title: Determining threshold values for barcoding fungi: lessons from Cortinarius (Basidiomycota), a highly diverse and widespread ectomycorrhizal genus.
abstract: Different distance-based threshold selection approaches were used to assess and compare use of the internal transcribed spacer (ITS) region to distinguish among 901 Cortinarius species represented by >3000 collections. Sources of error associated with genetic markers and selection approaches were explored and evaluated using MOTUs from genus and lineage based-alignments. Our study indicates that 1%-2% more species can be distinguished by using the full-length ITS barcode as compared to either the ITS1 or ITS2 regions alone. Optimal threshold values for different picking approaches and genetic marker lengths inferred from a subset of species containing major lineages ranged from 97.0% to 99.5% sequence similarity using clustering optimization and UNITE SH, and from 1% to 2% sequence dissimilarity with CROP. Errors for the optimal cutoff ranged from 0% to 70%, and these can be reduced to a maximum of 22% when excluding species lacking a barcode gap. A threshold value of 99% is suitable for distinguishing species in the majority of lineages in the genus using the entire ITS region but only 90% of the species could be identified using just the ITS1 or ITS2 region. Prior identification of species, lacking barcode gaps and their subsequent separate analyses, maximized the accuracy of threshold approaches.
authors.affiliation: Institute of Evolution and Ecology, Plant Evolutionary Ecology, University of Tübingen, Auf der Morgenstelle 5, D-72076 Tübingen, Germany sigisfredo.garnica@uni-tuebingen.de.
authors.forename: Sigisfredo
authors.initials: S
authors.lastname: Garnica
authors.affiliation: Institute of Evolution and Ecology, Plant Evolutionary Ecology, University of Tübingen, Auf der Morgenstelle 5, D-72076 Tübingen, Germany.
authors.forename: Max Emil
authors.initials: ME
authors.lastname: Schön
authors.affiliation: Institute of Ecology and Earth Sciences, University of Tartu, 51005 Tartu, Estonia.
authors.forename: Kessy
authors.initials: K
authors.lastname: Abarenkov
authors.affiliation: Institute of Evolution and Ecology, Plant Evolutionary Ecology, University of Tübingen, Auf der Morgenstelle 5, D-72076 Tübingen, Germany.
authors.forename: Kai
authors.initials: K
authors.lastname: Riess
authors.affiliation: Department of Biosciences, Plant Biology, University of Helsinki, PO Box 65, 00014 Lahti, Finland.
authors.forename: Kare
authors.initials: K
authors.lastname: Liimatainen
authors.affiliation: Jodrell Laboratory, Royal Botanic Gardens, Kew, TW9 3DS, UK.
authors.forename: Tuula
authors.initials: T
authors.lastname: Niskanen
authors.affiliation: Department of Biosciences, Plant Biology, University of Helsinki, PO Box 65, 00014 Lahti, Finland.
authors.forename: Bálint
authors.initials: B
authors.lastname: Dima
authors.affiliation: Honorary Research Associate, Swedish Museum of Natural History, Department of Cryptogamic Botany, Naturhistoriska riksmuseet, 104 05 Stockholm, Sweden.
authors.forename: Karl
authors.initials: K
authors.lastname: Soop
authors.affiliation: Natural History Museum of Denmark, Center for Geogenetics, University of Copenhagen, Øster Voldgade 5-7, 1350 Copenhagen K, Denmark.
authors.forename: Tobias Guldberg
authors.initials: TG
authors.lastname: Frøslev
authors.affiliation: Natural History Museum of Denmark, Collections, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen Ø, Denmark.
authors.forename: Thomas Stjernegaard
authors.initials: TS
authors.lastname: Jeppesen
authors.affiliation: Institute of Microbiology, University Innsbruck, Technikerstraße 25, 6020 Innsbruck, Austria.
authors.forename: Ursula
authors.initials: U
authors.lastname: Peintner
authors.affiliation: Institute of Microbiology, University Innsbruck, Technikerstraße 25, 6020 Innsbruck, Austria.
authors.forename: Regina
authors.initials: R
authors.lastname: Kuhnert-Finkernagel
authors.affiliation: Department of Landscape Ecology (Oslo), Norwegian Institute for Nature Research, N-Oslo 5, Norway.
authors.forename: Tor Erik
authors.initials: TE
authors.lastname: Brandrud
authors.affiliation: Dammenmühle 7, D-77933 Lahr-Sulz, Germany.
authors.forename: Günter
authors.initials: G
authors.lastname: Saar
authors.affiliation: INRES, University of Bonn, Auf dem Hügel 6, D-53121 Bonn, Germany.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Oertel
authors.affiliation: Department of Biology, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Joseph F
authors.initials: JF
authors.lastname: Ammirati
mesh_terms: D055363:Cortinarius / Q000145:classification* / Q000235:genetics*; D058893:DNA Barcoding, Taxonomic / Q000379:methods*; D021901:DNA, Intergenic / Q000235:genetics*; D005819:Genetic Markers / Q000235:genetics; D038821:Mycorrhizae / Q000235:genetics*; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D021901:DNA, Intergenic; D005819:Genetic Markers
keywords: Cortinarius; DNA barcoding; barcoding gap; internal transcribed spacer (ITS); molecular operational taxonomic unit (MOTU); species identification; threshold approaches