The European eel Anguilla anguilla (eel hereafter) is critically endangered and has a catadromous lifecycle, which means adult eels that live in pumped catchments must pass through pumps during their downstream spawning migration. We are currently lacking detailed site-by-site eel distribution information in order to estimate the overall impact of individual pumping stations on eel escapement, and as such lack the data to enable informed prioritisation of pumping station management and targeted mitigation. In this study, we investigated whether environmental DNA (eDNA) metabarcoding can provide increased detection sensitivity for eel and fish community structure in highly regulated pumped catchments, when compared directly to current standard practice fish survey protocols (seine netting/electric fishing). Eels were detected in 14/17 sites (82.4%) using eDNA metabarcoding in contrast to 3/17 (17.6%) using traditional catch methods. Additionally, when using eDNA monitoring species richness was higher in 16/17 sites (94.1%) and site occupancy ≥ traditional methods for 23/26 of the fish species detected (88.5%). While eDNA methods presented significantly higher average species richness and species site occupancy overall, eDNA and Catch methods were positively correlated in terms of species richness and site occupancy. We therefore found that eDNA metabarcoding was a high sensitivity method for detecting eels in pumped catchments, while also increasing the detection of overall fish community structure compared to traditional catch methods. In addition, we highlight how eDNA monitoring is especially suited to increased detection of particular species, with traditional methods sufficient for others. This high sensitivity, coupled with the ability to sample multiple sites in a short time frame suggests eDNA metabarcoding could be an invaluable tool when prioritising pumping station management.

The European eelAnguilla anguilla(eel hereafter) is critically endangered and has a catadromous life cycle, which means adult eels that live in pumped catchments must pass through pumps during their downstream spawning migration. Policy makers are currently lacking detailed site-by-site eel distribution information to estimate the overall impact of individual pumping stations on eel escapement, and as such lack the data to enable informed prioritisation of pumping station management and targeted mitigation. This study investigated whether environmental DNA (eDNA) metabarcoding can provide increased detection sensitivity for eel and fish community structure in highly regulated pumped catchments, when compared directly to current standard practice fish survey protocols (seine netting/electric fishing). Eels were detected in 14 of 17 sites (82.4%) using eDNA metabarcoding in contrast to 3 of 17 sites (17.6%) using traditional catch methods. In addition, when using eDNA monitoring, species richness was higher in 16 of 17 sites (94.1%), and site occupancy was greater than or equal to traditional methods for 23 of 26 of the fish species detected (88.5%). Although eDNA methods presented significantly higher average species richness and species site occupancy overall, eDNA and catch methods were positively correlated in terms of species richness and site occupancy. It was therefore found that eDNA metabarcoding was a high-sensitivity method for detecting eels in pumped catchments while also increasing the detection of overall fish community structure compared to traditional catch methods. In addition, this study highlights how eDNA monitoring is especially suited to increase the detection of particular species, with traditional methods sufficient for others. This high sensitivity, coupled with the ability to sample multiple sites in a short time frame, suggests that eDNA metabarcoding workflows could be invaluable tools when prioritising pumping station management.

In the UK, the native European otter (Lutra lutra) and invasive American mink (Neovison vison) have experienced concurrent declines and expansions. Currently, the otter is recovering from persecution and waterway pollution, whereas the mink is in decline due to population control and probable interspecific interaction with the otter. We explored the potential of DNA metabarcoding for investigating diet and niche partitioning between these mustelids. Otter spraints (n = 171) and mink scats (n = 19) collected from three sites (Malham Tarn, River Hull and River Glaven) in northern and eastern England were screened for vertebrates using high-throughput sequencing. Otter diet mainly comprised aquatic fishes (81.0%) and amphibians (12.7%), whereas mink diet predominantly consisted of terrestrial birds (55.9%) and mammals (39.6%). The mink used a lower proportion (20%) of available prey (n = 40 taxa) than the otter and low niche overlap (0.267) was observed between these mustelids. Prey taxon richness of mink scats was lower than otter spraints and beta diversity of prey communities was driven by taxon turnover (i.e. the otter and mink consumed different prey taxa). Considering otter diet only, prey taxon richness was higher in spraints from the River Hull catchment and beta diversity of prey communities was driven by taxon turnover (i.e. the otter consumed different prey taxa at each site). Studies using morphological faecal analysis may misidentify the predator as well as prey items. Faecal DNA metabarcoding can resolve these issues and provide more accurate and detailed dietary information. When scaled up across multiple habitat types, DNA metabarcoding should greatly improve future understanding of resource use and niche overlap between the otter and mink.

Environmental DNA (eDNA) metabarcoding is a promising tool for rapid, non-invasive biodiversity monitoring. In this study, eDNA metabarcoding is applied to explore the spatial and temporal distribution of eDNA in two ponds following the introduction and removal of two rare fish species. When two rare species were introduced and kept at a fixed location in the ponds, eDNA concentration (i.e., proportional read counts abundance) of the introduced species typically peaked after two days. Thereafter, it gradually declined and stabilised after six days. These findings are supported by the highest community dissimilarity of different sampling positions being observed on the second day after introduction, which then gradually decreased over time. On the sixth day, there was no longer a significant difference in community dissimilarity between sampling days. The introduced species were no longer detected at any sampling positions 48 hrs after removal from the ponds. The eDNA signal and detection probability of the introduced species were strongest near the keepnets, resulting in the highest community variance of different sampling events at this position. Thereafter, the eDNA signal significantly decreased with increasing distance, although the signal increased slightly again at 85 m position away from the keepnets. Collectively, these findings reveal that eDNA distribution in lentic ecosystems is highly localised in space and time, which adds to the growing weight of evidence that eDNA signal provides a good approximation of the presence and distribution of species in ponds. Moreover, eDNA metabarcoding is a powerful tool for detection of rare species alongside more abundant species due to the use of generic PCR primers, and can enable monitoring of spatial and temporal community variance.

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 different applications. Such standardisation will provide more robust, comparable, and ecologically meaningful data to enable effective conservation and management of pond biodiversity.

Early detection is paramount for attempts to remove invasive non-native species (INNS). Traditional methods rely on physical sampling and morphological identification, which can be problematic when species are in low densities and/or are cryptic. The use of environmental DNA (eDNA) as a monitoring tool in freshwater systems is becoming increasingly acceptable and widely used for the detection of single species. Here we demonstrate the development and application of standard PCR primers for the detection of four freshwater invasive species which are high priority for monitoring in the UK and elsewhere: Dreissenid mussels; Dreissena rostriformis bugensis (Andrusov, 1987) and D. polymorpha (Pallas, 1771), and Gammarid shrimps; Dikerogammarus villosus (Sowinsky, 1984) and D. haemobaphes (Eichwald, 1843). We carried out a rigorous validation process for testing the new primers, including DNA detection and degradation rate experiments in mesocosm, and a field comparison with traditional monitoring protocols. We successfully detected all four target species in mesocosms, but success was higher for mussels than shrimps. eDNA from single individuals of both mussel species could be detected within four hours of the start of the experiment. By contrast, shrimp were only consistently detected at higher densities (20 individuals) . In field trials, the two mussel species and D. haemobaphes were detected at all sites where the species are known to be present, and eDNA consistently outperformed traditional kick sampling for species detection. However, D. villosus eDNA was only detected in one of five sites where the species was confirmed by kick sampling. These results demonstrate the applicability of standard PCR for eDNA detection of freshwater invasive species, but also highlight the importance of differences between taxa in terms of the detection ability.