A global phylogenomic analysis of the shiitake genus Lentinula.

Lentinula is a broadly distributed group of fungi that contains the cultivated shiitake mushroom, L. edodes. We sequenced 24 genomes representing eight described species and several unnamed lineages of Lentinula from 15 countries on four continents. Lentinula comprises four major clades that arose in the Oligocene, three in the Americas and one in Asia-Australasia. To expand sampling of shiitake mushrooms, we assembled 60 genomes of L. edodes from China that were previously published as raw Illumina reads and added them to our dataset. Lentinula edodes sensu lato (s. lat.) contains three lineages that may warrant recognition as species, one including a single isolate from Nepal that is the sister group to the rest of L. edodes s. lat., a second with 20 cultivars and 12 wild isolates from China, Japan, Korea, and the Russian Far East, and a third with 28 wild isolates from China, Thailand, and Vietnam. Two additional lineages in China have arisen by hybridization among the second and third groups. Genes encoding cysteine sulfoxide lyase (lecsl) and γ-glutamyl transpeptidase (leggt), which are implicated in biosynthesis of the organosulfur flavor compound lenthionine, have diversified in Lentinula. Paralogs of both genes that are unique to Lentinula (lecsl 3 and leggt 5b) are coordinately up-regulated in fruiting bodies of L. edodes. The pangenome of L. edodes s. lat. contains 20,308 groups of orthologous genes, but only 6,438 orthogroups (32%) are shared among all strains, whereas 3,444 orthogroups (17%) are found only in wild populations, which should be targeted for conservation.

Global phylogeny of the Shiitake mushroom and related Lentinula species uncovers novel diversity and suggests an origin in the Neotropics.

Lentinula (Basidiomycota, Agaricales) includes the most widely cultivated mushroom in the world, Lentinula edodes, also known as shiitake (Japanese) or xiang-gu (Chinese). At present, nine species are recognized in the genus, based on morphology, mating criteria, and geographic distribution. However, analyses of internal transcribed spacers (ITS) of ribosomal RNA genes have suggested that there are cryptic lineages. We analyzed a global-scale phylogenetic dataset from 325 Lentinula individuals from 24 countries in Asia-Australasia and the Americas plus Madagascar, with 325 sequences of ITS, 80 LSU sequences, and 111 sequences of translation elongation factor (tef1-α) genes. We recovered 15 independent lineages (Groups 1-15) that may correspond to species. Lineages in Asia-Australasia (Groups 1-5) and the Americas plus Madagascar (Groups 6-15) formed sister clades. Four lineages are represented only by sequences from single individuals and require further molecular sampling, including L. aff. raphanica (Group 7), L. ixodes (Group 8), L. boryana (Group 12), and L. aff. aciculospora (Group 14). Groups 1 and 5 are here referred to L. edodes and L. aff. edodes, respectively. However, these groups most likely represent the same species and are only recognized as (unsupported) monophyletic lineages by maximum likelihood analyses of ITS alone. Other putative species resolved here include L. lateritia (Group 2), L. novae-zelandieae (Group 3), L. aff. lateritia (Group 4), L. raphanica (Group 6), L. aff. detonsa (Group 9), L. detonsa (Group 10), L. guzmanii sp. nov. (Group 11), L. aciculospora (Group 13), and L. madagasikarensis (Group 15). Groups 9-12 represent the "L. boryana complex". Molecular clock and historical biogeographic analyses suggest that the most recent common ancestor (MRCA) of Lentinula can be placed in the middle Oligocene, ca. 30 million years ago (Ma), and had a likely presence in neotropical America. The MRCA of Lentinula in the Americas and Madagascar lived ca. 22 Ma in the Neotropics and the MRCA of Lentinula in Asia-Australasia lived ca. 6 Ma in Oceania. Given the current knowledge about plate tectonics and paleoclimatic models of the last 30 Myr, our phylogenetic hypothesis suggests that the extant distribution of Lentinula is likely to have arisen, in large part, due to long-distance dispersal. Lentinula collections include at least four dubious taxa that need further taxonomic studies: L. reticeps from the USA (Ohio); L. guarapiensis from Paraguay; Lentinus puiggarii from Brazil (São Paulo); and "L. platinedodes" from Vietnam. Approximately ten of the fifteen Groups are reported on Fagaceae, which appears to be the ancestral substrate of Lentinula.

