Nils Hallenberg

Intraspecific ITS Variability in the Kingdom Fungi as Expressed in the International Sequence Databases and Its Implications for Molecular Species Identification

The internal transcribed spacer (ITS) region of the nuclear ribosomal repeat unit is the most popular locus for species identification and subgeneric phylogenetic inference in sequence-based mycological research. The region is known to show certain variability even within species, although its intraspecific variability is often held to be limited and clearly separated from interspecific variability. The existence of such a divide between intra- and interspecific variability is implicitly assumed by automated approaches to species identification, but whether intraspecific variability indeed is negligible within the fungal kingdom remains contentious. The present study estimates the intraspecific ITS variability in all fungi presently available to the mycological community through the international sequence databases. Substantial differences were found within the kingdom, and the results are not easily correlated to the taxonomic affiliation or nutritional mode of the taxa considered. No single unifying yet stringent upper limit for intraspecific variability, such as the canonical 3% threshold, appears to be applicable with the desired outcome throughout the fungi. Our results caution against simplified approaches to automated ITS-based species delimitation and reiterate the need for taxonomic expertise in the translation of sequence data into species names.

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.

A software pipeline for processing and identification of fungal ITS sequences

BackgroundFungi from environmental samples are typically identified to species level through DNA sequencing of the nuclear ribosomal internal transcribed spacer (ITS) region for use in BLAST-based similarity searches in the International Nucleotide Sequence Databases. These searches are time-consuming and regularly require a significant amount of manual intervention and complementary analyses. We here present software – in the form of an identification pipeline for large sets of fungal ITS sequences – developed to automate the BLAST process and several additional analysis steps. The performance of the pipeline was evaluated on a dataset of 350 ITS sequences from fungi growing as epiphytes on building material.ResultsThe pipeline was written in Perl and uses a local installation of NCBI-BLAST for the similarity searches of the query sequences. The variable subregion ITS2 of the ITS region is extracted from the sequences and used for additional searches of higher sensitivity. Multiple alignments of each query sequence and its closest matches are computed, and query sequences sharing at least 50% of their best matches are clustered to facilitate the evaluation of hypothetically conspecific groups. The pipeline proved to speed up the processing, as well as enhance the resolution, of the evaluation dataset considerably, and the fungi were found to belong chiefly to the Ascomycota, with Penicillium and Aspergillus as the two most common genera. The ITS2 was found to indicate a different taxonomic affiliation than did the complete ITS region for 10% of the query sequences, though this figure is likely to vary with the taxonomic scope of the query sequences.ConclusionThe present software readily assigns large sets of fungal query sequences to their respective best matches in the international sequence databases and places them in a larger biological context. The output is highly structured to be easy to process, although it still needs to be inspected and possibly corrected for the impact of the incomplete and sometimes erroneously annotated fungal entries in these databases. The open source pipeline is available for UNIX-type platforms, and updated releases of the target database are made available biweekly. The pipeline is easily modified to operate on other molecular regions and organism groups.

Hybridization among cryptic species of the cellar fungus Coniophora puteana (Basidiomycota)

In this study we have analysed the genetic variation and phylogeography in a global sample of the cellar fungus Coniophora puteana, which is an important destroyer of wooden materials indoor. Multilocus genealogies of three DNA regions (beta tubulin, nrDNA ITS and translation elongation factor 1α) revealed the occurrence of three cryptic species (PS1–3) in the morphotaxon C. puteana. One of the lineages (PS3) is apparently restricted to North America while the other two (PS1–2) have wider distributions on multiple continents. Interspecific hybridization has happened between two of the lineages (PS1 and PS3) in North America. In three dikaryotic isolates, two highly divergent beta tubulin alleles coexisted, one derived from PS1 and one from PS3. Furthermore, one isolate included a recombinant ITS sequence, where ITS1 resembled the ITS1 version of PS3 while ITS2 was identical to a frequent PS1 ITS2 version. This pattern must be due to hybridization succeeded by intralocus recombination in ITS. The results further indicated that introgression has happened between subgroups appearing in PS1. We hypothesize that the observed reticulate evolution is due to previous allopatric separation followed by more recent reoccurrence in sympatry, where barriers to gene flow have not yet evolved. A complex phylogeographical structure is observed in the morphotaxon C. puteana caused by (i) cryptic speciation; (ii) the interplay between natural migration and distribution patterns and probably more recent human mediated dispersal events; and (iii) hybridization and introgression.

Speciation and distribution in Corticiaceae (Basidiomy- cetes)

Evolutionary processes inCorticiaceae (wood-inhabiting basidiomycetes) are discussed on the basis of sibling species analysis, colonization strategies, and the known present distribution. It is proposed that the phylogenetical differentiation of basidiocarp structures may be very old and many species have remained unchanged. Subsequent evolution has to a great part been the effect of biological interaction with the environment, like colonization of new substrata, and the formation of species complexes may be a consequence of this. The amount of spores liberated from a single basidiocarp is regarded as an adaptive character for dispersal mainly in the immediate environment. The actual, wide distribution of many species is supposed to be associated with expansion of pertaining forest types in the past. When rapid expansion of a species occurs it is likely to be connected with occupation of a new ecological niche. Finally, the consequences for fungal communities in modern forestry are discussed.

