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

RESEARCH ARTICLE Spatial Representativeness of Environmental DNA Metabarcoding Signal for Fish Biodiversity Assessment in a Natural Freshwater System Raphaël Civade1*, Tony Dejean2, Alice Valentini2, Nicolas Roset3, JeanClaude Raymond3, Aurélie Bonin4,5, Pierre Taberlet4,5, Didier Pont1 a11111 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 * OPEN ACCESS Citation: Civade R, Dejean T, Valentini A, Roset N, Raymond J-C, Bonin A, et al. (2016) Spatial Representativeness of Environmental DNA Metabarcoding Signal for Fish Biodiversity Assessment in a Natural Freshwater System. PLoS ONE 11(6): e0157366. doi:10.1371/journal. pone.0157366 Editor: Carlos Garcia de Leaniz, Swansea University, UNITED KINGDOM Received: December 21, 2015 Accepted: May 27, 2016 Published: June 30, 2016 Copyright: © 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. Data Availability Statement: 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. 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. 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 PLOS ONE | DOI:10.1371/journal.pone.0157366 June 30, 2016 1 / 19 Spatial Fish Assemblages Using eDNA Metabarcoding financially supported this work (Contract IRSTEASPYGENONEMA, 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. Competing Interests: The authors have read the journal's policy and the authors of this manuscript have the following competing interests: PT is coinventor 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. Introduction Environmental DNA (eDNA) corresponds to the DNA extracted from an environmental sample such as soil, water or feces without first isolating any target organisms. Total eDNA contains cellular DNA originating from living cells or organisms, and extracellular DNA resulting from natural cell death and subsequent destruction of cell structure [1]. In the last few years, eDNA analysis has drawn the attention of many ecologists as it is non-invasive [2,3], cost-effective [4,5], more sensitive than traditional methods [6], and useful for monitoring and conservation purposes [3,6–10]. Environmental DNA can be used either to detect single invasive or endangered species with species-specific markers, or to describe species diversity for a given taxonomic group using a generalist molecular marker (eDNA metabarcoding [1]). In freshwater environments, the eDNA metabarcoding approach is increasingly adopted both in mesocosm and in situ experiments [5,11–14], and it is particularly interesting for large scale biomonitoring as recommended by recent European directives (e.g. Water Framework Directive, 2000 [15]). Recently, Valentini et al. [13] proposed a new eDNA metabarcoding workflow based on markers targeting fish and amphibians, which were validated in silico and in vitro. Compared with traditional in situ sampling, eDNA analysis was found to be more efficient to assess species richness. However, in addition to the issues inherent to any DNA study such as contaminations or PCR errors, eDNA studies suffer from specific limitations that should be acknowledged and properly assessed [16–18]. This is necessary to ensure reliability of the results and, ultimately, to interpret the eDNA signal detected in natural aquatic ecosystems [16–18]. In mesocosms, eDNA has been shown to remain detectable from a few days to a few weeks after its release in water [19–22]. Nonetheless, mesocosm studies remain imperfect, as it is difficult to model the complexity of natural ecosystems with all the factors potentially involved [16]. For example, eDNA of target species is usually in higher concentrations in mesocosms than in natural ecosystems. In the case of river ecosystems, the distance of detection depends not only on eDNA persistence, but also on the water flow and can therefore be highly variable. For example, (...truncated)