The UNITE database for molecular identification and taxonomic communication of fungi and other eukaryotes: sequences, taxa and classifications reconsidered.

UNITE (https://unite.ut.ee) is a web-based database and sequence management environment for molecular identification of eukaryotes. It targets the nuclear ribosomal internal transcribed spacer (ITS) region and offers nearly 10 million such sequences for reference. These are clustered into ∼2.4M species hypotheses (SHs), each assigned a unique digital object identifier (DOI) to promote unambiguous referencing across studies. UNITE users have contributed over 600 000 third-party sequence annotations, which are shared with a range of databases and other community resources. Recent improvements facilitate the detection of cross-kingdom biological associations and the integration of undescribed groups of organisms into everyday biological pursuits. Serving as a digital twin for eukaryotic biodiversity and communities worldwide, the latest release of UNITE offers improved avenues for biodiversity discovery, precise taxonomic communication and integration of biological knowledge across platforms.

The UNITE database for molecular identification of fungi: handling dark taxa and parallel taxonomic classifications.

UNITE (https://unite.ut.ee/) is a web-based database and sequence management environment for the molecular identification of fungi. It targets the formal fungal barcode-the nuclear ribosomal internal transcribed spacer (ITS) region-and offers all ∼1 000 000 public fungal ITS sequences for reference. These are clustered into ∼459 000 species hypotheses and assigned digital object identifiers (DOIs) to promote unambiguous reference across studies. In-house and web-based third-party sequence curation and annotation have resulted in more than 275 000 improvements to the data over the past 15 years. UNITE serves as a data provider for a range of metabarcoding software pipelines and regularly exchanges data with all major fungal sequence databases and other community resources. Recent improvements include redesigned handling of unclassifiable species hypotheses, integration with the taxonomic backbone of the Global Biodiversity Information Facility, and support for an unlimited number of parallel taxonomic classification systems.

North American matsutake: names clarified and a new species described.

Tricholoma matsutake, known widely as "matsutake," has great commercial and cultural significance in Japan. Because Japanese production is insufficient to meet the high domestic demand, morphologically similar mushrooms, thought by many to belong to T. magnivelare, are imported from western North America. However, molecular data produced since the early 2000s have indicated that more than one species of matsutake occur in North America and this raises the question of correct naming for the different species. To address this question, we assessed the phylogenetic diversity within North American matsutake based on nuc rDNA ITS1-5.8S-ITS2 (internal transcribed spacer [ITS] barcode) sequences, including newly obtained sequences from the type collections for Agaricus ponderosus and Armillaria arenicola, and morphological characters. Our results agree with earlier indications that three matsutake species occur in North America and allow us to clarify the correct application of names-T. magnivelare from the eastern USA and Canada, T. murrillianum from the western USA and Canada, and T. mesoamericanum from Mexico, newly described here. The existence of the three North American species is further supported by the results of evolutionary divergence analysis, geographical distributions, and morphological characters.

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.

Holarctic Species in the Pluteus podospileus Clade: Description of Six New Species and Reassessment of Old Names.

We studied the taxonomy of Pluteus podospileus and similar species using morphological and molecular (nrITS, TEF1-α) data, including a detailed study of the type collections of P. inflatus var. alneus, Pluteus minutissimus f. major, and P. granulatus var. tenellus. Within the P. podospileus complex, we phylogenetically confirmed six species in Europe, five in Asia, and eight in North America. Based on our results, we recognize P. seticeps as a separate species occurring in North America, while P. podospileus is limited to Eurasia. We describe six new species and a new variety: P. absconditus, P. fuscodiscus, P. gausapatus, P. inexpectatus, P. millsii, and P. notabilis and its variety, P. notabilis var. insignis. We elevate Pluteus seticeps var. cystidiosus to species rank as Pluteus cystidiosus. Based on the holotype of P. inflatus var. alneus, collections of P. inflatus identified by Velenovský, and several modern collections, we resurrect the name P. inflatus. Based on molecular analyses of syntypes of Pluteus minutissimus f. major and a holotype of Pluteus granulatus var. tenellus, we synonymize them under P. inflatus. We also increase our knowledge about the morphology and distribution of P. cutefractus.

DNA barcoding of fungal specimens using PacBio long-read high-throughput sequencing.

Molecular methods are increasingly used to identify species that lack conspicuous macro- or micromorphological characters. Taxonomic and ecological research teams barcode large numbers of collected voucher specimens annually. In this study we assessed the efficiency of long-read high throughput sequencing (HTS) as opposed to the traditionally used Sanger method for taxonomic identification of multiple vouchered fungal specimens. We also evaluated whether this method can provide reference information about intraindividual gene polymorphism. We developed a workflow based on a test set of 423 basidiomycete specimens (representing 195 species), the PacBio HTS method, and ribosomal rRNA operon internal transcribed spacer (ITS) and 28S rRNA gene (LSU) markers. The PacBio HTS had a higher success rate than Sanger sequencing at a comparable cost. Species identification based on PacBio reads was usually straightforward, because the dominant operational taxonomic unit (OTU) typically represented the targeted organism. The PacBio HTS also enabled us to detect widespread polymorphism within the ITS marker. We conclude that multiplex DNA barcoding of the fungal ITS and LSU markers using PacBio HTS is a useful tool for taxonomic identification of large amounts of collected voucher specimens at a competitive price. Furthermore, PacBio HTS accurately recovers various alleles and paralogues, which can provide crucial information for species delimitation and population-level studies.

