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Caveats of fungal barcoding: a case study in Trametes s.lat. (Basidiomycota: Polyporales) in Vietnam reveals multiple issues with mislabelled reference sequences and calls for third-party annotations Authors: Lücking, Robert, Truong, Ba Vuong, Huong, Dang Thi Thu, Le, Ngoc Han, Nguyen, Quoc Dat, et al. Source: Willdenowia, 50(3) : 383-403 Published By: Botanic Garden and Botanical Museum Berlin (BGBM) URL: https://doi.org/10.3372/wi.50.50302 BioOne Complete (complete.BioOne.org) is a full-text database of 200 subscribed and open-access titles in the biological, ecological, and environmental sciences published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Complete website, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/terms-of-use. Usage of BioOne Complete content is strictly limited to personal, educational, and non - commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. Downloaded From: https://bioone.org/journals/Willdenowia on 10 Feb 2021 Terms of Use: https://bioone.org/terms-of-use Willdenowia Annals of the Botanic Garden and Botanical Museum Berlin ROBERT LÜCKING1*, BA VUONG TRUONG2, DANG THI THU HUONG3, NGOC HAN LE3, QUOC DAT NGUYEN4, VAN DAT NGUYEN5, ECKHARD VON RAAB-STRAUBE1, SARAH BOLLENDORFF1, KIM GOVERS1 & VANESSA DI VINCENZO1 Caveats of fungal barcoding: a case study in Trametes s.lat. (Basidiomycota: Poly­ porales) in Vietnam reveals multiple issues with mislabelled reference sequences and calls for third-party annotations Version of record first published online on 15 September 2020 ahead of inclusion in December 2020 issue. Abstract: DNA barcoding using the nuclear internal transcribed spacer (ITS) has become prevalent in surveys of fungal diversity. This approach is, however, associated with numerous caveats, including the desire for speed, rather than accuracy, through the use of automated analytical pipelines, and the shortcomings of reference sequence re- positories. Here we use the case of a specimen of the bracket fungus Trametes s.lat. (which includes the common and widespread turkey tail, T. versicolor) to illustrate these problems. The material was collected in Vietnam as part of a biodiversity inventory including DNA barcoding approaches for arthropods, plants and fungi. The ITS barcod- ing sequence of the query taxon was compared against reference sequences in GenBank and the curated fungal ITS database UNITE, using BLASTn and MegaBLAST, and was subsequently analysed in a multiple alignment-based phylogenetic context through a maximum likelihood tree including related sequences. Our results initially indicated issues with BLAST searches, including the use of pairwise local alignments and sorting through Total score and E value, rather than Percentage identity, as major shortcomings of the DNA barcoding approach. However, after thor- ough analysis of the results, we concluded that the single most important problem of this approach was incorrect sequence labelling, calling for the implementation of third-party annotations or analogous approaches in primary se- quence repositories. In addition, this particular example revealed problems of improper fungal nomenclature, which required reinstatement of the genus name Cubamyces (= Leiotrametes), with three new combinations: C. flavidus, C. lactineus and C. menziesii. The latter was revealed as the correct identification of the query taxon, although the name did not appear among the best BLAST hits. While the best BLAST hits did correspond to the target taxon in terms of sequence data, their label names were misleading or unresolved, including [Fungal endophyte], [Uncultured fungus], Basidiomycota, Trametes cf. cubensis, Lenzites elegans and Geotrichum candidum (an unrelated ascomycetous con- taminant). Our study demonstrates that accurate identification of fungi through molecular barcoding is currently not a fast-track approach that can be achieved through automated pipelines. Key words: Basidiomycota, fungal barcoding, Polyporales, sequence contamination, Trametes, Trametes cubensis, Trametes menziesii, Vietnam Article history: Received 5 May 2020; peer-review completed 15 July 2020; received in revised form 10 August 2020; accepted for publication 20 August 2020. Citation: Lücking R., Truong B. V., Huong D. T. T., Le N. H., Nguyen Q. D., Nguyen V. D., Raab-Straube E. von, Bollendorff S., Govers K. & Di Vincenzo V. 2020: Caveats of fungal barcoding: a case study in Trametes s.lat. (Basidiomycota: Polyporales) in Vietnam reveals multiple issues with mislabelled reference sequences and calls for third-party annotations. – Willdenowia 50: 383 – 403. doi: https://doi.org/10.3372/wi.50.50302 1 Botanischer Garten und Botanisches Museum, Freie Universität Berlin, Königin-Luise-Straße 6–8, 14195 Berlin, Germany; *e-mail: [email protected] (author for correspondence). 2 Institute of Tropical Biology, Department of Biological Resources, Vietnam Academy of Science and Technology, 85 Tran Quoc Toan St., District 3, Ho Chi Minh City, Vietnam. 3 Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam. 4 Southern Institute of Ecology, Vietnam Academy of Science and Technology, 01 Mac Dinh Chi, Ben Nghe, District 1, Ho Chi Minh City, Vietnam. 