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.

Vincent Prié

and 14 more

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.

Vincent Prié

and 14 more

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.