Insights into the representativeness of biodiversity assessment in large reservoir through eDNA metabarcoding

S4 Fig. Probability of identifying fish by sampling for eDNA at different depths. General- ized Linear Model (GLM) showing the probability (y-axis) of sampling fish taxa in different depths (1 meter, 13 meters and 25 meters from the surface) after three samples (x-axis). Each line represents one depth according to the colors in the legend. (PNG) S5 Fig. Statistics from the Discriminant Analysis of Principal Components (DAPC). A) Sum of contribution of each sampled station for the DAPC clustering assignment. Given the differences in sampling size, the pilot sampling is not included. Station abbreviations as in S1 Table. Each bar corresponds to one station, and the value shows the sum of contribu- tion for the six first Principal Components (see Methods). The stations with the highest val- ues are C01, C11 and C10, respectively. B) Rarefaction curve constructed using only samples from C01, C11 and C10, where the curve reaches the plateau curve around eight samples. (PNG) S1 Table. Sampling locations’ information. Description, geographic coordinates, sampling date, water volume, number of replicates, depth and abbreviations. (XLSX) S2 Table. Pair of primers used to amplify the partial sequences of COI, 12SrRNA, and 16SrRNA genes. Sequences and references are shown for each marker. (XLSX) S3 Table. Summary of reads, Amplicon Sequence Variants (ASVs) and Molecular Opera- tional Taxonomic Units (MOTUs) statistics. Based on eDNA analyses of both pilot and sta- tion sampling, values are shown after and before trimming steps. (XLSX)

S4 Table. List of Amplicon Sequence Variants (ASVs) amplified using eDNA metabarcod- ing. For both pilot and sampling stations included, the molecular marker, frequency, number of samples, length and taxonomic classification information are displayed for each ASVs. (XLSX) S5 Table. Biodiversity inventory for non-fish taxa in the Três Irmãos (TI) reservoir. The inventory was built using previous literature, eDNA and GBIF information. For each taxon, the source of the record is indicated, as well as the reference, if needed. (XLSX) S6 Table. Biodiversity inventory for ichthyofauna in the Três Irmãos (TI) reservoir. The inventory was built using previous literature, eDNA and Global Biodiversity Information Facility (GBIF) information. For each taxon, the source of the record is indicated, as well as the reference, if needed. (XLSX) Acknowledgments We thank Ange´lica Beccato, from Environment and Land Manager at Tijoa´ Energia, for her support and collaboration in developing this work. Author Contributions Conceptualization: Danielle do Amaral, Mauro Rebelo. PLOS ONE eDNA metabarcoding reservoir PLOS ONE | https://doi.org/10.1371/journal.pone.0314210 January 24, 2025 18 / 22