ABSTRACT Biodiversity monitoring using environmental DNA (eDNA) metabarcoding has expanded rapidly, providing a noninvasive tool widely adopted by ecologists and stakeholders. However, eDNA surveys are prone to imperfect detection, and non‐detections are often misinterpreted as true absences, a critical issue when monitoring rare or elusive species. Despite its implications for biodiversity assessments, detection uncertainty is rarely quantified in eDNA‐based studies. Occupancy modeling offers a powerful solution to this limitation but remains underused, partly due to a lack of accessible and flexible tools. We developed NeMO (Nested eDNA Metabarcoding Occupancy), a user‐friendly R package for fitting multi‐species occupancy models in a Bayesian framework. NeMO explicitly accounts for the nested structure of eDNA metabarcoding workflows—typically involving multiple replication steps such as field samples and PCR replicates—while accommodating presence/absence or read‐count data. The framework estimates species occupancy, eDNA collection probability, amplification probability, and expected read counts, and allows users to assess the influence of environmental or methodological covariates on each process. Crucially, NeMO helps to rigorously assess detectability and optimize resource allocation in eDNA surveys. It estimates the minimum number of eDNA samples, PCR replicates, and sequencing depth required to reliably detect species when present, thereby guiding study design. We illustrate its utility using a fish biodiversity dataset from the Rhône River (France). NeMO integrates key modeling features into a single streamlined framework, providing researchers and practitioners with an accessible and effective tool to assess detectability and optimize resource allocation in eDNA metabarcoding surveys. Our results highlight the importance of quantifying detection uncertainty, which has major implications for conservation monitoring and for designing cost‐effective and reliable eDNA strategies.

Environmental DNA (eDNA) analysis allows efficient surveys of freshwater species and is being increasingly used. However, because different authors use different methods, eDNA data cannot easily be summarised for a global overview. One promising approach is to standardise methods for long-term monitoring. Standardised eDNA metabarcoding methods have been implemented in France for an elusive group: freshwater bivalves. All French bivalve species can be detected and identified at each sampling site. Here, we analyse this comprehensive dataset (280 sites across all France; 40 species) to investigate detection probabilities, infer species distributions and reassess conservation status, and finally examine community patterns (richness) and their determinants. Our method gave over 95 % probability of detection for most species. Species distributions inferred from eDNA sampling were compared with currently known distributions. Four invasive species and one species of conservation concern were found outside their known range. Others were found in a surprisingly small number of localities, although they are currently thought to be widespread and not of conservation concern. Indeed, the databases currently used to determine conservation status are biased by the inclusion of old data, mainly based on shell records only, and may include misidentified species. The main drivers of species richness were latitude and distance from the spring. Co-occurrence of species was investigated for the first time. The results highlight the valuable data that eDNA analysis can provide when used on a large scale, for monitoring and ecological studies, as well as providing improved species distributions, which are essential for conservation policymaking.

Les inventaires d’espèces dulçaquicoles par analyse de l’ADN environnemental (ADNe) se sont développés depuis une dizaine d’années. Nous présentons ici l’application de cette méthode à l’inventaire des Bivalves d’eau douce de France. De 2015 à 2018, nous avons réalisé plusieurs tests de terrain en France, en choisissant différentes conditions environnementales. Nous avons comparé les résultats des analyses ADNe avec les connaissances sur la répartition des Bivalves à l’échelle de la France métropolitaine, à l’échelle des bassins-versants de la Meuse et du Rhône, et avec des sites ayant fait l’objet d’inventaires poussés, impliquant plusieurs malacologues utilisant les méthodes traditionnelles (observations au bathyscope, plongée hyperbare, tri de sédiment). Nos résultats montrent l’efficacité des analyses d’ADNe pour la détection et la distinction de presque tous les taxons de Bivalves dulçaquicoles de France (à l’exception des différentes espèces de Corbicules et des sous-espèces de la Mulette méridionale Unio mancus Lamarck, 1819). La méthode est commercialisée depuis 2018 et notre base de données comporte fin 2020 plus de 300 sites inventoriés par analyse de l’ADNe. L’analyse de ces résultats montre que certaines espèces peu étudiées, en particulier des espèces des écosystèmes de l’aval, souffrent d’extinctions locales en France. Ils plaident en faveur de la mise en œuvre d’actions de conservation en particulier pour la Cyclade des fleuves Sphaerium solidum (Normand, 1844), la Grande Cyclade Sphaerium rivicola (Lamarck, 1818) et l’Anodonte comprimée Pseudanodonta complanata (Rossmässler, 1835).

