Ellen Larsson

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.

The phylogenetic distribution of resupinate forms across the major clades of mushroom-forming fungi (Homobasidiomycetes)

Abstract Phylogenetic relationships of resupinate Homobasidiomycetes (Corticiaceae s. lat. and others) were studied using ribosomal DNA (rDNA) sequences from a broad sample of resupinate and nonresupinate taxa. Two datasets were analysed using parsimony, a ‘core’ dataset of 142 species, each of which is represented by four rDNA regions (mitochondrial and nuclear large and small subunits), and a ‘full’ dataset of 656 species, most of which were represented only by nuclear large subunit rDNA sequences. Both datasets were analysed using traditional heuristic methods with bootstrapping, and the full dataset was also analysed with the Parsimony Ratchet, using equal character weights and six‐parameter weighted parsimony. Analyses of both datasets supported monophyly of the eight major clades of Homobasidiomycetes recognised by Hibbett and Thorn, as well as independent lineages corresponding to the Gloeophyllum clade, corticioid clade and Jaapia argillacea. Analyses of the full dataset resolved two additional groups, the athelioid clade and trechisporoid clade (the latter may be nested in the polyporoid clade). Thus, there are at least 12 independent clades of Homobasidiomycetes. Higher‐level relationships among the major clades are not resolved with confidence. Nevertheless, the euagarics clade, bolete clade, athelioid clade and Jaapia argillacea are consistently resolved as a monophyletic group, whereas the cantharelloid clade, gomphoid‐phalloid clade and hymenochaetoid clade are placed at the base of the Homobasidiomycetes, which is consistent with the preponderance of imperforate parenthesomes in those groups. Resupinate forms occur in each of the major clades of Homobasidiomycetes, some of which are composed mostly or exclusively of resupinate forms (athelioid clade, corticioid clade, trechisporoid clade, Jaapia). The largest concentrations of resupinate forms occur in the polyporoid clade, russuloid clade and hymenochaetoid clade. The cantharelloid clade also includes many resupinate forms, including some that have traditionally been regarded as heterobasidiomycetes (Sebacinaceae, Tulasnellales, Ceratobasidiales). The euagarics clade, which is by far the largest clade in the Homobasidiomycetes, has the smallest fraction of resupinate species. Results of the present study are compared with recent phylogenetic analyses, and a table summarising the phylogenetic distribution of resupinate taxa is presented, as well as notes on the ecology of resupinate forms and related Homobasidiomycetes.

High phylogenetic diversity among corticioid homobasidiomycetes

Homobasidiomycetes display a variety of fruit body morphologies. Examples include gilled mushrooms, coral fungi, polypores and puffballs but also species with simple crust-like basidiomata, usually called corticioid fungi. The latter group has largely been neglected in recent studies of homobasidiomycete evolution. The major goal of the present study was to explore the impact that the addition of a wide selection of species with crust-like basidiomata would have on homobasidiomycete phylogeny. Two genes, 5.8S and 28S in the nuclear rDNA repeats, were sequenced and a data set with 178 taxa analysed using neighbour-joining and maximum parsimony methods. Support for clades was evaluated by bootstrap. Basal nodes generally received weak support and branching order for major clades remained largely unresolved. Twelve major groups were recovered and corticioid fungi make up a major or important constituent in most of them. Nine groups are strongly supported but support for euagarics and polyporoid clades is poor. Phlebioid fungi were in earlier studies merged with the polyporoid clade but are here identified as a separate clade. Athelia is allied with ectomycorrhizal genera, inter alia Piloderma and Amphinema, in a separate clade forming a sister group to the boletes. We conclude that corticioid fungi hold a considerable share of the phylogenetic diversity displayed by homobasidiomycetes, and should always be considered when phylogenetic studies of larger basidiomycetes are designed.

Diversity and abundance of resupinate thelephoroid fungi as ectomycorrhizal symbionts in Swedish boreal forests.

