Genetic Analyses of Discrete Geographic Samples of a Golden Chanterelle in Canada Reveal Evidence for Recent Regional Differentiation.

The wild edible mushroom Cantharellus enelensis is a recently described species of the golden chanterelles found in eastern North America. At present, the genetic diversity and population structure of C. enelensis are not known. In this study, we analyzed a total of 230 fruiting bodies of C. enelensis that were collected from three regions of Canada: near the east and west coasts of Newfoundland (NFLD), with 110 fruiting bodies each, and around Hamilton, Ontario (10 fruiting bodies). Among the 110 fruiting bodies from each coast in NFLD, 10 from 2009 were without specific site information, while 100 sampled in 2010 were from each of five patches separated by at least 100 m from each other. Each fruiting body was genotyped at three microsatellite loci. Among the total 28 multilocus genotypes (MLGs) identified, 2 were shared among all three regions, 4 were shared between 2 of the 3 regions, and the remaining 22 were each found in only 1 region. Minimal spanning network analyses revealed several region-specific MLG clusters, consistent with geographic specific mutation and expansion. Though the most frequently observed MLGs were shared among local (patch) and regional populations, population genetic analyses revealed that both local and regional geographic separations contributed significantly to the observed genetic variation in the total sample. All three regional populations showed excess heterozygosity; for the eastern NFLD population, we reject the null hypothesis of Hardy-Weinberg equilibrium (HWE) at all three loci. However, the analyses of clone-corrected samples revealed that most loci were in HWE. Together, our results suggest that the three discrete regional populations of C. enelensis were likely colonized from a common refugium since the last ice age. However, the local and regional populations are diverging from each other through mutation, drift, and selection at least partly due to heterozygous advantage.

A phylogenetic overview of Squamanita, with descriptions of nine new species and four new combinations.

Nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS barcode) sequence data from eight type specimens of previously described Squamanita species were obtained. Phylogenetic analysis of ITS and partial nuc 28S rDNA data revealed Squamanita as paraphyletic splitting into two monophyletic groups, which we recognize as the genera Squamanita and Dissoderma. We accept 14 Squamanita and nine Dissoderma species, provide the first sequences of 13 of these, and describe six new species of Squamanita and three new species of Dissoderma. We transfer three species of Squamanita into Dissoderma, one into Cystoderma, and treat S. basii and S. umbilicata as synonyms of D. paradoxum. Squamanita can be distinguished from Dissoderma by the generally larger fleshier basidiomata with a tricholomatoid or amanitoid stature and yellowish to tawny brown pileus and often similarly colored stipe. Most species have cheilo- and pleurocystidia. Species of Dissoderma are small, collybioid or mycenoid, lack cystidia, and the pileus and often upper stipe are purplish gray. Both genera parasitize basidiomata of other agarics.

Multiple Fungi May Connect the Roots of an Orchid (Cypripedium reginae) and Ash (Fraxinus nigra) in Western Newfoundland.

Showy lady's slipper (Cypripedium reginae Walter, Orchidaceae) and black ash (Fraxinus nigra Marshall, Oleaceae) often co-occur in close proximity in fens in western Newfoundland, Canada. Metabarcoding of DNA extracted from root samples of both species following surface sterilization, and others without surface sterilization was used to determine if there were shared fungal endophytes in the roots of both species that could form a common mycorrhizal network between them. A wide variety of fungi were recovered from primers amplifying the nuclear ribosomal internal transcribed spacer region (ITS2). Sixty-six fungal sequences were shared by surface-sterilized roots of both orchid and ash, among them arbuscular mycorrhizal fungi (Claroideoglomus, Dominikia, Glomus and Rhizophagus), ectomycorrhizal fungi (Inocybe and Tomentella), the broad-host root endophyte Cadophora orchidicola, along with root pathogens (Dactylonectria, Ilyonectria, Pyricularia, and Xylomyces) and fungi of unknown function. There appear to be multiple fungi that could form a common mycorrhizal network between C. reginae and F. nigra, which might explain their frequent co-occurrence. Transfer of nutrients or carbon between the orchid and ash via one or more of the shared fungal endophytes remains to be demonstrated.

Simple Matching Using QIIME 2 and RDP Reveals Misidentified Sequences and an Underrepresentation of Fungi in Reference Datasets.

