Rescue of Scn5a mis-splicing does not improve the structural and functional heart defects of a DM1 heart mouse model.

Myotonic Dystrophy Type 1 (DM1) is an autosomal dominant multisystemic disorder for which cardiac features, including conduction delays and arrhythmias, are the second leading cause of disease mortality. DM1 is caused by expanded CTG repeats in the 3' untranslated region of the DMPK gene. Transcription of the expanded DMPK allele produces mRNAs containing long tracts of CUG repeats, which sequester the Muscleblind-Like family of RNA binding proteins, leading to their loss-of-function and the dysregulation of alternative splicing. A well-characterized mis-regulated splicing event in the DM1 heart is the increased inclusion of SCN5A exon 6A rather than the mutually exclusive exon 6B that normally predominates in adult heart. As previous work showed that forced inclusion of Scn5a exon 6A in mice recapitulates cardiac DM1 phenotypes, we tested whether rescue of Scn5a mis-splicing would improve the cardiac phenotypes in a DM1 heart mouse model. We generated mice lacking Scn5a exon 6A to force the expression of the adult SCN5A isoform including exon 6B and crossed these mice to our previously established CUG960 DM1 heart mouse model. We showed that correction Scn5a mis-splicing does not improve the DM1 heart conduction delays and structural changes induced by CUG repeat RNA expression. Interestingly, we found that in addition to Scn5a mis-splicing, Scn5a expression is reduced in heart tissues of CUG960 mice and DM1-affected individuals. These data indicate that Scn5a mis-splicing is not the sole driver of DM1 heart deficits and suggest a potential role for reduced Scn5a expression in DM1 cardiac disease.

Alternative splicing mediates the compensatory upregulation of MBNL2 upon MBNL1 loss-of-function.

Loss of gene function can be compensated by paralogs with redundant functions. An example of such compensation are the paralogs of the Muscleblind-Like (MBNL) family of RNA-binding proteins that are sequestered and lose their function in Myotonic Dystrophy Type 1 (DM1). Loss of MBNL1 increases the levels of its paralog MBNL2 in tissues where Mbnl2 expression is low, allowing MBNL2 to functionally compensate for MBNL1 loss. Here, we show that loss of MBNL1 increases the inclusion of Mbnl2 exon 6 and exon 9. We find that inclusion of Mbnl2 exon 6 increases the translocation of MBNL2 to the nucleus, while the inclusion of Mbnl2 exon 9 shifts the reading frame to an alternative C-terminus. We show that the C-terminus lacking exon 9 contains a PEST domain which causes proteasomal degradation. Loss of MBNL1 increases the inclusion of exon 9, resulting in an alternative C-terminus lacking the PEST domain and the increase of MBNL2. We further find that the compensatory mechanism is active in a mouse DM1 model. Together, this study uncovers the compensatory mechanism by which loss of MBNL1 upregulates its paralog MBNL2 and highlights a potential role of the compensatory mechanism in DM1.

Genome-scale phylogeny and comparative genomics of the fungal order Sordariales.

The order Sordariales is taxonomically diverse, and harbours many species with different lifestyles and large economic importance. Despite its importance, a robust genome-scale phylogeny, and associated comparative genomic analysis of the order is lacking. In this study, we examined whole-genome data from 99 Sordariales, including 52 newly sequenced genomes, and seven outgroup taxa. We inferred a comprehensive phylogeny that resolved several contentious relationships amongst families in the order, and cleared-up intrafamily relationships within the Podosporaceae. Extensive comparative genomics showed that genomes from the three largest families in the dataset (Chaetomiaceae, Podosporaceae and Sordariaceae) differ greatly in GC content, genome size, gene number, repeat percentage, evolutionary rate, and genome content affected by repeat-induced point mutations (RIP). All genomic traits showed phylogenetic signal, and ancestral state reconstruction revealed that the variation of the properties stems primarily from within-family evolution. Together, the results provide a thorough framework for understanding genome evolution in this important group of fungi.

Assessing Microbial Metabolic and Biological Diversity to Inform Natural Product Library Assembly.

