V. Robert

One fungus, which genes? Development and assessment of universal primers for potential secondary fungal DNA barcodes

The aim of this study was to assess potential candidate gene regions and corresponding universal primer pairs as secondary DNA barcodes for the fungal kingdom, additional to ITS rDNA as primary barcode. Amplification efficiencies of 14 (partially) universal primer pairs targeting eight genetic markers were tested across > 1 500 species (1 931 strains or specimens) and the outcomes of almost twenty thousand (19 577) polymerase chain reactions were evaluated. We tested several well-known primer pairs that amplify: i) sections of the nuclear ribosomal RNA gene large subunit (D1–D2 domains of 26/28S); ii) the complete internal transcribed spacer region (ITS1/2); iii) partial β -tubulin II (TUB2); iv) γ-actin (ACT); v) translation elongation factor 1-α (TEF1α); and vi) the second largest subunit of RNA-polymerase II (partial RPB2, section 5–6). Their PCR efficiencies were compared with novel candidate primers corresponding to: i) the fungal-specific translation elongation factor 3 (TEF3); ii) a small ribosomal protein necessary for t-RNA docking; iii) the 60S L10 (L1) RP; iv) DNA topoisomerase I (TOPI); v) phosphoglycerate kinase (PGK); vi) hypothetical protein LNS2; and vii) alternative sections of TEF1α. Results showed that several gene sections are accessible to universal primers (or primers universal for phyla) yielding a single PCR-product. Barcode gap and multi-dimensional scaling analyses revealed that some of the tested candidate markers have universal properties providing adequate infra- and inter-specific variation that make them attractive barcodes for species identification. Among these gene sections, a novel high fidelity primer pair for TEF1α, already widely used as a phylogenetic marker in mycology, has potential as a supplementary DNA barcode with superior resolution to ITS. Both TOPI and PGK show promise for the Ascomycota, while TOPI and LNS2 are attractive for the Pucciniomycotina, for which universal primers for ribosomal subunits often fail.

MycoBank gearing up for new horizons.

MycoBank, a registration system for fungi established in 2004 to capture all taxonomic novelties, acts as a coordination hub between repositories such as Index Fungorum and Fungal Names. Since January 2013, registration of fungal names is a mandatory requirement for valid publication under the International Code of Nomenclature for algae, fungi and plants (ICN). This review explains the database innovations that have been implemented over the past few years, and discusses new features such as advanced queries, registration of typification events (MBT numbers for lecto, epi- and neotypes), the multi-lingual database interface, the nomenclature discussion forum, annotation system, and web services with links to third parties. MycoBank has also introduced novel identification services, linking DNA sequence data to numerous related databases to enable intelligent search queries. Although MycoBank fills an important void for taxon registration, challenges for the future remain to improve links between taxonomic names and DNA data, and to also introduce a formal system for naming fungi known from DNA sequence data only. To further improve the quality of MycoBank data, remote access will now allow registered mycologists to act as MycoBank curators, using Citrix software.

The Genera of Fungi: fixing the application of type species of generic names.

To ensure a stable platform for fungal taxonomy, it is of paramount importance that the genetic application of generic names be based on their DNA sequence data, and wherever possible, not morphology or ecology alone. To facilitate this process, a new database, accessible at www.GeneraofFungi.org (GoF) was established, which will allow deposition of metadata linked to holo-, lecto-, neo- or epitype specimens, cultures and DNA sequence data of the type species of genera. Although there are presently more than 18 000 fungal genera described, we aim to initially focus on the subset of names that have been placed on the “Without-prejudice List of Protected Generic Names of Fungi” (see IMA Fungus 4(2): 381–443, 2013). To enable the global mycological community to keep track of typification events and avoid duplication, special MycoBank Typification identfiers (MBT) will be issued upon deposit of metadata in MycoBank. MycoBank is linked to GoF, thus deposited metadata of generic type species will be displayed in GoF (and vice versa), but will also be linked to Index Fungorum (IF) and the curated RefSeq Targeted Loci (RTL) database in GenBank at the National Center for Biotechnology Information (NCBI). This initial paper focuses on eight genera of appendaged coelomycetes, the type species of which are neo- or epitypified here: Bartalinia (Bartalinia robillardoides; Amphisphaeriaceae, Xylariales), Chaetospermum (Chaetospermum chaetosporum, incertae sedis, Sebacinales), Coniella (Coniella fragariae, Schizoparmaceae, Diaporthales), Crinitospora (Crinitospora pulchra, Melanconidaceae, Diaporthales), Eleutheromyces (Eleutheromyces subulatus, Helotiales), Kellermania (Kellermania yuccigena, Planistromataceae, Botryosphaeriales), Mastigosporium (Mastigosporium album, Helotiales), and Mycotribulus (Mycotribulus mirabilis, Agaricales). Authors interested in contributing accounts of individual genera to larger multi-authored papers to be published in IMA Fungus, should contact the associate editors listed below for the major groups of fungi on the List of Protected Generic Names for Fungi.

