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.

Diet analysis is an important aspect when investigating the ecology of fish‐eating animals and essential for assessing their functional role in food webs across aquatic and terrestrial ecosystems. The identification of fish remains in dietary samples, however, can be time‐consuming and unsatisfying using conventional morphological analysis of prey remains. Here, we present a two‐step multiplex PCR system, comprised of six assays, allowing for rapid, sensitive and specific detection of fish DNA in dietary samples. This approach encompasses 78 fish and lamprey species native to Central European freshwaters and enables the identification of 31 species, six genera, two families, two orders and two fish family clusters. All targeted taxa were successfully amplified from 25 template molecules, and each assay was specific when tested against a wide range of invertebrates and vertebrates inhabiting aquatic environments. The applicability of the multiplex PCR system was evaluated in a feeding trial, wherein it outperformed morphological prey analysis regarding species‐specific prey identification in faeces of Eurasian otters. Additionally, a wide spectrum of fish species was detected in field‐collected faecal samples and regurgitated pellets of Common Kingfishers and Great Cormorants, demonstrating the broad applicability of the approach. In conclusion, this multiplex PCR system provides an efficient, easy to use and cost‐effective tool for assessing the trophic ecology of piscivores in Central Europe. Furthermore, the multiplex PCRs and the primers described therein will be applicable wherever DNA of the targeted fish species needs to be detected at high sensitivity and specificity.

Potamodromous fish are considered important indicators of habitat connectivity in freshwater ecosystems, but they are globally threatened by anthropogenic impacts. Hence, non-invasive techniques are necessary for monitoring during spawning migrations. The use of environmental DNA (eDNA) potentially facilitates these efforts, albeit quantitative examinations of spawning migrations remain so far mostly uncharted. Here, we investigated spawning migrations of Danube bleak, Alburnus mento, and Vimba bream, Vimba vimba, and found a strong correlation between daily visual fish counts and downstream eDNA signals obtained from filtered water samples analysed with digital PCR and end-point PCR coupled with capillary electrophoresis. By accounting for daily discharge fluctuations, it was possible to predict eDNA signal strength from the number of migrating fish: first, the whole spawning reach was taken into account. Second, the model was validated using eDNA signals and fish counts obtained from the upper half of the examined river stretch. Consequently, fish counts and their day-to-day changes could be described via an eDNA-based time series model for the whole migration period. Our findings highlight the capability of eDNA beyond delivering simple presence/absence data towards efficient and informative monitoring of highly dynamic aquatic processes such as spawning migrations of potamodromous fish species.

32 33 1. Environmental DNA (eDNA) analysis utilises trace DNA released by organisms into their 34 environment for species detection and is revolutionising non-invasive species monitoring. The 35 use of this technology requires rigorous validation -from field sampling to interpretation of PCR-36 based results -for meaningful application and interpretation. Assays targeting eDNA released by 37 individual species are typically validated with no predefined criteria to answer specific research 38 questions in one ecosystem. Their general applicability, uncertainties and limitations often 39 remain undetermined. The absence of clear guidelines prevents targeted eDNA assays from 40 being incorporated into species monitoring and policy, thus their establishment will be key for the 41 future implementation of eDNA-based surveys. 42 2. We describe the measures and tests necessary for successful validation of targeted eDNA 43 assays and the associated pitfalls to form the basis of guidelines. A list of 122 variables was 44 compiled and consolidated into a scale to assess the validation status of individual assays. 45These variables were evaluated for 546 published single-species assays. The resulting dataset 46 was used to provide an overview of current validation practices and test the applicability of the 47 (30%) of investigated assays were classified as Level 1 (incomplete), and 15% did not achieve 52 this first level. These assays were characterised by minimal in silico and in vitro testing, but their 53 share in annually published eDNA assays has declined since 2014. The total number of reported 54 variables ranged from 20% to 76% and deviated both between and within levels. 55 4. The meta-analysis demonstrates the suitability of the 5-level validation scale for assessing 56 targeted eDNA assays. It is a user-friendly tool to evaluate previously published assays for future 57 4 research and routine monitoring, while also enabling appropriate interpretation of results. Finally, 58 it provides guidance on validation and reporting standards for newly developed assays. 59 60