Resupinate thelephoroid fungi (hereafter called tomentelloid fungi) have a world‐wide distribution and comprise ≈70 basidiomycete species with inconspicuous, resupinate sporocarps. It is only recently that their ability to form ectomycorrhizas (EM) has been realized, so their distribution, abundance and significance as mycobionts in forest ecosystems is still largely unexplored. In order to provide baseline data for future ecological studies of tomentelloid fungi, we explored their presence and abundance in nine Swedish boreal forests in which the EM communities had been analysed. Phylogenetic analyses were used to compare the internal transcribed spacer of nuclear ribosomal DNA (ITS rDNA) sequence data obtained from mycobionts on single ectomycorrhizal tips with that obtained from sporocarps of identified tomentelloid fungi. Five species of Tomentella and one species of Pseudotomentella were identified as ectomycorrhizal fungi. The symbiotic nature of Tomentella bryophila, T. stuposa, T. badia and T. atramentaria is demonstrated for the first time. T. stuposa and Pseudotomentellatristis were the most commonly encountered tomentelloid fungi, with the other species, including T. sublilacina, only being recorded from single stands. Overall, tomentelloid fungi were found in five of the studies, colonizing between 1 and 8% of the mycorrhizal root tips. Two of the five sites supported several tomentelloid species. Tomentelloid fungi appear to be relatively common ectomycorrhizal symbionts with a wide distribution in Swedish coniferous forests. The results are in accordance with accumulating data that fungal species which lack conspicuous sporocarps may be of considerable importance in EM communities.

Phylogenetic and phylogenomic overview of the Polyporales

We present a phylogenetic and phylogenomic overview of the Polyporales. The newly sequenced genomes of Bjerkandera adusta, Ganoderma sp., and Phlebia brevispora are introduced and an overview of 10 currently available Polyporales genomes is provided. The new genomes are 39 500 000–49 900 00 bp and encode for 12 910–16 170 genes. We searched available genomes for single-copy genes and performed phylogenetic informativeness analyses to evaluate their potential for phylogenetic systematics of the Polyporales. Phylogenomic datasets (25, 71, 356 genes) were assembled for the 10 Polyporales species with genome data and compared with the most comprehensive dataset of Polyporales to date (six-gene dataset for 373 taxa, including taxa with missing data). Maximum likelihood and Bayesian phylogenetic analyses of genomic datasets yielded identical topologies, and the corresponding clades also were recovered in the 373-taxa dataset although with different support values in some datasets. Three previously recognized lineages of Polyporales, antrodia, core polyporoid and phlebioid clades, are supported in most datasets, while the status of the residual polyporoid clade remains uncertain and certain taxa (e.g. Gelatoporia, Grifola, Tyromyces) apparently do not belong to any of the major lineages of Polyporales. The most promising candidate single-copy genes are presented, and nodes in the Polyporales phylogeny critical for the suprageneric taxonomy of the order are identified and discussed.

Mining metadata from unidentified ITS sequences in GenBank: A case study in Inocybe (Basidiomycota)

BackgroundThe lack of reference sequences from well-identified mycorrhizal fungi often poses a challenge to the inference of taxonomic affiliation of sequences from environmental samples, and many environmental sequences are thus left unidentified. Such unidentified sequences belonging to the widely distributed ectomycorrhizal fungal genus Inocybe (Basidiomycota) were retrieved from GenBank and divided into species that were identified in a phylogenetic context using a reference dataset from an ongoing study of the genus. The sequence metadata of the unidentified Inocybe sequences stored in GenBank, as well as data from the corresponding original papers, were compiled and used to explore the ecology and distribution of the genus. In addition, the relative occurrence of Inocybe was contrasted to that of other mycorrhizal genera.ResultsMost species of Inocybe were found to have less than 3% intraspecific variability in the ITS2 region of the nuclear ribosomal DNA. This cut-off value was used jointly with phylogenetic analysis to delimit and identify unidentified Inocybe sequences to species level. A total of 177 unidentified Inocybe ITS sequences corresponding to 98 species were recovered, 32% of which were successfully identified to species level in this study. These sequences account for an unexpectedly large proportion of the publicly available unidentified fungal ITS sequences when compared with other mycorrhizal genera. Eight Inocybe species were reported from multiple hosts and some even from hosts forming arbutoid or orchid mycorrhizae. Furthermore, Inocybe sequences have been reported from four continents and in climate zones ranging from cold temperate to equatorial climate. Out of the 19 species found in more than one study, six were found in both Europe and North America and one was found in both Europe and Japan, indicating that at least many north temperate species have a wide distribution.ConclusionAlthough DNA-based species identification and circumscription are associated with practical and conceptual difficulties, they also offer new possibilities and avenues for research. Metadata assembly holds great potential to synthesize valuable information from community studies for use in a species and taxonomy-oriented framework.

Phylogenetic relationships of russuloid basidiomycetes with emphasis on aphyllophoralean taxa.

