis. Many of the traditional, morphologically defined genera are not supported as monophyletic and additional sampling and taxonomic revisions are needed. The majority of the Boletineae are confirmed or putatively ectomycorrhizal (ECM), but two putatively mycoparasitic lineages (one lineage of Buchwaldoboletus lignicola and Chalciporus piperatus and the second Pseudoboletus parasiticus) are strongly supported.

Australasian sequestrate fungi 18: Solioccasus polychromus gen. & sp. nov., a richly colored, tropical to subtropical, hypogeous fungus.

Solioccasus polychromus gen. & sp. nov., the most brightly colored hypogeous fungus known, is described from Papua New Guinea and tropical northern Australia south into subtropical forests along the Queensland coast and coastal mountains to near Brisbane. Phylogenetic analysis of molecular data places it as a sister genus to Bothia in the Boletineae, a clade of predominantly ectomycorrhizal boletes. Ectomycorrhizal trees, such as members of the Myrtaceae (Eucalyptus, Corymbia, Lophostemon, Melaleuca spp.) and Allocasuarina littoralis, were present usually in mixture or in some cases dominant, so we infer some or all of them to be among the ectomycorrhizal hosts of S. polychromus.

Sutorius: a new genus for Boletus eximius.

Sutorius is described as a new genus of Boletaceae to accommodate Boletus robustus originally named illegitimately by C.C. Frost from eastern North America. The legitimate name, Boletus eximius, provided by C.H. Peck, has been used since for a dark purple to chocolate brown bolete with finely scaly stipe and reddish brown spore deposit. This iconic taxon has been documented on five continents. Despite the straightforward species identification from morphology, the interpretation of stipe macro-morphology and spore color has led to equivocal generic placement. Phylogenetic analyses of genes encoding large subunit rRNA and translation elongation factor 1α confirm Sutorius as a unique generic lineage in the Boletaceae. Two species are recognized based on multiple accessions: S. eximius, represented by collections from North America, Costa Rica, Guyana, Indonesia and Japan (molecular data are lacking for only the Guyanan and Japanese material); and S. australiensis, represented by material from Queensland, Australia. Additional collections from Zambia and Thailand represent independent lineages, but sampling is insufficient to describe new species for these entities.

Molecular phylogenetics of porcini mushrooms (Boletus section Boletus).

Porcini (Boletus section Boletus: Boletaceae: Boletineae: Boletales) are a conspicuous group of wild, edible mushrooms characterized by fleshy fruiting bodies with a poroid hymenophore that is "stuffed" with white hyphae when young. Their reported distribution is with ectomycorrhizal plants throughout the Northern Hemisphere. Little progress has been made on the systematics of this group using modern molecular phylogenetic tools because sampling has been limited primarily to European species and the genes employed were insufficient to resolve the phylogeny. We examined the evolutionary history of porcini by using a global geographic sampling of most known species, new discoveries from little explored areas, and multiple genes. We used 78 sequences from the fast-evolving nuclear internal transcribed spacers and are able to recognize 18 reciprocally monophyletic species. To address whether or not porcini form a monophyletic group, we compiled a broadly sampled dataset of 41 taxa, including other members of the Boletineae, and used separate and combined phylogenetic analysis of sequences from the nuclear large subunit ribosomal DNA, the largest subunit of RNA polymerase II, and the mitochondrial ATPase subunit six gene. Contrary to previous studies, our separate and combined phylogenetic analyses support the monophyly of porcini. We also report the discovery of two taxa that expand the known distribution of porcini to Australia and Thailand and have ancient phylogenetic connections to the rest of the group. A relaxed molecular clock analysis with these new taxa dates the origin of porcini to between 42 and 54 million years ago, coinciding with the initial diversification of angiosperms, during the Eocene epoch when the climate was warm and humid. These results reveal an unexpected diversity, distribution, and ancient origin of a group of commercially valuable mushrooms that may provide an economic incentive for conservation and support the hypothesis of a tropical origin of the ectomycorrhizal symbiosis.

Study on species of phylloporus I: Neotropics and North America.

Seventeen out of the 24 taxa of Phylloporus (Boletaceae) known from the Neotropics are presented here. Complete descriptions, illustrations and a key to the 17 species are provided. Phylloporus alborufus is newly described, and an unnamed species is also described from Costa Rican oak forests. Phylloporus colligatus was recently described from a Dicymbe forest in Guyana. A table of the 24 known New World Phylloporus taxa, their distribution and possible hosts also is presented.

Fistulinella cinereoalba sp. nov. and new distribution records for Austroboletus from Guyana.

