Rare but widespread: A systematic revision of the truffle-forming genera Destuntzia and Kjeldsenia and the formation of a new genus, Hosakaea.

Here we present the results of taxonomic and systematic study of the rare truffle-forming genera Destuntzia and Kjeldsenia. Truffle-forming fungi are difficult to study due to their reduced morphological features and their cryptic, hypogeous fruiting habits. The rare occurrence of Destuntzia and Kjeldsenia further compounds these difficulties due to the lack of adequate material for study. Recent forays in North Carolina and Tennessee recovered new specimens of another rarely collected fungus, Octaviania purpurea. Morphological and phylogenetic analysis revealed that Octaviania purpurea is a member of the genus Destuntzia, and this led us to reevaluate the taxonomic status and systematic relationships of other Destuntzia species. We performed a multilocus phylogenetic analysis of Destuntzia specimens deposited in public fungaria, including all available type material, and environmental sequences from animal scat and soil. Our analyses indicate that Destuntzia is a member of the family Claustulaceae within the order Phallales and is a close relative of Kjeldsenia. Results of our phylogenetic analysis infer that three species originally described in the genus Destuntzia are members of the genus Kjeldsenia. We propose three new combinations in Kjeldsenia to accommodate these species as well as a new combination in Destuntzia to accommodate Octaviania purpurea. We also describe a new genus in Claustulaceae, Hosakaea, to accommodate a closely affiliated species, Octaviania violascens. Finally, we transfer the genus Destunzia into the family Claustulaceae and emend the description of the family. The newly proposed combinations in Destuntzia and Kjeldsenia significantly expand the known geographic ranges of both genera. The data from metabarcode analysis of scat and soil also reveal several additional undescribed species that expand these ranges well beyond those suggested by basidiomata collections. Systematic placement of Destuntzia in the saprotrophic order Phallales suggests that this genus is not ectomycorrhizal, and the ecological implications of this systematic revision are discussed.

Thaxterogaster revisited: A phylogenetic and taxonomic overview of sequestrate Cortinarius from Patagonia.

In the Patagonian region, Cortinarius is the most diverse and abundant genus of ectomycorrhizal fungi with at least 250 species. Sequestrate forms were until recently documented within the genus Thaxterogaster, a genus now known to be polyphyletic, and many were consequently transferred to Cortinarius. Original descriptions were mostly available in German and Spanish and interpretations of morphological structures outdated. Despite recent advances in Cortinarius systematics, the current classification, diversity, and ecology of sequestrate "cortinarioid" fungi in Patagonia remain unclear. The objective of this study was to provide an update on sequestrate Cortinarius of southern South America. We documented each species with morphological descriptions, photographs, basidiospore scanning electron microscopy (SEM) images, and molecular characterization using nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS) and nuc 28S rDNA (28S) sequence data. Original descriptions of taxa were also translated to English and revised based on fresh collections. We documented 24 species from Patagonia based on molecular data and conducted morphological and phylogenetic analysis for 18 previously described species based on type and reference specimens. In addition, we formally described two new species. Four additional taxa were provisionally determined as new but require further study. New ITS sequence data were produced from eight type specimens. We also provide a new name, Cortinarius gloiodes, nom. nov., for the taxon previously described as Thaxterogaster gliocyclus. In addition to the species treated in detail, we provided additional reference information and discussion on six described species that remained incompletely known or for which no recent collections were found. Of the 24 taxa documented from Patagonia, 15 species were assigned to 12 current sections in the genus Cortinarius. Analysis of spore ultrastructure showed that sequestrate forms of Patagonian Cortinarius lack a true perisporium.

Brahmaculus gen. nov. (Leotiomycetes, Chlorociboriaceae).