The pressing need for novel chemical matter to support bioactive compound discovery has led natural product researchers to explore a wide range of source organisms and environments. One of the implicit guiding principles behind those efforts is the notion that sampling different environments is critical to accessing unique natural products. This idea was tested by comparing fungi from disparate biomes: aquatic sediments from Lake Michigan (USA) and terrestrial samples taken from the surrounding soils. Matched sets of Penicillium brevicompactum, Penicillium expansum, and Penicillium oxalicum from the two source environments were compared, revealing modest differences in physiological performance and chemical output. Analysis of LC-MS/MS-derived molecular feature data showed no source-dependent differences in chemical richness. High levels of scaffold homogeneity were also observed with 78-83% of scaffolds shared among the terrestrial and aquatic Penicillium spp. isolates. A comparison of the culturable fungi from the two biomes indicated that certain genera were more strongly associated with aquatic sediments (e.g., Trichoderma, Pseudeurotium, Cladosporium, and Preussia) versus the surrounding terrestrial environment (e.g., Fusarium, Pseudogymnoascus, Humicola, and Acremonium). Taken together, these results suggest that focusing efforts on sampling the microbial resources that are unique to an environment may have a more pronounced effect on enhancing the sought-after natural product diversity needed for chemical discovery and screening collections.

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.

Resolving the Mortierellaceae phylogeny through synthesis of multi-gene phylogenetics and phylogenomics.

Early efforts to classify Mortierellaceae were based on macro- and micromorphology, but sequencing and phylogenetic studies with ribosomal DNA (rDNA) markers have demonstrated conflicting taxonomic groupings and polyphyletic genera. Although some taxonomic confusion in the family has been clarified, rDNA data alone is unable to resolve higher level phylogenetic relationships within Mortierellaceae. In this study, we applied two parallel approaches to resolve the Mortierellaceae phylogeny: low coverage genome (LCG) sequencing and high-throughput, multiplexed targeted amplicon sequencing to generate sequence data for multi-gene phylogenetics. We then combined our datasets to provide a well-supported genome-based phylogeny having broad sampling depth from the amplicon dataset. Resolving the Mortierellaceae phylogeny into monophyletic groups led to the definition of 14 genera, 7 of which are newly proposed. Low-coverage genome sequencing proved to be a relatively cost-effective means of generating a well-resolved phylogeny. The multi-gene phylogenetics approach enabled much greater sampling depth and breadth than the LCG approach, but was unable to resolve higher-level organization of groups. We present this work to resolve some of the taxonomic confusion and provide a genus-level framework to empower future studies on Mortierellaceae diversity, biology, and evolution.

Emydomyces testavorans, a New Genus and Species of Onygenalean Fungus Isolated from Shell Lesions of Freshwater Aquatic Turtles.

The fungal order Onygenales includes many pathogens of humans and animals, and recent studies have shown some onygenalean fungi to be significant emerging pathogens of reptiles. Although many of these fungi have similar morphological features in histologic tissue sections, recent molecular analyses have revealed a genetically complex and diverse group of reptile pathogens comprising several genera, most notably Nannizziopsis, Ophidiomyces, and Paranannizziopsis Infections by members of these genera have been previously reported in a variety of reptile species, including crocodilians, lizards, snakes, and tuataras, with negative impacts on conservation efforts for some reptiles. Despite the well-documented pathogenicity of these fungi in all other extant reptile lineages, infection has not yet been reported in aquatic turtles. In this study, we report the isolation of an onygenalean fungus associated with shell lesions in freshwater aquatic turtles. The morphologic and genetic characteristics of multiple isolates (n = 21) are described and illustrated. Based on these features and results of a multigene phylogenetic analysis, a new genus and species, Emydomyces testavorans, are proposed for these fungi isolated from turtle shell lesions.

Secondary Metabolites from the Fungus Dictyosporium sp. and Their MALT1 Inhibitory Activities.

Bioassay-guided separation of an extract from a Dictyosporium sp. isolate led to the identification of six new compounds, 1-6, together with five known compounds, 7-11. The structures of the new compounds were primarily established by extensive 1D and 2D NMR experiments. The absolute configurations of compounds 3-6 were determined by comparison of their experimental electronic circular dichroism (ECD) spectra with DFT quantum mechanical calculated ECD spectra. Compounds 3-5 possess novel structural scaffolds, and biochemical studies revealed that oxepinochromenones 1 and 7 inhibited the activity of MALT1 protease.

