Comparative transcriptomics of fungal endophytes in co-culture with their moss host Dicranum scoparium reveals fungal trophic lability and moss unchanged to slightly increased growth rates.

Mosses harbor fungi whose interactions within their hosts remain largely unexplored. Trophic ranges of fungal endophytes from the moss Dicranum scoparium were hypothesized to encompass saprotrophism. This moss is an ideal host to study fungal trophic lability because of its natural senescence gradient, and because it can be grown axenically. Dicranum scoparium was co-cultured with each of eight endophytic fungi isolated from naturally occurring D. scoparium. Moss growth rates, and gene expression levels (RNA sequencing) of fungi and D. scoparium, were compared between axenic and co-culture treatments. Functional lability of two fungal endophytes was tested by comparing their RNA expression levels when colonizing living vs dead gametophytes. Growth rates of D. scoparium were unchanged, or increased, when in co-culture. One fungal isolate (Hyaloscyphaceae sp.) that promoted moss growth was associated with differential expression of auxin-related genes. When grown with living vs dead gametophytes, Coniochaeta sp. switched from having upregulated carbohydrate transporter activity to upregulated oxidation-based degradation, suggesting an endophytism to saprotrophism transition. However, no such transition was detected for Hyaloscyphaceae sp. Individually, fungal endophytes did not negatively impact growth rates of D. scoparium. Our results support the long-standing hypothesis that some fungal endophytes can switch to saprotrophism.

Evolutionary transition to the ectomycorrhizal habit in the genomes of a hyperdiverse lineage of mushroom-forming fungi.

The ectomycorrhizal (ECM) symbiosis has independently evolved from diverse types of saprotrophic ancestors. In this study, we seek to identify genomic signatures of the transition to the ECM habit within the hyperdiverse Russulaceae. We present comparative analyses of the genomic architecture and the total and secreted gene repertoires of 18 species across the order Russulales, of which 13 are newly sequenced, including a representative of a saprotrophic member of Russulaceae, Gloeopeniophorella convolvens. The genomes of ECM Russulaceae are characterized by a loss of genes for plant cell wall-degrading enzymes (PCWDEs), an expansion of genome size through increased transposable element (TE) content, a reduction in secondary metabolism clusters, and an association of small secreted proteins (SSPs) with TE 'nests', or dense aggregations of TEs. Some PCWDEs have been retained or even expanded, mostly in a species-specific manner. The genome of G. convolvens possesses some characteristics of ECM genomes (e.g. loss of some PCWDEs, TE expansion, reduction in secondary metabolism clusters). Functional specialization in ECM decomposition may drive diversification. Accelerated gene evolution predates the evolution of the ECM habit, indicating that changes in genome architecture and gene content may be necessary to prime the evolutionary switch.

Coalescent-based delimitation and species-tree estimations reveal Appalachian origin and Neogene diversification in Russula subsection Roseinae.

Numerous lineages of mushroom-forming fungi have been subject to bursts of diversification throughout their evolutionary history, events that can impact our ability to infer well-resolved phylogenies. However, groups that have undergone quick genetic change may have the highest adaptive potential. As the second largest genus of mushroom-forming fungi, Russula provides an excellent model for studying hyper-diversification and processes in evolution that drives it. This study focuses on the morphologically defined group - Russula subsection Roseinae. Species hypotheses based on morphological differentiation and multi-locus phylogenetic analyses are tested in the Roseinae using different applications of the multi-species coalescent model. Based on this combined approach, we recognize fourteen species in Roseinae including the Albida and wholly novel Magnarosea clades. Reconstruction of biogeographic and host association history suggest that parapatric speciation in refugia during glacial cycles of the Pleistocene drove diversification within the Roseinae, which is found to have a Laurasian distribution with an evolutionary origin in the Appalachian Mountains of eastern North America. Finally, we detect jump dispersal at a continental scale that has driven diversification since the most recent glacial cycles.

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.

Russulaceae: a new genomic dataset to study ecosystem function and evolutionary diversification of ectomycorrhizal fungi with their tree associates.