A second genus in Chlorociboriaceae is described here as Brahmaculus gen. nov. Macroscopically distinctive, all species have bright yellow apothecia with several apothecial cups held on short branches at the tip of a long stipe. The genus is widely distributed across the Southern Hemisphere; the four new species described here include two from Chile (B. magellanicus sp. nov., B. osornoensis sp. nov.) and one each from New Zealand (B. moonlighticus sp. nov.) and Australia (B. packhamiae sp. nov.). They differ from species referred to Chlorociboria, the only other genus in Chlorociboriaceae, in their terrestrial habitat and ascomata that are noticeably more hairy than the known Chlorociboria species, most of which have apothecia with short, macroscopically indistinct hair-like elements. Based on our analyses, Chlorociboria as accepted here is paraphyletic. Additional study is needed to clarify where alternative, monophyletic generic limits should be drawn and how these genera may be recognised morphologically. Also described here are three new Chlorociboria spp. from New Zealand (C. metrosideri sp. nov., C. solandri sp. nov., C. subtilis sp. nov.), distinctive in developing on dead leaves rather than wood and in two of them not forming the green pigmentation characteristic of most Chlorociboria species. New Zealand specimens previously incorrectly identified as Chlorociboria argentinensis are provided with a new name, C. novae-zelandiae sp. nov.

Effects of Field Fumigation and Inoculation With the Pecan Truffle (Tuber lyonii) on the Fungal Community of Pecan (Carya illinoinensis) Seedlings Over 5 Years.

Truffle fungi are esteemed for their aromatic qualities and are among the most widely cultivated edible ectomycorrhizal fungi. Here we document a successful method for establishing Tuber lyonii, the pecan truffle, on pecan (Carya illinoinensis) seedlings in a field setting. We assessed the impacts of soil fumigation and varying concentrations of truffle spore inoculum on the ectomycorrhizal fungal and the complete fungal communities as well as the colonization of T. lyonii on pecan roots at three nurseries in Georgia, United States. To identify fungal communities on pecan seedlings, we performed high-throughput amplicon sequencing of the fungal ITS1 rDNA region. Our 5-year long field experiment demonstrates that fumigation and inoculation together resulted in the highest persistence of T. lyonii on pecan roots. While fungal OTU numbers fluctuated over the years of our experiments, there was no statistical support to demonstrate diversification of communities when Shannon diversity metrics were used. However, we did find that older seedlings were less likely to be dominated by T. lyonii compared to younger ones, suggesting successional changes in the fungal community over time. This suggests that transplanting inoculated seedlings after 2 or 3 years post-inoculation is optimal for future truffle propagation efforts. Our results demonstrate that T. lyonii can be established in situ with methods that are compatible with current pecan nursery industry practices and that fungal communities on pecan seedlings vary depending on the experimental treatments used during planting. While the pecan truffle is not yet widely cultivated, our results provide insights for future large-scale cultivation of this and perhaps other Tuber species.

Cortinarius section Thaumasti in South American Nothofagaceae forests.

We studied the South American species of Cortinarius section Thaumasti based on morphological and molecular data. Members of this group can easily be identified in the field because the basidiomata are small and Phlegmacium-like with a bulbous stipe and the universal veil in most species forms a distinct volva at the base of the stipe. The phylogenetic delimitation of the clade was mostly in concordance with the earlier, morphology-based grouping of the South American taxa except that C. chrysophaeus was resolved outside of the clade. Altogether nine species were recognized in the section. Four species, C. chlorophanus, C. coleopus, C. cosmoxanthus, and C. vaginatus, were previously described by other authors, whereas three species, C. chlorosplendidus, C. olivaceovaginatus, and C. subcosmoxanthus, are described here as new. We were able to identify two remaining taxa, but we do not have sufficient morphological data to allow for a formal description. All of the species in C. section Thaumasti form ectomycorrhizal associations with Nothofagaceae. They have been documented from South America and New Zealand. The Patagonian species are considered endemic to the region. A key to the described species is provided.

Systematic study of truffles in the genus Ruhlandiella, with the description of two new species from Patagonia.

