Freshwater fauna are particularly sensitive to environmental change and disturbance. Management agencies frequently use fish and amphibian biodiversity as indicators of ecosystem health and a way to prioritize and assess management strategies. Traditional aquatic bioassessment that relies on capture of organisms via nets, traps and electrofishing gear typically has low detection probabilities for rare species and can injure individuals of protected species. Our objective was to determine whether environmental DNA (eDNA) sampling and metabarcoding analysis can be used to accurately measure species diversity in aquatic assemblages with differing structures. We manipulated the density and relative abundance of eight fish and one amphibian species in replicated 206‐L mesocosms. Environmental DNA was filtered from water samples, and six mitochondrial gene fragments were Illumina‐sequenced to measure species diversity in each mesocosm. Metabarcoding detected all nine species in all treatment replicates. Additionally, we found a modest, but positive relationship between species abundance and sequencing read abundance. Our results illustrate the potential for eDNA sampling and metabarcoding approaches to improve quantification of aquatic species diversity in natural environments and point the way towards using eDNA metabarcoding as an index of macrofaunal species abundance.

The foundation for any ecological study and for the effective management of biodiversity in natural systems requires knowing what species are present in an ecosystem. We assessed fish communities in a stream using two methods, depletion‐based electrofishing and environmental DNA metabarcoding (eDNA) from water samples, to test the hypothesis that eDNA provides an alternative means of determining species richness and species identities for a natural ecosystem. In a northern Indiana stream, electrofishing yielded a direct estimate of 12 species and a mean estimated richness (Chao II estimator) of 16.6 species with a 95% confidence interval from 12.8 to 42.2. eDNA sampling detected an additional four species, congruent with the mean Chao II estimate from electrofishing. This increased detection rate for fish species between methods suggests that eDNA sampling can enhance estimation of fish fauna in flowing waters while having minimal sampling impacts on fish and their habitat. Modern genetic approaches therefore have the potential to transform our ability to build a more complete list of species for ecological investigations and inform management of aquatic ecosystems.

Fisheries conservation requires accurate knowledge of species identities and distributions. Fisheries are typically assessed via capture‐based sampling, but managers frequently are unable to conduct extensive surveys due to budgetary constraints. Sampling of environmental DNA (eDNA) released by fish is a potentially cost‐effective approach that could improve species detection per unit effort. However, eDNA methods have not been widely adopted, in part because the cost and effort of eDNA versus traditional sampling are often unclear to managers. We compared the monetary costs and sampling effort required to assess the distribution of Brook Trout Salvelinus fontinalis in a Wisconsin watershed using both electrofishing and eDNA. We detected Brook Trout via both electrofishing and eDNA. The eDNA analysis required lower sampling effort and 67% less cost than triple‐pass electrofishing. However, eDNA was more expensive than presence–absence electrofishing, and no population structure information was obtained. Our study illustrates the potential of eDNA to complement traditional sampling methods during fish surveys.

Species richness is a metric of biodiversity that represents the number of species present in a community. Traditional fisheries assessments that rely on capture of organisms often underestimate true species richness. Environmental DNA (eDNA) metabarcoding is an alternative tool that infers species richness by collecting and sequencing DNA present in the ecosystem. Our objective was to determine how spatial distribution of samples and "bioinformatic stringency" affected eDNA-metabarcoding estimates of species richness compared with capture-based estimates in a 2.2 ha reservoir. When bioinformatic criteria required species to be detected only in a single sample, eDNA metabarcoding detected all species captured with traditional methods plus an additional 11 noncaptured species. However, when we required species to be detected with multiple markers and in multiple samples, eDNA metabarcoding detected only seven of the captured species. Our analysis of the spatial patterns of species detection indicated that eDNA was distributed relatively homogeneously throughout the reservoir, except near the inflowing stream. We suggest that interpretation of eDNA metabarcoding data must consider the potential effects of water body type, spatial resolution, and bioinformatic stringency.Résumé : La richesse spécifique est une mesure de la biodiversité qui représente le nombre d'espèces présentes dans une communauté. Les évaluations traditionnelles des ressources halieutiques qui reposent sur la capture d'organismes sousestiment souvent la richesse spécifique réelle. Les méta-codes à barres d'ADN environnemental) (ADNe) constituent un autre outil qui permet d'inférer la richesse spécifique en recueillant et en séquençant l'ADN présent dans l'écosystème. Notre objectif consistait à déterminer comment la répartition spatiale des échantillons et la « rigueur bioinformatique » influent sur les estimations de la richesse spécifique reposant sur les méta-codes à barres d'ADNe par rapport aux estimations reposant sur la capture, dans un réservoir de 2,2 ha. Quand les critères bioinformatiques exigeaient la détection d'une espèce dans un seul échantillon, la méthode des méta-codes à barres d'ADNe a détecté toutes les espèces capturées par les méthodes traditionnelles en plus de 11 autres espèces non capturées. Toutefois, quand il fallait que les espèces soient détectées sur la base de plus d'un marqueur et dans plus d'un échantillon, les méta-codes à barres d'ADNe n'ont détecté que sept des espèces capturées. Notre analyse de la répartition spatiale de la détection d'espèces indique que l'ADNe était réparti de manière assez uniforme dans tout le réservoir, sauf près de l'embouchure du cours d'eau qui l'alimente. Nous proposons que l'interprétation des données obtenues par la méthode des méta-codes à barres d'ADNe doit tenir compte des effets potentiels du type de plan d'eau, de la résolution spatiale et de la rigueur bioinformatique. [Traduit par la Rédaction]