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      Surveying Europe’s Only Cave-Dwelling Chordate Species ( Proteus anguinus) Using Environmental DNA

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          Abstract

          In surveillance of subterranean fauna, especially in the case of rare or elusive aquatic species, traditional techniques used for epigean species are often not feasible. We developed a non-invasive survey method based on environmental DNA (eDNA) to detect the presence of the red-listed cave-dwelling amphibian, Proteus anguinus, in the caves of the Dinaric Karst. We tested the method in fifteen caves in Croatia, from which the species was previously recorded or expected to occur. We successfully confirmed the presence of P. anguinus from ten caves and detected the species for the first time in five others. Using a hierarchical occupancy model we compared the availability and detection probability of eDNA of two water sampling methods, filtration and precipitation. The statistical analysis showed that both availability and detection probability depended on the method and estimates for both probabilities were higher using filter samples than for precipitation samples. Combining reliable field and laboratory methods with robust statistical modeling will give the best estimates of species occurrence.

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          Detection of a Diverse Marine Fish Fauna Using Environmental DNA from Seawater Samples

          Marine ecosystems worldwide are under threat with many fish species and populations suffering from human over-exploitation. This is greatly impacting global biodiversity, economy and human health. Intriguingly, marine fish are largely surveyed using selective and invasive methods, which are mostly limited to commercial species, and restricted to particular areas with favourable conditions. Furthermore, misidentification of species represents a major problem. Here, we investigate the potential of using metabarcoding of environmental DNA (eDNA) obtained directly from seawater samples to account for marine fish biodiversity. This eDNA approach has recently been used successfully in freshwater environments, but never in marine settings. We isolate eDNA from ½-litre seawater samples collected in a temperate marine ecosystem in Denmark. Using next-generation DNA sequencing of PCR amplicons, we obtain eDNA from 15 different fish species, including both important consumption species, as well as species rarely or never recorded by conventional monitoring. We also detect eDNA from a rare vagrant species in the area; European pilchard (Sardina pilchardus). Additionally, we detect four bird species. Records in national databases confirmed the occurrence of all detected species. To investigate the efficiency of the eDNA approach, we compared its performance with 9 methods conventionally used in marine fish surveys. Promisingly, eDNA covered the fish diversity better than or equal to any of the applied conventional methods. Our study demonstrates that even small samples of seawater contain eDNA from a wide range of local fish species. Finally, in order to examine the potential dispersal of eDNA in oceans, we performed an experiment addressing eDNA degradation in seawater, which shows that even small (100-bp) eDNA fragments degrades beyond detectability within days. Although further studies are needed to validate the eDNA approach in varying environmental conditions, our findings provide a strong proof-of-concept with great perspectives for future monitoring of marine biodiversity and resources.
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            GenBank

            GenBank® is a comprehensive database that contains publicly available nucleotide sequences for more than 300 000 organisms named at the genus level or lower, obtained primarily through submissions from individual laboratories and batch submissions from large-scale sequencing projects. Most submissions are made using the web-based BankIt or standalone Sequin programs, and accession numbers are assigned by GenBank® staff upon receipt. Daily data exchange with the European Molecular Biology Laboratory Nucleotide Sequence Database in Europe and the DNA Data Bank of Japan ensures worldwide coverage. GenBank is accessible through the National Center for Biotechnology Information (NCBI) Entrez retrieval system, which integrates data from the major DNA and protein sequence databases along with taxonomy, genome, mapping, protein structure and domain information, and the biomedical journal literature via PubMed. BLAST provides sequence similarity searches of GenBank and other sequence databases. Complete bimonthly releases and daily updates of the GenBank database are available by FTP. To access GenBank and its related retrieval and analysis services, begin at the NCBI Homepage: www.ncbi.nlm.nih.gov.
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              An In silico approach for the evaluation of DNA barcodes