Ruhlandiella is a genus of exothecial, ectomycorrhizal fungi in the order Pezizales. Ascomata of exothecial fungi typically lack a peridium and are covered with a hymenial layer instead. Ruhlandiella species have nonoperculate asci and highly ornamented ascospores. The genus was first described by Hennings in 1903 to include the single species, R. berolinensis. Since then, mycologists have uncovered Ruhlandiella species in many locations around the globe, including Australia, Spain, Italy, and the USA. Currently, there are four recognized species: R. berolinensis, R. peregrina, R. reticulata, and R. truncata. All were found near Eucalyptus or Melaleuca trees of Australasian origin. Recently, we discovered two new species of Ruhlandiella in Nothofagaceae forests in South America. They regularly form mitotic spore mats directly on soil in the forests of Patagonia. Here, we formally describe these new species and construct the phylogeny of Ruhlandiella and related genera using a multilocus phylogenetic analysis. We also revise the taxonomy of Ruhlandiella and provide an identification key to accepted species of Ruhlandiella.

The Cedrus-associated truffle Trappeindia himalayensis is a morphologically unique and phylogenetically divergent species of Rhizopogon.

In the northwestern Himalayan mountains of India, the hypogeous sequestrate fungus Trappeindia himalayensis is harvested from forests dominated by the ectomycorrhizal tree Cedrus deodara (Himalayan cedar). This truffle has basidiospores that are ornamented with raised reticulation. The original description of Trappeindia himalayensis suggested that the gleba of this species is similar to young specimens of Scleroderma (Boletales), whereas its basidiospores are ornamented with raised reticulation, suggesting a morphological affinity to Leucogaster (Russulales) or Strobilomyces (Boletales). Given this systematic ambiguity, we have generated DNA sequence data from type material and other herbarium specimens and present the first molecular phylogenetic analysis of this unusual Cedrus-associated truffle. Despite the irregular ornamented basidiospore morphology, T. himalayensis is resolved within the genus Rhizopogon (Suillineae, Boletales) and represents a unique lineage that has not been previously detected. All known Rhizopogon species possess an ectomycorrhizal trophic mode, and because of its placement in this lineage, it is likely that Trappeindia himalayensis is an ectomycorrhizal partner of Cedrus deodara. This study highlights the importance of generating sequence data from herbarium specimens in order to identify fungal biodiversity and clarify the systematic relationships of poorly documented fungi.

Unveiling new sequestrate Cortinarius species from northern Patagonian Nothofagaceae forests based on molecular and morphological data.

Because of systematic sampling campaigns in the northern Patagonian Nothofagaceae forests of Argentina, several specimens of sequestrate fungi were collected. Some of those collections showed phylogenetic affinities and morphological similarities to members of the formerly recognized sequestrate genus Thaxterogaster, currently a synonym of Cortinarius on the basis of molecular data. Comparisons of macro- and micromorphological features and sequences of nuc rDNA internal transcribed spacer (ITS) regions have revealed that these collections belong to formerly undescribed species. The sequences of the four new taxa presented here, Cortinarius flavopurpureus, C. translucidus, C. nahuelhuapensis, and C. infrequens, were combined into a data set including additional sequences generated from herbarium collections and retrieved from public gene databases and analyzed by maximum likelihood and Bayesian inference methods. The four new species were resolved as distinct clades with strong support; at the same time, they showed unique morphological characteristics (hypogeous to subhypogeous habit, complete gasteromycetation, and spore shape and ornamentation) that separate them from previously described Cortinarius species. In addition, several undescribed and/or not previously sequenced species from these forests were detected through phylogenetic analysis of ectomycorrhizal root tip sequences. A key of characters to identify the sequestrate Cortinarius from Patagonia is provided.

Ectomycorrhizal fungi and soil enzymes exhibit contrasting patterns along elevation gradients in southern Patagonia.

