Mark A. Renshaw

Environmental Conditions Influence eDNA Persistence in Aquatic Systems

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.

Quantification of mesocosm fish and amphibian species diversity via environmental DNA metabarcoding.

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 room temperature preservation of filtered environmental DNA samples and assimilation into a phenol-chloroform-isoamyl alcohol DNA extraction

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 Longmires, 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 Longmires preservation buffer with a PCI DNA extraction.

Environmental DNA (eDNA) detects the invasive rusty crayfish Orconectes rusticus at low abundances

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.

Estimating species richness using environmental DNA

Abstract 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.

Environmental DNA (eDNA) Detection Probability Is Influenced by Seasonal Activity of Organisms

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.

Environmental DNA (eDNA) detects the invasive crayfishes Orconectes rusticus and Pacifastacus leniusculus in large lakes of North America

We report results of a study that made reciprocal comparisons of environmental DNA (eDNA) assays for two major invasive crayfishes between their disparate invasive ranges in North America. Specifically, we tested for range expansions of the signal crayfish Pacifastacus leniusculus (Dana, 1852) into the Laurentian Great Lakes region known to be invaded by the rusty crayfish Orconectes rusticus (Girard, 1852), as well as for the invasion of O. rusticus into large lakes of California and Nevada, US known to be invaded by P. leniusculus. We compared eDNA detections to historic localities for O. rusticus within the Great Lakes, and to recent sampling for presence/absence and relative abundance of P. leniusculus in California and Nevada via overnight sets of baited traps. We successfully detected O. rusticus eDNA at six sites from the Great Lakes and P. leniusculus from six of seven lakes where it was known to occur in California and Nevada, but did not detect any range expansions by either species across the North American continent. eDNA appears suitable to detect benthic arthropods from exceptionally large lakes, and will likely be useful in applications for monitoring of new biological invasions into these and other freshwater and marine habitats.

A sensitive environmental DNA (eDNA) assay leads to new insights on Ruffe (Gymnocephalus cernua) spread in North America

Detection of invasive species before or soon after they establish in novel environments is critical to prevent widespread ecological and economic impacts. Environmental DNA (eDNA) surveillance and monitoring is an approach to improve early detection efforts. Here we describe a large-scale conservation application of a quantitative polymerase chain reaction assay with a case study for surveillance of a federally listed nuisance species (Ruffe, Gymnocephalus cernua) in the Laurentian Great Lakes. Using current Ruffe distribution data and predictions of future Ruffe spread derived from a recently developed model of ballast-mediated dispersal in US waters of the Great Lakes, we designed an eDNA surveillance study to target Ruffe at the putative leading edge of the invasion. We report a much more advanced invasion front for Ruffe than has been indicated by conventional surveillance methods and we quantify rates of false negative detections (i.e. failure to detect DNA when it is present in a sample). Our results highlight the important role of eDNA surveillance as a sensitive tool to improve early detection efforts for aquatic invasive species and draw attention to the need for an improved understanding of detection errors. Based on axes that reflect the weight of eDNA evidence of species presence and the likelihood of secondary spread, we suggest a two-dimensional conceptual model that management agencies might find useful in considering responses to eDNA detections.

Microsatellite Fragment Analysis Using the ABI P rism ® 377 DNA Sequencer

The ABI PRISM (®) 377 DNA Sequencer is used for a variety of microsatellite-based research. The platform provides researchers with a cost-effective means for high-throughput genotyping, which can be further optimized by multiplexing microsatellite loci or by using a tail-labeling approach to screen large sets of markers. The goals of this chapter are to present a protocol for performing microsatellite-based analyses on the ABI 377 and to provide researchers with information on how to troubleshoot common issues associated with running the ABI 377 sequencers.

Effects of sampling effort on biodiversity patterns estimated from environmental DNA metabarcoding surveys

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.