Worldwide Engagement for Digitizing Biocollections (WeDigBio): The Biocollections Community's Citizen-Science Space on the Calendar.

The digitization of biocollections is a critical task with direct implications for the global community who use the data for research and education. Recent innovations to involve citizen scientists in digitization increase awareness of the value of biodiversity specimens; advance science, technology, engineering, and math literacy; and build sustainability for digitization. In support of these activities, we launched the first global citizen-science event focused on the digitization of biodiversity specimens: Worldwide Engagement for Digitizing Biocollections (WeDigBio). During the inaugural 2015 event, 21 sites hosted events where citizen scientists transcribed specimen labels via online platforms (DigiVol, Les Herbonautes, Notes from Nature, the Smithsonian Institution's Transcription Center, and Symbiota). Many citizen scientists also contributed off-site. In total, thousands of citizen scientists around the world completed over 50,000 transcription tasks. Here, we present the process of organizing an international citizen-science event, an analysis of the event's effectiveness, and future directions-content now foundational to the growing WeDigBio event.

What lies beneath? Fungal diversity at the bottom of Lake Michigan and Lake Superior.

Fungi are phylogenetically diverse organisms found in nearly every environment as key contributors to the processes of nutrient cycling and decomposition. To date, most fungal diversity has been documented from terrestrial habitats leaving aquatic habitats underexplored. In particular, comparatively little is known about fungi inhabiting freshwater lakes, particularly the benthic zone, which may serve as an untapped resource for fungal biodiversity. Advances in technology allowing for direct sequencing of DNA from environmental samples provide a new opportunity to investigate freshwater benthic fungi. In this study, we employed both culture-dependent and culture-independent methods to evaluate the diversity of fungi in one of the largest freshwater systems on Earth, the North American Laurentian Great Lakes. This study presents the first comprehensive survey of fungi from sediment from Lake Michigan and Lake Superior, resulting in 465 fungal taxa with only 7% of sequence overlap between these two methods. Additionally, culture-independent analyses of the ITS1 and ITS2 regions revealed 49% and 72%, respectively, of the OTUs did not match a described fungal taxonomic group below kingdom Fungi. The low level of sequence overlap between methods and high percentage of fungal taxa that can only be classified at the kingdom level suggests an immense amount of fungal diversity remains to be studied in these aquatic fungal communities.

Fungal Identification Using Molecular Tools: A Primer for the Natural Products Research Community.

Fungi are morphologically, ecologically, metabolically, and phylogenetically diverse. They are known to produce numerous bioactive molecules, which makes them very useful for natural products researchers in their pursuit of discovering new chemical diversity with agricultural, industrial, and pharmaceutical applications. Despite their importance in natural products chemistry, identification of fungi remains a daunting task for chemists, especially those who do not work with a trained mycologist. The purpose of this review is to update natural products researchers about the tools available for molecular identification of fungi. In particular, we discuss (1) problems of using morphology alone in the identification of fungi to the species level; (2) the three nuclear ribosomal genes most commonly used in fungal identification and the potential advantages and limitations of the ITS region, which is the official DNA barcoding marker for species-level identification of fungi; (3) how to use NCBI-BLAST search for DNA barcoding, with a cautionary note regarding its limitations; (4) the numerous curated molecular databases containing fungal sequences; (5) the various protein-coding genes used to augment or supplant ITS in species-level identification of certain fungal groups; and (6) methods used in the construction of phylogenetic trees from DNA sequences to facilitate fungal species identification. We recommend that, whenever possible, both morphology and molecular data be used for fungal identification. Our goal is that this review will provide a set of standardized procedures for the molecular identification of fungi that can be utilized by the natural products research community.

Studies in the genus Glutinoglossum.