The biological and functional diversity of ectomycorrhizal (ECM) associations remain largely unknown in South America. In Patagonia, the ECM tree Nothofagus pumilio forms monospecific forests along mountain slopes without confounding effects of vegetation on plant-fungi interactions. To determine how fungal diversity and function are linked to elevation, we characterized fungal communities, edaphic variables, and eight extracellular enzyme activities along six elevation transects in Tierra del Fuego (Argentina and Chile). We also tested whether pairing ITS1 rDNA Illumina sequences generated taxonomic biases related to sequence length. Fungal community shifts across elevations were mediated primarily by soil pH with the most species-rich fungal families occurring mostly within a narrow pH range. By contrast, enzyme activities were minimally influenced by elevation but correlated with soil factors, especially total soil carbon. The activity of leucine aminopeptidase was positively correlated with ECM fungal richness and abundance, and acid phosphatase was correlated with nonECM fungal abundance. Several fungal lineages were undetected when using exclusively paired or unpaired forward ITS1 sequences, and these taxonomic biases need reconsideration for future studies. Our results suggest that soil fungi in N. pumilio forests are functionally similar across elevations and that these diverse communities help to maintain nutrient mobilization across the elevation gradient.

New species of Cortinarius sect. Austroamericani, sect. nov., from South American Nothofagaceae forests.

In this study, we document and describe the new Cortinarius section Austroamericani. Our results reveal high species diversity within this clade, with a total of 12 recognized species. Of these, only C. rufus was previously documented. Seven species are described as new based on basidiomata collections. The four remaining species are only known from environmental sequences. All examined species form ectomycorrhizal associations with species of Nothofagaceae and are currently only known from Argentinean and Chilean Patagonia. The phylogenetic analysis based on the nuc rDNA internal transcriber spacer (ITS1-5.8S-ITS2 = ITS) and partial 28S gene (28S) sequences shows that this section is related to other taxa from the Southern Hemisphere. Species in this group do not belong to subg. Telamonia, where C. rufus was initially placed. Cortinarius rufus and the newly described C. subrufus form a basal clade within sect. Austroamericani that has a weakly supported relationship with the core clade. Because the two species are morphologically similar to species from the core clade and share their distribution and Nothofagaceae associations, we include them here as part of sect. Austroamericani sensu lato (s.l.) until more material is available to refine the delimitation.

Phylogenetic studies in Genabea, Myrmecocystis, and related genera.

A multigene phylogenetic analysis of Genabea, Myrmecocystis, and related genera in Pyronemataceae was conducted to establish genetically supported generic limits. The nuc rDNA 28S gene, translation elongation factor 1-α gene (TEF1), and RNA polymerase II second-largest subunit gene (RPB2) significantly supported the monophyly of several distinct genera of hypogeous Pyronemataceae. The genetic identity of Genabea fragilis is established, and the new species Genabea hyalospora, G. urbana, Myrmecocystis mediterranea, and M. microspora are proposed to accommodate undescribed lineages. Two rare species, M. sphaerospora and M. spinospora, are tentatively identified based on collections that are morphologically similar to the original descriptions. The genus Genea was found to be monophyletic, except for G. cazaresii, which is nested among epigeous species of Humaria. The new combination Humaria cazaresii is proposed to accommodate this species.

A systematic overview of Descolea (Agaricales) in the Nothofagaceae forests of Patagonia.

The descolea clade includes species of ectomycorrhizal basidiomycetes in the genera Descolea, Setchelliogaster, Descomyces, and Timgrovea that are known primarily from the Southern Hemisphere. Taxa in this group produce basidiomes that range in morphology from typical epigeous mushrooms (Descolea) and secotioid taxa (Setchelliogaster) to fully gasteroid species (Descomyces and Timgrovea). High intraspecific morphological variation has been reported in several species within this clade, suggesting that careful morphological and molecular studies are needed to refine species concepts. Molecular analyses of fresh Patagonian collections in conjunction with taxonomic studies have confirmed high variability in key morphological features, including overall sporocarp form, spore shape and dimensions, universal veil remnants, and cuticle configuration. Based on our synthesis, we emend the genus Descolea to include sequestrate species. We describe the new sequestrate taxon Descolea inferna sp. nov. from Nothofagaceae forests in Patagonia and we propose Cortinarius squamatus as a synonym of our new combination Descolea brunnea. We also formalize the identity of Descolea pallida as a synonym of Descolea antarctica and provide new specimens of Cortinarius archeuretus, a species that has not been encountered since the original discovery during the expeditions of Roland Thaxter in 1905-1906. Here we re-describe and transfer this species to Descolea as D. archeureta. We also discuss diagnostic features that can be used to delimitate the four known South American taxa in the descolea clade.

