Environmental DNA (eDNA) analysis is a rapid, cost‐effective, non‐invasive biodiversity monitoring tool which utilises DNA left behind in the environment by organisms for species detection. The method is used as a species‐specific survey tool for rare or invasive species across a broad range of ecosystems. Recently, eDNA and “metabarcoding” have been combined to describe whole communities rather than focusing on single target species. However, whether metabarcoding is as sensitive as targeted approaches for rare species detection remains to be evaluated. The great crested newt Triturus cristatus is a flagship pond species of international conservation concern and the first UK species to be routinely monitored using eDNA. We evaluate whether eDNA metabarcoding has comparable sensitivity to targeted real‐time quantitative PCR (qPCR) for T. cristatus detection. Extracted eDNA samples (N = 532) were screened for T. cristatus by qPCR and analysed for all vertebrate species using high‐throughput sequencing technology. With qPCR and a detection threshold of 1 of 12 positive qPCR replicates, newts were detected in 50% of ponds. Detection decreased to 32% when the threshold was increased to 4 of 12 positive qPCR replicates. With metabarcoding, newts were detected in 34% of ponds without a detection threshold, and in 28% of ponds when a threshold (0.028%) was applied. Therefore, qPCR provided greater detection than metabarcoding but metabarcoding detection with no threshold was equivalent to qPCR with a stringent detection threshold. The proportion of T. cristatus sequences in each sample was positively associated with the number of positive qPCR replicates (qPCR score) suggesting eDNA metabarcoding may be indicative of eDNA concentration. eDNA metabarcoding holds enormous potential for holistic biodiversity assessment and routine freshwater monitoring. We advocate this community approach to freshwater monitoring to guide management and conservation, whereby entire communities can be initially surveyed to best inform use of funding and time for species‐specific surveys.

Environmental DNA (eDNA) metabarcoding has revolutionized biomonitoring in both marine and freshwater ecosystems. However, for semi‐aquatic and terrestrial animals, the application of this technique remains relatively untested. We first assess the efficiency of eDNA metabarcoding in detecting semi‐aquatic and terrestrial mammals in natural lotic ecosystems in the UK by comparing sequence data recovered from water and sediment samples to the mammalian communities expected from historical data. Secondly, using occupancy modelling we compared the detection efficiency of eDNA metabarcoding to multiple conventional non‐invasive survey methods (latrine surveys and camera trapping). eDNA metabarcoding detected a large proportion of the expected mammalian community within each area. Common species in the areas were detected at the majority of sites. Several key species of conservation concern in the UK were detected by eDNA sampling in areas where authenticated records do not currently exist, but potential false positives were also identified. Water‐based eDNA metabarcoding provided comparable results to conventional survey methods in per unit of survey effort for three species (water vole, field vole and red deer) using occupancy models. The comparison between survey ‘effort’ to reach a detection probability of ≥.95 revealed that 3–6 water replicates would be equivalent to 3–5 latrine surveys and 5–30 weeks of single camera deployment, depending on the species. Synthesis and applications. eDNA metabarcoding can be used to generate an initial ‘distribution map’ of mammalian diversity at the landscape level. If conducted during times of peak abundance, carefully chosen sampling points along multiple river courses provide a reliable snapshot of the species that are present in a catchment area. In order to fully capture solitary, rare and invasive species, we would currently recommend the use of eDNA metabarcoding alongside other non‐invasive surveying methods (i.e. camera traps) to maximize monitoring efforts.

Environmental DNA (eDNA) analysis is a rapid, non-invasive, cost-efficient biodiversity monitoring tool with enormous potential to inform aquatic conservation and management. Development is ongoing, with strong commercial interest, and new uses are continually being discovered. General applications of eDNA and guidelines for best practice in freshwater systems have been established, but habitat-specific assessments are lacking. Ponds are highly diverse, yet understudied systems that could benefit from eDNA monitoring. However, eDNA applications in ponds and methodological constraints specific to these environments remain unaddressed. Following a stakeholder workshop in 2017, researchers combined knowledge and expertise to review these applications and challenges that must be addressed for the future and consistency of eDNA monitoring in ponds. The greatest challenges for pond eDNA surveys are representative sampling, eDNA capture, and potential PCR inhibition. We provide recommendations for sampling, eDNA capture, inhibition testing, and laboratory practice, which should aid new and ongoing eDNA projects in ponds. If implemented, these recommendations will contribute towards an eventual broad standardisation of eDNA research and practice, with room to tailor workflows for optimal analysis and

The use of environmental DNA (eDNA) analysis for species monitoring requires rigorous validation-from field sampling to the analysis of PCR-based results-for meaningful application and interpretation. Assays targeting eDNA released by individual species are typically validated with no predefined criteria to answer specific research questions in one ecosystem. Hence, the general applicability of assays, as well as associated uncertainties and limitations, often remain undetermined. The absence of clear guidelines for assay validation prevents targeted eDNA assays from being incorporated into species monitoring and policy; thus, their establishment is essential for realizing the potential of eDNA-based surveys. We describe the measures and tests necessary for successful validation of targeted eDNA assays and the associated pitfalls to form the basis of guidelines. A list of 122 variables was compiled, consolidated into 14 thematic blocks (e.g., "in silico analysis"), and arranged on a 5-level validation scale from "incomplete" to "operational" with defined minimum validation criteria for each level. These variables were evaluated for 546 published single-species assays. The resulting dataset was used to provide an overview of current validation practices and test the applicability of the validation scale for future assay rating. Of the 122 variables, 20% to 76% were reported; the majority (30%) of investigated assays were classified as Level 1 (incomplete), and 15% did not achieve this first level. These assays were characterized by minimal in silico and in vitro testing, but their share in annually published eDNA assays has declined since 2014. The meta-analysis demonstrates the suitability of the 5-level validation scale for assessing targeted eDNA assays. It is a user-friendly tool to evaluate previously published assays for future research and routine monitoring, while also enabling the appropriate interpretation of results. Finally, it provides guidance on validation and reporting standards for newly developed assays.