Fistulinella cinereoalba sp. nov., Austroboletus rostrupii, previously known from southeastern Asia, and Austroboletus festivus from Brazilian Amazonia are described for the first time from the Guiana Shield. These boletes were collected from tropical forests dominated by ectomycorrhizal Dicymbe spp. (Fabaceae subfam. Caesalpinioideae) in the Pakaraima Mountains of western Guyana.

New species of Boletellus from Guyana.

Boletellus exiguus sp. nov. and Boletellus dicymbophilus sp. nov. (Boletaceae, Boletales, Basidiomycota) are described as new to science. These boletes were collected from tropical forests dominated by ectomycorrhizal Dicymbe corymbosa (Caesalpiniaceae) in the Pakaraima Mountains of western Guyana.

Pacific boletes: implications for biogeographic relationships.

The obligate association of boletes with their plant partners is critical to understanding biogeographic distribution of these fungi. Only in rare instances are boletes not obligatory associates of plants; the majority are presumed or proven partners in obligate symbioses with a variety of plants. The array of plant-associated distributions provides a potential handle for evaluating bolete distribution on a global scale. However, migration processes remain unclear and distributions are often disjunct. As an illustration of phylogeographic studies of putatively widespread bolete taxa, we present preliminary analyses for Tylopilus ballouii using LSU rDNA and RPB1 sequence data. The LSU data suggest geographic structuring of the tested accessions. However, RPB1 data indicate that long-distance dispersal events (possibly mediated by humans) are possible, or that selection or other factors have obscured geographical patterns. Molecular divergence between samples in RPB1 argues against panmixis, and indicates that populations have been isolated for long periods.

Tylopilus orsonianus sp. nov. and Tylopilus eximius from Guyana.

Tylopilus orsonianus sp. nov. and Tylopilus eximius (Boletaceae, Basidiomycota) are described for the first time from the Pakaraima Mountains of Guyana. Both boletes occur in forests dominated by ectomycorrhizal trees in the genus Dicymbe (Caesalpiniaceae). A key to Tylopilus species distinguishes those known to occur in Guyana.

A new genus of Boletaceae from eastern North America.

Bothia is described as a new genus in the Boletaceae based on Boletinus castanellus described by C.H. Peck from eastern North America. A widespread, occasionally encountered taxon, Bothia castanella possesses a combination of macro- and microscopic features that has prompted past placement in seven different genera. Yet, as a species it is readily recognizable with its chestnut brown, dry pileus, decurrent, pale brown hymenophore with radially elongated tubes, a short, sometimes eccentric, exannulate stipe, yellow brown spore deposit and constant association with Quercus. Phylogenetic analyses of large subunit rDNA and BLAST searches using the ITS region confirm the placement of B. castanella as a unique generic lineage in the Boletaceae.

Morphological and molecular evidence supporting an arbutoid mycorrhizal relationship in the Costa Rican páramo.

This study examines evidence for a particular arbutoid mycorrhizal interaction in páramo, a high-altitude neotropical ecosystem important in hydrological regulation but poorly known in terms of its fungal communities. Comarostaphylis arbutoides Lindley (Ericaceae) often forms dense thickets in Central American páramo habitats. Based on phylogenetic classification, it has been suggested that C. arbutoides forms arbutoid mycorrhizae with diverse Basidiomycetes and Ascomycetes; however, this assumption has not previously been confirmed. Based on field data, we hypothesized an arbutoid mycorrhizal association between C. arbutoides and the recently described bolete Leccinum monticola Halling & G.M. Mueller; in this study, we applied a rigorous approach using anatomical and molecular data to examine evidence for such an association. We examined root samples collected beneath L. monticola basidiomes for mycorrhizal structures, and we also compared rDNA internal transcribed spacer (ITS) sequences between mycorrhizal root tips and leaf or basidiome material of the suspected symbionts. Root cross sections showed a thin hyphal sheath and intracellular hyphal coils typical of arbutoid mycorrhizae. DNA sequence comparisons confirmed the identity of C. arbutoides and L. monticola as the mycorrhizal symbionts. In addition, this paper provides additional evidence for the widespread presence of minisatellite-like inserts in the ITS1 spacer in Leccinum species (including a characterization of the insert in L. monticola) and reports the use of an angiosperm-specific ITS primer pair useful for amplifying plant DNA from mycorrhizal roots without co-amplifying fungal DNA.

Leccinum (boletaceae) in costa rica.

Complete descriptions, illustrations, distributions and a key to species are provided for the eight known Costa Rican species of Leccinum. Leccinum tablense is newly described from oak forests, and Leccinum monticola is newly described from the páramo and adjacent timberline oak forests where it appears to be associated with Comarostaphylis arbutoides-a member of the Ericaceae. All are compared to similar taxa.