Susceptibility and Diversity in the Therapy-Refractory Genus Scedosporium

ABSTRACT Scedosporium species show decreased susceptibility to the majority of systemic antifungal drugs. Acquired resistance is likely to disseminate differentially with the mode of exchange of genetic material between lineages. Inter- and intraspecific diversities of Scedosporium species were analyzed for three partitions (rDNA internal transcribed spacer gene [ITS], partial β-tubulin gene, and amplified fragment length polymorphism profiles), with the aim to establish distribution of resistance between species, populations, and strains. Heterogeneity of and recombination between lineages were determined, and distances between clusters were calculated using a centroid approach. Clinical, geographic, and antifungal data were plotted on diversity networks. Scedosporium minutisporum, Scedosporium desertorum, and Scedosporium aurantiacum were distinguished unambiguously in all partitions and had differential antifungal susceptibility profiles (ASP). Pseudallescheria fusoidea and Pseudallescheria ellipsoidea were indistinguishable from Scedosporium boydii. Pseudallescheria angusta took an intermediate position between Scedosporium apiospermum and S. boydii. Scedosporium boydii and S. apiospermum had identical ASP. Differences in (multi)resistance were linked to individual strains. S. apiospermum and S. boydii showed limited interbreeding and were recognized as valid, sympatric species. The S. apiospermum/S. boydii group, comprising the main clinically relevant Scedosporium species, consists of separate lineages and is interpreted as a complex undergoing sympatric evolution with incomplete lineage sorting. In routine diagnostics, the lineages in S. apiospermum/S. boydii are indicated with the umbrella descriptor “S. apiospermum complex”; individual species can be identified with rDNA ITS with 96.3% confidence. Voriconazole is recommended as the first-line treatment; resistance against this compound is rare.

DNA barcoding analysis of more than 9 000 yeast isolates contributes to quantitative thresholds for yeast species and genera delimitation

DNA barcoding is a global initiative for species identification through sequencing of short DNA sequence markers. Sequences of two loci, ITS and LSU, were generated as barcode data for all (ca. 9k) yeast strains included in the CBS collection, originally assigned to ca. 2u2008000 species. Taxonomic sequence validation turned out to be the most severe bottleneck due to the large volume of generated trace files and lack of reference sequences. We have analysed and validated CBS strains and barcode sequences automatically. Our analysis shows that there were 6 and 9.5 % of CBS yeast species that could not be distinguished by ITS and LSU, respectively. Among them, ∼3 % were indistinguishable by both loci. Except for those species, both loci were successfully resolving yeast species as the grouping of yeast DNA barcodes with the predicted taxonomic thresholds was more than 90 % similar to the grouping with respect to the expected taxon names. The taxonomic thresholds predicted to discriminate yeast species were 98.41 % for ITS and 99.51 % for LSU. To discriminate current yeast genera, thresholds were 96.31 % for ITS and 97.11 % for LSU. Using ITS and LSU barcodes, we were also able to show that the recent reclassifications of basidiomycetous yeasts in 2015 have made a significant improvement for the generic taxonomy of those organisms. The barcodes of 4u2008730 (51 %) CBS yeast strains of 1u2008351 (80 %) accepted yeast species that were manually validated have been released to GenBank and the CBS-KNAW website as reference sequences for yeast identification.

Making moulds meet. Information retrieval as a basis for understanding Pseudallescheria and Scedosporium.

