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      Spatial Representativeness of Environmental DNA Metabarcoding Signal for Fish Biodiversity Assessment in a Natural Freshwater System

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          Abstract

          In the last few years, the study of environmental DNA (eDNA) has drawn attention for many reasons, including its advantages for monitoring and conservation purposes. So far, in aquatic environments, most of eDNA research has focused on the detection of single species using species-specific markers. Recently, species inventories based on the analysis of a single generalist marker targeting a larger taxonomic group (eDNA metabarcoding) have proven useful for bony fish and amphibian biodiversity surveys. This approach involves in situ filtering of large volumes of water followed by amplification and sequencing of a short discriminative fragment from the 12S rDNA mitochondrial gene. In this study, we went one step further by investigating the spatial representativeness (i.e. ecological reliability and signal variability in space) of eDNA metabarcoding for large-scale fish biodiversity assessment in a freshwater system including lentic and lotic environments. We tested the ability of this approach to characterize large-scale organization of fish communities along a longitudinal gradient, from a lake to the outflowing river. First, our results confirm that eDNA metabarcoding is more efficient than a single traditional sampling campaign to detect species presence, especially in rivers. Second, the species list obtained using this approach is comparable to the one obtained when cumulating all traditional sampling sessions since 1995 and 1988 for the lake and the river, respectively. In conclusion, eDNA metabarcoding gives a faithful description of local fish biodiversity in the study system, more specifically within a range of a few kilometers along the river in our study conditions, i.e. longer than a traditional fish sampling site.

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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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            ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases

            Background During the last 15 years the internal transcribed spacer (ITS) of nuclear DNA has been used as a target for analyzing fungal diversity in environmental samples, and has recently been selected as the standard marker for fungal DNA barcoding. In this study we explored the potential amplification biases that various commonly utilized ITS primers might introduce during amplification of different parts of the ITS region in samples containing mixed templates ('environmental barcoding'). We performed in silico PCR analyses with commonly used primer combinations using various ITS datasets obtained from public databases as templates. Results Some of the ITS primers, such as ITS1-F, were hampered with a high proportion of mismatches relative to the target sequences, and most of them appeared to introduce taxonomic biases during PCR. Some primers, e.g. ITS1-F, ITS1 and ITS5, were biased towards amplification of basidiomycetes, whereas others, e.g. ITS2, ITS3 and ITS4, were biased towards ascomycetes. The assumed basidiomycete-specific primer ITS4-B only amplified a minor proportion of basidiomycete ITS sequences, even under relaxed PCR conditions. Due to systematic length differences in the ITS2 region as well as the entire ITS, we found that ascomycetes will more easily amplify than basidiomycetes using these regions as targets. This bias can be avoided by using primers amplifying ITS1 only, but this would imply preferential amplification of 'non-dikarya' fungi. Conclusions We conclude that ITS primers have to be selected carefully, especially when used for high-throughput sequencing of environmental samples. We suggest that different primer combinations or different parts of the ITS region should be analyzed in parallel, or that alternative ITS primers should be searched for.
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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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                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
                30 June 2016
                2016
                : 11
                : 6
                : e0157366
                Affiliations
                [1 ]Hydrosystems and Bioprocesses Research unit, IRSTEA, Antony, France
                [2 ]SPYGEN, Le Bourget du Lac, France
                [3 ]Rhône-Alpes Regional Direction, ONEMA, Bron, France
                [4 ]Laboratoire d'Ecologie Alpine (LECA), CNRS, Grenoble, France
                [5 ]Laboratoire d'Ecologie Alpine (LECA), Université Grenoble-Alpes, Grenoble, France
                Swansea University, UNITED KINGDOM
                Author notes

                Competing Interests: The authors have read the journal's policy and the authors of this manuscript have the following competing interests: PT is co-inventor of a patent on "teleo" primers and on the use of the amplified fragment for identifying fish species from environmental samples. This patent only restricts commercial applications and has no impact on the use of this method by academic researchers. AV and TD are research scientists in a private company (SPYGEN), specialized on the use of eDNA for species detection. The private companies EDF and SPYGEN participated at this study, but this does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

                Conceived and designed the experiments: RC AV TD JCR NR DP. Performed the experiments: RC TD JCR NR. Analyzed the data: RC JCR NR DP. Wrote the paper: RC AV TD JCR NR AB PT DP. Provided guidance during the study: AV PT DP. Performed the metabarcoding analysis: RC AV TD.

                Article
                PONE-D-15-54691
                10.1371/journal.pone.0157366
                4928825
                27359116
                703657ec-fd40-4875-9b52-125256b64193
                © 2016 Civade 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
                : 21 December 2015
                : 27 May 2016
                Page count
                Figures: 2, Tables: 7, Pages: 19
                Funding
                In addition to their participation to field work, data collection and preparation of the manuscript, ONEMA (French National Agency for Water and Aquatic Environments) and SPYGEN financially supported this work (Contract IRSTEA-SPYGENONEMA, 2012-2015). EDF (Electricity of France) also supported financially this study. 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
                Lakes
                Ecology and Environmental Sciences
                Aquatic Environments
                Freshwater Environments
                Lakes
                Earth Sciences
                Marine and Aquatic Sciences
                Aquatic Environments
                Freshwater Environments
                Lakes
                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
                Organisms
                Animals
                Vertebrates
                Fishes
                Freshwater Fish
                Earth Sciences
                Hydrology
                Surface Water
                Biology and Life Sciences
                Organisms
                Animals
                Vertebrates
                Fishes
                Osteichthyes
                Trout
                Biology and Life Sciences
                Ecology
                Biodiversity
                Ecology and Environmental Sciences
                Ecology
                Biodiversity
                Earth Sciences
                Hydrology
                Fresh Water
                Research and Analysis Methods
                Database and Informatics Methods
                Biological Databases
                Sequence Databases
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Sequence Databases
                Research and Analysis Methods
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Sequence Databases
                Custom metadata
                Sequences for the reference are available on Dryad doi: 10.5061/dryad.jm58p. All Illumina raw sequences data are available on Dryad doi: 10.5061/dryad.kp6n4. Inventories from traditional sampling sessions are available on http://www.image.eaufrance.fr/poisson/cours/p-ce-resultats.htm.

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