Comparative Genomics of the Ectomycorrhizal Sister Species Rhizopogon vinicolor and Rhizopogon vesiculosus (Basidiomycota: Boletales) Reveals a Divergence of the Mating Type B Locus.

Divergence of breeding system plays an important role in fungal speciation. Ectomycorrhizal fungi, however, pose a challenge for the study of reproductive biology because most cannot be mated under laboratory conditions. To overcome this barrier, we sequenced the draft genomes of the ectomycorrhizal sister species Rhizopogon vinicolor Smith and Zeller and R. vesiculosus Smith and Zeller (Basidiomycota, Boletales)-the first genomes available for Basidiomycota truffles-and characterized gene content and organization surrounding their mating type loci. Both species possess a pair of homeodomain transcription factor homologs at the mating type A-locus as well as pheromone receptor and pheromone precursor homologs at the mating type B-locus. Comparison of Rhizopogon genomes with genomes from Boletales, Agaricales, and Polyporales revealed synteny of the A-locus region within Boletales, but several genomic rearrangements across orders. Our findings suggest correlation between gene content at the B-locus region and breeding system in Boletales with tetrapolar species possessing more diverse gene content than bipolar species. Rhizopogon vinicolor possesses a greater number of B-locus pheromone receptor and precursor genes than R. vesiculosus, as well as a pair of isoprenyl cysteine methyltransferase genes flanking the B-locus compared to a single copy in R. vesiculosus Examination of dikaryotic single nucleotide polymorphisms within genomes revealed greater heterozygosity in R. vinicolor, consistent with increased rates of outcrossing. Both species possess the components of a heterothallic breeding system with R. vinicolor possessing a B-locus region structure consistent with tetrapolar Boletales and R. vesiculosus possessing a B-locus region structure intermediate between bipolar and tetrapolar Boletales.

Competitive avoidance not edaphic specialization drives vertical niche partitioning among sister species of ectomycorrhizal fungi.

Soil depth partitioning is thought to promote the diversity of ectomycorrhizal (EM) fungal communities, but little is known about whether it is controlled by abiotic or biotic factors. In three bioassay experiments, we tested the role of vertical soil heterogeneity in determining the distributions and competitive outcomes of the EM sister species Rhizopogon vinicolor and Rhizopogon vesiculosus. We planted Pseudotsuga menziesii seedlings into soils that were either a homogenized mix of upper and lower depths or vertically stratified combinations mimicking natural field conditions. We found that both species colonized the upper or lower soil depths in the absence of competition, suggesting that their distributions were not limited by abiotic edaphic factors. In competition within homogeneous soils, R. vesiculosus completely excluded colonization by R. vinicolor, but R. vinicolor was able to persist when soils were stratified. The amount of colonization by R. vinicolor in the stratified soils was also significantly correlated with the number of multilocus genotypes present. Taken together, our findings suggest that the differential vertical distributions of R. vinicolor and R. vesiculosus in natural settings are probably attributable to competition rather than edaphic specialization, but that soil heterogeneity may play a key role in promoting EM fungal diversity.

First detection of the larval chalkbrood disease pathogen Ascosphaera apis (Ascomycota: Eurotiomycetes: Ascosphaerales) in adult bumble bees.

