We present a genome assembly from an individual male Gasterosteus aculeatus (three-spined stickleback; Chordata; Actinopteri; Perciformes; Gasterosteidae). The assembly contains two haplotypes with total lengths of 511.14 megabases and 457.35 megabases. Most of haplotype 1 (97.82%) is scaffolded into 22 chromosomal pseudomolecules, including the X and Y sex chromosomes. Haplotype 2 was assembled to scaffold level. The mitochondrial genome has also been assembled, with a length of 16.56 kilobases. This assembly was generated as part of the Darwin Tree of Life project, which produces genomes for eukaryotic species found in Britain and Ireland.

Environmental DNA (eDNA) sequencing from water samples has emerged as a promising and cost-effective approach to collect comprehensive freshwater biodiversity data. However, critics might highlight potential shortcomings, such as the possibility of false positives (detection of absent species) and false negatives (failing to detect a species that is present). Misconceptions and misunderstandings may also stem from the complexity of the scientific approach and technical language, with implications for decision-makers and potentially hindering conservation. In the present article, we propose an analogy of eating a pizza to simplify messaging and increase understanding of detection probability within typical eDNA metabarcoding workflows. The pizza represents a site, slices represent water samples, bites represent PCR replicates, toppings represent species and olives represent a low-abundance species. Overall, the pizza analogy provides a novel, lighthearted and memorable way to communicate complex eDNA workflows to a broad spectrum of biological scientists and practitioners.

Reintroduction of keystone species is considered part of the solution to the current biodiversity crisis. The Eurasian beaver (Castor fiber) is one such species, shaping its habitat by felling trees, building dams and creating wetlands. However, whilst the potential benefits to aquatic biodiversity and ecological functioning have been studied on a local scale, the impacts of beavers on catchment-scale processes such as fish migration remain understudied. Sequencing of environmental DNA (eDNA metabarcoding) from water samples is a cost-effective method to study species distributions across large geographical scales. Here, eDNA samples (n = 426) were collected from 142 sites across Britain's oldest and largest established wild beaver population, located on Tayside, East Scotland and analysed using a vertebrate-specific metabarcoding assay. We combined detection/non-detection data from eDNA results with other environmental and anthropogenic variables to model the effects of beaver eDNA detections on the distribution of three migratory fish species. Using generalised linear models, we found no effects of the current beaver eDNA detections on the distribution of Atlantic salmon or lamprey, but a positive co-occurrence with European eel at the catchment scale. Model outputs also reinforced previous findings on the impact of barriers to migration and other abiotic and biotic factors on fish species, demonstrating the effectiveness of eDNA sampling in rivers for understanding species distributions at a catchment scale. Synthesis and applications. This study provides novel insights into the catchment-scale co-distribution of beavers and migratory fish, and there was no evidence of negative effects on the catchment-scale distribution of migratory fish species. More generally, this study highlights how catchment-wide eDNA monitoring can be applied by environmental managers to aid decision-making and impact assessment of multiple priority species at the catchment scale.

Robust methods to monitor species distributions are vital to ensuring successful conservation strategies, particularly in the case of conservation translocations. Environmental DNA (eDNA) from water samples is a cost-effective method to monitor species distributions without physical capture or disturbance. However, eDNA is vulnerable to long-distance transport depending on the hydrological and environmental characteristics which can lead to spatially false positives and ultimately inaccurate species distributions. Recently, the development of particle transport models has allowed researchers to integrate hydrological and environmental variables to predict how far eDNA will transport from a source point. Here, eDNA samples (n=218) were collected and quantified using digital PCR (dPCR) to study monthly changes in Eurasian beaver ( Castor fiber ) eDNA concentrations downstream of an enclosure which contained 4 - 5 beavers located in Scotland. The shortest eDNA transport distances (< 2 km) were observed in the summer which correlated with the lowest flows and highest temperatures. In contrast, throughout the winter eDNA was consistently detected up to 5.8 km downstream correlating with the highest discharge and lowest temperature. The eDNA transport model reliably reproduced the decrease in eDNA concentrations downstream of the enclosure, however there were challenges surrounding stream-specific decay rates following a confluence. To study localised species distributions, samples should be collected during summer low flow conditions. Conversely, to maximise species detections sampling should be conducted in winter which had the longest eDNA transport and highest detectability. ### Competing Interest Statement The authors have declared no competing interest. University of the Highlands and Islands, https://ror.org/02s08xt61

