Introducing ribosomal tandem repeat barcoding for fungi

Sequence comparison and analysis of the various ribosomal genetic markers are the dominant molecular methods for identification and description of fungi. However, new environmental fungal lineages known only from DNA data reveal significant gaps in our sampling of the fungal kingdom in terms of both taxonomy and marker coverage in the reference sequence databases. To facilitate the integration of reference data from all of the ribosomal markers, we present three sets of general primers that allow for amplification of the complete ribosomal operon from the ribosomal tandem repeats. The primers cover all ribosomal markers: ETS, SSU, ITS1, 5.8S, ITS2, LSU and IGS. We coupled these primers successfully with third-generation sequencing (PacBio and Nanopore sequencing) to showcase our approach on authentic fungal herbarium specimens (Basidiomycota), aquatic chytrids (Chytridiomycota) and a poorly understood lineage of early diverging fungi (Nephridiophagidae). In particular, we were able to generate high-quality reference data with Nanopore sequencing in a high-throughput manner, showing that the generation of reference data can be achieved on a regular desktop computer without the involvement of any large-scale sequencing facility. The quality of the Nanopore generated sequences was 99.85%, which is comparable with the 99.78% accuracy described for Sanger sequencing. With this work, we hope to stimulate the generation of a new comprehensive standard of ribosomal reference data with the ultimate aim to close the huge gaps in our reference datasets

Solving the taxonomic identity of Pseudotomentella tristis s.l. (Thelephorales, Basidiomycota) – a multi-gene phylogeny and taxonomic review, integrating ecological and geographical data

P. tristis is an ectomycorrhizal, corticioid fungus whose name is frequently assigned to collections of basidiomata as well as root tip and soil samples from a wide range of habitats and hosts across the northern hemisphere. Despite this, its identity is unclear; eight heterotypic taxa have in major reviews of the species been considered synonymous with or morphologically similar to P. tristis, but no sequence data from type specimens have been available. With the aim to clarify the taxonomy, systematics, morphology, ecology and geographical distribution of P. tristis and its morphologically similar species, we studied their type specimens as well as 147 basidiomata collections of mostly North European material. We used gene trees generated in BEAST 2 and PhyML and species trees estimated in STACEY and ASTRAL to delimit species based on the ITS, LSU, Tef1α and mtSSU regions. We enriched our sampling with environmental ITS sequences from the UNITE database. We found the P. tristis group to contain 13 molecularly and morphologically distinct species. Three of these, P. tristis, P. umbrina and P. atrofusca, are already known to science, while ten species are here described as new: P. sciastra sp. nov., P. tristoides sp. nov., P. umbrinascens sp. nov., P. pinophila sp. nov., P. alnophila sp. nov., P. alobata sp. nov., P. pluriloba sp. nov., P. abundiloba sp. nov., P. rotundispora sp. nov. and P. media sp. nov. We discovered P. rhizopunctata and P. atrofusca to form a sister clade to all other species in P. tristis s.l. These two species, unlike all other species in the P. tristis complex, are dimitic. In this study, we designate epitypes for P. tristis, P. umbrina and Hypochnopsis fuscata and lectotypes for Auricularia phylacteris and Thelephora biennis. We show that the holotype of Hypochnus sitnensis and the lectotype of Hypochnopsis fuscata are conspecific with P. tristis, but in the absence of molecular information we regard Pseudotomentella longisterigmata and Hypochnus rhacodium as doubtful taxa due to their aberrant morphology. We confirm A. phylacteris, Tomentella biennis and Septobasidium arachnoideum as excluded taxa, since their morphology clearly show that they belong to other genera. A key to the species of the P. tristis group is provided. We found P. umbrina to be a common species with a wide, Holarctic distribution, forming ectomycorrhiza with a large number of host species in habitats ranging from tropical forests to the Arctic tundra. The other species in the P. tristis group were found to be less common and have narrower ecological niches

How, not if, is the question mycologists should be asking about DNA-based typification