5 Vietnam National Museum of Nature, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam. Downloaded From: https://bioone.org/journals/Willdenowia on 10 Feb 2021 Terms of Use: https://bioone.org/terms-of-use 384 Lücking & al.: Caveats of fungal barcoding: a case study in Trametes s.lat. Introduction species delimitation do not exist; the actual threshold is lineage-specific and depends on the phylogenetic con- The true fungi (Fungi) represent the third largest king- text. In some groups, species can be distinguished by dom in terms of known species and the second largest pairwise similarity thresholds as narrow as 99.5 % (Vu & with respect to estimated richness, with between 2.2 al. 2016; Lücking & al. 2017). Based on observed iden- and 3.8 million species (Hawksworth & Lücking 2018). tity values alone, it is therefore not possible to decide Due to their simple body plan, fungi have few diagnos- whether the closest BLAST hit is actually the species in tic characters compared to plants and animals (Nagy question or a closely related taxon. & al. 2017; Lücking 2019). This renders their accurate Finally, a substantial proportion of ITS reference se- identification based on phenotype often difficult or even quences deposited in GenBank and other databases are impossible (Lücking & al. 2020). Molecular barcoding wrongly or incompletely labelled (Harris 2003; Vilgalys has therefore become an important tool in the identifi- 2003; Nilsson & al. 2005, 2006, 2012, 2014; Meier 2008; cation of fungi, as well as other organisms, and is even Bidartondo & al. 2008; Lücking & al. 2012; Kõljalg & being implemented in citizen science projects (Geiger al. 2013; Hofstetter & al. 2019). As of 25 March 2020, & al. 2016; Beenken & al. 2017; Bubner & al. 2019; GenBank returned 1 367 715 fungal ITS sequences using Chiovitti & al. 2019). the structured query <Fungi[organism] AND (5.8S[title] For fungi, the mycological community has agreed on OR ITS1[title] OR ITS2[title] OR ITS[title] OR “inter- the nuclear internal transcribed spacer of the nuclear ri- nal transcribed spacer”[title])>. Of these, only 443 645 bosomal DNA cistron (ITS) as universal barcoding mark- (32 %) were fully identified to species with a Latin bino- er (Schoch & al. 2012). While this marker works rather mial, though not necessarily correctly so. Consequently, well in most fungal groups, in some lineages it does not the best BLAST matches may not have species-level and provide sufficient resolution and secondary barcoding often not even genus-level identifications. Sequence la- markers such as TEF1 and COX1 (COI) have been pro- bels are often incorrect, either due to misidentifications posed (Chen & al. 2009; O’Donnell & al. 2015; Al-Hatmi or outdated taxonomic concept. In some cases, taxon & al. 2016; Raja & al. 2017; Tepkinar & Kalmer 2019). names are associated with sequences that represent un- In some cases, phenomena such as hybridization, intro- related contaminants (Lücking & Nelsen 2018). As a gression or gene duplication also complicate the use of result, a species-level clade can contain numerous differ- the ITS barcoding marker (Lindner & Banki 2013; Li & ent names, and the same name can appear in various and al. 2013, 2017; Lücking & al. 2020). even unrelated clades. Problems in sequence annotation In addition to these technical difficulties, identifica- can even perpetuate themselves when using erroneous tion of fungi through molecular barcoding faces other annotations of BLAST hits to annotate newly generated issues. One is the quality and completeness of existing sequences (e.g. Gilks & al. 2002). Using curated sec- reference databases (Nilsson & al. 2006; Meier 2008; ondary databases and/or improved data standards have Begerow & al. 2010; Tedersoo & al. 2011; Kõljalg & al. been proposed
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  • Funghi E Natura Gruppo Di Padova

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    FUNGHI E NATURA www.ambpadova.it Anno 46° ~ 2° semestre 2019 Gruppo di Padova notiziario micologico semestrale riservato agli associati FUNGHI E NATURA www.ambpadova.it Anno 46° ~ 2° semestre 2019 Associazione Micologica Bresadola Gruppo di Padova Foto di Copertina www.ambpadova.it Boletus Notizie Utili reticulatus e-mail: [email protected] Foto di Sede a Padova Via Bezzecca 17 Riccardo Menegazzo C/C/ Postale 14153357 C.F. 00738410281 Quota associativa anno 2019: € 25,00 incluse ricezioni di: “Rivista di Micologia” Gruppo di Padova edita da AMB Nazionale e “Funghi e Natura” notiziario micologico semestrale riservato agli associati del Gruppo di Padova. Incontri e serate ad Albignasego (PD) nella Casa delle Associazioni, in via Damiano Chiesa, angolo Via Fabio Filzi Presidente Riccardo Novella (tel.335 7783745) SOMMARIO Vice Pres. Rossano Giolo (tel. 049 9714147). Segretario Funghi e Natura 31 Luglio 2019 Paolo Bordin (tel. 049 8725104). Tesoriere: Ida Varotto (tel. 347 9212708). Dalla segreteria pag. 3 Direttore Gruppo di Studio: Paolo Di Piazza(tel. 349 4287268). di Paolo Bordin Vicedirettore Gruppo di Studio: Riccardo Menegazzo. Gomphidius tyrrhenicus Resp. attività ricreative: D. Antonini & M. Antonini Ennio Albertin (tel. 049 811681). Resp. organizzazione mostre ed erbario: di Rossano Giolo pag. 5 Andrea Cavalletto Resp. pubbliche relazioni: Cronaca di una serata da Ida Varotto (tel. 347 9212708) e Gino Segato. ricordare Gestione materiale e allestimento mostre: Ennio Albertin. di Alberto Parpajola pag. 8 Coordinatore Funghi e Natura: Lepiota andegavensis: Alberto Parpajola e-mail: [email protected] rarissima specie raccolta Consiglio Direttivo: sui Colli Euganei R. Novella ,E. Albertin, P. Bordin, A. Cavalletto, R.