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      A DNA barcode library for 5,200 German flies and midges (Insecta: Diptera) and its implications for metabarcoding‐based biomonitoring

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          Abstract

          This study summarizes results of a DNA barcoding campaign on German Diptera, involving analysis of 45,040 specimens. The resultant DNA barcode library includes records for 2,453 named species comprising a total of 5,200 barcode index numbers (BINs), including 2,700 COI haplotype clusters without species‐level assignment, so called “dark taxa.” Overall, 88 out of 117 families (75%) recorded from Germany were covered, representing more than 50% of the 9,544 known species of German Diptera. Until now, most of these families, especially the most diverse, have been taxonomically inaccessible. By contrast, within a few years this study provided an intermediate taxonomic system for half of the German Dipteran fauna, which will provide a useful foundation for subsequent detailed, integrative taxonomic studies. Using DNA extracts derived from bulk collections made by Malaise traps, we further demonstrate that species delineation using BINs and operational taxonomic units (OTUs) constitutes an effective method for biodiversity studies using DNA metabarcoding. As the reference libraries continue to grow, and gaps in the species catalogue are filled, BIN lists assembled by metabarcoding will provide greater taxonomic resolution. The present study has three main goals: (a) to provide a DNA barcode library for 5,200 BINs of Diptera; (b) to demonstrate, based on the example of bulk extractions from a Malaise trap experiment, that DNA barcode clusters, labelled with globally unique identifiers (such as OTUs and/or BINs), provide a pragmatic, accurate solution to the “taxonomic impediment”; and (c) to demonstrate that interim names based on BINs and OTUs obtained through metabarcoding provide an effective method for studies on species‐rich groups that are usually neglected in biodiversity research projects because of their unresolved taxonomy.

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          The integrative future of taxonomy

          Background Taxonomy is the biological discipline that identifies, describes, classifies and names extant and extinct species and other taxa. Nowadays, species taxonomy is confronted with the challenge to fully incorporate new theory, methods and data from disciplines that study the origin, limits and evolution of species. Results Integrative taxonomy has been proposed as a framework to bring together these conceptual and methodological developments. Here we review perspectives for an integrative taxonomy that directly bear on what species are, how they can be discovered, and how much diversity is on Earth. Conclusions We conclude that taxonomy needs to be pluralistic to improve species discovery and description, and to develop novel protocols to produce the much-needed inventory of life in a reasonable time. To cope with the large number of candidate species revealed by molecular studies of eukaryotes, we propose a classification scheme for those units that will facilitate the subsequent assembly of data sets for the formal description of new species under the Linnaean system, and will ultimately integrate the activities of taxonomists and molecular biologists.
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            A new versatile primer set targeting a short fragment of the mitochondrial COI region for metabarcoding metazoan diversity: application for characterizing coral reef fish gut contents

            Introduction The PCR-based analysis of homologous genes has become one of the most powerful approaches for species detection and identification, particularly with the recent availability of Next Generation Sequencing platforms (NGS) making it possible to identify species composition from a broad range of environmental samples. Identifying species from these samples relies on the ability to match sequences with reference barcodes for taxonomic identification. Unfortunately, most studies of environmental samples have targeted ribosomal markers, despite the fact that the mitochondrial Cytochrome c Oxidase subunit I gene (COI) is by far the most widely available sequence region in public reference libraries. This is largely because the available versatile (“universal”) COI primers target the 658 barcoding region, whose size is considered too large for many NGS applications. Moreover, traditional barcoding primers are known to be poorly conserved across some taxonomic groups. Results We first design a new PCR primer within the highly variable mitochondrial COI region, the “mlCOIintF” primer. We then show that this newly designed forward primer combined with the “jgHCO2198” reverse primer to target a 313 bp fragment performs well across metazoan diversity, with higher success rates than versatile primer sets traditionally used for DNA barcoding (i.e. LCO1490/HCO2198). Finally, we demonstrate how the shorter COI fragment coupled with an efficient bioinformatics pipeline can be used to characterize species diversity from environmental samples by pyrosequencing. We examine the gut contents of three species of planktivorous and benthivorous coral reef fish (family: Apogonidae and Holocentridae). After the removal of dubious COI sequences, we obtained a total of 334 prey Operational Taxonomic Units (OTUs) belonging to 14 phyla from 16 fish guts. Of these, 52.5% matched a reference barcode (>98% sequence similarity) and an additional 32% could be assigned to a higher taxonomic level using Bayesian assignment. Conclusions The molecular analysis of gut contents targeting the 313 COI fragment using the newly designed mlCOIintF primer in combination with the jgHCO2198 primer offers enormous promise for metazoan metabarcoding studies. We believe that this primer set will be a valuable asset for a range of applications from large-scale biodiversity assessments to food web studies.
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              DNA barcoding and taxonomy in Diptera: a tale of high intraspecific variability and low identification success.