Fungal metabarcoding of substrates such as soil, wood, and water is uncovering an unprecedented number of fungal species that do not seem to produce tangible morphological structures and that defy our best attempts at cultivation, thus falling outside the scope of the International Code of Nomenclature for algae, fungi, and plants. The present study uses the new, ninth release of the species hypotheses of the UNITE database to show that species discovery through environmental sequencing vastly outpaces traditional, Sanger sequencing-based efforts in a strongly increasing trend over the last five years. Our findings chal-lenge the present stance of some in the mycological community - that the current situation is satisfactory and that no change is needed to "the code" - and suggest that we should be discussing not whether to allow DNA-based descriptions (typifications) of species and by extension higher ranks of fungi, but what the precise requirements for such DNA-based typifications should be. We submit a tentative list of such criteria for further discussion. The present authors hope for a revitalized and deepened discussion on DNA-based typification, because to us it seems harmful and counter-productive to intentionally deny the overwhelming majority of extant fungi a formal standing under the International Code of Nomenclature for algae, fungi, and plants

Taxonomic annotation of public fungal ITS sequences from the built environment - A report from an April 10-11, 2017 workshop (Aberdeen, UK)

The UNITE database community gratefully acknowledges support from the Alfred P. Sloan Foundation. HN and CW gratefully acknowledges financial support from Stiftelsen Olle Engkvist Byggmästare, Stiftelsen Lars Hiertas Minne, Kapten Carl Stenholms Donationsfond, and Birgit och Birger Wålhströms Minnesfond. CW gratefully acknowledges a Marie Skłodowska-Curie post doctoral grant from the ERC. Leho Tedersoo is gratefully acknowledged for providing helpful feedback on an earlier draft of this manuscript.Peer reviewedPublisher PD

Taxonomy and Systematics of Thelephorales – Glimpses Into its Hidden Hyperdiversity

The order Thelephorales is a widespread group of many thousands of species of ecologically important, ectomycorrhizal fungi, of which only a fraction have been described to date. Most species are corticioid (skin-like) and form complexes of morphologically similar, closely related species. At the same time the names that do exist are often old, have unclear synonymy and their common presence within such complexes often hinders the description of new species. For the comparatively few stipitate (with cap and stipe) Thelephorales species taxonomic knowledge is more complete but the phylogenetic relationships between taxa is largely unknown; most existing genera have been circumscribed based on macromorphology. Many stipitate species occurring in the Nordic countries are dependent on old growth forest and are hence included in the national Red Lists, while the conservational situation for nearly all corticioid species is unknown, due to their unclear taxonomy. Pseudotomentella tristis s.l. is a seemingly common, widespread and ecologically very plastic, corticioid morphospecies with an old name and nine heterotypic synonyms. Through a combination of type studies, precise spore measurements, ecological data and a multi-gene phylogeny, three species are identified under already existing names and another ten are described as new. One species, P. umbrina, is found to indeed be a common and widespread species with a wide ecological amplitude, while the remaining 12 are less common, possibly less widespread, have narrower ecological niches and in a few cases seem to be host-restricted. In similarity to stipitate species, a large proportion of the newly described species seem to only occur in old growth forest. Three corticioid species from the Scandes mountains, two Pseudotomentella species and one Tomentella, are described as new, based on ITS-LSU phylogenies. The Pseudotomentella species belong to the P. tristis group, where they are more or less cryptic with another newly described species. A new, stipitate species in the hitherto corticioid genus Amaurodon is described, the stipitate genera Hydnellum and Sarcodon are delimited against each other and the stipitate genus Polyozellus is delimited against the corticioid genus Pseudotomentella – the former two with phylogenies based on ITS and LSU sequences and the latter based on a multi-gene dataset. Hydnellum is found to make Sarcodon paraphyletic, as does Polyozellus Pseudotomentella. To amend this, twelve species are recombined from Hydnellum to Sarcodon, while all species, including the type, are moved from Pseudotomentella to Polyozellus. In conclusion, this thesis demonstrates that corticioid species complexes in Thelephorales with many taxa and old names can be successfully disentangled and presents a method for doing so; it identifies molecular markers and sets a standard of measuring spores and collating ecological data that will facilitate further taxonomic work within the order. In addition, it shows that basidiomata shape is a poor predictor of generic affinity, even when derived from such striking differences as the separation of stipitate and corticioid forms. Consequently, the extinction threat previously documented for stipitate species is likely not restricted to such, and this is also tentatively shown for corticioid Polyozellus species

Reassessment of the generic limits for Hydnellum and Sarcodon (Thelephorales, Basidiomycota)

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