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The terminal reservoirs of water transfer projects directly supply water for domestic, agricultural, and industrial applications, and the water quality of these reservoirs produce crucial effects on the achievement of project targets. Typically, fish assemblages are monitored as indicators of reservoir water quality, and can also be regulated for its improvement. In the present study, we compared traditional fish landing (TFL) and environmental DNA (eDNA) metabarcoding methods for monitoring fish assemblages in three terminal reservoirs of the East Route of the South-to-North Water Transfer Project, China. Results of TFL and eDNA showed similar assemblage structures and patterns of diversity and spatial distribution with obvious differences in fish composition across three examined reservoirs. Demersal and small fish were dominant in all reservoirs. In addition, a strong association between water transfer distance and assemblages and distribution of non-native fish was found. Our findings highlight the necessity of the fish assemblage monitoring and managing for water quality and revealed the impact of water diversion distance on the structure of fish assemblages and dispersal of alien species along the water transfer project.

The implementation of DNA metabarcoding and environmental DNA (eDNA) to the biodiversity assessment and biomonitoring of aquatic ecosystems has great potential worldwide. However, DNA metabarcoding and eDNA are highly reliant on the coverage of the DNA barcode reference libraries that are currently hindered by the substantial lack of reference sequences. The main objective of this study was to analyze the current coverage of DNA barcode reference libraries for phytoplankton species of the aquatic Mediterranean ecoregion in the southeast of Italy (Apulia Region) in order to assess the applicability of DNA metabarcoding and eDNA in this area. To do so, we investigated three main DNA barcode reference libraries, BOLD Systems, GenBank and SILVA, for the availability of DNA barcodes of the examined phytoplankton species. The gap analysis was conducted for three molecular gene markers, 18S, 16S and COI. The results showed a considerable lack of barcodes for all three markers. However, among the three markers, 18S had a greater coverage in the reference libraries. For the 18S gene marker, the barcode coverage gap across the three types of ecosystems examined was 32.21–39.68%, 60.12–65.19% for the 16S marker gene, and 72.44–80.61 for the COI marker gene. Afterwards, the interspecific genetic distance examined on the most represented molecular marker, 18S, was able to distinguish 80% of the species mined for lakes and 70% for both marine and transitional waters. Conclusively, this work highlights the importance of filling the gaps in the reference libraries, and constitutes the basis towards the advancement of DNA metabarcoding and eDNA application for biodiversity assessment and biomonitoring.

Background The use of environmental DNA analysis has revolutionized biodiversity monitoring. Initially, eDNA monitoring surveys in aquatic environments utilized a targeted approach, but there has been a steady shift toward whole community assessments (eDNA metabarcoding). Both approaches can increase the detection sensitivity for rare and elusive species, compared to more conventional methods. However, it is important to understand the benefits and limitations of targeted and whole community eDNA monitoring to tailor surveys to research questions and management objectives. Aims Here, we aimed to test the relative merits of targeted eDNA analysis versus eDNA metabarcoding in an intermittent river system. Methods First, samples collected during different seasons were used to assess the influence of seasonality on the detection probabilities of both methods. Second, detection probabilities from the two monitoring approaches for one focal species were compared to evaluate the sensitivity of both methods. Finally, the data from an eDNA metabarcoding survey conducted across the outer distribution limits of an invasive species were used to evaluate whether species interactions can be inferred by this method. Results Analyses showed that sampling intermittent river systems during low flow events increases the performance of the targeted eDNA surveys, while sampling season does not influence the performance of eDNA metabarcoding surveys. Environmental DNA metabarcoding was found to be less sensitive than a targeted monitoring approach, thus making the latter more suitable for generating detailed distribution data. Nevertheless, eDNA metabarcoding survey data can be interpreted in a semiquantitative manner and can provide insights into biological interactions. A comparison of targeted eDNA monitoring and eDNA metabarcoding showed a higher detection sensitivity for targeted surveys. Environment DNA metabarcoding surveys can, however, be used to monitor species interaction.

