Environmental DNA metabarcoding is becoming a key tool for biodiversity monitoring over large geographical or taxonomic scales and for elusive taxa like soil organisms. Increasing sample sizes and interest in remote or extreme areas often require the preservation of soil samples and thus deviations from optimal standardized protocols. However, we still ignore the impact of different methods of soil sample preservation on the results of metabarcoding studies and there is no guidelines for best practices so far. Here, we assessed the impact of four methods of soil sample preservation commonly used in metabarcoding studies (preservation at room temperature for 6h, preservation at 4°C for three days, desiccation immediately after sampling and preservation for 21 days, and desiccation after 6h at room temperature and preservation for 21 days). For each preservation method, we benchmarked resulting estimates of taxon diversity and community composition of three different taxonomic groups (bacteria, fungi and eukaryotes) in three different habitats (forest, river bank and grassland) against results obtained under optimal conditions (i.e. extraction of eDNA right after sampling). Overall, the different preservation methods only marginally impaired results and only under certain conditions. When rare taxa were considered, we detected small but significant changes in MOTU richness of bacteria, fungi and eukaryotes across treatments, while the exclusion of rare taxa led to robust results across preservation methods. The differences in community structure among habitats were evident for all treatments, and the communities retrieved using the different preservation conditions were extremely similar. We propose guidelines on the selection of the optimal soil sample preservation conditions for metabarcoding studies, depending on the practical constraints, costs and ultimate research goals.

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.