In Vitro Observations of the Interactions between Pholiota carbonaria and Polytrichum commune and Its Potential Environmental Relevance.

Wildfires play a critical role in maintaining biodiversity and shaping ecosystem structure in fire-prone regions, and successional patterns involving numerous plant and fungal species in post-fire events have been elucidated. Evidence is growing to support the idea that some post-fire fungi can form endophytic/endolichenic relationships with plants and lichens. However, no direct observations of fire-associated fungal-moss interactions have been visualized to date. Therefore, physical interactions between a post-fire fungus, Pholiota carbonaria, and a moss, Polytrichum commune, were visually examined under laboratory conditions. Fungal appressoria were visualized on germinating spores and living protonemata within two weeks of inoculation in most growth chambers. Appressoria were pigmented, reddish gold to braun, and with a penetration peg. Pigmented, reddish gold to braun fungal hyphae were associated with living tissue, and numerous mature rhizoids contained fungal hyphae at six months. Inter-rhizoidal hyphae were pigmented and reddish gold to braun, but no structures were visualized on mature gametophyte leaf or stem tissues. Based on our visual evidence and previous work, we provide additional support for P. carbonaria having multiple strategies in how it obtains nutrients from the environment, and provide the first visual documentation of these structures in vitro.

Secret lifestyles of pyrophilous fungi in the genus Sphaerosporella.

PREMISE: Pyrophilous fungi form aboveground fruiting structures (ascocarps) following wildfires, but their ecology, natural history, and life cycles in the absence of wildfires are largely unknown. Sphaerosporella is considered to be pyrophilous. This study explores Sphaerosporella ascocarp appearance following a rare 2016 wildfire in the Great Smoky Mountains National Park (GSMNP), compares the timing of ascocarp formation with recovery of Sphaerosporella DNA sequences in soils, and explores the association of Sphaerosporella with post-fire Table Mountain pine (Pinus pungens) seedlings. METHODS: Burned sites in the GSMNP were surveyed for pyrophilous fungal ascocarps over 2 years. Ascocarps, mycorrhizae, and endophyte cultures were evaluated morphologically and by Sanger sequencing of the nuclear ribosomal ITS gene region (fungal barcode; Schoch et al., 2012). DNA from soil cores was subjected to Illumina sequencing. RESULTS: The timing and location of post-fire Sphaerosporella ascocarp formation was correlated with recovery of Sphaerosporella DNA sequences in soils. Genetic markers (fungal barcode) of Sphaerosporella were also recovered from mycorrhizal root tips and endophyte cultures from seedlings of Pinus pungens. CONCLUSIONS: This study demonstrates that Sphaerosporella species, in the absence of fire, are biotrophic, forming both mycorrhizal and endophytic associations with developing Pinus pungens seedlings and may persist in nature in the absence of wildfire as a conifer symbiont. We speculate that Sphaerosporella may fruit only after the host plant is damaged or destroyed and that after wildfires, deep roots, needle endophytes, or heat-resistant spores could serve as a source of soil mycelium.

Pyrophilous fungi detected after wildfires in the Great Smoky Mountains National Park expand known species ranges and biodiversity estimates.

Following a late fall wildfire in 2016 in the Great Smoky Mountains National Park, pyrophilous fungi in burn zones were documented over a 2-y period with respect to burn severity and phenology. Nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) barcodes were obtained to confirm morphological evaluations. Forty-one taxa of Ascomycota and Basidiomycota were identified from burn sites and categorized as fruiting only in response to fire or fruiting enhanced by fire. Twenty-two species of Pezizales (Ascomycota) were among the earliest to form ascomata in severe burn zones, only one of which had previously been documented in the Great Smoky Mountains National Park. Nineteen species of Basidiomycota, primarily Agaricales, were also documented. Among these, only five species (Coprinellus angulatus, Gymnopilus decipiens, Lyophyllum anthracophilum, Pholiota carbonicola, and Psathyrella pennata) were considered to be obligate pyrophilous taxa, but fruiting of two additional taxa (Hygrocybe conica and Mycena galericulata) was clearly enhanced by fire. Laccaria trichodermophora was an early colonizer of severe burn sites and persisted through the winter of 2017 and into spring and summer of 2018, often appearing in close association with Pinus pungens seedlings. Fruiting of pyrophilous fungi peaked 4-6 mo post fire then diminished, but some continued to fruit up to 2.5 y after the fire. In all, a total of 27 previously unrecorded taxa were added to the All Taxa Biodiversity Inventory (ATBI) database (~0.9%). Most pyrophilous fungi identified in this study are either cosmopolitan or have a Northern Hemisphere distribution, but cryptic endemic lineages were detected in Anthracobia and Sphaerosporella. One new combination, Hygrocybe spadicea var. spadicea f. odora, is proposed.

