PacBio high-throughput multi-locus sequencing reveals high genetic diversity in mushroom-forming fungi.

Multi-locus sequence data are widely used in fungal systematic and taxonomic studies to delimit species and infer evolutionary relationships. We developed and assessed the efficacy of a multi-locus pooled sequencing method using PacBio long-read high-throughput sequencing. Samples included fresh and dried voucher specimens, cultures and archival DNA extracts of Agaricomycetes with an emphasis on the order Cantharellales. Of the 283 specimens sequenced, 93.6% successfully amplified at one or more loci with a mean of 3.3 loci amplified. Our method recovered multiple sequence variants representing alleles of rDNA loci and single copy protein-coding genes rpb1, rpb2 and tef1. Within-sample genetic variation differed by locus and taxonomic group, with the greatest genetic divergence observed among sequence variants of rpb2 and tef1 from corticioid Cantharellales. Our method is a cost-effective approach for generating accurate multi-locus sequence data coupled with recovery of alleles from polymorphic samples and multi-organism specimens. These results have important implications for understanding intra-individual genomic variation among genetic loci commonly used in species delimitation of fungi.

Does One Size Fit All? Variations in the DNA Barcode Gaps of Macrofungal Genera.

The nuclear ribosomal internal transcribed spacer (nrITS) region has been widely used in fungal diversity studies. Environmental metabarcoding has increased the importance of the fungal DNA barcode in documenting fungal diversity and distribution. The DNA barcode gap is seen as the difference between intra- and inter-specific pairwise distances in a DNA barcode. The current understanding of the barcode gap in macrofungi is limited, inhibiting the development of best practices in applying the nrITS region toward research on fungal diversity. This study examined the barcode gap using 5146 sequences representing 717 species of macrofungi from eleven genera, eight orders and two phyla in datasets assembled by taxonomic experts. Intra- and inter-specific pairwise distances were measured from sequence and phylogenetic data. The results demonstrate that barcode gaps are influenced by differences in intra- and inter-specific variance in pairwise distances. In terms of DNA barcode behavior, variance is greater in the ITS1 than ITS2, and variance is greater in both relative to the combined nrITS region. Due to the difference in variance, the barcode gaps in the ITS2 region are greater than in the ITS1. Additionally, the taxonomic approach of "splitting" taxa into numerous taxonomic units produces greater barcode gaps when compared to "lumping". The results show variability in the barcode gaps between fungal taxa, demonstrating a need to understand the accuracy of DNA barcoding in quantifying species richness. For taxonomic studies, variability in nrITS sequence data supports the application of multiple molecular markers to corroborate the taxonomic and systematic delineation of species.

New reports, new species, and high diversity of Cantharellus in the southern Appalachians.

Chanterelles (genus Cantharellus) are among the most popular wild edible mushrooms worldwide. Efforts to understand chanterelle diversity have yielded numerous new species in recent years, particularly in eastern North America. We constructed a multilocus phylogeny including all described temperate species of Cantharellus and newly collected specimens from the eastern United States with an emphasis on southern Appalachia. We describe a new species, Cantharellus vicinus, an oak-associated chanterelle known only from lower-elevation areas in east Tennessee, based on phylogenetic and morphological data. Cantharellus vicinus is characterized by a compact stature, bright yellow hymenophore that turns salmon when mature, white stipe, and pale yellow pileus with a whitish bloom. The southeastern Cantharellus minor f. intensissimus is elevated to species level based on morphological and molecular evidence. The taxon is epitypified due to the sterile state of the holotype and ambiguity concerning application of the name. Evaluation of genetic diversity and gene conflict within Cantharellus camphoratus shows that it is a widespread species with populations in Atlantic Canada, the southeastern United States, and Japan. Similarly, C. cibarius and C. tenuithrix form complexes and may be more geographically widespread than previously thought. Additionally, we report the first known instances of Cantharellus betularum, C. corallinus, and C. altipes from the southern Appalachian Mountains.

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.

Six new species and reports of Hydnum (Cantharellales) from eastern North America.