Abstract Context Pumping stations pose a threat to fish globally through land use change, habitat fragmentation and entrainment risk, with the catadromous and critically endangered European eel particularly impacted. Objectives/methods Establish, model, assess and understand the present-day distribution of European eel and resident fishes in 152 pumping station catchments in a once extensive wetland (The Fens) using eDNA metabarcoding (855 samples over two and half years), with specific focus on anthropogenic influences on hydrological connectivity and habitat quality. A removal survey design maximised confidence in negative results while minimising time and consumable costs. Results Eel occurrence upstream of pumping stations was low (occupancy = 28.3%) and positively associated with catchment area, fish species richness and natural hydrological connectivity (gravity drainage or flooding) and negatively associated with distance from the tidal limit. Fish species richness replaced catchment area and improved model performance, potentially acting as a biotic indicator of habitat quality and connectivity. Pumped catchments with manually operated upstream water transfers had reduced eel presence, potentially linked to the direction of water flow or the timing of operation. By contrast, fish species richness increased in these catchments during summer, suggesting displacement into unsuitable long-term habitats. Physical habitat maintenance had no detectable effect on eel occurrence or fish species richness. Conclusions This study provides the first landscape-scale assessment of European eel distribution and drivers of occurrence in pumped river catchments. The highly novel and comprehensive insights have implications for European eel conservation as well as infrastructure and catchment management, including compliance with legislation (EC Regulation No. 1100/2007).

Environmental DNA (eDNA) analysis enables non-invasive detection of aquatic species, but no established framework integrates eDNA best practices with forensic validation standards. The critically endangered freshwater pearl mussel (Margaritifera margaritifera) exemplifies the need for forensically validated eDNA assays as traditional survey methods are invasive and time-consuming. This research provides a framework that bridges both ecological monitoring standards and forensic legal admissibility requirements in wildlife crime investigations. Here we describe the validation of a multiplex quantitative PCR (qPCR) assay targeting the mitochondrial cytochrome c oxidase subunit I (COI) gene, using the highest forensic standards and eDNA best practice.In silico and in vitro testing confirmed species specificity, with alignment against reference sequences showing 100% identity. The multiplex assay LOD and LOQ were calculated at 2.5 and 3.14 copies/µL respectively, determined by the performance of the short fragment. The assay demonstrated robust repeatability (R², 0.986–1.000), precision (coefficient of variation, 6–28%), and accuracy (recovery, 62–137%), meeting forensic thresholds. Field validation detected a previously undetected Welsh population through intelligence-led sampling, while blind proficiency testing in the Lake District achieved 95.8–100% detection rates across juvenile release sites and successfully detected distribution patterns later confirmed by stakeholders.We establish the first forensic eDNA validation of a protected species, providing a transferable methodology for conservation, regulatory enforcement, and wildlife crime investigations, ready for deployment in an operational environment. We believe that this integrated approach can be transferred to similar forensic and conservation scenarios across a broad range of species and geographical contexts.

Policy‐driven decision‐making is an important aspect of environmental management globally, often focused on protecting priority species. However, declining trends in freshwater biodiversity have resulted in uncertainty regarding the present‐day distribution of rare and elusive species. This is problematic for applied ecologists and environmental managers, since when dealing with priority species, it is generally more challenging to provide a confident assessment of absence than merely confirm their presence. Without such confident assessments, resource‐intensive management plans may be misplaced and not adequately targeted to conserve important remaining populations. We present a framework to estimate confidence in absence, referred to as “Confidence in Absence for Decision‐Making” (CIADM), based on single‐visit environmental DNA metabarcoding data obtained from water samples. It uses a case study of European eel presence/absence upstream of 44 water pumping stations. Through a high degree of biological (sample) and technical (PCR) replication, we retrospectively assigned ‘confidence in absence’ values and proposed various strategies to achieve the required confidence levels in future surveys. Seventeen out of 44 pumping stations tested positive for eel, and we were able to assign a >99% confidence level that the remaining 27 sites were negative for eel DNA at the time of sampling. Increasing both biological and technical replication increased ‘confidence in absence’ values. For example, using three PCR replicates per sample, required four replicate biological samples to achieve >95% and six to achieve >99% confidence in eel absence given non‐detection. However, we estimate that by using seven PCR replicates per sample a >99% confidence in eel absence following non‐detection could be achieved from only three replicate biological samples. Furthermore, we found that eel positive sites had significantly higher species richness, and fish communities differed between eel positive and eel negative sites. Synthesis and applications . This study highlights the importance of optimising workflow specific replication and provides an adaptable and multi‐level framework (CIADM) to produce confidence in priority species absence given non‐detection. Thereby enabling applied ecologists and decision‐makers to collaboratively design surveys and make decisions, such as policy compliance or management interventions to meet conservation objectives while effectively prioritising resources.

