Metabarcodes for the monitoring of freshwater benthic biodiversity
through environmental DNA
Abstract
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.