Two additional species of Gymnopus (Euagarics, Basidiomycotina).

For more than a decade, a combination of molecular phylogenetic analyses and morphological characterisation has led to a renovation of the Omphalotaceae, especially of Gymnopus sensu lato. Numerous new genera have been proposed, but Gymnopus sensu stricto has also seen an accretion of species and species complexes. In this manuscript, two species are added to Gymnopus sensu stricto within Section Androsacei.

Revision of pyrophilous taxa of Pholiota described from North America reveals four species-P. brunnescens, P. castanea, P. highlandensis, and P. molesta.

A systematic reevaluation of North American pyrophilous or "burn-loving" species of Pholiota is presented based on molecular and morphological examination of type and historical collections. Confusion surrounds application of the names P. brunnescens, P. carbonaria, P. castanea, P. fulvozonata, P. highlandensis, P. molesta, and P. subsaponacea, with multiple names applied to a single species and multiple species described more than once. Molecular annotations using nuc rDNA ITS1-5.8S-ITS2 (internal transcribed spacer [ITS] barcode) and RPB2 (RNA polymerase II second largest subunit) are used to aid in application of these names in a phylogenetic context. Based on ITS molecular annotations of 13 types, the following heterotypic synonymies are proposed: P. highlandensis (syn. P. carbonaria and P. fulvozonata); P. molesta (syn. P. subsaponacea); and P. brunnescens (syn. P. luteobadia). In addition, we observed that the species P. castanea, known previously only from the type collection in Tennessee, is found commonly on burned sites near the Gulf Coast and other southeast regions of the United States. Overall, the pyrophilous trait is evolutionarily derived in Pholiota. Endophytic and endolichenic stages were deduced for P. highlandensis, the most widely distributed of the pyrophilous Pholiota. As a result, we introduce the "body snatchers" hypothesis that explains the maintenance of some pyrophilous fungi in ecosystems as endophytes and/or endolichenic fungi. Photographs, taxonomic descriptions, and a dichotomous key to pyrophilous species of Pholiota that occur in North America are presented.

Intragenomic nuclear RNA variation in a cryptic Amanita taxon.

Amanita cf. lavendula collections in eastern North America, Mexico, and Costa Rica were found to consist of four cryptic taxa, one of which exhibited consistently unreadable nuclear rDNA ITS1-5.8S-ITS2 (fungal barcode) sequences after ITS1 base 130. This taxon is designated here as Amanita cf. lavendula taxon 1. ITS sequences from dikaryotic basidiomata were cloned, but sequences recovered from cloning did not segregate into distinct haplotypes. Rather, there was a mix of haplotypes that varied among themselves predominantly at 28 ITS positions. Analysis of each of these 28 variable bases showed predominantly two alternate bases at each position. Based on these findings and additional sequence data from the nuclear rDNA 28S, RNA polymerase II subunit 2 (RPB2) and mitochondrial rDNA small subunit (SSU) and 23S genes, we speculate that taxon 1 represents an initial hybridization event between two divergent taxa followed by failure of the ribosomal repeat to homogenize. Homogenization failure may be a result of repeated hybridization between divergent internal transcribed spacer (ITS) types with inadequate time for concerted evolution of the ribosomal repeat or, alternately, a complete failure of the ribosomal homogenization process. To our knowledge, this finding represents the first report of a geographically widespread taxon (Canada, eastern USA, Costa Rica) with apparent homogenization failure across all collections. Findings such as these have implications for fungal barcoding efforts and the application of fungal barcodes in identifying environmental sequences.

