The crucian carp (Carassius carassius) is one of few fish species associated with small ponds in the UK. These populations contain genetic diversity not found in Europe and are important to conservation efforts for the species which has declined across its range in Europe. Detection and monitoring of extant crucian carp populations are crucial for conservation success. Environmental DNA (eDNA) analysis could be very useful in this respect as a rapid, cost‐efficient monitoring tool. We developed a species‐specific quantitative polymerase chain reaction (qPCR) assay for eDNA surveillance of crucian carp to enable non‐invasive, large‐scale distribution monitoring. We compared fyke netting and eDNA analysis at ponds with (n = 10) and without (n = 10) crucian carp for presence–absence detection. We examined biotic (crucian carp density represented by catch‐per‐unit‐effort [CPUE] estimate) and abiotic influences on eDNA detection probability using a hierarchical occupancy model, and eDNA quantification using a mixed‐effects model. eDNA analysis achieved 90% detection for crucian carp (n = 10), failing in only one pond where presence was known. CPUE estimate and conductivity had positive and negative influences on eDNA detection probability in qPCR replicates respectively. Similarly, conductivity had a negative effect on DNA copy number, whereas copy number increased with CPUE estimate. Our results demonstrate that eDNA analysis could enable detection of crucian carp populations in ponds and benefit ongoing conservation efforts, but imperfect species detection in relation to biotic and abiotic factors and eDNA workflow requires further investigation. Nonetheless, we have established an eDNA framework for the crucian carp as well as sources of imperfect detection which future investigations can build upon.

The European eel Anguilla 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.

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.

24 25 1. The crucian carp (Carassius carassius) is one of few fish species associated with small ponds in the UK. These 26 populations contain genetic diversity not found in Europe and are important to conservation efforts for the 27 species, which has declined across its range. Detection and monitoring of extant crucian carp populations are 28 crucial for conservation success. Environmental DNA (eDNA) analysis could be very useful in this respect as a 29 rapid, cost-efficient monitoring tool.30 2. We developed a species-specific quantitative PCR (qPCR) assay for eDNA surveillance of crucian carp to 31 enable non-invasive, large-scale distribution monitoring. We compared fyke netting and eDNA at ponds with 32 (N = 10) and without (N = 10) crucian carp for presence-absence detection and relative abundance estimation, 33 specifically whether DNA copy number reflected catch-per-unit-effort (CPUE) estimate. We examined biotic 34 and abiotic influences on eDNA detection and quantification, and compared qPCR to standard PCR. Notably, 35 eDNA occurrence and detection probabilities in relation to biotic and abiotic factors were estimated using a 36 hierarchical occupancy model.37 3. eDNA analysis achieved 90% detection for crucian carp (N = 10), failing in only one pond where presence was 38 known. We observed an overall positive trend between DNA copy number and CPUE estimate, but this was not 39 significant. Macrophyte cover decreased the probability of eDNA occurrence at ponds, whereas CPUE and 40 conductivity had positive and negative influences on eDNA detection probability in qPCR replicates 41 respectively. Conductivity also had a negative effect on DNA copy number, but copy number increased with 42 temperature and percentage of macrophyte cover. PCR was comparable to qPCR for species detection and may 43 provide semi-quantitative information.44 4. Our results demonstrate that eDNA could enable detection of crucian carp populations in ponds and benefit 45 ongoing conservation efforts, but imperfect species detection in relation to biotic and abiotic factors and eDNA 46 workflow requires further investigation. Nonetheless, we have established an eDNA framework for crucian carp 47 and sources of imperfect detection which future investigations can build upon.

