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

of reads observed in the 42 negative controls is 3 for these 16 species. Contamination can therefore be excluded during the collection and / or laboratory steps. For each identified taxon we reported their percentages of identity with the COI reference to which they are assigned, as well as the total read number (S6 Table). This table also reports the records of these taxa in the Amazon and the Beni region. These 252 taxa are distributed among 149 different genera in 11 orders: 45.2% of the taxa are Siluriformes, 34.5% Characiformes, 9.1% Gymnotiformes, 6.0% Cichliformes. The orders Clupeiformes, Myliobatiformes, Perciformes, Synbranchiformes, Salmoniformes, Osteoglossi- formes, Cyprinodontiformes together account for the remaining 5.2% of the identified taxa. The number of taxa per site averaged 51.03 (SD = 23.1) and ranged between 2 to 105. Over- all, 53.5% of taxa were identified at four sites or less (S2 Fig). A single taxon, Prochilodus nigri- cans, was observed at all sites except Pelechuco which is located at 4,508 m a.s.l. At this site only Orestias aff. ascotanensis (agassizii group) and Oncorhynchus mykiss were identified. A significant correlation was observed between taxa richness observed and altitude (r = 0.85, p-value = 0.011), with a regular pattern of increasing richness towards downstream sites (Fig 3). In rivers, taxa richness ranged from 2 at Pelechuco (4,508 m a.s.l.) to 95 in the Beni river (210 m a.s.l.). In lakes, the two most upstream have the lowest richness values (21

and 22 taxa identified in Chalala´n and Santa Rosa, respectively) and the highest was observed in the Colorada lake with 105 taxa identified (see S3 Fig and S3 Table for details). Geographic distribution patterns of species detected in the Tuichi basin and the Beni lakes. The hierarchical clustering of the sampled sites shows four clusters, representing four biogeo- graphic entities that are supported by bootstrap values greater than 81% (Fig 4). The nine lakes constitute a first exclusive biogeographic entity, separating the three most-upstream lakes (Ruta, Chalala´n and Santa Rosa) from the downstream lakes. The second (Alto Tuichi) brings together the four upstream sites collected on the Tuichi river, the third includes all the sites collected on the rank 3 rivers, and finally the fourth entity groups the tributaries of rank 1 and 2. The calculated coverage rates (number of observed taxa / estimated number of taxa using Jack2 estimator) for each of the four biogeographic entities (lakes, Alto Tuichi, rank 1 and 2 rivers and rank 3 rivers) range from 67.9% to 79.7% and are not significantly different from each other (p-value = 0.09) showing that our sampling effort is similar among these biogeo- graphic entities and therefore allows for comparison. In rivers, 172 taxas were identified, of which 52.3% and 32.6% belonged to Siluriformes and Characiformes, respectively. In lakes, 153 taxa were identified, including 32% of Siluriformes and 39.9% of Characiformes. As illustrated in Fig 4, Siluriformes (brown lines) had a signifi-

cantly stronger occurrence (p-value < 0.001) in rivers than lakes whereas Characiformes (red lines) had a stronger occurrence in lakes, although this trend was not significant (p- value > 0.1). Taxa belonging to the Clupeiformes and Osteoglossiformes orders were detected only in lakes, whereas Cyprinodontiformes and Salmoniformes were only detected in rivers. In the Madidi NP alone, of the 186 taxa identified, 17 taxa are common to lakes and rivers, 14 taxa identified only in lakes and 140 taxa identified only in rivers (Fig 5). Considering the four biogeographic entities established by the hierarchical analysis, their difference in terms of species composition remains strongly marked. Only 7 taxa are common to these groups. Six taxa were identified only in the Alto Tuichi group, six in the rank 3 rivers, and 54 in the rank 1 and 2 rivers group (see S7 Table for taxa lists in each area of the Venn diagram). Between biogeographic entities the number of taxa shows a clear increase between the Alto Tuichi (46 taxa) above 700 m a.s.l and those at lower altitude (Fig 3). Below 700 m a.s.l., species diversity follows the increase in rank and therefore in size of the sub-basins, going from 98 taxa (rank 3 tributaries) to 155 taxa (rank 1 or 2 tributaries). PLOS ONE Fish diversity and distribution in the Tuichi basin PLOS ONE | https://doi.org/10.1371/journal.pone.0262357 January 4, 2022 10 / 25