Glutinoglossum is a recently described genus in Geoglossomycetes, characterized by viscid black fruiting bodies. Glutinoglossum was described based on molecular evidence showing that the fungus formerly known as Geoglossum glutinosum formed a well-supported clade separate from Geoglossum. This study provides a comprehensive examination of Glutinoglossum based on maximum-likelihood and Bayesian analyses of a dataset that includes nuc rDNA internal transcribed spacer and partial 28S regions and the nuc genes for RNA polymerase II largest subunit (RPB1) and minichromosome maintenance complex component 7 (MCM7). Four new species are described, an emended description of Glutinoglossum glutinosum and a key to the genus Glutinoglossum is provided.

A re-evaluation of the genus Myceliophthora (Sordariales, Ascomycota): its segregation into four genera and description of Corynascus fumimontanus sp. nov.

Based on a number of isolates of Myceliophthora (Chaetomiaceae, Sordariales, Ascomycota) recently isolated from soil samples collected in USA, the taxonomy of the genus was re-evaluated through phylogenetic analyses of sequences from the nuc rDNA internal transcribed spacer region and genes for the second largest subunit of RNA polymerase II and translation elongation factor 1α. Members of Myceliophthora were split into four monophyletic clades strongly supported by molecular and phenotypic data. Such clades correspond with Myceliophthora, now restricted only to the type species of the genus Corynascus, which is re-established with five species, the new monotypic genus Crassicarpon and also the new genus Thermothelomyces (comprising four species). Myceliophthora lutea is mesophilic and a permanently asexual morph compared to the members of the other three mentioned genera, which also are able to sexually reproduce morphs with experimentally proven links to their asexual morphs. The asexual morph of M. lutea is characterized by broadly ellipsoidal, smooth-walled conidia with a wide, truncate base. Crassicarpon thermophilum is thermophilic and heterothallic and produces spherical to cuneiform, smooth-walled conidia and cleistothecial ascomata of smooth-walled, angular cells and ascospores with a germ pore at each end. Corynascus spp. are homothallic and mesophilic and produce spherical, mostly ornamented conidia and cleistothecial ascomata with textura epidermoidea composed of ornamented wall cells, and ascospores with one germ pore at each end. Thermothelomyces spp. are thermophilic, heterothallic and characterized by similar ascomata and conidia as Corynascus spp., but its ascospores exhibit only a single germ pore. A dichotomous key to distinguish Myceliophthora from the other mentioned genera are provided, as well as dichotomous keys to identify the species of Corynascus and Thermothelomyces. A new species, namely Corynascus fumimontanus, characterized by verrucose ascomatal wall cells and irregularly shaped ascospores, is described and illustrated.

Minutisphaerales (Dothideomycetes, Ascomycota): a new order of freshwater ascomycetes including a new family, Minutisphaeraceae, and two new species from North Carolina, USA.

Minutisphaera is a recently established genus of freshwater Dothideomycetes characterized by small, globose to subglobose or apothecioid, erumpent to superficial, brown ascomata; fissitunicate, eight-spored, ovoid to obclavate asci; and 1-2-septate, clavate to broadly fusiform, hyaline to pale brown ascospores with or without a gelatinous sheath and filamentous appendages. The genus currently contains two species: M. fimbriatispora, the type species, and M. japonica. The higher-level phylogenetic relationship of Minutisphaera within the Dothideomycetes currently is unresolved. To establish the phylogenetic position of Minutisphaera within the Dothideomycetes and evaluate the phylogenetic affinities of newly collected Minutisphaera-like taxa, we sequenced three rDNA regions-18S, ITS1-5.8SITS2 (ITS) and 28S nuc rDNA, and a protein-coding gene, MCM7, for newly collected strains of Minutisphaera. Based on maximum likelihood and Bayesian analyses of a combined dataset (18S and 28S) composed of 167 taxa, a more refined dataset (28S and MCM7) comprising 52 taxa and a separate ITS dataset, and an examination of morphology, we describe and illustrate two new species of Minutisphaera. The Minutisphaera clade was strongly supported within the Dothideomycetes with likelihood and Bayesian statistics but did not share phylogenetic affinities with any existing taxonomic group within the Dothideomycetes. We therefore establish a new order, Minutisphaerales, and new family, Minutisphaeraceae, for this monophyletic clade of freshwater ascomycetes. Chemical analysis of the organic extract M. aspera (G427) resulted in isolation and characterization of five known secondary metabolites, of which four were dipeptides (1-4) and one an aromatic polyketide (5). Conversely, two aromatic polyketides (5, 6) were isolated and identified from the organic extract of M. parafimbriatispora (G156-4). The isolated compounds were tested for their antimicrobial activity against an array of bacteria and fungi. Compound 6 showed promising activity against Staphylococcus aureus and Mycobacterium smegmatis with minimal inhibitory concentration values of 30 and 60 µg/mL, respectively.