We present a genome assembly from an individual Scardinius erythrophthalmus (Rudd; Chordata; Actinopteri; Cypriniformes; Leuciscidae). The assembly contains two haplotypes with total lengths of 1 229.65 megabases and 1 189.97 megabases. Most of haplotype 1 (95.02%) is scaffolded into 25 chromosomal pseudomolecules. Haplotype 2 was assembled to scaffold level. The mitochondrial genome has also been assembled, with a length of 16.61 kilobases. This assembly was generated as part of the Darwin Tree of Life project, which produces reference genomes for eukaryotic species found in Britain and Ireland.

We present a genome assembly from an individual Rutilus rutilus (Roach; Chordata; Actinopteri; Cypriniformes; Leuciscidae). The genome sequence has a total length of 1 100.18 megabases. Most of the assembly (99.56%) is scaffolded into 25 chromosomal pseudomolecules. The mitochondrial genome has also been assembled, with a length of 16.61 kilobases. Gene annotation of this assembly on Ensembl identified 25 358 protein-coding genes. This assembly was generated as part of the Darwin Tree of Life project, which produces reference genomes for eukaryotic species found in Britain and Ireland.

We present a genome assembly from an individual Rutilus rutilus (Roach; Chordata; Actinopteri; Cypriniformes; Leuciscidae). The genome sequence has a total length of 1 100.18 megabases. Most of the assembly (99.56%) is scaffolded into 25 chromosomal pseudomolecules. The mitochondrial genome has also been assembled, with a length of 16.61 kilobases. Gene annotation of this assembly on Ensembl identified 25 358 protein-coding genes. This assembly was generated as part of the Darwin Tree of Life project, which produces reference genomes for eukaryotic species found in Britain and Ireland.

We present a genome assembly from a specimen of Abramis brama (bronze bream; Chordata; Actinopteri; Cypriniformes; Leuciscidae). The genome sequence has a total length of 1 108.20 megabases. Most of the assembly (99.13%) is scaffolded into 25 chromosomal pseudomolecules. The mitochondrial genome has also been assembled, with a length of 16.61 kilobases. Gene annotation of this assembly on Ensembl identified 25 268 protein-coding genes.

We present a genome assembly from a specimen of Abramis brama (bronze bream; Chordata; Actinopteri; Cypriniformes; Leuciscidae). The genome sequence has a total length of 1 108.20 megabases. Most of the assembly (99.13%) is scaffolded into 25 chromosomal pseudomolecules. The mitochondrial genome has also been assembled, with a length of 16.61 kilobases. Gene annotation of this assembly on Ensembl identified 25 268 protein-coding genes.