Five species of Hydnum have been generally recognized from eastern North America based on morphological recognition: H.albidum, H.albomagnum, H.repandum and varieties, H.rufescens, and H.umbilicatum. Other unique North American species, such as H.caespitosum and H.washingtonianum, are either illegitimately named or considered synonymous with European taxa. Here, seventeen phylogenetic species of Hydnum are detected from eastern North America based on a molecular phylogenetic survey of ITS sequences from herbarium collections and GenBank data, including environmental sequences. Based on current distribution results, sixteen of these species appear endemic to North America. Of these, six species are described as new: H.alboaurantiacum, H.cuspidatum, H.ferruginescens, H.subconnatum, H.subtilior, and H.vagabundum. Geographic range extensions and taxonomic notes are provided for five additional species recently described as new from eastern North America. A new name, H.geminum, is proposed for H.caespitosum Banning ex Peck, non Valenti. Overall, species of Hydnum are best recognized by a combination of morphological and molecular phylogenetic analyses. Taxonomic descriptions are provided for seventeen species, including epitype designations for H.albidum, H.albomagnum, and H.umbilicatum, taxa described more than 100 years ago, and molecular annotation of the isotype of H.washingtonianum. Photographs and a key to eastern North American Hydnum species are presented.

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.

Stable isotope analyses reveal previously unknown trophic mode diversity in the Hymenochaetales.

PREMISE OF THE STUDY: The Hymenochaetales are dominated by lignicolous saprotrophic fungi involved in wood decay. However, the group also includes bryophilous and terricolous taxa, but their modes of nutrition are not clear. Here, we investigate patterns of carbon and nitrogen utilization in numerous non-lignicolous Hymenochaetales and provide a phylogenetic context in which these non-canonical ecological guilds arose. METHODS: We combined stable isotope analyses of δ13 C and δ15 N and phylogenetic analyses to explore assignment and evolution of nutritional modes. Clustering procedures and statistical tests were performed to assign trophic modes to Hymenochaetales and test for differences between varying ecologies. Genomes of Hymenochaetales were mined for presence of enzymes involved in plant cell wall and lignin degradation and sucrolytic activity. KEY RESULTS: Three different trophic clusters were detected - biotrophic, saprotrophic, and a second biotrophic cluster including many bryophilous Hymenochaetales and mosses. Non-lignicolous Hymenochaetales are generally biotrophic. All lignicolous Hymenochaetales clustered as saprotrophic and most terricolous Hymenochaetales clustered as ectomycorrhizal. Overall, at least 15 species of Hymenochaetales are inferred as biotrophic. Bryophilous species of Rickenella can degrade plant cell walls and lignin, and cleave sucrose to glucose consistent with a parasitic or endophytic life style. CONCLUSIONS: Most non-lignicolous Hymenochaetales are biotrophic. Stable isotope values of many bryophilous Hymenochaetales cluster as ectomycorrhizal or in a biotrophic cluster indicative of parasitism or an endophytic life style. Overall, trophic mode diversity in the Hymenochaetales is greater than anticipated, and non-lignicolous ecological traits and biotrophic modes of nutrition are evolutionarily derived features.

The Inocybe geophylla group in North America: a revision of the lilac species surrounding I. lilacina.

The Inocybe geophylla group is circumscribed based on phylogenetic analysis of DNA sequences largely sampled from North America and Europe. Twenty-nine phylogenetic species are uncovered after analysis of combined nuc 28S rDNA (28S) and RNA polymerase II second largest subunit (rpb2) DNA sequence data. Species in the I. geophylla group share the presence of a cortina, silky-fibrillose pileus and stipe, pruinose stipe apex, spermatic odor, thick-walled hymenial cystidia, and smooth amygdaliform or elliptical basidiospores. Within the group, as many as five phylogenetic species attributable to I. lilacina and allies form a strongly supported clade based on analysis of nuc ITS1-5.8S-ITS2 rDNA (ITS [internal transcribed spacer]), 28S, and rpb2 data. However, all lilac-colored species do not form a monophyletic group. Sufficient morphological and ecological data are present to document four of the I. lilacina subgroup species, two of which are described from North America as new: I. ionocephala and I. sublilacina. Inocybe lilacina is recircumscribed based on sequencing the holotype and is distributed in the eastern United States under pines and/or hardwoods. Inocybe pallidicremea is a widespread and common conifer associate in mostly northern parts of North America, to which the name I. lilacina was previously applied. Descriptions, photographs, line drawings, and a taxonomic key to lilac species in the I. lilacina subgroup from North America are provided. Well-documented collections, especially notes on gross morphology and ecology, are needed to continue to assess and describe the high taxonomic variation in the I. lilacina subgroup and its allies worldwide.