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      Hidden in our pockets: building of a DNA barcode library unveils the first record of Myotis alcathoe for Portugal

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          Abstract

          Background

          The advent and boom of DNA barcoding technologies have provided a powerful tool for the fields of ecology and systematics. Here, we present the InBIO Barcoding Initiative Database: Portuguese Bats ( Chiroptera ) dataset containing DNA sequences of 63 specimens representing the 25 bat species currently known for continental Portugal. For that, we sequenced tissues samples obtained in a vast array of projects spanning the last two decades.

          New information

          We added four new Barcoding Index Numbers (BINs) to existing Chiroptera barcodes on BOLD, two belonging to Myotis escalerai , one to Plecotus auritus and the other to Rhinolophus hipposideros . Surprisingly, one of the samples initially identified in the field as Myotis mystacinus turned out to be Myotis alcathoe , which represents the first record of this species for Portugal. The presence of Nyctalus noctula in Portugal was also genetically confirmed for the first time. This case study shows the power and value of DNA barcoding initiatives to unravel new data that may be hidden on biological collections.

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          Most cited references24

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          obitools: a unix-inspired software package for DNA metabarcoding.

          DNA metabarcoding offers new perspectives in biodiversity research. This recently developed approach to ecosystem study relies heavily on the use of next-generation sequencing (NGS) and thus calls upon the ability to deal with huge sequence data sets. The obitools package satisfies this requirement thanks to a set of programs specifically designed for analysing NGS data in a DNA metabarcoding context. Their capacity to filter and edit sequences while taking into account taxonomic annotation helps to set up tailor-made analysis pipelines for a broad range of DNA metabarcoding applications, including biodiversity surveys or diet analyses. The obitools package is distributed as an open source software available on the following website: http://metabarcoding.org/obitools. A Galaxy wrapper is available on the GenOuest core facility toolshed: http://toolshed.genouest.org.
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            Robust Detection of Rare Species Using Environmental DNA: The Importance of Primer Specificity

            Environmental DNA (eDNA) is being rapidly adopted as a tool to detect rare animals. Quantitative PCR (qPCR) using probe-based chemistries may represent a particularly powerful tool because of the method’s sensitivity, specificity, and potential to quantify target DNA. However, there has been little work understanding the performance of these assays in the presence of closely related, sympatric taxa. If related species cause any cross-amplification or interference, false positives and negatives may be generated. These errors can be disastrous if false positives lead to overestimate the abundance of an endangered species or if false negatives prevent detection of an invasive species. In this study we test factors that influence the specificity and sensitivity of TaqMan MGB assays using co-occurring, closely related brook trout (Salvelinus fontinalis) and bull trout (S. confluentus) as a case study. We found qPCR to be substantially more sensitive than traditional PCR, with a high probability of detection at concentrations as low as 0.5 target copies/µl. We also found that number and placement of base pair mismatches between the Taqman MGB assay and non-target templates was important to target specificity, and that specificity was most influenced by base pair mismatches in the primers, rather than in the probe. We found that insufficient specificity can result in both false positive and false negative results, particularly in the presence of abundant related species. Our results highlight the utility of qPCR as a highly sensitive eDNA tool, and underscore the importance of careful assay design.
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              Validation and Development of COI Metabarcoding Primers for Freshwater Macroinvertebrate Bioassessment

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                Author and article information

                Contributors
                Journal
                Biodivers Data J
                Biodivers Data J
                1
                urn:lsid:arphahub.com:pub:F9B2E808-C883-5F47-B276-6D62129E4FF4
                urn:lsid:zoobank.org:pub:245B00E9-BFE5-4B4F-B76E-15C30BA74C02
                Biodiversity Data Journal
                Pensoft Publishers
                1314-2836
                1314-2828
                2020
                28 July 2020
                : 8
                : e54479
                Affiliations
                [1 ] CIBIO-InBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Universidade do Porto, Vairão, Portugal CIBIO-InBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Universidade do Porto Vairão Portugal
                [2 ] CIBIO-InBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Instituto Superior de Agronomia, Lisboa, Portugal CIBIO-InBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Instituto Superior de Agronomia Lisboa Portugal
                [3 ] Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Porto, Portugal Departamento de Biologia, Faculdade de Ciências, Universidade do Porto Porto Portugal
                Author notes
                Corresponding author: Vanessa A. Mata ( vanessamata@ 123456hotmail.com ).

                Academic editor: Ricardo Moratelli

                Author information
                https://orcid.org/0000-0002-6884-3966
                https://orcid.org/0000-0001-8164-0760
                https://orcid.org/0000-0003-3005-9030
                Article
                54479 13828
                10.3897/BDJ.8.e54479
                7403162
                eff10ad0-fc99-4596-8552-513d24f23c8c
                Hugo Rebelo, Sónia Ferreira, Francisco Amorim, Pedro Horta, Helena Raposeira, Helena Santos, Pedro Beja, Vanessa A. Mata

                This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 19 May 2020
                : 29 June 2020
                Page count
                Figures: 4, Tables: 1, References: 32
                Funding
                Funding: The FCT - Fundação para Ciência e Tecnologia, I.P. funded V.M. (PD/BD/113462/2015) and F.A. (PD/BD/52606/2014). S.F. was funded by the project PORBIOTA - Portuguese E-Infrastructure for Information and Research on Biodiversity (POCI-01-0145-FEDER-022127), supported by Operational Thematic Program for Competitiveness and Internationalization (POCI), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). This research also received funding from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No 668981.Fieldwork and sample collection were funded under the scope of the FCT projects PTDC/BIA-BIC/110587/2009, LTER/BIA-BEC/0004/2009 and PTDC/BIA-ECO/31731/2017, and from EDP – Energias de Portugal.
                Categories
                Data Paper (Biosciences)
                Chiroptera
                Animalia
                Molecular biology
                Zoology & Animal Biology
                Genetics
                Europe

                dna barcoding,bats,myotis alcathoe,museum collections,species identification,coi

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