IntroductionMangroves play a vital role in stabilizing coastlines, supporting biodiversity, and delivering essential ecosystem services.MethodsTo assess animal biodiversity in Avicennia marina (grey mangrove) habitats along Egypt’s Red Sea governorate’s coast, seawater samples were collected for environmental DNA (eDNA) analysis from six key mangrove sites: 17 km and 40 km south of Safaga City, El-Sharm El-Bahary, Wadi El-Gemal, El-Qolaan, and Hamata. For comparison, eDNA was also obtained from two non-mangrove sites in the same region. The eDNA was analyzed via COI gene metabarcoding, and diversity metrics were used to compare habitats.ResultsThirteen fish species were identified as exclusive to mangroves, alongside diverse arthropods, poriferans, and cnidarians. Moolgarda sp. (mullets) and Psettodes erumei (Indian halibut) were the most abundant fish species detected. Relative read abundances differed significantly between mangrove and non-mangrove sites, with some species exhibiting dual habitat use (e.g., transitioning between coral and mangrove systems). Diversity and evenness indices showed that biodiversity in mangrove habitats, especially Wadi El-Gemal location, overtook that in non-mangrove locations. Notably, DNA from two marine alien species could be identified, i.e., Anthopleura fuscoviridis (Cnidaria) in a mangrove site, and Callinectes sapidus (Decapoda) in non-mangrove sites. The presence of C. sapidus could be verified by conventional PCR.DiscussionThis study underscores the high biodiversity supported by Red Sea mangroves, emphasizing their ecological and economic value. These findings can inform sustainable management strategies to protect these critical ecosystems for future generations.

Environmental DNA (eDNA) has the potential to provide more comprehensive biodiversity assessments, particularly for vertebrates in species-rich regions. However, this method requires the completeness of a reference database (i.e. a list of DNA sequences attached to each species), which is not currently achieved for many taxa and ecosystems. As an alternative, a range of operational taxonomic units (OTUs) can be extracted from eDNA metabarcoding. However, the extent to which the diversity of OTUs provided by a limited eDNA sampling effort can predict regional species diversity is unknown. Here, by modelling OTU accumulation curves of eDNA seawater samples across the Coral Triangle, we obtained an asymptote reaching 1531 fish OTUs, while 1611 fish species are recorded in the region. We also accurately predict ( R ² = 0.92) the distribution of species richness among fish families from OTU-based asymptotes. Thus, the multi-model framework of OTU accumulation curves extends the use of eDNA metabarcoding in ecology, biogeography and conservation.

Marine biodiversity is threatened by human activities. To understand the changes happening in aquatic ecosystems and to inform management, detailed, synoptic monitoring of biodiversity across large spatial extents is needed. Such monitoring is challenging due to the time, cost, and specialized skills that this typically requires. In an unprecedented study, we combined citizen science with eDNA metabarcoding to map coastal fish biodiversity at a national scale. We engaged 360 citizen scientists to collect filtered seawater samples from 100 sites across Denmark over two seasons (1 p.m. on September 29th 2019 and May 10th 2020), and by sampling at nearly the exact same time across all 100 sites, we obtained an overview of fish biodiversity largely unaffected by temporal variation. This would have been logistically impossible for the involved scientists without the help of volunteers. We obtained a high return rate of 94% of the samples, and a total richness of 52 fish species, representing approximately 80% of coastal Danish fish species and approximately 25% of all Danish marine fish species. We retrieved distribution patterns matching known occurrence for both invasive, endangered, and cryptic species, and detected seasonal variation in accordance with known phenology. Dissimilarity of eDNA community compositions increased with distance between sites. Importantly, comparing our eDNA data with National Fish Atlas data (the latter compiled from a century of observations) we found positive correlation between species richness values and a congruent pattern of community compositions. These findings support the use of eDNA-based citizen science to detect patterns in biodiversity, and our approach is readily scalable to other countries, or even regional and global scales. We argue that future large-scale biomonitoring will benefit from using citizen science combined with emerging eDNA technology, and that such an approach will be important for data-driven biodiversity management and conservation.