Effect of Trans, Trans-Farnesol on Pseudogymnoascus destructans and Several Closely Related Species.

Bat white-nose syndrome, caused by the psychrophilic fungus Pseudogymnoascus destructans, has dramatically reduced the populations of many hibernating North American bat species. The search for effective biological control agents targeting P. destructans is of great importance. We report that the sesquiterpene trans, trans-farnesol, which is also a Candida albicans quorum sensing compound, prevented in vitro conidial germination for at least 14 days and inhibited growth of preexisting hyphae of five P. destructans isolates in filtered potato dextrose broth at 10 °C. Depending on the inoculation concentrations, both spore and hyphal inhibition occurred upon exposure to concentrations as low as 15-20 µM trans, trans-farnesol. In contrast, most North American Pseudogymnoascus isolates were more tolerant to the exposure of trans, trans-farnesol. Our results suggest that some Candida isolates may have the potential to inhibit the growth of P. destructans and that the sesquiterpene trans, trans-farnesol has the potential to be utilized as a biological control agent.

Phylogenetic relationships of five uncommon species of Lasiosphaeria and three new species in the Helminthosphaeriaceae (Sordariomycetes).

In an ongoing effort to monograph the genus Lasiosphaeria, it is desirable to obtain estimates of the phylogenetic relationships for five uncommon species, L. coacta, L. munkii, L. punctata, L. sphagnorum and L. stuppea. Three additional species previously placed in Lasiosphaeria, Echinosphaeria canescens, Hilberina caudata and Ruzenia spermoides, also were included in this study as well as three undescribed species. These species were believed to have relations elsewhere based on various ambiguous morphological characters, so an independent dataset from one or more genes was used to resolve their phylogenetic affinities. Sequences from the nuclear ribosomal 28S large subunit (LSU) and ß-tubulin genes were generated for these taxa. Maximum likelihood and Bayesian analyses indicated these taxa form a well supported monophyletic group with members of the Helminthosphaeriaceae and therefore, should be transferred out of Lasiosphaeria. Except for Helminthosphaeria gibberosa, Hilberina elegans, Ruzenia spermoides and Synaptospora plumbea, all taxa within this clade possess ascomata with distinct thick-walled setae. Based on a combination of morphological and molecular data, Helminthosphaeria tomaculum, Helminthosphaeria triseptata and Hilberina robusta are described as new and four new combinations are proposed: Helminthosphaeria ludens, Hel. stuppea, Hilberina punctata and H. sphagnorum. Ten new combinations are proposed based on morphological data: Echinosphaeria heterostoma, Helminthosphaeria flavocompta, Hel. gibberosa, Hel. heterotricha, Hilberina breviseta, H. elegans, H. foliicola, H. meznaensis, H. moseri and H. rhynchospora. Lasiosphaeria coacta is placed in synonymy with Hel. ludens and the previous transfer of Hilberina munkii is accepted. Synaptospora plumbea was found to belong in the family. Illustrations are provided for most Helminthosphaeriaceae taxa seen in this study.

Naming and outline of Dothideomycetes-2014 including proposals for the protection or suppression of generic names.

Article 59.1, of the International Code of Nomenclature for Algae, Fungi, and Plants (ICN; Melbourne Code), which addresses the nomenclature of pleomorphic fungi, became effective from 30 July 2011. Since that date, each fungal species can have one nomenclaturally correct name in a particular classification. All other previously used names for this species will be considered as synonyms. The older generic epithet takes priority over the younger name. Any widely used younger names proposed for use, must comply with Art. 57.2 and their usage should be approved by the Nomenclature Committee for Fungi (NCF). In this paper, we list all genera currently accepted by us in Dothideomycetes (belonging to 23 orders and 110 families), including pleomorphic and nonpleomorphic genera. In the case of pleomorphic genera, we follow the rulings of the current ICN and propose single generic names for future usage. The taxonomic placements of 1261 genera are listed as an outline. Protected names and suppressed names for 34 pleomorphic genera are listed separately. Notes and justifications are provided for possible proposed names after the list of genera. Notes are also provided on recent advances in our understanding of asexual and sexual morph linkages in Dothideomycetes. A phylogenetic tree based on four gene analyses supported 23 orders and 75 families, while 35 families still lack molecular data.