The white-rotting genus Phanerochaete is polyphyletic and distributed throughout the phleboid clade of the Polyporales (Basidiomycota)

The genus Phanerochaete (Polyporales, Basidiomycota) has traditionally been delimited based on the gross morphology of the fruiting body and on the nature of the hyphal structure, cystidia, and spores. However, several recent studies based on molecular data indicate that the genus is polyphyletic as presently circumscribed, although most of its species are found in the phlebioid clade of the Polyporales. To further our understanding of the genus, 54 new sequences from the large subunit of the nuclear ribosomal DNA of 45 previously unsequenced Phanerochaete species were obtained and analyzed jointly with a large selection of taxa in the phlebioid clade. The results show that there is a well-supported Phanerochaete core group that includes the type species Phanerochaete velutina (the currently accepted name of the generic type, Thelephora alnea). Representatives of a few satellite genera, including Hjortstamia, Phlebiopsis, and Rhizochaete, are found in the immediate topological vicinity of Phanerochaete.Outside the core group but still within the phlebioid clade are several taxa that have been referred to as members of Phanerochaete by some authors but that are here assigned to at least five different monophyletic clades. Phanerochaete viticola is found in the Hymenochaetales and is shown to have phylogenetic affinities to the partially symbiotic Rickenellaceae. A new genus, Ginnsia, is erected for this species, and the new combinations Ginnsia viticola, Hjortstamia brunneocystidiata, H. laxa, and Phlebiopsis lamprocystidiata are proposed. A denser taxon and gene sampling in the phleboid clade will be needed to settle the precise taxonomic affiliation of many of the species presently referred to as Phanerochaete, and numerous nomenclatural changes are doubtlessly looming on the horizon.

Phylogeography of Hyphoderma setigerum (Basidiomycota) in the Northern Hemisphere

Previous studies of morphological variation in the homobasidiomycete Hyphoderma setigerum have lead to suspicions of a species complex. This study explores variation in DNA sequences from the nuclear ribosomal ITS region of 45 specimens from America, Asia, and Europe in a phylogeographic context. Based on molecular analysis, morphological studies, and crossing tests, nine preliminary taxa are shown to exist inside the species complex, and the two previously described segregate species H. subsetigerum and H. nudicephalum are confirmed. The molecular analysis shows evidence of allopatric differentiation over intercontinental distances. Only one of the nine well-supported clades has a geographic distribution spanning more than one continent, probably indicating the importance of vicariance in the evolution of this species complex. The basionym of H. setigerum, Thelephora setigera, is neotypified to fix the application of that name.

Phylogenetic studies in species of Corticiaceae growing on branches

Parsimony analysis based on ribosomal DNA sequence and morphological data was per- formed for 28 species of Acanthophysium, Amyloster- eum, Aleurodiscus, Corticium, Cytidia, Dendrocorti- cium, Duportella, Peniophora, Stereum, and Vuilleminia (Corticiaceae, Basidiomycota). Sequence data from a portion of the ribosomal large subunit (nDNA) and morphological data were analyzed separately as well

A phylogenetic study of the genus Schizopora (Basidiomycota) based on ITS DNA sequences.

A phylogenetic analysis based on internal transcribed spacer regions of the nuclear ribosomal RNA region (ITS) sequences has been undertaken for species in the genus Schizopora. Four known species were studied, viz. S. radula, S. paradoxa, S. flavipora, S. nothofagi, and a hitherto undescribed species from Taiwan. Allopatric differentiation was apparent in S. radula, for which several geographically representive samples were available. Among these, ITS sequence data obtained from S. radula isolates from British Columbia and Australasia were identical. All previously recognised Schizopora species were well separated from each other with respect to their ITS sequences. Mating compatibility tests also showed complete reproductive separation between species, but intercompatibility between cultures from the same species, irrespective of geographical origin. Overall, the morphology of basidiomes of S. radula from New Zealand differs only in minor respects from those of European and Argentinean origin, but shows a higher degree of variability than described for European specimens.

Phylogenetic studies in Peniophora

Phylogenetic relationships in Peniophora were investigated using ITS sequences from nuclear rDNA. The result was compared with parsimony analysis based on morphological characters, and a traditional classification based on basidiome morphology. Sixty-four specimens belonging to 24 species in Peniophora were sequenced. In particular, ITS2 sequences gave a good resolution in phylogenetic analyses and 3 distinct clades were distinguished. Intraspecific variation was constantly low in ITS2, and rarely exceeded 2%. In ITS1, the variation in certain species was considerable and hence most confidence was placed on the results from ITS2. Intercompatibility tests were done for the representatives of each taxon. In some species, intersterility had been found between representatives regarded as conspecific on morphological criteria. Such sibling species were not separated from each other in ITS2 sequences.