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      High-throughput sequencing and morphology perform equally well for benthic monitoring of marine ecosystems

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          Abstract

          Environmental diversity surveys are crucial for the bioassessment of anthropogenic impacts on marine ecosystems. Traditional benthic monitoring relying on morphotaxonomic inventories of macrofaunal communities is expensive, time-consuming and expertise-demanding. High-throughput sequencing of environmental DNA barcodes (metabarcoding) offers an alternative to describe biological communities. However, whether the metabarcoding approach meets the quality standards of benthic monitoring remains to be tested. Here, we compared morphological and eDNA/RNA-based inventories of metazoans from samples collected at 10 stations around a fish farm in Scotland, including near-cage and distant zones. For each of 5 replicate samples per station, we sequenced the V4 region of the 18S rRNA gene using the Illumina technology. After filtering, we obtained 841,766 metazoan sequences clustered in 163 Operational Taxonomic Units (OTUs). We assigned the OTUs by combining local BLAST searches with phylogenetic analyses. We calculated two commonly used indices: the Infaunal Trophic Index and the AZTI Marine Biotic Index. We found that the molecular data faithfully reflect the morphology-based indices and provides an equivalent assessment of the impact associated with fish farms activities. We advocate that future benthic monitoring should integrate metabarcoding as a rapid and accurate tool for the evaluation of the quality of marine benthic ecosystems.

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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 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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              DNA metabarcoding and the cytochrome c oxidase subunit I marker: not a perfect match.

              DNA metabarcoding enables efficient characterization of species composition in environmental DNA or bulk biodiversity samples, and this approach is making significant and unique contributions in the field of ecology. In metabarcoding of animals, the cytochrome c oxidase subunit I (COI) gene is frequently used as the marker of choice because no other genetic region can be found in taxonomically verified databases with sequences covering so many taxa. However, the accuracy of metabarcoding datasets is dependent on recovery of the targeted taxa using conserved amplification primers. We argue that COI does not contain suitably conserved regions for most amplicon-based metabarcoding applications. Marker selection deserves increased scrutiny and available marker choices should be broadened in order to maximize potential in this exciting field of research. © 2014 The Author(s) Published by the Royal Society. All rights reserved.
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                Author and article information

                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group
                2045-2322
                10 September 2015
                2015
                : 5
                : 13932
                Affiliations
                [1 ]Department of Genetics and Evolution, University of Geneva , Switzerland
                [2 ]IRCAM, UMR 9912, Université Pierre et Marie Curie , Paris, France
                [3 ]ADMM UMR 7144, CNRS, Station Biologique de Roscoff , 29682 Roscoff, France
                [4 ]SAMS, Scottish Marine Institute , Oban, Argyll, UK
                Author notes
                [*]

                Present address: Department of Genetics and Evolution, University of Geneva, Sciences 3, 30, Quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland.

                Article
                srep13932
                10.1038/srep13932
                4564730
                26355099
                3c2e2491-510d-4001-a107-e5a7ae0d80b3
                Copyright © 2015, Macmillan Publishers Limited

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 01 April 2015
                : 12 August 2015
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