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

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

Global diversity and distribution of mushroom-inhabiting bacteria.

Mushroom-forming fungi are important sources of food and medicine in many regions of the world, and their development and health are known to depend on various microbes. Recent studies have examined the structure of mushroom-inhabiting bacterial (MIB) communities and their association with local environmental variables, but global-scale diversity and determinants of these communities remain poorly understood. Here we examined the MIB global diversity and community composition in relation to climate, soil and host factors. We found a core global mushroom microbiome, accounting for 30% of sequence reads, while comprising a few bacterial genera such as Halomonas, Serratia, Bacillus, Cutibacterium, Bradyrhizobium and Burkholderia. Our analysis further revealed an important role of host phylogeny in shaping the communities of MIB, whereas the effects of climate and soil factors remained negligible. The results suggest that the communities of MIB and free-living bacteria are structured by contrasting community assembly processes and that fungal-bacterial interactions are an important determinant of MIB community structure.

Pluteus insidiosus Complex, Four New Species Described and Pluteus reisneri Resurrected.

We studied the taxonomy of Pluteus insidiosus and similar species using morphological and molecular (nrITS, TEF1-α) data, including a detailed study of the type collection of P. insidiosus. Based on our results, we recognize five species in this group: P. insidiosus sensu stricto and four other taxa: P. assimilatus; P. farensis; P. flavostipitatus; and P. pseudoinsidiosus; described here as new. All these taxa are distinct from each other based on molecular data, but some of them are semi-cryptic based on morphology and co-occur in the Palaearctic region. An additional molecular lineage, phylogenetically separates from the P. insidiosus complex, but with many morphological similarities, was recognized in the molecular phylogenies. Based on the revision of available type collections, the name Pluteus reisneri Velen., was adopted for this Clade. Pluteus reisneri was validly published in 1921, but it has barely been used since its original description. A modern epitype, with molecular data, was selected for P. reisneri.

454 Pyrosequencing and Sanger sequencing of tropical mycorrhizal fungi provide similar results but reveal substantial methodological biases.

• Compared with Sanger sequencing-based methods, pyrosequencing provides orders of magnitude more data on the diversity of organisms in their natural habitat, but its technological biases and relative accuracy remain poorly understood. • This study compares the performance of pyrosequencing and traditional sequencing for species' recovery of ectomycorrhizal fungi on root tips in a Cameroonian rain forest and addresses biases related to multi-template PCR and pyrosequencing analyses. • Pyrosequencing and the traditional method yielded qualitatively similar results, but there were slight, but significant, differences that affected the taxonomic view of the fungal community. We found that most pyrosequencing singletons were artifactual and contained a strongly elevated proportion of insertions compared with natural intra- and interspecific variation. The alternative primers, DNA extraction methods and PCR replicates strongly influenced the richness and community composition as recovered by pyrosequencing. • Pyrosequencing offers a powerful alternative for the identification of ectomycorrhizal fungi in pooled root samples, but requires careful selection of molecular tools. A well-populated backbone database facilitates the detection of biological and technical artifacts. The pyrosequencing pipeline is available at http://unite.ut.ee/454pipeline.tgz.

New species and reports of Cuphophyllus from northern North America compared with related Eurasian species.

This study describes four gray or brown species of Cuphophyllus (Hygrophoraceae, Agaricales), two of them new species, restricted to arctic-alpine and northern boreal zones of North America, and relates them morphologically and phylogenetically using multigene and nuc rDNA internal transcribed spacer ITS1-5.8S-ITS (ITS barcode) analyses to their similar, known counterparts. Cuphophyllus cinerellus, epitypified here, is shown to be a pan-palearctic species with sequence-confirmed collections from Fennoscandia and easternmost Asia. Occupying a similar habitat in the Nearctic is its sister species, the morphologically similar but novel C. esteriae, so far known only from eastern North America, including Greenland. Sister to the C. cinerellus-C. esteriae lineage, and known only from boreal raised Sphagnum bogs in Newfoundland, is a new medium-sized light cinereous brown species, C. lamarum. It has a yellow stipe but is phylogenetically distant from the yellow-stiped European C. flavipes and its North American sister species, Hygrophorus pseudopallidus. As cryptic speciation was discovered within C. flavipes, we lecto- and epitypify the name and transfer H. pseudopallidus to Cuphophyllus based on ITS analysis of the holotype. We also transfer the small European Hygrocybe comosa to Cuphophyllus based on morphology. Cuphophyllus hygrocyboides is reported from North America with the first sequence-confirmed collections from arctic-alpine British Columbia and Greenland. In addition, sequencing the holotype of C. subviolaceus identifies it as the sister species to the putative C. lacmus. Both species seem to have an intercontinental distribution. In total, we add new sequences to GenBank from 37 Cuphophyllus collections, including the holotypes of C. hygrocyboides and C. subviolaceus, the two new epitypes, and the two novel species.

