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

Introduction The Amazon basin hosts the largest freshwater fish diversity on earth with 2,406 native species [1, 2] as well as a high genetic diversity [3], but is facing increasing threats from anthropogenic activities such as deforestation, habitat degradation, overexploitation, hydropower dam build- ing, invasive species and pollution, and from climate change [4–6]. Yet, the high number of new fish species described every year suggest that, as impressive as it already is, the species list of the Amazon basin is far from complete [7]. The Madeira is one of the most species-rich basins of the Amazon [2]. It includes the Beni watershed, already considered vulnerable to anthropic pressure and where knowledge gaps remain, such as in the Tuichi River [1]. The Tuichi basin is part of the Madidi National Park and Natural Area of Integrated Management (hereafter abbreviated Madidi NP), which is included in the Tropical Andes hotspot of biodi- versity [8–10] and also hosts one of the few identified spawning area for many fish species [11]. It therefore appears urgent to implement conservation and management measures to protect this biodiversity and to fill known gaps in terms of collection and knowledge of fish species [1]. Recently, environmental DNA (eDNA) sampling has been developed as an efficient and reliable method for understanding the biodiversity of all taxonomic groups in an ecosystem [12–15]. Environmental DNA sampling is a rapid and non-destructive method. It can target small taxonomic groups and even detect rare or cryptic species found at low abundances. For

example, in the Brazilian Atlantic forest, a single eDNA sampling detected the nine known species of Anuran, from the most abundant to the rarest, demonstrating the sensitivity of the approach [16]. In herpetofauna, eDNA sampling detected different species of turtles and amphibians in lakes and rivers in North America including the rarest species [17]. In the streams of central Idaho rivers, the giant salamander (Dicamptodon aterrimus) and the Rocky Mountain tailed frog (Ascaphus montanus), two rare species of amphibian, were detected thanks to eDNA sampling [18]. The eradication of an invasive species often requires early detection, and eDNA has proven more effective than traditional methods for detecting intro- duced buffalo toads in France [19], and the python, Python bivittatus, in Florida [20]. Simi- larly, species that were believed to be extinct or residual in a marine ecosystem, including large shark species, were detected using the eDNA approach, thereby facilitating the development of conservation strategies [21]. Aquatic mammals can also be detected: three endangered species of manatees (Trichechus ssp.) have been successfully detected in African, Amazonian and Indian rivers, helping to refine known distributions and establish conservation management strategies [22]. eDNA can also complement acoustic conservation monitoring for marine ceta- ceans in vast oceans [23]. So far, however, the eDNA method has been more rarely used to assess general biodiversity, especially in extremely biodiverse areas [24]. The Neotropics,

which harbour about 30% of all described freshwater ichthyofauna, is one such extremely bio- diverse area for fish species [7, 25]. Very few eDNA studies were carried out so far on the ich- thyofauna of the Neotropics (e.g. [25–28]), and even fewer in the most species-rich basin of the Neotropics: the Amazon basin [27, 28]. All these studies were limited by the size of the ref- erence database. Nevertheless, with suitable database development, eDNA will revolutionize research on rare or very discrete species, but also our understanding of biodiversity, both cru- cial aspects for the sustainable management of aquatic ecosystems. Here, we tested the effectiveness of eDNA assessment (1) to fill the knowledge gap regard- ing the ichthyological diversity in the hydrological basin of the Tuichi River [1], hypothesizing that eDNA would be more efficient than traditional assessment methods for a quick inventory of species diversity and to unravel rare or cryptic species, and (2) to investigate the bio- geographical structure of the Tuichi’s ichthyofauna. The information provided will be used for PLOS ONE Fish diversity and distribution in the Tuichi basin PLOS ONE | https://doi.org/10.1371/journal.pone.0262357 January 4, 2022 2 / 25 (BioProject ID: PRJNA661268, BioSample accessions: SAMN16049263- SAMN16049300). Funding: CGD has been supported by Programa Nacional de Investigacio´n Cientı´fica y Estudios Avanzados (PROCIENCA) [grant number 017- 2018-FONDECYT]. GM, RW, GT, CR has been supported by Gordon and Betty Moore Foundation [grant number GBMF331.07]. CM, FD, JFR, CDG

has been supported by Laboratoire Mixte International - Evolution et Domestication de l’Ichtyofaune Amazonienne (No grant number). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.