Simple nucleotide matching identification methods are not as accurate as once thought at identifying environmental fungal sequences. This is largely because of incorrect naming and the underrepresentation of various fungal groups in reference datasets. Here, we explore these issues by examining an environmental metabarcoding dataset of partial large subunit rRNA sequences of Basidiomycota and basal fungi. We employed the simple matching method using the QIIME 2 classifier and the RDP Classifier in conjunction with the latest releases of the SILVA (138.1, 2020) and RDP (11, 2014) reference datasets and then compared the results with a manual phylogenetic binning approach. Of the 71 query sequences tested, 21 and 42% were misidentified using QIIME 2 and the RDP Classifier, respectively. Of these simple matching misidentifications, more than half resulted from the underrepresentation of various groups of fungi in the SILVA and RDP reference datasets. More comprehensive reference datasets with fewer misidentified sequences will increase the accuracy of simple matching identifications. However, we argue that the phylogenetic binning approach is a better alternative to simple matching since, in addition to better accuracy, it provides evolutionary information about query sequences.

Identification and analyses of the chemical composition of a naturally occurring albino mutant chanterelle.

White chanterelles (Basidiomycota), lacking the orange pigments and apricot-like odour of typical chanterelles, were found recently in the Canadian provinces of Québec (QC) and Newfoundland & Labrador (NL). Our phylogenetic analyses confirmed the identification of all white chanterelles from NL and QC as Cantharellus enelensis; we name these forma acolodorus. We characterized carotenoid pigments, lipids, phenolics, and volatile compounds in these and related chanterelles. White mutants of C. enelensis lacked detectable ß-carotene, confirmed to be the primary pigment of wild-type, golden-orange individuals, and could also be distinguished by their profiles of fatty acids and phenolic acids, and by the ketone and terpene composition of their volatiles. We detected single base substitutions in the phytoene desaturase (Al-1) and phytoene synthase (Al-2) genes of the white mutant, which are predicted to result in altered amino acids in their gene products and may be responsible for the loss of ß-carotene synthesis in that form.

Competing sexual-asexual generic names in Agaricomycotina (Basidiomycota) with recommendations for use.

With the change to one scientific name for fungal taxa, generic names typified by species with sexual or asexual morph types are being evaluated to determine which names represent the same genus and thus compete for use. In this paper generic names of the Agaricomycotina (Basidiomycota) were evaluated to determine synonymy based on their type. Forty-seven sets of sexually and asexually typified names were determined to be congeneric and recommendations are made for which generic name to use. In most cases the principle of priority is followed. However, 16 generic names are recommended for use that do not have priority and thus need to be protected: Aleurocystis over Matula; Armillaria over Acurtis and Rhizomorpha; Asterophora over Ugola; Botryobasidium over Acladium, Allescheriella, Alysidium, Haplotrichum, Physospora, and Sporocephalium; Coprinellus over Ozonium; Coprinopsis over Rhacophyllus; Dendrocollybia over Sclerostilbum and Tilachlidiopsis; Diacanthodes over Bornetina; Echinoporia over Echinodia; Neolentinus over Digitellus; Postia over Ptychogaster; Riopa over Sporotrichum; Scytinostroma over Artocreas, Michenera, and Stereofomes; Tulasnella over Hormomyces; Typhula over Sclerotium; and Wolfiporia over Gemmularia and Pachyma. Nine species names are proposed for protection: Botryobasidium aureum, B. conspersum, B. croceum, B. simile, Pellicularia lembosporum (syn. B. lembosporum), Phanerochaete chrysosporium, Polyporus metamorphosus (syn. Riopa metamorphosa), Polyporus mylittae (syn. Laccocephalum mylittae), and Polyporus ptychogaster (syn. Postia ptychogaster). Two families are proposed for protection: Psathyrellaceae and Typhulaceae. Three new species names and 30 new combinations are established, and one lectotype is designated.

Exidia qinghaiensis, a new species from China.

