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

the management of the Madidi NP and serve as a reference for the management of other hydrographic basins in the tropical Andean foothills. Methods Sampling Study area and collecting sites. The sampling area is located mainly in the Tuichi sub- basin in the Beni watershed, sub-basin of the Madeira River, the largest tributary of the Ama- zon (Fig 1). It is part of the Madidi NP, one of the largest protected areas in Bolivia covering 1,895,750 ha [29]. The Tuichi sub-basin constitutes a hydrological network of more than 7,100 km (including all tributaries) that extends into the Andes at an altitude of more than 6,000 m a.s.l. and flows 270 km to its confluence with the Beni River, 25 km upstream of the town of Rurrenabaque. Environmental DNA samples were collected from lakes and rivers between September and October in 2017 (2 sites), 2018 (10 sites) and 2019 (22 sites); each site being sampled only once (see S1 Table for details and geographical coordinates). This period of the dry season with low water levels allows for easier navigation. But more important, during this period the connectivity between lakes and rivers is interrupted, which ensures that eDNA col- lected in the lakes provide information on their own ichthyofauna without eDNA input from the main stream. In the Tuichi basin 22 sites were collected: 12 on the Tuichi River, eight on its main tributaries and two on tectonic lakes (Chalala´n and Santa Rosa). Additionally, in order to assess a possible peculiarity of fish compositions in the Tuichi basin, we also sampled

two sites on the Beni River, one upstream and one downstream of the confluence with the Tui- chi, three tributaries of the Beni (Hondo, Quende´que´ and Quiquibe´ rivers) upstream of the confluence with the Tuichi, one oxbow lake (Ruta) on the Hondo River, as well as 6 other lakes located downstream in the Beni basin, outside of the Madidi NP. A total of 34 sites were col- lected, 25 in rivers and 9 in oxbow lakes (Fig 1A and 1B) covering an altitudinal range from 200 to 4,505 m a.s.l. The average river network distance between sites collected in the Madidi NP is 14.4 km (SD = 21.8). Overall, the time required to complete eDNA collections was approximately four weeks. Following an ordering system of drainage patterns [30], the Beni River, the main river in the watershed, receives the rank 1; the Tuichi, Quiquibe´, Quende´que´ and Hondo rivers were put in rank 2 and all the Tuichi’s tributaries in rank 3 (Fig 1). According to Wasson et al. (2002) [31] geomorphological and climatic structures delineate nine geoclimatic domains sub-structured into 40 hydro-ecoregions (HER2) in the Bolivian Amazonian basin. HER are congruent with geology, relief, climate (temperature and precipita- tion), vegetation, soils and hydrography. Our sampling sites are distributed across 7 HERs (Fig 1 and S1 Table for details) corresponding to Glacial mountain (Cg, 1 site), High peri-Amazo- nian Yungas (Ya-a, 1 site), Amazonian gallery forest (Ab-a, 2 sites), Beni Gallery Forest (Ab-b, 4 sites), Dry valleys of the Yungas (Ay, 2 sites), High humid sub-Andean (Sa-a, 1 site) and Low

humid sub-Andean (Sa-b, 23 sites). Ethic statement. The Wildlife Conservation Society (WCS) is authorized by the Bolivian government to study and sample the Bolivian Amazonian biodiversity. The Ministerio de Medio Ambiente y Agua (MMAyA) of Bolivia approved the study and provided permits (auto- rization numbers: FCPV-IE-0196/2017; FCPV-IE-0030/2018; FCPV-IE-0064/2019) for collec- tion, export and analysis of the filter cartridges to the French National Institute of Research for Sustainable Development (IRD) laboratories in France. eDNA field sampling. Due to variations in water turbidity in these areas, particularly in streams, and in order to ensure sufficient filtered volume before clogging, we used sterile Envirocheck HV filter cartridge with a porosity of 1 μm (Pall Corporation, Ann Arbor, MI, USA). One cartridge per river sampling site was used, and two to five cartridges for lakes. For PLOS ONE Fish diversity and distribution in the Tuichi basin PLOS ONE | https://doi.org/10.1371/journal.pone.0262357 January 4, 2022 3 / 25