Crowdsourcing natural products discovery to access uncharted dimensions of fungal metabolite diversity.

A fundamental component for success in drug discovery is the ability to assemble and screen compounds that encompass a broad swath of biologically relevant chemical-diversity space. Achieving this goal in a natural-products-based setting requires access to a wide range of biologically diverse specimens. For this reason, we introduced a crowdsourcing program in which citizen scientists furnish soil samples from which new microbial isolates are procured. Illustrating the strength of this approach, we obtained a unique fungal metabolite, maximiscin, from a crowdsourced Alaskan soil sample. Maximiscin, which exhibits a putative combination of polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS), and shikimate pathway components, was identified as an inhibitor of UACC-62 melanoma cells (LC50=0.93 µM). The metabolite also exhibited efficacy in a xenograft mouse model. These results underscore the value of building cooperative relationships between research teams and citizen scientists to enrich drug discovery efforts.

Ramomarthamyces octomerus sp. nov. and Insights into the Evolution and Diversification of Ramomarthamyces (Ascomycota, Leotiomycetes, Marthamycetales).

The apothecial fungus Ramomarthamyces octomerus sp. nov. is described from specimens collected in Mediterranean climate regions in southern Portugal, Spain (Canary Islands), and the Dalmatian region of Croatia. Presumably saprobic, R. octomerus occurs on intact, decorticated wood of Laurus novocanariensis and Olea europaea. Ascospores are cylindric-ellipsoid and seven-septate. Surprisingly, in our four-locus phylogenetic analysis (nuSSU, ITS1-5.8S-ITS2, LSU, mtSSU), this fungus clusters among species of Cyclaneusma, Marthamyces, Naemacyclus, and Ramomarthamyces in a core Marthamycetaceae clade that circumscribes primarily leaf-inhabiting, filiform-spored species. In addition, the asci of R. octomerus possess an amyloid pore, but the reaction varies between specimens collected in the Canary Islands and those collected in Portugal and Croatia. The occurrence of an amyloid reaction in the asci of R. octomerus challenges the characterization of Marthamycetales taxa as possessing inamyloid asci. In our discussion we provide background and analysis of these notable observations.

Psychrophilic and psychrotolerant fungi on bats and the presence of Geomyces spp. on bat wings prior to the arrival of white nose syndrome.

Since 2006, Geomyces destructans, the causative agent of white nose syndrome (WNS), has killed over 5.7 million bats in North America. The current hypothesis suggests that this novel fungus is an invasive species from Europe, but little is known about the diversity within the genus Geomyces and its distribution on bats in the United States. We documented the psychrophilic and psychrotolerant fungal flora of hibernating bats prior to the arrival of WNS using culture-based techniques. A total of 149 cultures, which were obtained from 30 bats in five bat hibernacula located in four caves and one mine, were sequenced for the entire internal transcribed spacer (ITS) nuclear ribosomal DNA (nrDNA) region. Approximately 53 operational taxonomic units (OTUs) at 97% similarity were recovered from bat wings, with the community dominated by fungi within the genera Cladosporium, Fusarium, Geomyces, Mortierella, Penicillium, and Trichosporon. Eleven Geomyces isolates were obtained and placed in at least seven distinct Geomyces clades based on maximum-likelihood phylogenetic analyses. Temperature experiments revealed that all Geomyces strains isolated are psychrotolerant, unlike G. destructans, which is a true psychrophile. Our results confirm that a large diversity of fungi, including several Geomyces isolates, occurs on bats prior to the arrival of WNS. Most of these isolates were obtained from damaged wings. Additional studies need to be conducted to determine potential ecological roles of these abundant Geomyces strains isolated from bats.