The European green toad (Bufotes viridis) is currently in decline and considered endangered across the northern extent of its native range, with large investments in ongoing conservation and translocation efforts. To assist conservation efforts, survey methods must be established that are cost-effective, non-invasive, and rapidly deployable. Here we evaluated the effectiveness of eDNA metabarcoding for amphibian conservation across three objectives: (1) Test B. viridis probability of detection before and after translocation efforts in 3 ponds in & Ouml;land, Sweden. (2) Assess pond biodiversity and biotic interactions across & Ouml;land and Kalmar using eDNA metabarcoding. (3) Determine which surveyed sites are suitable for future translocation efforts. We found that the detection probability of B. viridis increased 100% 24 h after the translocation was initiated, whereby they were undetected prior to release. Additionally, we detected 11 fish species, 14 bird species, 9 mammal species, and 4 amphibian species across the translocated sites. The results from the 37 pond eDNA surveys resulted in the detection of 15 fish species, 38 bird species, 8 amphibian species, and 17 mammal species. Species richness of the surveyed ponds ranged from 1 to 24, with an average richness of 8. Co-occurrence analysis found significant associations between several species, including a significant negative association between amphibian occurrence and cattle and gray heron and positive associations with duck and common crane. Multi-Criteria Decision Analysis (MCDA) suggests 6 sites had consistent lower site rankings, indicating them as more favorable locations for future amphibian translocation efforts. Overall, these findings showcase eDNA high-throughput sequencing as a viable means to non-invasively assess European green toads and simultaneously assess wider community dynamics that may help evaluate the sustainability of reintroduced and endemic populations.

Environmental DNA (eDNA) refers to the DNA derived from species and found in environmental samples such as water, air and soil. eDNA has been used in biodiversity and conservation studies but not routinely implemented in forensic investigations. Currently physical surveys are implemented at freshwater pearl mussel, Margaritifera margaritifera, crime scenes which are costly, invasive, reliant on practitioner experience and susceptible to false negative results. The aim of this study was to identify key end-user requirements for a novel eDNA sampling method collected through stakeholder discussion and questionnaire. Stakeholder discussions identified the following key criteria for method development: the end-user group, ease of collection, risk to end-user, speed of collection, cost of collection, and data accuracy. These criteria were used to develop three possible eDNA collection approaches: basic (2L water bottles for bankside collection), intermediate (Whirl-Pak® bags for bankside collection), and advanced (onsite Sterivex™ filtration equipment to use on samples collected by entering the watercourse). To compare the three possible methods in respect to their ease and risk, a questionnaire was disseminated to government officers, police officers, postgraduate students and university staff with expertise in either wildlife crime and/or eDNA approaches (N = 63). The time taken to collect samples together with the associated costs of equipment and analysis time were also calculated. Finally, impact of collection method on data accuracy was assessed through the development of a qPCR assay to measure the sensitivity, specificity and concentrations across two different DNA fragment sizes from hatchery reared populations of M. margaritifera. End-user questionnaire data reveals a preference for the basic and intermediate methods in respect to their ease of use and identified risks. Furthermore, these sampling strategies were quicker and cheaper to use although the advanced method could offer logistical benefits in terms of longer preservation times and reduced laboratory processing time. In respect to impact on data accuracy, the advanced method also showed poor detection rates of eDNA, a surprising result given the popularity of the method in conservation studies. Together, these data show the benefits of a co-developed and usable method for police and conservation first responders to use at wildlife crime investigations concerning M. margaritifera habitat destruction. Further research to develop a robust qPCR assay and assess the usability of the basic and intermediate collection methods on wild populations is recommended to determine the overall effectiveness of the described methods.

Freshwater biodiversity is critically affected by human modifications of terrestrial land use and land cover (LULC). Yet, knowledge of the spatial extent and magnitude of LULC-aquatic biodiversity linkages is still surprisingly limited, impeding the implementation of optimal management strategies. Here, we compiled fish diversity data using environmental DNA (eDNA) sampling across a 160,000-km2 subtropical river catchment in Thailand characterized by exceptional biodiversity yet intense anthropogenic alterations, and attributed fish species richness and community composition to contemporary terrestrial LULC across the catchment. We estimated a spatial range of LULC effects extending up to about 20 km upstream from sampling sites, and explained nearly 60% of the variance in the observed species richness, associated with major LULC categories including croplands, forest, and urban areas. We find that integrating both the spatial range and magnitude of LULC effects is needed to accurately predict fish species richness. Further, projected LULC changes showcase future gains and losses of fish species richness across the river network and offer a scalable basis for riverine biodiversity conservation and land management, allowing for potential mitigation of biodiversity loss in highly diverse yet data-deficient tropical to sub-tropical riverine habitats.

