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      Evaluating freshwater macroinvertebrates from eDNA metabarcoding: A river Nalón case study

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          Abstract

          Rivers are a vital resource for human wellbeing. To reduce human impact on water bodies, the European Union has established an essential regulatory framework for protection and sustainable management (WFD; 2000/60/EC). In this strategy, reliable and economic bioindicators are a fundamental component. Benthic macroinvertebrates are the group most commonly used as bioindicators through all European countries. However, their conventional assessment currently entails serious cost-efficiency limitations. In this study, we have tested the reliability of metabarcoding as a tool to record river macroinvertebrates using samples from a mock community ( in vitro validation) and eDNA extracted for field validation from water from six sites within a north Iberian river (River Nalón, Asturias, Spain). Two markers (V4 region within the nuclear 18S rDNA and a fragment of the mitochondrial COI gene) were amplified and sequenced using an Illumina platform. The molecular technique has proven to be more sensitive than the visual one. A cost-benefit analysis shows that the metabarcoding approach is more expensive than conventional techniques for determining macroinvertebrate communities but requires fewer sampling and identification efforts. Our results suggest metabarcoding is a useful tool for alternative assessment of freshwater quality.

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          Most cited references 35

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          Environmental DNA for wildlife biology and biodiversity monitoring.

          Extraction and identification of DNA from an environmental sample has proven noteworthy recently in detecting and monitoring not only common species, but also those that are endangered, invasive, or elusive. Particular attributes of so-called environmental DNA (eDNA) analysis render it a potent tool for elucidating mechanistic insights in ecological and evolutionary processes. Foremost among these is an improved ability to explore ecosystem-level processes, the generation of quantitative indices for analyses of species, community diversity, and dynamics, and novel opportunities through the use of time-serial samples and unprecedented sensitivity for detecting rare or difficult-to-sample taxa. Although technical challenges remain, here we examine the current frontiers of eDNA, outline key aspects requiring improvement, and suggest future developments and innovations for research. Copyright © 2014 Elsevier Ltd. All rights reserved.
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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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              Environmental DNA metabarcoding: Transforming how we survey animal and plant communities

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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: SoftwareRole: ValidationRole: Writing – original draft
                Role: Data curationRole: Formal analysisRole: MethodologyRole: Software
                Role: Methodology
                Role: ConceptualizationRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: SupervisionRole: Writing – review & editing
                Role: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                8 August 2018
                2018
                : 13
                : 8
                Affiliations
                [1 ] Department of Functional Biology, University of Oviedo, Oviedo, Asturias, Spain
                [2 ] Unit of DNA Analysis, Scientific-Technical Services, University of Oviedo, Oviedo, Asturias, Spain
                Oklahoma State University, UNITED STATES
                Author notes

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

                Article
                PONE-D-17-43575
                10.1371/journal.pone.0201741
                6082553
                30089147
                © 2018 Fernández 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.

                Page count
                Figures: 3, Tables: 5, Pages: 17
                Product
                Funding
                Funded by: AMBER project
                Award ID: 689682
                Award Recipient : Sara Fernández Fernández
                This study was funded from the European Union’s Horizon 2020 Research and Innovation program under Grant Agreement No 689682, Adaptive Management of Barriers in European Rivers (AMBER) project. The study was also partially funded by the Juan de la cierva Fellowship to AA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Earth Sciences
                Marine and Aquatic Sciences
                Bodies of Water
                Rivers
                Ecology and Environmental Sciences
                Aquatic Environments
                Freshwater Environments
                Rivers
                Earth Sciences
                Marine and Aquatic Sciences
                Aquatic Environments
                Freshwater Environments
                Rivers
                Biology and Life Sciences
                Taxonomy
                Computer and Information Sciences
                Data Management
                Taxonomy
                Research and Analysis Methods
                Database and Informatics Methods
                Biological Databases
                Sequence Databases
                Research and Analysis Methods
                Database and Informatics Methods
                Bioinformatics
                Sequence Analysis
                Sequence Databases
                Research and analysis methods
                Database and informatics methods
                Bioinformatics
                Sequence analysis
                BLAST algorithm
                Research and Analysis Methods
                Database and Informatics Methods
                Bioinformatics
                Sequence Analysis
                Sequence Alignment
                Earth Sciences
                Hydrology
                Surface Water
                Biology and life sciences
                Molecular biology
                Molecular biology techniques
                Sequencing techniques
                DNA sequencing
                Next-Generation Sequencing
                Research and analysis methods
                Molecular biology techniques
                Sequencing techniques
                DNA sequencing
                Next-Generation Sequencing
                Biology and Life Sciences
                Computational Biology
                Genome Analysis
                Transcriptome Analysis
                Next-Generation Sequencing
                Biology and Life Sciences
                Genetics
                Genomics
                Genome Analysis
                Transcriptome Analysis
                Next-Generation Sequencing
                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
                Custom metadata
                Raw NGS sequences are available on NCBI’s Sequence Read Archive (SRA) with the Study number SRP124881. All other relevant data are within the paper and its Supporting Information files.

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