Many homobasidiomycetes are characterized by a combination of gloeocystidia and amyloid basidiospores. They display a great variation in basidioma morphology, including erect and effused forms and gilled and nongilled forms. Earlier studies have shown these taxa to be related, and the group has been named the russuloid clade. Phylogenetic relationships among russuloid basidiomycetes were investigated using sequence data from the nuclear 5.8S, ITS2 and large-subunit rDNA genes. A dataset including 127 ingroup sequences representing 43 genera and ca 120 species were analyzed by maximum-parsimony and neighbor-joining methods. The sampling of taxa had an emphasis on nongilled taxa and two-thirds of the species possessed corticioid basidiomata. Thirteen major well-supported clades were identified within the russuloid clade. All clades except one include corticioid species. Ten characters from basidioma morphology and cultured mycelium were observed and evaluated. Results suggest that gloeocystidia are a synapomorphy for taxa within the russuloid clade while the amyloidity of spores is inconsistent. The ornamentation of spores and type of nuclear behavior seems to be informative characters at genus level. The agaricoid genera Lactarius and Russula are nested in a clade with corticioid species at the basal position. The new combinations Boidinia aculeata, Gloeodontia subasperispora, Gloeocystidiopsis cryptacantha and Megalocystidium wakullum are proposed.

A Comprehensive, Automatically Updated Fungal ITS Sequence Dataset for Reference-Based Chimera Control in Environmental Sequencing Efforts

The nuclear ribosomal internal transcribed spacer (ITS) region is the most commonly chosen genetic marker for the molecular identification of fungi in environmental sequencing and molecular ecology studies. Several analytical issues complicate such efforts, one of which is the formation of chimeric—artificially joined—DNA sequences during PCR amplification or sequence assembly. Several software tools are currently available for chimera detection, but rely to various degrees on the presence of a chimera-free reference dataset for optimal performance. However, no such dataset is available for use with the fungal ITS region. This study introduces a comprehensive, automatically updated reference dataset for fungal ITS sequences based on the UNITE database for the molecular identification of fungi. This dataset supports chimera detection throughout the fungal kingdom and for full-length ITS sequences as well as partial (ITS1 or ITS2 only) datasets. The performance of the dataset on a large set of artificial chimeras was above 99.5%, and we subsequently used the dataset to remove nearly 1,000 compromised fungal ITS sequences from public circulation. The dataset is available at http://unite.ut.ee/repository.php and is subject to web-based third-party curation.

Improving ITS sequence data for identification of plant pathogenic fungi

SummaryPlant pathogenic fungi are a large and diverse assemblage of eukaryotes with substantial impacts on natural ecosystems and human endeavours. These taxa often have complex and poorly understood life cycles, lack observable, discriminatory morphological characters, and may not be amenable to in vitro culturing. As a result, species identification is frequently difficult. Molecular (DNA sequence) data have emerged as crucial information for the taxonomic identification of plant pathogenic fungi, with the nuclear ribosomal internal transcribed spacer (ITS) region being the most popular marker. However, international nucleotide sequence databases are accumulating numerous sequences of compromised or low-resolution taxonomic annotations and substandard technical quality, making their use in the molecular identification of plant pathogenic fungi problematic. Here we report on a concerted effort to identify high-quality reference sequences for various plant pathogenic fungi and to re-annotate incorrectly or insufficiently annotated public ITS sequences from these fungal lineages. A third objective was to enrich the sequences with geographical and ecological metadata. The results – a total of 31,954 changes – are incorporated in and made available through the UNITE database for molecular identification of fungi (http://unite.ut.ee), including standalone FASTA files of sequence data for local BLAST searches, use in the next-generation sequencing analysis platforms QIIME and mothur, and related applications. The present initiative is just a beginning to cover the wide spectrum of plant pathogenic fungi, and we invite all researchers with pertinent expertise to join the annotation effort.

Ectomycorrhizal Diversity on Dryas octopetala and Salix reticulata in an Alpine Cliff Ecosystem

Abstract The ectomycorrhizal communities in alpine habitats have been relatively little studied. As global change is predicted to have a large impact in Arctic and alpine environments, it is important to document the fungi of these climatic regions to monitor changes and to understand upcoming successions. This study investigates the ectomycorrhizal community of Dryas octopetala and Salix reticulata on cliff ledges in a mid-alpine setting using the internal transcribed spacer region of nuclear ribosomal DNA for the identification of the fungal component of ectomycorrhizal root tips. It is shown that the community is relatively species rich, with 74 molecular operational taxonomic units (MOTUs)/species, and that it is dominated by Cenococcum geophilum, Thelephoraceae spp., Cortinarius spp., and Sebacinales spp. Furthermore, the dominating species have low specificity regarding the tested hosts and seem likely to be able to facilitate the succession of the alpine tundra to subalpine forest by serving as mycorrhizal partners for establishing pioneer trees.