Due to the societal reliance on goods and services provided by river systems and their close proximity to settlements, few modern-day rivers are without significant anthropogenic modifications. The natural river hydrology is often altered as a consequence of pumping water for flood alleviation, retaining water for irrigation, and modifying channels for navigation. In recent years, water pumping stations have been found to have several adverse impacts, including fish mortality (direct and indirect) and habitat fragmentation. More broadly, modern-day river systems face a myriad of anthropogenic flow and channel modifications, with varying impacts on different fish life stages. To manage such risks in line with policy, knowledge of the overall fish community and priority species present is required. It is therefore important to understand the robustness of developing survey strategies across differently managed river systems. This study investigates the seasonal patterns of environmental DNA (eDNA) metabarcoding fish detections from water samples taken across three differently managed river types over a one-year period. We observed some significant seasonal variation in detection rates and fish communities; however, this variation was not consistent among river types. Despite this, we found comparatively poor fish communities upstream of pumping stations all year round, with pumped catchments containing significantly fewer species than the adjacent main river channel and our regional control site. Finally, we highlight that seasonal variation in detectability for the overall fish community may not always reflect that of priority species. In our case, we found favorable European eel (Anguilla anguilla) detection in the summer months across all river types. It is therefore recommended that rather than focusing on overall detectability, policy-driven targeted surveys should be designed with priority species ecology in mind.

We present a genome assembly from an individual female Gobio gobio (the gudgeon; Chordata; Actinopteri; Cypriniformes; Gobionidae). The genome sequence spans 1,460.70 megabases. Most of the assembly is scaffolded into 25 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 16.61 kilobases in length.

Environmental DNA (eDNA) is widely used in biodiversity, conservation, and ecological studies but despite its successes, similar approaches have not yet been regularly applied to assist in wildlife crime investigations. The purpose of this paper is to review current eDNA methods and assess their potential forensic application in freshwater environments considering collection, transport and persistence, analysis, and interpretation, while identifying additional research required to present eDNA evidence in court. An extensive review of the literature suggests that commonly used collection methods can be easily adapted for forensic frameworks providing they address the appropriate investigative questions and take into consideration the uniqueness of the target species, its habitat, and the requirements of the end user. The use of eDNA methods to inform conservationists, monitor biodiversity and impacts of climate change, and detect invasive species and pathogens shows confidence within the scientific community, making the acceptance of these methods by the criminal justice system highly possible. To contextualise the potential application of eDNA on forensic investigations, two test cases are explored involving i) species detection and ii) species localisation. Recommendations for future work within the forensic eDNA discipline include development of suitable standardised collection methods, considered collection strategies, forensically validated assays and publication of procedures and empirical research studies to support implementation within the legal system.

We present a genome assembly from an individual Squalius cephalus (the European chub; Chordata; Actinopteri; Cypriniformes; Cyprinidae). The genome sequence is 1,101.9 megabases in span. Most of the assembly is scaffolded into 25 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 16.61 kilobases in length.

Hybridization plays a pivotal role in evolution, influencing local adaptation and speciation. However, it can also reduce biodiversity, which is especially damaging when native and non-native species meet. Hybridization can threaten native species via competition (with vigorous hybrids), reproductive resource wastage and gene introgression. The latter, in particular, could result in increased fitness in invasive species, decreased fitness of natives and compromise reintroduction or recovery conservation practices. In this study, we use a combination of RAD sequencing and microsatellites for a range-wide sample set of 1366 fish to evaluate the potential for hybridization and introgression between native crucian carp (Carassius carassius) and three non-native taxa (Carassius auratus auratus, Carassius auratus gibelio and Cyprinus carpio) in European water bodies. We found hybridization between native and non-native taxa in 82% of populations with non-natives present, highlighting the potential for substantial ecological impacts from hybrids on crucian carp populations. However, despite such high rates of hybridization, we could find no evidence of introgression between these taxa. The presence of triploid backcrosses in at least two populations suggests that the lack of introgression among these taxa is likely due to meiotic dysfunction in hybrids, leading to the production of polyploid offspring which are unable to reproduce sexually. This result is promising for crucian reintroduction programs, as it implies limited risk to the genetic integrity of source populations. Future research should investigate the reproductive potential of triploid hybrids and the ecological pressures hybrids impose on C. carassius.