Human activities have significantly modified habitats, resulting in a global biodiversity crisis. In this study, we leveraged the first national‐scale biodiversity survey based on airborne environmental DNA, comparing the effects of three human pressure indices increasing in complexity and scope – a binary urban–rural index, an index integrating land cover and pollutant concentrations as a proxy of human activity, and the composite human footprint index – across mammals, birds, insects, plants and fungi. While most taxa exhibited higher diversity in urban areas compared to rural ones, we uncovered more complex patterns using the landscape‐pollution and human footprint indices, including dual diversity maxima at both high and moderate levels of human pressure. We also show an effect of human pressure on community composition even when local species richness remained stable: regardless of the human pressure index, anthropogenic sites were mostly characterized by synanthropic and invasive species. Overall, our results underscore the complex interactions among anthropogenic pressures, taxon diversity and community composition, demonstrating the value of multi‐taxon analyses and multiple indices to better understand biodiversity patterns at large scales.

Invertebrate richness and community composition are key indicators of aquatic ecosystem health. Compared with morphotaxonomy‐based survey methods, DNA metabarcoding is a highly efficient and scalable biomonitoring approach that has become increasingly used in invertebrate surveys. However, the accuracy and efficacy of this approach depend on the performance of universal primers; yet systematic comparative assessments of universal primer performance are lacking. Here, we evaluated the performance of 18 commonly used invertebrate metabarcoding primer pairs targeting the mitochondrial COI and nuclear 18S rRNA genes. We compared the taxonomic specificity, coverage and resolution of the primers via in silico and in vitro assessments using environmental DNA (eDNA) samples from both natural freshwater (rivers and lakes) and marine coastal habitats. We also assessed the effects of sequence reference databases (e.g. GenBank, BOLD, SILVA) on taxonomic assignments. The primers were highly variable in their specificity both in silico and in vitro. The in silico results showed that invertebrate sequences accounted for greater proportions of the total sequences amplified using the COI primers compared with the 18S primers; similar results were obtained in the in vitro assessments, but the proportions of invertebrate sequences were lower. Each primer pair detected 91–1524 invertebrate taxa from freshwater eDNA and 74–624 from marine eDNA and the taxonomic resolution was limited. The COI primers recovered substantially greater taxonomic richness than the 18S primers, but additional phyla were detected by certain 18S primers. The COI primers also preferentially recovered specific invertebrate orders such as bioindicator insects. We provide recommendations for primer and sequence database selection for the metabarcoding of invertebrate biodiversity. Overall, our findings have implications for improving the efficacy of (e)DNA‐based invertebrate surveillance and ecosystem bioassessments. Our results also highlight the gaps within public invertebrate sequence databases.

Because significant global changes are currently underway in the Arctic, creating a large‐scale standardized database for Arctic marine biodiversity is particularly pressing. This study evaluates the potential of aquatic environmental DNA (eDNA) metabarcoding to detect Arctic coastal biodiversity changes and characterizes the local spatio‐temporal distribution of eDNA in two locations. We extracted and amplified eDNA using two COI primer pairs from ~80 water samples that were collected across two Canadian Arctic ports, Churchill and Iqaluit, based on optimized sampling and preservation methods for remote regions surveys. Results demonstrate that aquatic eDNA surveys have the potential to document large‐scale Arctic biodiversity change by providing a rapid overview of coastal metazoan biodiversity, detecting nonindigenous species, and allowing sampling in both open water and under the ice cover by local northern‐based communities. We show that DNA sequences of ~50% of known Canadian Arctic species and potential invaders are currently present in public databases. A similar proportion of operational taxonomic units was identified at the species level with eDNA metabarcoding, for a total of 181 species identified at both sites. Despite the cold and well‐mixed coastal environment, species composition was vertically heterogeneous, in part due to river inflow in the estuarine ecosystem, and differed between the water column and tide pools. Thus, COI‐based eDNA metabarcoding may quickly improve large‐scale Arctic biomonitoring using eDNA, but we caution that aquatic eDNA sampling needs to be standardized over space and time to accurately evaluate community structure changes. Despite the cold and well‐mixed coastal environment, eDNA composition was vertically heterogeneous, in part due to eDNA river inflow in the estuarine ecosystem, and differed between water column and tide pools. eDNA metabarcoding may quickly improve large‐scale Arctic biomonitoring, but we caution that water eDNA biomonitoring needs to be standardized over space and time to accurately evaluate community structure changes.