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.

Current research targeting filtered macrobial environmental DNA (eDNA) often relies upon cold ambient temperatures at various stages, including the transport of water samples from the field to the laboratory and the storage of water and/or filtered samples in the laboratory. This poses practical limitations for field collections in locations where refrigeration and frozen storage is difficult or where samples must be transported long distances for further processing and screening. This study demonstrates the successful preservation of eDNA at room temperature (20 °C) in two lysis buffers, CTAB and Longmire's, over a 2-week period of time. Moreover, the preserved eDNA samples were seamlessly integrated into a phenol–chloroform–isoamyl alcohol (PCI) DNA extraction protocol. The successful application of the eDNA extraction to multiple filter membrane types suggests the methods evaluated here may be broadly applied in future eDNA research. Our results also suggest that for many kinds of studies recently reported on macrobial eDNA, detection probabilities could have been increased, and at a lower cost, by utilizing the Longmire's preservation buffer with a PCI DNA extraction.

Advances in detection of genetic material from species in aquatic ecosystems, including environmental DNA (eDNA), have improved species monitoring and management. eDNA from target species can readily move in streams and rivers and the goal is to measure it, and with that infer where and how abundant species are, adding great value to delimiting species invasions, monitoring and protecting rare species, and estimating biodiversity. To date, we lack an integrated framework that identifies environmental factors that control eDNA movement in realistic, complex, and heterogeneous flowing waters. To this end, using an empirical approach and a simple conceptual model, we propose a framework of how eDNA is transported, retained, and resuspended in stream systems. Such an understanding of eDNA dispersal in streams will be essential for designing optimized sampling protocols and subsequently estimating biomass or organismal abundance. We also discuss guiding principles for more effective use of eDNA methods, highlighting the necessity of understanding these parameters for use in future predictive modeling of eDNA transport.

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.

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]

While environmental DNA (eDNA) is now being regularly used to detect rare and elusive species, detection in lotic environments comes with a caveat: The species being detected is likely some distance upstream from the point of sampling. Here, we conduct a series of seminatural stream experiments to test the sensitivity of new digital droplet PCR (ddPCR) to detect low concentrations of eDNA in a lotic system, measure the residence time of eDNA compared to a conservative tracer, and we model the transport of eDNA in this system. We found that while ddPCR improves our sensitivity of detection, the residence time and transport of eDNA does not follow the same dynamics as the conservative tracer and necessitates a more stochastic framework for modeling eDNA transport. There was no evidence for differences in the transport of eDNA due to substrate type. The relatively large amount of unexplained variability in eDNA transport reveals the need for uncovering mechanisms and processes by which eDNA is transported downstream leading to species detections, particularly when inferences are to be made in natural systems where eDNA is being used for conservation management.

Environmental DNA (eDNA) metabarcoding can greatly enhance our understanding of global biodiversity and our ability to detect rare or cryptic species. However, sampling effort must be considered when interpreting results from these surveys. We explored how sampling effort influenced biodiversity patterns and nonindigenous species (NIS) detection in an eDNA metabarcoding survey of four commercial ports. Overall, we captured sequences from 18 metazoan phyla with minimal differences in taxonomic coverage between 18 S and COI primer sets. While community dissimilarity patterns were consistent across primers and sampling effort, richness patterns were not, suggesting that richness estimates are extremely sensitive to primer choice and sampling effort. The survey detected 64 potential NIS, with COI identifying more known NIS from port checklists but 18 S identifying more operational taxonomic units shared between three or more ports that represent un-recorded potential NIS. Overall, we conclude that eDNA metabarcoding surveys can reveal global similarity patterns among ports across a broad array of taxa and can also detect potential NIS in these key habitats. However, richness estimates and species assignments require caution. Based on results of this study, we make several recommendations for port eDNA sampling design and suggest several areas for future research.

Abstract1. As environmental DNA (eDNA) from macro-organisms is often assumed to be highly degraded, current eDNA assays target small DNA fragments to estimate species richness by metabarcoding. A limitation of this approach is the inherent lack of unique species-specific single-nucleotide polymorphisms available for unequivocal species identification.2. We designed a novel primer pair capable of amplifying whole mitochondrial genomes and evaluated it in silico for a wide range of ray-finned fishes (Class: Actinopterygii). We tested the primer pair using long-range PCR and Illumina sequencing in vitro on a mock community of fish species assembled from pooling genomic DNA extracted from tissues. In situ we utilized long-range PCR and Illumina sequencing to generate fragments between 16 and 17 kb from eDNA extracted from filtered water samples. Water samples were sourced from a mesocosm experiment and from a natural stream.3. We validated our method in silico for 61 orders of Actinopterygii; we successfully sequenced mitogenomes in vitro from all six species in our mock community. In situ we recovered mitogenomes for all species present in our mesocosms. We additionally recovered mitogenomes from 10 of 12 species caught at the time of water sampling and two species previously only detected from eDNA metabarcoding of short DNA fragments from a natural stream.4. Successful amplification of large fragments (>16 kb) from eDNA demonstrates that not all eDNA is highly degraded. Sequencing whole mitogenomes from filtered water samples will alleviate many problems associated with identification of species from short-fragment PCR amplicon-based methods. K E Y W O R D SActinopterygii, eDNA, mitogenome sequencingThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.