Unveiling biogeographical patterns of the ichthyofauna in the Tuichi basin, a biodiversity hotspot in the Bolivian Amazon, using environmental DNA

effectivenesswere identified. These mock samples were used to set a minimum frequency at which a present taxon was declared. This threshold was calculated to maximize the rate of true positive and to minimize the rate of false positive and set a minimum value of 0.01% (MaxSSS) from which a taxon identified in a library is kept. In the "alien" sample, (included to control index breaks), the four expected taxa (which are absent in the Amazon) were identified. A maximum of three reads belonging to a species from this alien control were found in an Ama- zonian sample, which shows a low level of leakage or contamination occurred during library preparation or the sequencing process. On the basis of this estimated leak rate, an additional filter was applied and only the taxa represented by at least four reads per site were kept. Biodiversity. Taxonomic diversity in the study area. On all the sites, 252 taxa were identi- fied covering 79.8% of the estimated number of identifiable taxa (Jack2 = 315) given the limita- tions of our database (Fig 2). Among these taxa, 22 are identified to the genus level, 207 at the species level and 23 at the level of undescribed species (noted sp.) and all are described in South America (FishBase, [57]. Among the 207 species identified 150 have already been identi- fied in Bolivia [2, 43]. The 57 species never identified to date in Bolivia were verified on the basis of systematics and biogeography. Of these, 16 were named affinis (although they had a hit of more than 97% identity) because they were never identified in the Amazon basin.

Among these 16 species, 11 have an abundance of more than 193 reads after treatment with microDecon (deletion of reads on the basis of negative controls), while the maximum number Fig 2. Number of taxa observed (green bars) and second order Jackknife estimate of taxa richness (blue bars) computed per biogeographical entities, lakes, rivers and overall sites (given the COI database). The orange bars represent the percentage coverage (ratio between the number of taxa observed and the Jack2 estimator) with black vertical error bars indicating the 95% confidence interval. https://doi.org/10.1371/journal.pone.0262357.g002 PLOS ONE Fish diversity and distribution in the Tuichi basin PLOS ONE | https://doi.org/10.1371/journal.pone.0262357 January 4, 2022 9 / 25