The family Russulaceae is considered an iconic lineage of mostly mushroom-forming basidiomycetes due to their importance as edible mushrooms in many parts of the world, and their ubiquity as ectomycorrhizal symbionts in both temperate and tropical forested biomes. Although much research has been focused on this group, a comprehensive or cohesive synthesis by which to understand the functional diversity of the group has yet to develop. Interest in ectomycorrhizal fungi, of which Russulaceae is a key lineage, is prodigious due to the important roles they play as plant root mutualists in ecosystem functioning, global carbon sequestration, and a potential role in technology development toward environmental sustainability. As one of the most species-diverse ectomycorrhizal lineages, the Russulaceae has recently been the focus of a dense sampling and genome sequencing initiative with the Joint Genome Institute aimed at untangling their functional roles and testing whether functional niche specialization exists for independent lineages of ectomycorrhizal fungi. Here we present a review of important studies on this group to contextualize what we know about its members' evolutionary history and ecosystem functions, as well as to generate hypotheses establishing the Russulaceae as a valuable experimental system.

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.

Into and out of the tropics: global diversification patterns in a hyperdiverse clade of ectomycorrhizal fungi.

Ectomycorrhizal (ECM) fungi, symbiotic mutualists of many dominant tree and shrub species, exhibit a biogeographic pattern counter to the established latitudinal diversity gradient of most macroflora and fauna. However, an evolutionary basis for this pattern has not been explicitly tested in a diverse lineage. In this study, we reconstructed a mega-phylogeny of a cosmopolitan and hyperdiverse genus of ECM fungi, Russula, sampling from annotated collections and utilizing publically available sequences deposited in GenBank. Metadata from molecular operational taxonomic unit cluster sets were examined to infer the distribution and plant association of the genus. This allowed us to test for differences in patterns of diversification between tropical and extratropical taxa, as well as how their associations with different plant lineages may be a driver of diversification. Results show that Russula is most species-rich at temperate latitudes and ancestral state reconstruction shows that the genus initially diversified in temperate areas. Migration into and out of the tropics characterizes the early evolution of the genus, and these transitions have been frequent since this time. We propose the 'generalized diversification rate' hypothesis to explain the reversed latitudinal diversity gradient pattern in Russula as we detect a higher net diversification rate in extratropical lineages. Patterns of diversification with plant associates support host switching and host expansion as driving diversification, with a higher diversification rate in lineages associated with Pinaceae and frequent transitions to association with angiosperms.

Multilocus phylogenetic reconstruction of the Clavariaceae (Agaricales) reveals polyphyly of agaricoid members.

The genus Camarophyllopsis contains species with lamellate (agaricoid) basidiomes in the family Clavariaceae (Agaricales), a group otherwise dominated by club-like (clavarioid) or branched (coralloid) forms. Previous studies have suggested that species classified in Camarophyllopsis occur in two independent lineages. We reconstructed a multilocus phylogeny of the Clavaria-Camarophyllopsis-Clavicorona clade in the Clavariaceae using RNA polymerase II second largest subunit (rpb2), nuclear ribosomal 28S, and nuclear ribosomal ITS1-5.8S-ITS2 regions data and detected three independent groups of agaricoid fungi, including the genera Camarophyllopsis, Hodophilus, and Lamelloclavaria gen. nov, which distinctly differ in their pileipellis structure. In all, nine major lineages within the Clavaria-Camarophyllopsis-Clavicorona clade were recovered: Clavaria sensu stricto, Camarophyllopsis sensu stricto, Hodophilus, the Clavaria pullei clade, the Clavaria fumosa clade, Lamelloclavaria gen. nov., the Clavaria atrofusca clade, Holocoryne (= Clavaria sect. Holocoryne), and Clavicorona Clavaria is paraphyletic and represented by five clades. Additional gene sampling is necessary to determine and confirm relatedness of these lineages before splitting Clavaria into additional genera.

Long-distance dispersal and speciation of Australasian and American species of Cortinarius sect. Cortinarius.

We present a multigene phylogeny (partial nuc rDNA and RPB2) of Cortinarius sect. Cortinarius (i.e. the C. violaceus group), which reveals eight species distributed in Europe, Australasia, South America, Central America and North America. Relaxed molecular clock analyses suggested that diversification began during the Miocene, thus rejecting more ancient Gondwanan origin scenarios among the taxa currently occurring in the northern and southern hemispheres. There was strong support for an Australasian origin of the C. violaceus group with initial dispersal to the Neotropics, followed by migration into North America and Europe. A dispersal-extinction cladogenesis model that includes a parameter for founder effects was the most highly supported biogeographic model in the program BioGeoBEARS. A maximum likelihood analysis showed the most recent common ancestor of sect. Cortinarius was an angiosperm ectomycorrhizal associate. Ancestral associations at the plant family level, however, were ambiguous. Of eight recovered species-level lineages, C. violaceus is the only one that associates with Pinaceae and the only species to associate with both Pinaceae and angiosperms. This analysis showed that long-distance dispersal and founder event speciation have been important factors during evolution of the C. violaceus group.