Fungi in the genus Ascosphaera (Ascomycota: Eurotiomycetes: Ascosphaerales) cause chalkbrood disease in larvae of bees. Here, we report the first-ever detection of the fungus in adult bumble bees that were raised in captivity for studies on colony development. Wild queens of Bombus griseocollis, B. nevadensis and B. vosnesenskii were collected and maintained for establishment of nests. Queens that died during rearing or that did not lay eggs within one month of capture were dissected, and tissues were examined microscopically for the presence of pathogens. Filamentous fungi that were detected were plated on artificial media containing broad spectrum antibiotics for isolation and identification. Based on morphological characters, the fungus was identified as Ascosphaera apis (Maasen ex Claussen) Olive and Spiltoir, a species that has been reported earlier only from larvae of the European honey bee, Apis mellifera, the Asian honey bee, Apis cerana, and the carpenter bee Xylocopa californica arizonensis. The identity of the fungus was confirmed using molecular markers and phylogenetic analysis. Ascosphaera apis was detected in queens of all three bumble bee species examined. Of 150 queens dissected, 12 (8%) contained vegetative and reproductive stages of the fungus. Both fungal stages were also detected in two workers collected from colonies with Ascosphaera-infected B. nevadensis queens. In this study, wild bees could have been infected prior to capture for rearing, or, the A. apis infection could have originated via contaminated European honey bee pollen fed to the bumble bees in captivity. Thus, the discovery of A. apis in adult bumble bees in the current study has important implications for commercial production of bumble bee colonies and highlights potential risks to native bees via pathogen spillover from infected bees and infected pollen.

Rhizopogon togasawariana sp. nov., the first report of Rhizopogon associated with an Asian species of Pseudotsuga.

Rhizopogon subgenus Villosuli are the only members of the genus known to form an ectomycorrhizal relationship exclusively with Pseudotsuga. The specificity of this host relationship is unusual in that Rhizopogon is broadly associated with several tree genera within the Pinaceae and relationships with a host genus are typically distributed across Rhizopogon subgenera. Naturally occurring specimens of R. subg. Villosuli have been described only from North American collections, and the unique host relationship with Pseudotsuga is demonstrated only for Rhizopogon associated with P. menziesii (Douglas-fir), the dominant species of Pseudotsuga in North America. Species of Pseudotsuga are naturally distributed around the northern Pacific Rim, and Rhizopogon associates of other Pseudotsuga spp. are not yet described. Here we present the results of field sampling conducted in P. japonica forests throughout the Japanese archipelago and describe Rhizopogon togasawariana sp. nov., which occurs in ectomycorrhizal association with P. japonica. Placement of this new species within R. subg. Villosuli is supported by morphological and molecular phylogenetic analysis, and its implications to Pseudotsuga-Rhizopogon biogeography are discussed.

Within-population genetic structure differs between two sympatric sister-species of ectomycorrhizal fungi, Rhizopogon vinicolor and R. vesiculosus.

Using spatial autocorrelation analysis, we examined the within-population genetic structure of Rhizopogon vinicolor and R. vesiculosus, two hypogeous ectomycorrhizal (EM) species that are sympatric sister taxa known to differ in their clonal structure. We collected 121 sporocarps and 482 tuberculate EM of both species from a 20 ha forest stand dominated by Douglas-fir (Pseudotsuga menziesii). Field collections were identified to species with restriction fragment length polymorphism analysis of the nuclear ribosomal internal transcribed spacer. Five and six microsatellite markers were used to characterize the genetic diversity of EM and sporocarp samples from R. vesiculosus and R. vinicolor respectively. After correcting for genet structure, spatial autocorrelation analyses of the EM samples were used to test the null hypothesis that multilocus genotypes characterized from each species were randomly distributed within the study area. We detected positive and statistically significant fine-scale genetic structure up to 120 m within the R. vesiculosus sample. In contrast, no spatial genetic structure was evident for R. vinicolor, indicating that the genotypes characterized for this species were randomly distributed throughout the study area. Differences in statistical power or the nuclear count of basidiospores are unlikely agents of the genetic patterns observed. Our results suggest that differences in reproductive output or competitive ability may act individually or in combination to create clusters of similar genotypes for R. vesiculosus throughout the study area.