A novel, wood-inhabiting jelly fungus from China is described as a new species, Exidia qinghaiensis (Basidiomycota: Auriculariaceae). Phylogenetic analyses were based on sequences of the nuclear ribosomal DNA internal transcribed spacer (nrITS) and large subunit (nrLSU), RNA polymerase II second largest subunit (RPB2), and translation elongation factor 1-α (Tef1) regions. Sequences of the new taxon formed a sister group to Exidia thuretiana, a species known from Europe and Asia, and distant to sequences of Exidia repanda from Europe. Fruiting bodies are cushion-shaped to irregularly lobed and yellowish brown, basidiospores are hyaline, allantoid (averaging 12.7 × 3.4 µm; average length/width is 3.7), and the host is Betula. The new species also can be distinguished by nrITS, nrLSU, RPB2, and Tef1 sequences. Our multigene phylogeny supports an Exidia including Exidia japonica, type species of Tremellochaete, but defining generic limits in Auriculariaceae will require more extensive taxon sampling.

Experimental Climate Change Modifies Degradative Succession in Boreal Peatland Fungal Communities.

Peatlands play an important role in global climate change through sequestration of atmospheric CO2. Climate-driven changes in the structure of fungal communities in boreal peatlands that favor saprotrophic fungi can substantially impact carbon dynamics and nutrient cycling in these crucial ecosystems. In a mesocosm study using a full factorial design, 100 intact peat monoliths, complete with living Sphagnum and above-ground vascular vegetation, were subjected to three climate change variables (increased temperature, reduced water table, and elevated CO2 concentrations). Peat litterbags were placed in mesocosms, and fungal communities in litterbags were monitored over 12 months to assess the impacts of climate change variables on peat-inhabiting fungi. Changes in fungal richness, diversity, and community composition were assessed using Illumina MiSeq sequencing of ribosomal DNA (rDNA). While general fungal richness reduced under warming conditions, Ascomycota exhibited higher diversity under increased temperature treatments over the course of the experiment. Both increased temperature and lowered water table position drove shifts in fungal community composition with a strong positive effect on endophytic and mycorrhizal fungi (including one operational taxonomic unit (OTU) tentatively identified as Barrenia panicia) and different groups of saprotrophs identified as Mortierella, Galerina, and Mycena. These shifts were observed during a predicted degradative succession in the decomposer community as different carbon substrates became available. Since fungi play a central role in peatland communities, increased abundances of saprotrophic fungi under warming conditions, at the expense of reduced fungal richness overall, may increase decomposition rates under future climate scenarios and could potentially aggravate the impacts of climate change.

New Primers for Discovering Fungal Diversity Using Nuclear Large Ribosomal DNA.

Metabarcoding has become an important tool in the discovery of biodiversity, including fungi, which are the second most speciose group of eukaryotes, with diverse and important ecological roles in terrestrial ecosystems. We have designed and tested new PCR primers that target the D1 variable region of nuclear large subunit (LSU) ribosomal DNA; one set that targets the phylum Ascomycota and another that recovers all other fungal phyla. The primers yield amplicons compatible with the Illumina MiSeq platform, which is cost-effective and has a lower error rate than other high throughput sequencing platforms. The new primer set LSU200A-F/LSU476A-R (Ascomycota) yielded 95-98% of reads of target taxa from environmental samples, and primers LSU200-F/LSU481-R (all other fungi) yielded 72-80% of target reads. Both primer sets have fairly low rates of data loss, and together they cover a wide variety of fungal taxa. We compared our results with these primers by amplifying and sequencing a subset of samples using the previously described ITS3_KYO2/ITS4_KYO3 primers, which amplify the internal transcribed spacer 2 (ITS2) of Ascomycota and Basidiomycota. With approximately equivalent read depth, our LSU primers recovered a greater number and phylogenetic diversity of sequences than the ITS2 primers. For instance, ITS3_KYO2/ITS4_KYO3 primers failed to pick up any members of Eurotiales, Mytilinidiales, Pezizales, Saccharomycetales, or Venturiales within Ascomycota, or members of Exobasidiomycetes, Microbotryomycetes, Pucciniomycetes, or Tremellomycetes within Basidiomycota, which were retrieved in good numbers from the same samples by our LSU primers. Among the OTUs recovered using the LSU primers were 127 genera and 28 species that were not obtained using the ITS2 primers, although the ITS2 primers recovered 10 unique genera and 16 species that were not obtained using either of the LSU primers These features identify the new primer sets developed in this study as useful complements to other universal primers for the study of fungal diversity and community composition.

Gymnopanella nothofagi, a new genus and species of gymnopoid fungi (Omphalotaceae) from Chilean Nothofagus forest.