              DNA barcoding and DNA taxonomy have recently been proposed as solutions to the crisis of taxonomy and received significant attention from scientific journals, grant agencies, natural history museums, and mainstream media. Here, we test two key claims of molecular taxonomy using 1333 mitochondrial COI sequences for 449 species of Diptera. We investigate whether sequences can be used for species identification ("DNA barcoding") and find a relatively low success rate (< 70%) based on tree-based and newly proposed species identification criteria. Misidentifications are due to wide overlap between intra- and interspecific genetic variability, which causes 6.5% of all query sequences to have allospecific or a mixture of allo- and conspecific (3.6%) best-matching barcodes. Even when two COI sequences are identical, there is a 6% chance that they belong to different species. We also find that 21% of all species lack unique barcodes when consensus sequences of all conspecific sequences are used. Lastly, we test whether DNA sequences yield an unambiguous species-level taxonomy when sequence profiles are assembled based on pairwise distance thresholds. We find many sequence triplets for which two of the three pairwise distances remain below the threshold, whereas the third exceeds it; i.e., it is impossible to consistently delimit species based on pairwise distances. Furthermore, for species profiles based on a 3% threshold, only 47% of all profiles are consistent with currently accepted species limits, 20% contain more than one species, and 33% only some sequences from one species; i.e., adopting such a DNA taxonomy would require the redescription of a large proportion of the known species, thus worsening the taxonomic impediment. We conclude with an outlook on the prospects of obtaining complete barcode databases and the future use of DNA sequences in a modern integrative taxonomy.
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                Author and article information

                Contributors
                moriniere@snsb.de
                doczkal@snsb.de
                Journal
                Mol Ecol Resour
                Mol Ecol Resour
                10.1111/(ISSN)1755-0998
                MEN
                Molecular Ecology Resources
                John Wiley and Sons Inc. (Hoboken )
                1755-098X
                1755-0998
                14 May 2019
                July 2019
                : 19
                : 4 ( doiID: 10.1111/men.2019.19.issue-4 )
                : 900-928
                Affiliations
                [ 1 ] SNSB‐Zoologische Staatssammlung München Germany
                [ 2 ] Zoological Research Museum Alexander Koenig ‐ Leibniz Institute for Animal Biodiversity Bonn Germany
                [ 3 ] Centre for Biodiversity Genomics University of Guelph Guelph Ontario Canada
                Author notes
                [*] [* ] Correspondence

                Jérôme Morinière and Dieter Doczkal, SNSB‐Zoologische Staatssammlung, München, Germany.

                Emails: moriniere@ 123456snsb.de ; doczkal@ 123456snsb.de

                Author information
                https://orcid.org/0000-0001-9167-6409
                https://orcid.org/0000-0001-5751-8706
                Article
                MEN13022
                10.1111/1755-0998.13022
                6851627
                30977972
                99c55d19-eb4c-4540-a9da-918e8a92df00
                © 2019 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 19 December 2018
                : 22 March 2019
                : 25 March 2019
                Page count
                Figures: 3, Tables: 3, Pages: 29, Words: 19561
                Funding
                Funded by: Bavarian State Ministry of Science and the Arts
                Funded by: German Federal Ministry of Education and Research
                Award ID: BMBF FKZ 01LI1101
                Award ID: BMBF FKZ 01LI1501
                Categories
                Resource Article
                RESOURCE ARTICLES
                Molecular and Statistical Advances
                Custom metadata
                2.0
                July 2019
                Converter:WILEY_ML3GV2_TO_JATSPMC version:5.7.1 mode:remove_FC converted:13.11.2019

                Ecology
                barcode library,biodiversity monitoring,co1,cryptic diversity,diptera,dna barcoding,germany,metabarcoding,mitochondrial dna

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