              Background DNA barcoding is a key tool for assessing biodiversity in both taxonomic and environmental studies. Essential features of barcodes include their applicability to a wide spectrum of taxa and their ability to identify even closely related species. Several DNA regions have been proposed as barcodes and the region selected strongly influences the output of a study. However, formal comparisons between barcodes remained limited until now. Here we present a standard method for evaluating barcode quality, based on the use of a new bioinformatic tool that performs in silico PCR over large databases. We illustrate this approach by comparing the taxonomic coverage and the resolution of several DNA regions already proposed for the barcoding of vertebrates. To assess the relationship between in silico and in vitro PCR, we also developed specific primers amplifying different species of Felidae, and we tested them using both kinds of PCR Results Tests on specific primers confirmed the correspondence between in silico and in vitro PCR. Nevertheless, results of in silico and in vitro PCRs can be somehow different, also because tuning PCR conditions can increase the performance of primers with limited taxonomic coverage. The in silico evaluation of DNA barcodes showed a strong variation of taxonomic coverage (i.e., universality): barcodes based on highly degenerated primers and those corresponding to the conserved region of the Cyt-b showed the highest coverage. As expected, longer barcodes had a better resolution than shorter ones, which are however more convenient for ecological studies analysing environmental samples. Conclusions In silico PCR could be used to improve the performance of a study, by allowing the preliminary comparison of several DNA regions in order to identify the most appropriate barcode depending on the study aims.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                27 January 2017
                2017
                : 12
                : 1
                : e0170945
                Affiliations
                [1 ]Department of Zoology, Hungarian Natural History Museum, Budapest, Hungary
                [2 ]Molecular Taxonomy Laboratory, Hungarian Natural History Museum, Budapest, Hungary
                [3 ]Institute for Soil Sciences and Agricultural Chemistry, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungary
                [4 ]Department of Evolutionary Biology and Environment Studies, University of Zurich, Zurich, Switzerland
                [5 ]Koordinationsstelle für Amphibien- und Reptilienschutz in der Schweiz, Neuchâtel, Switzerland
                [6 ]Croatian Institute for Biodiversity, Zagreb, Croatia
                National Zoological Park, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceptualization: JV JTG DJ.

                • Data curation: JV OM.

                • Formal analysis: BRS.

                • Funding acquisition: JV DJ.

                • Investigation: JV JTG OM DJ.

                • Methodology: JV OM JTG DJ.

                • Project administration: JV DJ.

                • Resources: JV DJ JTG OM BRS.

                • Visualization: JV OM BRS DJ.

                • Writing – original draft: JV OM BRS DJ.

                • Writing – review & editing: JV OM BRS DJ.

                Author information
                http://orcid.org/0000-0001-9707-1443
                Article
                PONE-D-16-44547
                10.1371/journal.pone.0170945
                5271363
                28129383
                89244ea6-d292-430c-9508-10c31369589a
                © 2017 Vörös et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 9 November 2016
                : 12 January 2017
                Page count
                Figures: 3, Tables: 1, Pages: 14
                Funding
                Funded by: MAVA Foundation
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100009470, Zoological Society of London;
                Award Recipient :
                Funded by: Krka National Park
                Award Recipient :
                Funded by: Bolyai János Research Scholarship of the Hungarian Academy of Sciences
                Award ID: BO/00579/14/8
                Award Recipient :
                DJ was supported by the MAVA Foundation, Zoological Society of London and Krka National Park. JV was supported by the Bolyai János Research Scholarship of the Hungarian Academy of Sciences (BO/00579/14/8). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Earth Sciences
                Geomorphology
                Topography
                Landforms
                Caves
                Research and Analysis Methods
                Precipitation Techniques
                People and Places
                Geographical Locations
                Europe
                Croatia
                Research and Analysis Methods
                Equipment Preparation
                Equipment Sterilization
                Filter Sterilization
                Research and analysis methods
                Database and informatics methods
                Bioinformatics
                Sequence analysis
                DNA sequence analysis
                Research and analysis methods
                Extraction techniques
                DNA extraction
                Biology and Life Sciences
                Ecology
                Biodiversity
                Ecology and Environmental Sciences
                Ecology
                Biodiversity
                Custom metadata
                All relevant data are in the paper and its Supporting Information files.

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