A novel, lignicolous agaric from Nothofagus forests of southern Chile is described as a new genus and species, Gymnopanella nothofagi This taxon falls within the family Omphalotaceae as a sister group to Gymnopus in phylogenetic analyses based on sequences spanning the internal transcribed spacer region and D1/D2 region of nuclear 28S rDNA. Morphologically it is characterized by convex to flabellate basidiomata with distinctly gelatinized trama, pileipellis in the form of a cutis with erect fascicles of cylindrical, spirally incrusted hyphae and nonamyloid, broadly ellipsoid basidiospores. This combination of features, in particular the lack of a rameales structure, serve to distinguish Gymnopanella from Gymnopus, Marasmiellus and other similar genera of the Omphalotaceae or Marasmiaceae. The new taxon is known only from Chilean Nothofagus forests at approximately 45-46° south latitude, but concerted searching in similar habitats in surrounding areas or in New Zealand may extend the known range considerably.

Detection of phytohormones in temperate forest fungi predicts consistent abscisic acid production and a common pathway for cytokinin biosynthesis.

The phytohormones, abscisic acid and cytokinin, once were thought to be present uniquely in plants, but increasing evidence suggests that these hormones are present in a wide variety of organisms. Few studies have examined fungi for the presence of these "plant" hormones or addressed whether their levels differ based on the nutrition mode of the fungus. This study examined 20 temperate forest fungi of differing nutritional modes (ectomycorrhizal, wood-rotting, saprotrophic). Abscisic acid and cytokinin were present in all fungi sampled; this indicated that the sampled fungi have the capacity to synthesize these two classes of phytohormones. Of the 27 cytokinins analyzed by HPLC-ESI MS/MS, seven were present in all fungi sampled. This suggested the existence of a common cytokinin metabolic pathway in fungi that does not vary among different nutritional modes. Predictions regarding the source of isopentenyl, cis-zeatin and methylthiol CK production stemming from the tRNA degradation pathway among fungi are discussed.

Diversity of basidiomycetes in michigan agricultural soils.

We analyzed the communities of soil basidiomycetes in agroecosystems that differ in tillage history at the Kellogg Biological Station Long-Term Ecological Research site near Battle Creek, Michigan. The approach combined soil DNA extraction through a bead-beating method modified to increase recovery of fungal DNA, PCR amplification with basidiomycete-specific primers, cloning and restriction fragment length polymorphism screening of mixed PCR products, and sequencing of unique clones. Much greater diversity was detected than was anticipated in this habitat on the basis of culture-based methods or surveys of fruiting bodies. With "species" defined as organisms yielding PCR products with > or =99% identity in the 5' 650 bases of the nuclear large-subunit ribosomal DNA, 241 "species" were detected among 409 unique basidiomycete sequences recovered. Almost all major clades of basidiomycetes from basidiomycetous yeasts and other heterobasidiomycetes through polypores and euagarics (gilled mushrooms and relatives) were represented, with a majority from the latter clade. Only 24 of 241 "species" had 99% or greater sequence similarity to named reference sequences in GenBank, and several clades with multiple "species" could not be identified at the genus level by phylogenetic comparisons with named sequences. The total estimated "species" richness for this 11.2-ha site was 367 "species" of basidiomycetes. Since >99% of the study area has not been sampled, the accuracy of our diversity estimate is uncertain. Replication in time and space is required to detect additional diversity and the underlying community structure.

A new poroid species of Resupinatus from Puerto Rico, with a reassessment of the cyphelloid genus Stigmatolemma.

A fungus with gelatinous poroid fruiting bodies was found in Puerto Rico and determined by macro- and micromorphology to be most similar to members of the lamellate agaric genus Resupinatus. This species is described as a new species, Resupinatus porosus. Phylogenetic analyses of ribosomal DNA sequences support the inclusion of this fungus in the clade containing Resupinatus, and indicate that this monophyletic group also includes members of Asterotus and the cyphelloid genus Stigmatolemma. Resupinatus porosus is another example of tropical poroid representatives of lamellate agaric genera. Resupinatus is emended to include species with poroid (R. porosus) or merulioid (R. merulioides) hymenophore as well as those with laterally stipitate (Asterotus) or cyphelloid (Stigmatolemma) fruiting bodies. Seven new combinations in Resupinatus are proposed to accommodate well-known species of Stigmatolemma.