First report of the post-fire morel Morchella exuberans in eastern North America.

Reports of true morels (Morchella) fruiting on conifer burn sites are common in western North America where five different fire-adapted species of black morels (Elata Clade) have been documented based on multilocus phylogenetic analyses. Fruiting of post-fire morels in eastern North America, by comparison, are rare and limited to a report from Minnesota in 1977 and eastern Ontario in 1991. Here, nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) sequences were used to identify the post-fire morel that fruited in great abundance the year following the 2012 Duck Lake Fire in the Upper Peninsula of Michigan and after the 2016 large-scale fire in the Great Smoky Mountains National Park in Tennessee as M. exuberans. A preliminary phylogenetic analysis suggests that the collections from eastern North America may be more closely related to those from Europe than from western North America, Europe, and China.

Lignomyces, a new genus of pleurotoid Agaricomycetes.

Collections of a pleurotoid fungus from dead aspen in eastern Russia were initially identified as Lentinus sp., then as Phyllotopsis nidulans. DNA sequencing of cultures derived from these specimens using the nuclear ribosomal 28S (nrLSU) and nuclear ribosomal ITS1-5.8S-ITS2 regions (nrITS) showed that they were neither Lentinus nor Phyllotopsis and were not related to other pleurotoid genera Hohenbuehelia and Pleurotus. Subsequent investigation showed that the Russian fungus was the same as Pleurotus vetlinianus described from Poland. A new genus, Lignomyces, is described and characterized and L. vetlinianus comb. nov. is proposed.

The Xeromphalina campanella/kauffmanii complex: species delineation and biogeographical patterns of speciation.

European, North American and northeastern Asian collections of Xeromphalina section Xeromphalina were studied by sequencing the nuc rDNA ITS1-5.8SITS2 and 28S 5' regions and partial RNA polymerase II second largest subunit gene (RPB2). Previously designated Xeromphalina campanella I is designated X. campanella s. str. and a neotype for this species from the topotype region is established. This species is shown to be a discrete, cold-tolerant organism that is distributed across North America and Eurasia and does not exhibit significant geographical partitioning. A second closely related phylogenetic species previously designated X. campanella II, proposed as X. enigmatica, cannot be distinguished from X. campanella morphologically but is reproductively isolated and is sympatric with X. campanella across much of Eurasia and North America. Unlike X. campanella it shows geographical partitioning and some of the geographical populations likely have become reproductively isolated. Phylogenetic and geographical evidence suggests that X. enigmatica may have given rise to the eastern North American endemic, Xeromphalina kauffmanii, which also is reproductively isolated and is characterized by a hardwood substrate and a difference in basidiospore shape. Two putatively interbreeding haplotypes are evident for both eastern North American X. kauffmanii and eastern North American X. enigmatica and might be contributions from different glacial refugia. Cryptic taxa related to X. enigmatica are identified but not named due to small sample sizes including Asian taxa 1-5 and an apparent endemic from Idaho and British Columbia. Several species-delineation procedures were attempted and compared with this complex molecular dataset. Rosenberg's PAB statistic and PID (liberal) were the most liberal, assigning species status to haplotypes or interbreeding clades within species. PID (strict) and PRD (randomly distinct) were more stringent. Ability to intercross was the most stringent criterion for species delineation and did not correlate well with PAB, PID and PRD delineations.

Evolutionary consequences of putative intra-and interspecific hybridization in agaric fungi.