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.

Three new species in Russula subsection Xerampelinae supported by genealogical and phenotypic coherence.

Xerampelinae is a subsection composed of species of ectomycorrhizal fungi belonging to the hyperdiverse and cosmopolitan genus Russula (Russulales). Species of Xerampelinae are recognized by their fishy or shrimp odor, browning context, and a green reaction to iron sulfate. However, species delimitation has traditionally relied on morphology and analysis of limited molecular data. Prior taxonomic work in Xerampelinae has led to the description of as many as 59 taxa in Europe and 19 in North America. Here we provide the first multilocus phylogeny of European and North American members based on two nrDNA loci and two protein-coding genes. The resulting phylogeny supports the recognition of 17 species-rank Xerampelinae clades; however, higher species richness (~23) is suggested by a more inclusive nuclear rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS barcode) analysis. Phylogenetic and morphological analyses support three new species with restricted geographic distributions: R. lapponica, R. neopascua, and R. olympiana. We confirm that the European species R. subrubens is present in North America and the North American species R. serissima (previously known as R. favrei) is present in Europe. Most other Xerampelinae appear restricted to either North America or Eurasia, which indicates a high degree of regional endemism; this includes R. xerampelina, a name widely applied to North American taxa, but a species restricted to Eurasia.

Towards a unified paradigm for sequence-based identification of fungi.

The nuclear ribosomal internal transcribed spacer (ITS) region is the formal fungal barcode and in most cases the marker of choice for the exploration of fungal diversity in environmental samples. Two problems are particularly acute in the pursuit of satisfactory taxonomic assignment of newly generated ITS sequences: (i) the lack of an inclusive, reliable public reference data set and (ii) the lack of means to refer to fungal species, for which no Latin name is available in a standardized stable way. Here, we report on progress in these regards through further development of the UNITE database (http://unite.ut.ee) for molecular identification of fungi. All fungal species represented by at least two ITS sequences in the international nucleotide sequence databases are now given a unique, stable name of the accession number type (e.g. Hymenoscyphus pseudoalbidus|GU586904|SH133781.05FU), and their taxonomic and ecological annotations were corrected as far as possible through a distributed, third-party annotation effort. We introduce the term 'species hypothesis' (SH) for the taxa discovered in clustering on different similarity thresholds (97-99%). An automatically or manually designated sequence is chosen to represent each such SH. These reference sequences are released (http://unite.ut.ee/repository.php) for use by the scientific community in, for example, local sequence similarity searches and in the QIIME pipeline. The system and the data will be updated automatically as the number of public fungal ITS sequences grows. We invite everybody in the position to improve the annotation or metadata associated with their particular fungal lineages of expertise to do so through the new Web-based sequence management system in UNITE.

A common new species of Inocybe in the Pacific Northwest with a diagnostic PDAB reaction.

A species of Inocybe common in Washington, Oregon and British Columbia is documented and described as new. The species, I. chondroderma, is characterized by these features: pileus with a fulvous disk and ochraceous to chamois margin, presence of a cortina, densely mycelioid stipe base, smooth spores and fall phenology. The most reliable and distinctive feature of the species is a blue-green or turquoise reaction in response to application of a solution of p-dimethylaminobenzaldehyde (PDAB), indicating the presence of what is most likely an indole alkaloid. PDAB use provides a quick and diagnostic character easily implemented in a laboratory setting. ITS sequences from recent collections of I. chondroderma and from materials collected in the 1940s in Washington and Oregon fully match numerous mislabeled sequences from specimens in British Columbia and Oregon. The species is most closely related to an unclarified taxon from Colorado and Japan (I. cf. chondroderma) and a rare European species, I. subnudipes. Nine different species names in Inocybe and one in Hebeloma attributed to I. chondroderma based on GenBank BLASTN searches of the ITS locus match with 99-100% similarity, reinforcing concerns about taxonomic inaccuracies in public DNA sequence databases. A complete morphological description, illustrations and phylogenetic assessment are provided.

Inocybe section Rimosae in Utah: phylogenetic affinities and new species.