The European eel (Anguilla anguilla) is a catadromous fish species, with population trends presenting significant declines over the last four decades. These declines throughout their range have resulted in their classification as ‘critically endangered’ by the IUCN. In addition, the European Union has implemented specific legislation surrounding A. anguilla, requiring member states to develop eel management plans [The EC Eel Regulation (1100/2007)]. Aimed to facilitate increased recruitment, these regulations state >40% of historic eel biomass should be allowed safe passage between inland waters and the sea. Cyprus however, applied and were granted an exemption from this, on the basis that there are no rivers on the island of suitable habitat and flow regimes which naturally host A. anguilla (2009/310/EC). Following this decision, recent findings have suggested that historically eels were more widespread in Cyprus than previously recognised. Indeed, a study by Zogaris et al. (2012) indicated that eels are likely the island’s most widespread native species. Cyprus’ freshwater lotic systems are dominated by intermittent rivers and streams, however the natural state and fish populations of these systems are poorly understood. The freshwaters of the island are now heavily fragmented, and with an estimated 108 dams retaining water are host to one of the highest densities of dam reservoirs in Europe. These interruptions to longitudinal connectivity lead to degraded freshwater systems in the lowlands, but despite this the island does have perennial freshwaters, particularly in the western side of the island and at higher elevations. If A. anguilla are indeed present in inland Cyprus, one key deterministic factor on their survival could be access to perennial summer refugia. Here, multiple monitoring methods were applied to build knowledge on present day eel distribution in Cyprus. By increasing knowledge regarding distribution, we can re-evaluate whether conservation initiatives are in fact justified and worthwhile. In 2020 environmental DNA metabarcoding was applied, 130 samples were taken across 26 freshwater sites to provide an up-to-date snapshot of eel distribution. In addition to this, temporal trends were considered based on an island wide fish monitoring programme spanning 2009 - 2019 which predominantly used electric fishing. Overall the results suggest that A. anguilla is widespread in western lowland Cyprus; 11/26 study sites (31/130 samples) tested positive for eel using eDNA metabarcoding, while eels were captured in 61/299 surveys (355 individuals) over the 10 year fish monitoring programme. The trends in eel distribution are highly concordant across methods, although not all sites were monitored with both methods (Fig. 1). These data indicate widespread eel recruitment in lowland freshwaters, but a lack of eels at higher elevation perennial areas. Lowland perennial areas are few and far between, however results here suggest they are abundant in A. anguilla. While higher elevation areas had higher overall freshwater fish species richness suggesting good habitat quality, the lack of the migratory eel in upper reaches indicates that barriers including dam structures may be preventing access for such migratory fish species. Environmental DNA detected eels in intermittent outlet flows of dams, but only in 2/9 reservoirs surveyed. We provide evidence for present day widespread eel recruitment in Cyprus’ inland freshwaters, however highlight the need to increase connectivity to inland perennial systems in order to enhance survival of this critically endangered species at its eastern most range.

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Aim: Despite significant population declines and targeted EU regulations aimed at A. anguilla conservation, little attention has been given to their status at their easternmost range. This study applies wide scale integrated monitoring to uncover the present-day eel distribution in Cyprus inland freshwaters. These are subject to increasing pressures from water supply requirements and dam construction, as seen throughout the Mediterranean. Location: Cyprus. Methods: We applied environmental DNA metabarcoding of water samples to determine A. anguilla distribution in key freshwater catchments. In addition, we present this alongside ten years of electrofishing/netting data. Refuge traps were also deployed to establish the timing of glass eel recruitment. These outputs are used together, alongside knowledge of the overall fish community and barriers to connectivity, to provide eel conservation and policy insights. Results: We confirm the presence of A. anguilla in Cyprus inland freshwaters, with recruitment occurring in March. Eel distribution is restricted to lower elevation areas, and is negatively associated with distance from coast and barriers to connectivity. Many barriers to connectivity are identified, though eels were detected in 2 reservoirs upstream of dams. The overall fish community varies between freshwater habitat types. Main Conclusions: Eels are much more widespread in Cyprus than previously thought, yet mostly restricted to lowland intermittent systems. This makes for a case to reconsider the requirement for eel management plans. Environmental DNA based data collected in 2020 indicate that present-day eel distribution is representative of 10 year survey trends. Suggesting that inland freshwaters may act as an unrealised refuge at A. anguilla's easternmost range. Conservation efforts in Mediterranean freshwaters should focus on improving connectivity, therefore enabling eels to access inland perennial refugia. Thus, mitigating the impact of climate change and the growing number of fragmented artificially intermittent river systems.

Despite significant population declines and targeted European Union regulations aimed at Anguilla anguilla conservation, little attention has been given to their status at their easternmost range. This study applies wide‐scale integrated monitoring to uncover the present‐day eel distribution in Cyprus' inland freshwaters. These are subject to increasing pressures from water supply requirements and dam construction, as seen throughout the Mediterranean. We applied environmental DNA metabarcoding of water samples to determine A. anguilla distribution in key freshwater catchments. In addition, we present this alongside 10 years of electrofishing/netting data. Refuge traps were also deployed to establish the timing of glass eel recruitment. These outputs are used together, alongside knowledge of the overall fish community and barriers to connectivity, to provide eel conservation and policy insights. This study confirm the presence of A. anguilla in Cyprus' inland freshwaters, with recruitment occurring in March. Eel distribution is restricted to lower elevation areas, and is negatively associated with distance from coast and barriers to connectivity. Many barriers to connectivity are identified, though eels were detected in two reservoirs upstream of dams. The overall fish community varies between freshwater habitat types. Eels are much more widespread in Cyprus than previously thought, yet mostly restricted to lowland intermittent systems. These findings make a case to reconsider the requirement for eel management plans. Environmental DNA‐based data collected in 2020 indicate that “present‐day” eel distribution is representative of 10‐year survey trends. Suggesting that inland freshwaters may act as an unrealized refuge at A. anguilla's easternmost range. Conservation efforts in Mediterranean freshwaters should focus on improving connectivity, therefore enabling eels to access inland perennial refugia. Thus, mitigating the impact of climate change and the growing number of fragmented artificially intermittent river systems.