The Taxon Hypothesis Paradigm-On the Unambiguous Detection and Communication of Taxa.

Here, we describe the taxon hypothesis (TH) paradigm, which covers the construction, identification, and communication of taxa as datasets. Defining taxa as datasets of individuals and their traits will make taxon identification and most importantly communication of taxa precise and reproducible. This will allow datasets with standardized and atomized traits to be used digitally in identification pipelines and communicated through persistent identifiers. Such datasets are particularly useful in the context of formally undescribed or even physically undiscovered species if data such as sequences from samples of environmental DNA (eDNA) are available. Implementing the TH paradigm will to some extent remove the impediment to hastily discover and formally describe all extant species in that the TH paradigm allows discovery and communication of new species and other taxa also in the absence of formal descriptions. The TH datasets can be connected to a taxonomic backbone providing access to the vast information associated with the tree of life. In parallel to the description of the TH paradigm, we demonstrate how it is implemented in the UNITE digital taxon communication system. UNITE TH datasets include rich data on individuals and their rDNA ITS sequences. These datasets are equipped with digital object identifiers (DOI) that serve to fix their identity in our communication. All datasets are also connected to a GBIF taxonomic backbone. Researchers processing their eDNA samples using UNITE datasets will, thus, be able to publish their findings as taxon occurrences in the GBIF data portal. UNITE species hypothesis (species level THs) datasets are increasingly utilized in taxon identification pipelines and even formally undescribed species can be identified and communicated by using UNITE. The TH paradigm seeks to achieve unambiguous, unique, and traceable communication of taxa and their properties at any level of the tree of life. It offers a rapid way to discover and communicate undescribed species in identification pipelines and data portals before they are lost to the sixth mass extinction.

Strong host preference of ectomycorrhizal fungi in a Tasmanian wet sclerophyll forest as revealed by DNA barcoding and taxon-specific primers.

Ectomycorrhizal (ECM) symbiosis is a widespread plant nutrition strategy in Australia, especially in semiarid regions. This study aims to determine the diversity, community structure and host preference of ECM fungi in a Tasmanian wet sclerophyll forest. Ectomycorrhizal fungi were identified based on anatomotyping and rDNA internal transcribed spacer (ITS)-large subunit (LSU) sequence analysis using taxon-specific primers. Host tree roots were identified based on root morphology and length differences of the chloroplast trnL region. A total of 123 species of ECM fungi were recovered from root tips of Eucalyptus regnans (Myrtaceae), Pomaderris apetala (Rhamnaceae) and Nothofagus cunninghamii (Nothofagaceae). The frequency of two thirds of the most common ECM fungi from several lineages was significantly influenced by host species. The lineages of Cortinarius, Tomentella-Thelephora, Russula-Lactarius, Clavulina, Descolea and Laccaria prevailed in the total community and their species richness and relative abundance did not differ by host species. This study demonstrates that strongly host-preferring, though not directly specific, ECM fungi may dominate the below-ground community. Apart from the richness of Descolea, Tulasnella and Helotiales and the lack of Suillus-Rhizopogon and Amphinema-Tylospora, the ECM fungal diversity and phylogenetic community structure is similar to that in the Holarctic realm.

Polyozellus multiplex (Thelephorales) is a species complex containing four new species.

Geographic, morphological, and internal transcribed spacer (ITS)-based molecular review of collections identified as Polyozellus multiplex revealed that it is a complex of five phylogenetic species. Average spore size-either less or more than 7 × 6 µm-splits the complex into a small-spored group of two (P. multiplex and P. atrolazulinus) and a large-spored group of three (P. mariae, P. marymargaretae, and P. purpureoniger). Basidiocarps of the small-spored species are somewhat smaller than the large-spored ones, are various shades of blue, dark all the way to black, with brownish tomentum only in early growth, have dark context, and have pilei that tend to flare out at the edge. The large-spored species produce somewhat larger sporocarps, have light or lighter context than the pileipelis, and usually retain some brown on the mature pileipellis, the edge of which tends to curl like a cabbage leaf. All will darken or blacken with age. The species of the P. multiplex complex are distributed in the northern coniferous region, with the exception of Europe. One species (P. atrolazulinus) is known from three regions, eastern Asia, western North America, and northeastern North America. Two species are known from two regions: P. purpureoniger in eastern Asia and northwestern North America and P. multiplex in eastern Asia and eastern North America. Two species have been documented in one region only: P. mariae in northeastern North America and P. marymargaretae in western North America. A combination of location, macromorphology, and spore size will usually differentiate the species of the complex.