Results of a study on species of Inocybe section Rimosae sensu lato in Utah are presented. Eight species, seven from the Pseudosperma clade (section Rimosae sensu stricto) and one from the Inosperma clade (section Rimosae pro parte), are documented morphologically and phylogenetically. Five of the eight species, I. aestiva, I. breviterincarnata, I. cercocarpi, I. niveivelata and I. occidentalis-all members of the Pseudosperma clade-are described as new from Utah and other western states. Two European species, I. spuria and I. obsoleta, are confirmed from Utah. Inocybe aurora, originally described from Nova Scotia, is synonymized with I. obsoleta. The only member of the Inosperma clade recorded from Utah is I. lanatodisca, a widely distributed species for which three geographical clusters were detected. The phylogenetic analyses indicate that the Pseudosperma clade includes 53 clusters or species worldwide and that the Inosperma clade includes 47 such clusters. Many of these probably correspond to undescribed species. A key to species of section Rimosae sensu lato from Utah is provided together with illustrations of the eight species found in the state.

A systematic, morphological and ecological overview of the Clavariaceae (Agaricales).

The Clavariaceae is a diverse family of mushroom-forming fungi composed of species that produce simple clubs, coralloid, lamellate-stipitate, hydnoid and resupinate sporocarps. Here we present a systematic and ecological overview of the Clavariaceae based on phylogenetic analysis of sequences of the nuclear large subunit ribosomal RNA (nLSU), including nine from type collections. Forty-seven sequences from sporocarps of diverse taxa across the Clavariaceae were merged with 243 environmental sequences from GenBank and analyzed phylogenetically to determine major clades within the family. Four major clades or lineages were recovered: (i) Mucronella, (ii) Ramariopsis-Clavulinopsis, (iii) Hyphodontiella and (iv) Clavaria-Camarophyllopsis-Clavicorona. Clavaria is paraphyletic, within which the lamellate and pileate-stipitate genus Camarophyllopsis is derived and composed of two independent lineages. The monotypic genus Clavicorona also appears nested within Clavaria. The monophyly of Clavaria and Camarophyllopsis, however, cannot be statistically rejected. We compared differing classification schemes for the genera Ramariopsis and Clavulinopsis, most of which are inconsistent with the molecular phylogeny and are statistically rejected. Scytinopogon, a genus classified in the Clavariaceae by several authors, shares phylogenetic affinities with the Trechisporales. Overall 126 molecular operational taxonomic units can be recognized in the Clavariaceae, roughly half of which are known only from environmental sequences, an estimate that exceeds the known number of species in the family. Stable isotope ratios of carbon and nitrogen were measured from specimens representing most major phylogenetic lineages to predict trophic strategies. These results suggest that most non-lignicolous species feature a biotrophic mode of nutrition. Ancestral state reconstruction analysis highlights the taxonomic significance of at least nine morphological traits at various depths in the family tree.

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.

Asynchronous origins of ectomycorrhizal clades of Agaricales.

The ectomycorrhizal (ECM) symbiosis is the most widespread biotrophic nutritional mode in mushroom-forming fungi. ECM fungi include, though are not limited to, about 5000 described species of Agaricales from numerous, independently evolved lineages. Two central hypotheses suggest different explanations for the origin of ECM fungal diversity: (i) dual origins, initially with the Pinaceae in the Jurassic and later with angiosperms during the Late Cretaceous, and (ii) a simultaneous and convergent radiation of ECM lineages in response to cooling climate during the Palaeogene and advancing temperate ECM plant communities. Neither of these hypotheses is supported here. While we demonstrate support for asynchronous origins of ECM Agaricales, the timing of such events appears to have occurred more recently than suggested by the first hypothesis, first during the Cretaceous and later during the Palaeogene. We are also unable to reject models of rate constancy, which suggests that the diversity of ECM Agaricales is not a consequence of convergent rapid radiations following evolutionary transitions from saprotrophic to ECM habits. ECM lineages of Agaricales differ not only in age, but also in rates of diversification and rate of substitution at nuclear ribosomal RNA loci. These results question the biological uniformity of the ECM guild.

DivBayes and SubT: exploring species diversification using Bayesian statistics.

SUMMARY: DivBayes is a program to estimate diversification rates from species richness and ages of a set of clades. SubT estimates diversification rates from node heights within a clade. Both programs implement Bayesian statistics and provide the ability to account for uncertainty in the ages of taxa in the underlying data, an improvement over more commonly used maximum likelihood methods. AVAILABILITY: DivBayes and SubT are released as C++ source code under the GNU GPL v. 3 software license in Supplementary information 1 and 2, respectively, and at http://web.utk.edu/~kryberg/. They have been successfully compiled on various Linux, MacOS X and Windows systems. CONTACT: kryberg@utk.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.