The analysis of environmental DNA (eDNA) allows efficient surveys of freshwater species and is being increasingly used. However, most studies generally have a limited sampling plan. Because different methods are used by different authors, all the data produced with eDNA cannot be easily pooled together for a global overview. Nevertheless, one of the promising perspectives is the standardization of the methods and protocols, for long-term monitoring, early detection of invasive species, and rare species detection. We here present the biggest dataset using eDNA metabarcoding with standardized methods for freshwater bivalves. Sampling was performed mainly in France, with over 350 localities spread over all major basins. Metabarcoding was performed using two new sets of primers, one for the Unionida and one for the Venerida. The species distributions inferred from eDNA sampling are then compared with those currently known. Results give an insight into the valuable data that eDNA analysis can provide if used at a large scale. Some species were found outside of their known range, especially invasive species but also species of conservation interest. Others were found in a surprisingly low number of localities, although currently considered widespread and of no conservation interest. This can be explained by the databases currently used to establish conservation status, which are often biased by including ancient data, shell-only data, and potentially misidentified species. The present study shows that our eDNA metabarcoding approach will be an essential tool to unveil the true species distributions, to better evaluate their status and improve conservation policymaking.

As fish communities are a major concern in rivers ecosystems, we investigated if their environmental (e)DNA signals vary according to the sampling period or hydromorphological conditions. Three rivers were studied over a year using eDNA metabarcoding approach. The majority of the species (c. 80%) were detected all year round in two rivers having similar hydromorphological conditions, whereas in the river affected by an upstream lake waterflow, more species were detected sporadically (42%). For all the rivers, in more than 98% of the occasional detections, the reads abundance represented <0.4% of the total reads per site and per sampling session. Even if the majority of the fish communities remained similar over the year for each of the three rivers, specific seasonal patterns were observed. We studied if the waterflow or the reproduction period had an effect on the observed dynamics. Waterflow, which influences eDNA downstream transportation, had a global influence in taxonomic richness, while the fishes' reproductive period had only an influence on certain species. Our results may help selecting the best sampling strategy according to research objectives. To study fish communities at local scale, seasons of low waterflow periods are recommended. This particularly helps to restraint effects of external eDNA coming from connections with other aquatic environment (tributaries, lakes, wetlands, sewage effluents, etc.). To obtain a more integrative overview of the fish community living in a river basin, high waterflow or breeding seasons are preferable for enhancing species detection probability, especially for rare species.

Despite the ecological and societal importance of large rivers, fish sampling remains costly and limited to specific habitats (e.g., river banks). Using an eDNA metabarcoding approach, we regularly sampled 500 km of a large river (Rhône River). Comparisons with long-term electrofishing surveys demonstrated the ability of eDNA metabarcoding to qualitatively and quantitatively reveal fish assemblage structures (relative species abundance) but eDNA integrated a larger space than the classical sampling location. Combination of a literature review and field data showed that eDNA behaves in the water column like fine particulate organic matter. Its detection distance varied from a few km in a small stream to more than 100 km in a large river. To our knowledge, our results are the first demonstration of the capacity of eDNA metabarcoding to describe longitudinal fish assemblage patterns in a large river, and metabarcoding appears to be a reliable, cost-effective method for future monitoring.