Agaric fungi of the southern Appalachian Mountains including Great Smoky Mountains National Park are often heterozygous for the rDNA internal transcribed spacer region (ITS) with >42% of collections showing some heterozygosity for indels and/or base-pair substitutions. For these collections, intra-individual haplotype divergence is typically less than 2%, but for 3% of these collections intra-individual haplotype divergence exceeds that figure. We hypothesize that high intra-individual haplotype divergence is due to hybridization between agaric fungi with divergent haplotypes, possibly migrants from geographically isolated glacial refugia. Four species with relatively high haplotype divergence were examined: Armillaria mellea, Amanita citrina f. lavendula, Gymnopus dichrous and the Hygrocybe flavescens/chlorophana complex. The ITS region was sequenced, haplotypes of heterozygotes were resolved through cloning, and phylogenetic analyses were used to determine the outcome of hybridization events. Within Armillaria mellea and Amanita citrina f. lavendula, we found evidence of interbreeding and recombination. Within G. dichrous and H. flavescens/chlorophana, hybrids were identified but there was no evidence for F2 or higher progeny in natural populations suggesting that the hybrid fruitbodies might be an evolutionary dead end and that the genetically divergent Mendelian populations from which they were derived are, in fact, different species. The association between ITS haplotype divergence of less than 5% (Armillaria mellea = 2.6% excluding gaps; Amanita citrina f. lavendula = 3.3%) with the presence of putative recombinants and greater than 5% (Gymnopus dichrous = 5.7%; Hygrocybe flavescens/chlorophana = 14.1%) with apparent failure of F1 hybrids to produce F2 or higher progeny in populations may suggest a correlation between genetic distance and reproductive isolation.

Evidence of natural hybridization among homothallic members of the basidiomycete Armillaria mellea sensu stricto.

Populations of Armillaria mellea (Basidiomycota, Agaricales) across much of its range are heterothallic; homothallic populations occur only in Africa (A. mellea ssp. africana), China (China Biological Species CBS G), and Japan (A. mellea ssp. nipponica). Monosporous isolates of heterothallic A. mellea are haploid and their mating behaviour is consistent with the requirement of two different alleles at two mating-type loci (tetrapolar mating system) to create a diploid individual. In contrast, monosporous isolates of homothallic A. mellea are putatively diploid; they bypass the haploid phase by undergoing karyogamy in the basidium (a unique type of secondary homothallism/pseudohomothallism). In order to determine the genetic origin of this homothallism, we analyzed genetic variation of 47 heterothallic isolates from China, Europe, and North America, and 14 homothallic isolates from Africa, China, and Japan. Gene trees and mutational networks were constructed for partial mitochondrial gene ATP synthase subunit 6 (ATP6) and for the following nuclear genes: actin (ACTIN), elongation factor subunit 1-alpha (EFA), glyceraldehyde 3-phosphate dehydrogenase (GPD), and the RNA polymerase subunit II (RPB2). Homothallic isolates from Africa and Japan shared a common mitochondrial ATP6 haplotype with homothallic isolates from China, and are likely introductions. Homothallic isolates from China that shared a common mitochondrial haplotype with all European isolates did not share European nuclear haplotypes, as revealed by median-joining networks, but instead clustered with haplotypes from China or were intermediate between those of China and Europe. Such mitochondrial-nuclear discordance in homothallic isolates from China is indicative of hybridization between lineages originating from China and Europe.

A new genus to accommodate Gymnopus acervatus (Agaricales).

Phylogenies based on ITS and LSU nrDNA sequences show Agaricus (Gymnopus) acervatus as unique within the Gymnopus/Rhodocollybia complex. These phylogenies imply that a separate genus is necessary, and Connopus is proposed. Infraspecific morphological and DNA-based variation within C. acervatus suggests that a western North American clade might be reproductively isolated from the eastern North American/Scandinavian clade and that in this species complex the European and eastern North American clade might be conspecific. A Scandinavian exemplar is selected for bar-coding. Two GenBank sequences with name-phylogenetic placement inconsistencies are identified.

Contributions of rpb2 and tef1 to the phylogeny of mushrooms and allies (Basidiomycota, Fungi).

