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

eDNA metabarcoding uncovers 171 MOTUs in the TI reservoir, including 29 previously unrecorded species As described in the Methods section, following the pilot assessments, a sampling campaign covering the entire reservoir through 14 stations was performed. After sequencing the 42 sam- ples from 14 stations, 67.9 million paired reads for 12S, 31.1 million for 16S, and 74.9 million for COI were obtained (S3 Table). The raw sequence data are also publicly available at the SRA database under the BioProject accession code PRJNA1105563. Following trimming and clus- tering steps, 2.9 million amplicons were identified for 12S, 22 million for 16S, and 14.8 million for COI. A total of 1187, 3687, and 7437 ASVs remained for 12S, 16S, and COI datasets, respectively. Subsequently, the classification using the Bayesian classifier from QIIME2 and removal of all off-target species, the ASVs clusters that share 97% of identity presented 34, 41, and 190 MOTUs, respectively. The biodiversity inventory of TI Reservoir, combining historical and eDNA-based data, encompasses 577 taxa (S5 and S6 Tables). The dataset was divided into non-fish (S5 Table) and fish (S6 Table) to permit a broader view of the fishes detected, the main group of interest. The final dataset of non-fish metazoan resulted in 422 taxa, where 36 (~8%) were also detected in this study (S5 Table). Of these, 20 taxa (~5% of the 422) were exclusively detected using eDNA, with species belonging to Annelida (Clitellata and Polychaeta), Arthropoda (Cope-

poda, Ostracoda, Insecta, and Branchiopoda), Chordata (Avians) and Rotifera. The remaining taxa, previously reported in the literature, include Annelida, Arthropoda, Chordata, and Mol- lusca species (Fig 2 and S5 Table). Arthropods, especially copepods, were highly abundant across all stations, including the pilot station. We also found four non-fish taxa never previously reported in the TI reservoir, even though they had been reported in upstream reservoirs and other rivers of the Parana´ Basin: an Olygochaeta (Chaetogaster diastrophus), a copepod (Acanthocyclops vernalis) and two rotifers (Polyarthra remata and Synchaeta sp.). SAR (Stramenopila, Alveolata, and Rhi- zaria) MOTUs belonging to the Stramenopila clade were not identified beyond this level because of the lack of reference sequences. Metabarcoding eDNA matched 12 species previously identified in TI through traditional taxonomy: two annelids (Branchiura sowerbyi and Limnodrilus sp.), one arthropoda (Diapha- nosoma spinulosum), three birds (Nycticorax nycticorax, Mycteria americana and Phalacro- corax brasilianus), one mammal (Hydrochoerus hydrochaeris), one mollusc (Limnoperna fortunei), and four rotifers (Asplanchna sp., Euchlanis dilatata, Polyarthra sp. and Trichocerca sp.). Although some taxa remained unclassified, most belong to groups previously found in TI and other rivers of the Parana´ Basin (S6 Table). Nonetheless, a few species present in previous reports in the literature were not detected through DNA sequences obtained in this work, such

as Cyanophyta, predominant during 2011 [43, 44]. No macro-crustacean was detected, includ- ing the exotic Macrobrachium amazonicum and Macrobrachium jelskii, both previously found in TI [45]. Insects [46], Molluscs [46], except for L. fortunei, and Platyhelminthes already reported in this area were not detected either. For the ichthyofauna, the resulting inventory comprised 135 species (S6 Table), with 35 spe- cies being identified through both literature review and eDNA surveys. In comparison, 81 spe- cies (~60%) were only documented in the literature over the past 50 years. Nine species detected with eDNA metabarcoding had not previously been recorded in TI: one cypriniform (Gobio gobio), seven characiforms (Hemiodus orthonops, Hoplias aimara, Hyphessobrycon ery- throstigma, Leporinus piau, Schizodon fasciatus, Schizodon knerii, Metynnis melanogrammus), and one cichliform (Crenicichla cyanonotus), here found in multiple sites (Fig 3). Approxi- mately 60% of all species were captured through both eDNA and traditional monitoring in PLOS ONE eDNA metabarcoding reservoir PLOS ONE | https://doi.org/10.1371/journal.pone.0314210 January 24, 2025 8 / 22