G. S. de Hoog, V. Robert, M. Lackner, M. J. G. T. Vehreschild, J. J. Vehreschild, F. Symoens, E. Gottlich-Fligg, D. Garcia-Hermoso, A. Harun, W. Meyer, S. C. A. Chen, A. Hamprecht, G. Fischer, W. Buzina, O. A. Cornely, J. Guarro, J. Cano and R. Horre* CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, The Netherlands, Peking University Health Science Center, Research Center for Medical Mycology, Beijing, China, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China, Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Innsbruck, Austria, 1st Department of Internal Medicine, University of Cologne, Cologne, Germany, Scientific Institute of Public Health, Brussels, Belgium, BZH Deutsches Beratungszentrum fur Hygiene des Universitatsklinikums Freiburg, Freiburg, Germany, Institut Pasteur, Unite de Mycologie Moleculaire, Centre National de Reference Mycologie et Antifongiques, Paris, France, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia, Molecular Mycology Research Laboratory, Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, Sydney Medical School Westmead Hospital, Sydney, The University of Sydney, Australia, Landesgesundheitsamt Baden-Wurttemberg, Stuttgart, Germany, Institute of Hygiene, Microbiology and Environmental Medicine, Medical University Graz, Graz, Austria, Clinical Trials Centre Cologne, ZKS Koln, University of Cologne, Cologne, Germany, Center for Integrated Oncology CIO Koln-Bonn, University of Cologne, Cologne, Germany, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany, Facultad de Medicina, Universitat Rovira i Virgili, Reus, Spain, Institute for Medical Microbiology, Immunology and Parasitology, University of Bonn, Bonn, Germany and Federal Institute for Drugs and Medical Devices (BfArM), Bonn, Germany

BioloMICS Software: Biological Data Management, Identification, Classification and Statistics

The BioloMICS software is briefly described in this application note. BioloMICS is a tool allowing specialized and scientific biological databases to be created to fit the specific needs of researchers working on any organisms (from viruses, bacteria, fungi, plants, insects to animals) by abroad base of users such as taxonomists, ecologists, human, animal or plant pathologists, molecular biologists, pharmacists, industrial researchers, etc. They can all use the system in completely different ways and with different goals in mind. One can create own custom databases without any prior knowledge of either databasing or programming. The system is completely dynamic and can evolve with the needs of the users. A number of tools for data retrieval and analysis are also included in the system. The system also allows linking with many other databases. BioloMICS is proposed as a suite of tools capable of archiving, analyzing and publishing data on local computers and Internet servers.

The Quest for a General and Reliable Fungal DNA Barcode

DNA sequences are key elements for both identification and classification of living organisms. Mainly for historical reasons, a limited number of genes are currently used for this purpose. From a mathematical point of view, any DNA segment, at any location, even outside of coding regions and even if they do not align, could be used as long as PCR primers could be designed to amplify them. This paper describes two methods to search genomic data for the most efficient DNA segments that can be used for identification and classification.

Large-scale generation and analysis of filamentous fungal DNA barcodes boosts coverage for kingdom fungi and reveals thresholds for fungal species and higher taxon delimitation

Species identification lies at the heart of biodiversity studies that has in recent years favoured DNA-based approaches. Microbial Biological Resource Centres are a rich source for diverse and high-quality reference materials in microbiology, and yet the strains preserved in these biobanks have been exploited only on a limited scale to generate DNA barcodes. As part of a project funded in the Netherlands to barcode specimens of major national biobanks, sequences of two nuclear ribosomal genetic markers, the Internal Transcribed Spaces and 5.8S gene (ITS) and the D1/D2 domain of the 26S Large Subunit (LSU), were generated as DNA barcode data for ca. 100u2008000 fungal strains originally assigned to ca. 17u2008000 species in the CBS fungal biobank maintained at the Westerdijk Fungal Biodiversity Institute, Utrecht. Using more than 24u2008000 DNA barcode sequences of 12u2008000 ex-type and manually validated filamentous fungal strains of 7u2008300 accepted species, the optimal identity thresholds to discriminate filamentous fungal species were predicted as 99.6 % for ITS and 99.8 % for LSU. We showed that 17 % and 18 % of the species could not be discriminated by the ITS and LSU genetic markers, respectively. Among them, ∼8 % were indistinguishable using both genetic markers. ITS has been shown to outperform LSU in filamentous fungal species discrimination with a probability of correct identification of 82 % vs. 77.6 %, and a clustering quality value of 84 % vs. 77.7 %. At higher taxonomic classifications, LSU has been shown to have a better discriminatory power than ITS. With a clustering quality value of 80 %, LSU outperformed ITS in identifying filamentous fungi at the ordinal level. At the generic level, the clustering quality values produced by both genetic markers were low, indicating the necessity for taxonomic revisions at genus level and, likely, for applying more conserved genetic markers or even whole genomes. The taxonomic thresholds predicted for filamentous fungal identification at the genus, family, order and class levels were 94.3 %, 88.5 %, 81.2 % and 80.9 % based on ITS barcodes, and 98.2 %, 96.2 %, 94.7 % and 92.7 % based on LSU barcodes. The DNA barcodes used in this study have been deposited to GenBank and will also be publicly available at the Westerdijk Institutes website as reference sequences for fungal identification, marking an unprecedented data release event in global fungal barcoding efforts to date.