A phylogeny of the fungal phylum Basidiomycota is presented based on a survey of 160 taxa and five nuclear genes. Two genes, rpb2, and tef1, are presented in detail. The rpb2 gene is more variable than tef1 and recovers well-supported clades at shallow and deep taxonomic levels. The tef1 gene recovers some deep and ordinal-level relationships but with greater branch support from nucleotides compared to amino acids. Intron placement is dynamic in tef1, often lineage-specific, and diagnostic for many clades. Introns are fewer in rpb2 and tend to be highly conserved by position. When both protein-coding loci are combined with sequences of nuclear ribosomal RNA genes, 18 inclusive clades of Basidiomycota are strongly supported by Bayesian posterior probabilities and 16 by parsimony bootstrapping. These numbers are greater than produced by single genes and combined ribosomal RNA gene regions. Combination of nrDNA with amino acid sequences, or exons with third codon positions removed, produces strong measures of support, particularly for deep internodes of Basidiomycota, which have been difficult to resolve with confidence using nrDNA data alone. This study produces strong boostrap support and significant posterior probabilities for the first time for the following monophyletic groups: (1) Ustilaginomycetes plus Hymenomycetes, (2) an inclusive cluster of hymenochaetoid, corticioid, polyporoid, Thelephorales, russuloid, athelioid, Boletales, and euagarics clades, (3) Thelephorales plus the polyporoid clade, (4) the polyporoid clade, and (5) the cantharelloid clade. Strong support is also recovered for the basal position of the Dacrymycetales in the Hymenomycetidae and paraphyly of the Exobasidiomycetidae.

Major clades of Agaricales: a multilocus phylogenetic overview.

An overview of the phylogeny of the Agaricales is presented based on a multilocus analysis of a six-gene region supermatrix. Bayesian analyses of 5611 nucleotide characters of rpb1, rpb1-intron 2, rpb2 and 18S, 25S, and 5.8S ribosomal RNA genes recovered six major clades, which are recognized informally and labeled the Agaricoid, Tricholomatoid, Marasmioid, Pluteoid, Hygrophoroid and Plicaturopsidoid clades. Each clade is discussed in terms of key morphological and ecological traits. At least 11 origins of the ectomycorrhizal habit appear to have evolved in the Agaricales, with possibly as many as nine origins in the Agaricoid plus Tricholomatoid clade alone. A family-based phylogenetic classification is sketched for the Agaricales, in which 30 families, four unplaced tribes and two informally named clades are recognized.

Variability and phylogenetic incongruence of an SSU nrDNA group I intron in Artomyces, Auriscalpium, and Lentinellus (Auriscalpiaceae: Homobasidiomycetes).

Previous research has shown that a group I intron occurs in the SSU ribosomal DNA gene of isolates of Artomyces (Clavicorona, in part) and Lentinellus, but apparently it is absent in an Auriscalpium isolate. However, further investigation revealed that the intron is apparently absent in some species of Artomyces and Lentinellus and is present in at least one species of Auriscalpium. To examine this further, the presence or absence of the group I intron is reported for 13 species of Lentinellus, two species of Auriscalpium, and 16 species of Artomyces. The presence of the intron among the species was variable and is documented for seven species of Lentinellus, one species of Auriscalpium, and 12 species of Artomyces. Furthermore, the presence of the intron was variable among the isolates of several species, and variability of its presence was observed within single isolates, indicating inter-ribosomal repeat heterogeneity. Independent phylogenetic estimations were generated for the intron and nuclear ribosomal internal transcribed spacer regions (ITS). Tests of congruence for the two trees indicated that the data were heterogeneous. Some of the discontinuity between the two phylogenies is due to placement of the Ar. austropiperatus intron within the Lentinellus intron clade. Variability in the length of the intron was observed in populations of the pan-Northern Temperate species Ar. pyxidatus. This was due to the presence of an additional unknown insertional element found only within North American collections of Ar. pyxidatus and absent from European and Asian collections.

Biogeographical patterns in Artomyces pyxidatus.

Artomyces pyxidatus (Auriscalpiaceae) is a lignicolous, coralloid basidiomycete found throughout temperate regions of the Northern Hemisphere. Previous studies established that populations from the eastern United States, Sweden, and China were conspecific based on mating compatibility and enzyme profiles. In this study, mating compatibility was extended to include collections from Russia, Costa Rica, Mexico, and Utah. The molecular diversity of A. pyxidatus was examined by DNA sequence and restriction site analyses of the nuclear ribosomal internally transcribed spacer region (ITS1-5.8S-ITS2). A phylogenetic analysis of twelve isolates based on ITS sequences revealed a broad geographical pattern in which Eurasian isolates comprise a sister clade to North American isolates. North American isolates appear to be further subdivided into northeastern and southwestern clades. A survey of 255 A. pyxidatus isolates using restriction enzymes revealed variable RFLP patterns that follow similar geographical patterns.