Environmental DNA (eDNA) surveillance holds great promise for improving species conservation and management. However, few studies have investigated eDNA dynamics under natural conditions, and interpretations of eDNA surveillance results are clouded by uncertainties about eDNA degradation. We conducted a literature review to assess current understanding of eDNA degradation in aquatic systems and an experiment exploring how environmental conditions can influence eDNA degradation. Previous studies have reported macrobial eDNA persistence ranging from less than 1 day to over 2 weeks, with no attempts to quantify factors affecting degradation. Using a SYBR Green quantitative PCR assay to observe Common Carp ( Cyprinus carpio ) eDNA degradation in laboratory mesocosms, our rate of Common Carp eDNA detection decreased over time. Common Carp eDNA concentration followed a pattern of exponential decay, and observed decay rates exceeded previously published values for aquatic macrobial eDNA. Contrary to our expectations, eDNA degradation rate declined as biochemical oxygen demand, chlorophyll, and total eDNA (i.e., from any organism) concentration increased. Our results help explain the widely divergent, previously published estimates for eDNA degradation. Measurements of local environmental conditions, consideration of environmental influence on eDNA detection, and quantification of local eDNA degradation rates will help interpret future eDNA surveillance results.

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.

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.

Environmental DNA (eDNA) is a sensitive technique for early detection of rare species, including bighead (Hypophthalmichthys nobilis) and silver (Hypophthalmichthys molitrix) carp, which are incipient invaders of the Great Lakes. Since 2009, 2822 samples have been collected from the Great Lakes basin to delimit the extent of Asian carp incursions. Samples collected in the Chicago Area Waterway System and in the western basin of Lake Erie indicate the presence of Asian carp DNA in the Great Lakes. These positive eDNA detections are within 6 and 4 km from where bighead carps were recovered in Lake Calumet, near Lake Michigan (2010), and from Sandusky Bay, Lake Erie (2000), respectively. To implement a Great Lakes surveillance plan for protecting imperiled species and reducing damages from invasive species, federal, state, and provincial agencies will need to cooperatively plan and implement a surveillance program that employs the unique strengths of multiple sampling tools, including eDNA methods.

Summary Early detection is invaluable for the cost‐effective control and eradication of invasive species, yet many traditional sampling techniques are ineffective at the low population abundances found at the onset of the invasion process. Environmental DNA (eDNA) is a promising and sensitive tool for early detection of some invasive species, but its efficacy has not yet been evaluated for many taxonomic groups and habitat types.We evaluated the ability of eDNA to detect the invasive rusty crayfish Orconectes rusticus and to reflect patterns of its relative abundance, in upper Midwest, USA, inland lakes. We paired conventional baited trapping as a measure of crayfish relative abundance with water samples for eDNA, which were analysed in the laboratory with a qPCR assay. We modelled detection probability for O. rusticus eDNA using relative abundance and site characteristics as covariates and also tested the relationship between eDNA copy number and O. rusticus relative abundance.We detected O. rusticus eDNA in all lakes where this species was collected by trapping, down to low relative abundances, as well as in two lakes where trap catch was zero. Detection probability of O. rusticus eDNA was well predicted by relative abundance of this species and lake water clarity. However, there was poor correspondence between eDNA copy number and O. rusticus relative abundance estimated by trap catches. Synthesis and applications. Our study demonstrates a field and laboratory protocol for eDNA monitoring of crayfish invasions, with results of statistical models that provide guidance of sampling effort and detection probabilities for researchers in other regions and systems. We propose eDNA be included as a tool in surveillance for invasive or imperilled crayfishes and other benthic arthropods.

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]

Environmental DNA (eDNA) holds great promise for conservation applications like the monitoring of invasive or imperiled species, yet this emerging technique requires ongoing testing in order to determine the contexts over which it is effective. For example, little research to date has evaluated how seasonality of organism behavior or activity may influence detection probability of eDNA. We applied eDNA to survey for two highly imperiled species endemic to the upper Black Warrior River basin in Alabama, US: the Black Warrior Waterdog (Necturus alabamensis) and the Flattened Musk Turtle (Sternotherus depressus). Importantly, these species have contrasting patterns of seasonal activity, with N. alabamensis more active in the cool season (October-April) and S. depressus more active in the warm season (May-September). We surveyed sites historically occupied by these species across cool and warm seasons over two years with replicated eDNA water samples, which were analyzed in the laboratory using species-specific quantitative PCR (qPCR) assays. We then used occupancy estimation with detection probability modeling to evaluate both the effects of landscape attributes on organism presence and season of sampling on detection probability of eDNA. Importantly, we found that season strongly affected eDNA detection probability for both species, with N. alabamensis having higher eDNA detection probabilities during the cool season and S. depressus have higher eDNA detection probabilities during the warm season. These results illustrate the influence of organismal behavior or activity on eDNA detection in the environment and identify an important role for basic natural history in designing eDNA monitoring programs.