eDNA metabarcoding is an effective method for studying fish communities but allows only an estimation of relative species abundance (density / biomass). Here, we combine metabarcoding with an estimation of the total abundance of eDNA amplified by our universal marker (teleo) using a qPCR approach to infer the absolute abundance of fish species. We carried out a 2850 km eDNA survey within the Danube catchment using a spatial integrative sampling protocol coupled with traditional electrofishing for fish biomass and density estimation. Total fish eDNA concentrations and total fish abundance were highly correlated. The correlation between eDNA concentrations per taxon and absolute specific abundance was of comparable strength when all sites were pooled and remained significant when the sites were considered separately. Furthermore, a non-linear mixed model showed that species richness was underestimated when the amount of teleo-DNA extracted from a sample was below a threshold of 0.65.106 copies of eDNA. This result, combined with the decrease in teleo-DNA concentration by several orders of magnitude with river size, highlights the need to increase sampling effort in large rivers. Our results show a comprehensive description of longitudinal changes in fish communities and underline our combined metabarcoding/qPCR approach for biomonitoring and bioassessment surveys when a rough estimate of absolute species abundance is sufficient.

Human activities can degrade the quality of coral reefs, cause a decline in fish species richness and functional diversity and an erosion of the ecosystem services provided. Environmental DNA metabarcoding (eDNA) has been proposed as an alternative to Underwater Visual Census (UVC) to offer more rapid assessment of marine biodiversity to meet management demands for ecosystem health indices. Taxonomic information derived from sequenced eDNA can be combined with functional traits and phylogenetic positions to generate a variety of ecological indices describing ecosystem functioning. Here, we inventoried reef fish assemblages of two contrasted coastal areas of Curaçao, (i) in close proximity to the island’s capital city and (ii) in a more remote area under more limited anthropogenic pressure. We sampled eDNA by filtering large volumes of sea water (2 x 30L) along 2km boat transects, which we coupled with species ecological properties related to habitat use, trophic level and body size to investigate the difference in fish taxonomic composition, functional and phylogenetic indices recovered from eDNA metabarcoding between these two distinct coastal areas. Despite no marked difference in species richness, we found a higher phylogenetic diversity in proximity to the city, but a higher functional diversity on the more isolated reef. Composition differences between coastal areas were associated with different frequencies of reef fish families. Because of a partial reference database, eDNA only partly matched those detected with UVC, but eDNA surveys nevertheless provided rapid and robust species occupancy responses to contrasted environments. eDNA metabarcoding coupled with functional and phylogenetic diversity assessment can serve the management of coastal habitats under increasing threat from global changes.

Environmental DNA (eDNA) metabarcoding has emerged as one of the most efficient method to assess aquatic species presence. While the method could in theory be used to investigate non-aquatic fauna, its development for inventorying semi-aquatic and terrestrial fauna is still at its early stages. Here we aimed at investigating the reliability of aquatic eDNA metabarcoding for inventorying mammals in Neotropical environments, be they aquatic, semi-aquatic or terrestrial. We collected aquatic eDNA in 96 sites distributed along three Guianese watersheds and compared our inventories to expected species distributions and field observations derived from line transect samples. Species occurrences and emblematic mammals’ richness patterns were consistent with the expected distribution of the fauna and our results revealed that aquatic eDNA metabarcoding brings additional data to line transect samples for diurnal non-aquatic (terrestrial and arboreal) species. eDNA also provided data on species not detectable in line transect surveys such as semi-aquatic, aquatic and nocturnal terrestrial and arboreal species. While wise application of the eDNA method to inventory mammals still needs some developments to optimize sampling efficiency, it can now be used as a complement to traditional surveys.

Environmental DNA and metabarcoding have great potential for the biomonitoring of freshwater environments. However, successful application of metabarcoding to biodiversity monitoring requires universal primers with high taxonomic coverage that amplify highly-variable, short metabarcodes with high taxonomic resolution. Moreover, reliable and extensive reference databases are essential to match the outcome of metabarcoding analyses with the available taxonomy and biomonitoring indices. Benthic invertebrates, particularly insects, are key taxa for freshwater biomonitoring. Nevertheless, so far, no formal comparison has assessed primers for metabarcoding of freshwater macrobenthos. Here we combined in vitro and in silico analyses to test the performance of metabarcoding primers amplifying regions in the 18S rDNA (Euka02 metabarcode), 16S rDNA (Inse01), and COI (BF1_BR2-COI) genes, and developed an extensive database of benthic invertebrates of France and Europe, with a special focus on three key insect orders (Ephemeroptera, Plecoptera and Trichoptera). In vitro analyses on 1514 individuals, belonging to 578 different taxonomic units showed very different amplification rates across primer combinations. The Euka02 marker showed the highest universality, while the Inse01 marker showed excellent performance for the amplification of insects. The BF1_BR2-COI metabarcode showed the highest resolution, while the resolution of Euka02 was often limited. By combining in vitro data with GenBank information, we developed a curated database including sequences representing 822 genera. The heterogeneous performance of the different metabarcodes highlights the complexity of the identification of the best markers, and advocates for the integration of multiple metabarcodes for a more comprehensive and accurate understanding of ecological impacts on freshwater biodiversity.

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.

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.

Environmental DNA (eDNA) has the potential to provide more comprehensive biodiversity assessments particularly for vertebrates in species-rich regions. Yet, this method requires the completeness of a reference database, i.e. a list of DNA sequences attached to each species, which is never met. As an alternative, a diversity 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 1,531 fish OTUs while 1,611 fish species are recorded in the region. Besides, we also accurately predict (R² = 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.