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The coastal tailed frog (Ascaphus truei) is endemic to the Pacific Northwest of North America and is listed as a species of Special Concern under the Canadian Species at Risk Act. Its range is limited to British Columbia where it occurs widely west of the Coast Mountain Ranges extending north almost to the Alaskan Panhandle. The present study focused on surveying within the Cayoosh, Bridge (Shulaps), Seton, Anderson, Carpenter, and Downton Lake drainages. Four years of previous inventory efforts using conventional time-constrained search (TCS) methods detected tailed frog at 23/292 discrete sites (7.9% detection rate) in seven watersheds. Non-invasive environmental DNA (eDNA) methods hold promise for cryptic and low-abundance species detection. We rigorously validated a quantitative real-time polymerase chain reaction (qPCR)-based tool for detecting coastal tailed frog eDNA in water samples. This eASTR4 test is highly specific and sensitive. We applied a two-step targeted eDNA analysis approach on duplicate filtered water samples from a total of 72 sites collected over five days. The first IntegritE-DNA step mitigates false negative results and tests all DNA samples for the ability to support amplification from endogenous plant chloroplast DNA as a measure of sample viability. Three DNA samples failed this step even after inhibitor clean up suggesting that these samples were poor quality and not reliable for targeted species' DNA analyses. All other DNA samples were deemed viable and were then tested for species-specific DNA. Coastal tailed frog eDNA was detected in 55/72 (76%) discrete stream reaches; nine sites with historical known occurrence were all eDNA positive. The false negative rate for TCS compared to eDNA methods was 58%. The results expand known coastal tailed frog distribution to 24 watersheds effectively more than tripling extant occurrences and confirm a previously suspected, apparently isolated coastal tailed frog metapopulation in the Shulaps drainage., Competing Interests: We have read the journal's policy and the authors of this manuscript have the following competing interests: JH is an employee of Hemmera Envirochem Inc. JH was involved with all aspects of the study with the exception of qPCR assay design, validation, data collection, and analysis. A patent is pending on some of the concepts presented in the manuscript (US provisional application number 62/406,802). The patent is owned by the University of Victoria who will handle commercial and academic licensing agreements. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Objective: Environmental DNA (eDNA) detection is a transformative tool for ecological surveys which in many cases offers greater accuracy and cost-effectiveness for tracking low-density, cryptic species compared to conventional methods. For the use of targeted quantitative PCR (qPCR)-based eDNA detection, protocols typically require freshly prepared reagents for each sample, necessitating systematic evaluation of reagent stability within the functional context of eDNA standard curve preparation and environmental sample evaluation. Herein, we assessed the effects of long-term storage and freeze–thaw cycles on qPCR reagents for eDNA analysis across six assays. Results: Results demonstrate qPCR plates (containing pre-made PCR mix, primer-probe, and DNA template) remain stable at 4 °C for three days before thermocycling without fidelity loss irrespective of qPCR assay used. Primer-probe mixes remain stable for five months of − 20 °C storage with monthly freeze-thaw cycles also irrespective of qPCR assay used. Synthetic DNA stocks maintain consistency in standard curves and sensitivity for three months under the same conditions. These findings enhance our comprehension of qPCR reagent stability, facilitating streamlined eDNA workflows by minimizing repetitive reagent preparations. [ABSTRACT FROM AUTHOR]

A current challenge for environmental DNA (eDNA) applications is how to account for an environmental (or false-positive) background in surveys. We performed two controlled experiments in the Goldstream Hatchery in British Columbia using a validated coho salmon (Oncorhynchus kisutch) eDNA assay (eONKI4). In the density experiment at high copy number, eDNA in 2 L water samples was measured from four 10 kL tanks containing 1 to 65 juvenile coho salmon. At these densities, we obtained a strong positive 1:1 relationship between predicted copy number/L and coho salmon biomass (g/L). The dilution experiment simulated a situation where fish leave a pool environment, and water from upstream continues to flow through at rates of 141–159 L/min. Here, three coho salmon were placed in four 10 kL tanks, removed after nine days, and the amount of remaining eDNA was measured at times coinciding with dilutions of 20, 40, 80, 160, and 1000 kL. The dilution experiment demonstrates a novel method using Binomial–Poisson distributions to detect target species eDNA at low copy number in the presence of an environmental background. This includes determination of the limit of blank with background (LOB-B) with a controlled false positive rate, and limit of detection with background (LOD-B) with a controlled false negative rate, which provides a statistically robust "Detect" or "No Detect" assessment for eDNA surveys. [ABSTRACT FROM AUTHOR]

Objective: Silica gel beads have promise as a non-toxic, cost-effective, portable method for storing environmental DNA (eDNA) immobilized on filter membranes. Consequently, many ecological surveys are turning to silica bead filter desiccation rather than ethanol preservation. However, no systematic evaluation of silica bead storage conditions or duration past 1 week has been published. The present study evaluates the quality of filter-immobilized eDNA desiccated with silica gel under different storage conditions for over a year using targeted quantitative real-time polymerase chain reaction (qPCR)-based assays. Results: While the detection of relatively abundant eDNA target was stable over 15 months from either ethanol- or silica gel-preserved filters at − 20 and 4 °C, silica gel out-performed ethanol preservation at 23 °C by preventing a progressive decrease in eDNA sample quality. Silica gel filter desiccation preserved low abundance eDNA equally well up to 1 month regardless of storage temperature (18, 4, or − 20 °C). However only storage at − 20 °C prevented a noticeable decrease in detectability at 5 and 12 months. The results indicate that brief storage of eDNA filters with silica gel beads up to 1 month can be successfully accomplished at a range of temperatures. However, longer-term storage should be at − 20 °C to maximize sample integrity. [ABSTRACT FROM AUTHOR]