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

Fish are both consumers and prey, and as such part of a dynamic trophic network. Measuring how they are trophically linked, both directly and indirectly, to other species is vital to comprehend the mechanisms driving alterations in fish communities in space and time. Moreover, this knowledge also helps to understand how fish communities respond to environmental change and delivers important information for implementing management of fish stocks. DNA‐based methods have significantly widened our ability to assess trophic interactions in both marine and freshwater systems and they possess a range of advantages over other approaches in diet analysis. In this review we provide an overview of different DNA‐based methods that have been used to assess trophic interactions of fish as consumers and prey. We consider the practicalities and limitations, and emphasize critical aspects when analysing molecular derived trophic data. We exemplify how molecular techniques have been employed to unravel food web interactions involving fish as consumers and prey. In addition to the exciting opportunities DNA‐based approaches offer, we identify current challenges and future prospects for assessing fish food webs where DNA‐based approaches will play an important role.

This publication is based upon work from COST Action DNAqua-Net, supported by COST (European Cooperation in Science and Technology).COST (European Cooperation in Science and Technology) is a funding agency for research and innovation networks.Our Actions help connect research initiatives across Europe and enable scientists to grow their ideas by sharing them with their peers. This boosts their research, career and innovation.

When using the eddy covariance (EC) method for measuring the ecosystem-atmosphere exchange of sensible and latent heat, it is not uncommon to fi nd that these two energy fl uxes fall short of the available energy by 20 to 30%. As the causes for the energy imbalance are s ll under discussion, it is currently not clear how the energy balance should be closed. The objec ve of this study was to use independent measurements of evapotranspira on (ET) for empirically devising how to best close the energy balance. To this end, the ET of a temperate mountain grassland was quan fi ed during two measurement campaigns using both an open-and a closed-path EC system, lysimeters, and an approach scaling up leaf-level stomatal conductance to canopy-level transpira on. Our study showed that both EC systems underes mated ET measured independently by lysimeters and the upscaling approach. The best correspondence with independently measured ET was achieved by assigning the en re energy imbalance to ET and by adjus ng the ET according to the average energy balance ra o during the fi rst and second measurement campaigns, respec vely. Due to a large spaal variability in ET during the fi rst measurement campaign and given large diff erences in spa al scale between the EC and the independent methods, we are more confi dent with the comparison of approaches during the second measurement campaign and thus recommend forcing energy balance closure by adjus ng for the average energy balance ra o.Abbrevia ons: CP, closed path; EC, eddy covariance; ET, evapotranspira on; IRGA, infrared gas analyzer; OP, open path; PAI plant area index.The energy imbalance, that is the widespread shortfall of measured sensible (H) and latent (λE) heat fl uxes with respect to available energy on the order of 20 to 30% (Wilson et al., 2002;Foken, 2008), is the thorn in the side of EC fl ux measurements (Baldocchi et al., 1988;Aubinet et al., 2000). Mathematically, the energy imbalance may be expressed aswhere Res is the residual energy or energy imbalance, R n is the net radiation, and G is the soil heat fl ux. We note that Eq.[1] neglects other forms of heat fl uxes such as biomass storage or metabolic reactions (Meyers and Hollinger, 2004;Oncley et al., 2007;Jacobs et al., 2008).Because the causes of the energy imbalance are still under discussion (for a recent synthesis, see Foken, 2008), EC measurements of ET must be suspected of being biased low-in the most extreme case, the entire energy imbalance may be attributed to the latent heat fl ux . Th e resulting uncertainty not only may be prohibitive for using EC ET measurements for practical applications such as irrigation scheduling (Twine et al., 2000) but poses problems if models, which assume the energy balance to be closed, are to be calibrated with EC fl ux data (Williams et al., 2009). Along this line, Wohlfahrt et al. (2009) recently showed that surface conductance estimates derived by inverting the Penman-Monteith combination equation (Campbell and Norman, 1998) may diff er widely depending on how ener...

The quantitative measurement of environmental DNA (eDNA) from field-collected water samples is gaining importance for the monitoring of fish communities and populations. The interpretation of these signal strengths depends, among other factors, on the amount of target eDNA shed into the water. However, shedding rates are presumably associated with species-specific traits such as physiology and behavior. Although such differences between juvenile and adult fish have been previously detected, the general impact of movement and energy use in a resting state on eDNA release into the surrounding water remains hardly addressed. In an aquarium experiment, we compared eDNA shedding between seven fish species occurring in European freshwaters. The investigated salmonids, cyprinids, and sculpin exhibit distinct adaptions to microhabitats, diets, and either solitary or schooling behavior. The fish were housed in aquaria with constant water flow and their activity was measured by snapshots taken every 30 s. Water samples for eDNA analysis were taken every 3 h and energy use was determined in an intermittent flow respirometer. After controlling for the effect of fish mass, our results demonstrate a positive correlation between target eDNA quantities as measured with digital PCR, fish activity, and energy use, as well as species-specific differences. For cyprinids, the model based on data from individual fish was only partly transferable to groups, which showed lower activity and higher energy use. Our findings highlight the importance of fish physiology and behavior for the comparative interpretation of taxon-specific eDNA quantities. Species traits should therefore be incorporated into eDNA-based monitoring and conservation efforts.