Global biodiversity in freshwater and the oceans is declining at high rates. Reliable tools for assessing and monitoring aquatic biodiversity, especially for rare and secretive species, are important for efficient and timely management. Recent advances in DNA sequencing have provided a new tool for species detection from DNA present in the environment. In this study, we tested whether an environmental DNA ( eDNA ) metabarcoding approach, using water samples, can be used for addressing significant questions in ecology and conservation. Two key aquatic vertebrate groups were targeted: amphibians and bony fish. The reliability of this method was cautiously validated in silico, in vitro and in situ. When compared with traditional surveys or historical data, eDNA metabarcoding showed a much better detection probability overall. For amphibians, the detection probability with eDNA metabarcoding was 0.97 ( CI = 0.90–0.99) vs. 0.58 ( CI = 0.50–0.63) for traditional surveys. For fish, in 89% of the studied sites, the number of taxa detected using the eDNA metabarcoding approach was higher or identical to the number detected using traditional methods. We argue that the proposed DNA ‐based approach has the potential to become the next‐generation tool for ecological studies and standardized biodiversity monitoring in a wide range of aquatic ecosystems.

In the last few years, the study of environmental DNA (eDNA) has drawn attention for many reasons, including its advantages for monitoring and conservation purposes. So far, in aquatic environments, most of eDNA research has focused on the detection of single species using species-specific markers. Recently, species inventories based on the analysis of a single generalist marker targeting a larger taxonomic group (eDNA metabarcoding) have proven useful for bony fish and amphibian biodiversity surveys. This approach involves in situ filtering of large volumes of water followed by amplification and sequencing of a short discriminative fragment from the 12S rDNA mitochondrial gene. In this study, we went one step further by investigating the spatial representativeness (i.e. ecological reliability and signal variability in space) of eDNA metabarcoding for large-scale fish biodiversity assessment in a freshwater system including lentic and lotic environments. We tested the ability of this approach to characterize large-scale organization of fish communities along a longitudinal gradient, from a lake to the outflowing river. First, our results confirm that eDNA metabarcoding is more efficient than a single traditional sampling campaign to detect species presence, especially in rivers. Second, the species list obtained using this approach is comparable to the one obtained when cumulating all traditional sampling sessions since 1995 and 1988 for the lake and the river, respectively. In conclusion, eDNA metabarcoding gives a faithful description of local fish biodiversity in the study system, more specifically within a range of a few kilometers along the river in our study conditions, i.e. longer than a traditional fish sampling site.

Summary Data‐rich restoration experiments offer opportunities to test the ability of bioassessment tools, such as those currently used to assess the ‘ecological status’ of waterbodies targeted by the European Water Framework Directive, to detect observed ecological changes. Minimum flow increases in four regulated reaches of the French Rhône River modified the invertebrate and fish communities in a predictable way, as detailed in other articles of this Special Issue. We tested the ability of several fish and macroinvertebrate metrics currently used in bioassessment to detect these changes. In addition, we considered changes in metrics that are expected to respond specifically to flow increase. These metrics were related to the habitat requirements of species, the ecological specialisation of communities and the abundance of macroinvertebrate functional groups (seen as surrogates for ecosystem attributes). For invertebrate communities, bioassessment metrics based on richness had equivocal responses to restoration and the Potamon‐Type Index demonstrated no or contradictory responses to restoration. The French biotic index was not sensitive to restoration and instead depicted spatial differences in biological quality. For fish communities, the French fish index was marginally sensitive in the reach with the largest minimum flow increase and some of its metrics were sensitive in other reaches. Contrasting with commonly used bioassessment indices and metrics, several metrics related to habitat requirements appropriately indicated the observed changes in community structure. Large flow changes increased the proportion of fish and macroinvertebrate individuals with preferences for midstream habitats, fast currents, deep waters and/or coarse substrates. However, these changes did not translate into the expected increase in ecological specialisation. In addition, functional metrics indicated that restoration led to higher proportions of grazers and higher availability of suspended food for filtering collectors, suggesting a return to the ecological conditions of a large river. The mixed and potentially contradictory responses of the different metrics confirm the difficulty of establishing benchmarks for ecological indicators in large‐regulated rivers and the need to design appropriate bioassessment metrics.

Freshwater is a basic need for the mankind. Effective biological tools (ecologically based, efficient, rapid and consistently applicable to different ecological regions) are needed to measure the "health" of rivers. Adapting such tools over a broad geographic area requires a detailed understanding of both the patterns of organisms assemblage composition and distribution within and among water bodies under natural conditions, and the nature of the major environmental gradients that cause or explain these patterns. A comprehensive review of the available litterature dealing with the identification of environmental factors structuring riverine fish assemblages under natural conditions permits to identify the most consistent ones.