The Himalayan uplift and evolution of aquatic biodiversity across Asia: Snowtrout (Cyprininae: Schizothorax) as a test case

& Forests (MoAF), Royal Government of Bhutan. The export of fin clips was authorized through a Material Transfer Agreement (MTA) provided by the Bhutan Agricultural and Food Regulatory Authority (BAFRA). Sampling protocols were approved by the University of Arkansas Institutional Animal Care and Use Committee (UA_IACUC_17064). Specifically, sampling was accomplished using standard fisheries methods, to include hand- held seines, dip nets, backpack shockers, as well as passive gear such as hoop/ trammel nets and minnow traps. These methods and devices are approved for use by the American Society of Ichthyologists and Herpetologists (ASIH; https://www.asih.org/resources) and the Ameri- can Fisheries Society (AFS; https://fisheries.org/books-journals/online-resources/). In addi- tion, educational resources [Association for the Advancement of Laboratory Animal Science (AALAS): https://www.aalas.org/] were employed to assist in the instruction of Bhutanese cadre. Small-bodied fishes (<10 cm total length) were removed from capture devices with <20 placed concurrently within a 19-liter bucket of well-aerated stream water. Direct handling of fish was minimized, and individuals were retrieved via dip net for identification and subse- quent release. Larger fishes were gently removed from capture devices and identified/ released at site of capture. Care was taken in all cases to ensure that water temperatures were identical between capture site and holding bucket. Once captured, fishes were continuously

monitored, and euthanasia immediately employed if sampling injuries were detected. Fishes were so recognized if they could not maintain equilibrium in the holding bucket, and/or dis- played erratic or impaired opercular movements (<20 respiratory cycles/minute). Euthana- sia was conducted via an overdose of clove oil (Eugenol), followed by fixation in 10% formalin. Clove oil satisfies specified criteria and is considered acceptable when used at high concen- trations (>400 mg/L), as it causes a reliable and rapid loss of consciousness and induces hyp- oxia (critical components for eliminating potential pain; American Veterinary Medical Association Guidelines on Euthanasia; https://www.avma.org/sites/default/files/2020-02/ Guidelines-on-Euthanasia-2020.pdf). A concentration of 50–150 mg/L was initially employed, then gradually increased to >400 mg/L through periodical additions (~50 mg/L). Complete cessation of opercular movement was used as an indicator of death, with the fish then being retained in solution for >10 additional min. Tissues for DNA analysis were non-lethally sampled by removing a small piece of anal fin (a non-propulsive appendage). The fish was first placed in a small bucket of stream water treated with clove oil, and subsequently removed via dipnet immediately following a loss of righting ability. A small piece of fin was removed with surgical scissors cleaned in 100% EtOH. The clip was transferred to a Whatman FTA© card for preservation and storage, and the fish revived in a small bucket of untreated stream water until righting ability was regained, at

which time it was released at the collecting site. Wounds caused by clipping of fins will nor- mally heal satisfactorily without application of antibiotics or topical analgesics, which are not recommended for use in wild fishes (per AFS resources). Sample sites: Drainages in Nepal and Bhutan Nepal is drained by three major east-to-west rivers (Koshi, Gandaki, and Karnali; Fig 2A) that collectively support eight tributaries, several of which originate on the QTP. In eastern Nepal the stronger effects of the monsoon are reflected by more eroded and abruptly-elevated moun- tainous slopes of the Koshi River. Central Nepal, dominated by the Gandaki Basin and its west- ern tributary (Kali Gandaki), serves as a distinct east-west vicariant break. Western Nepal is drained by the Karnali Basin and includes Rara Lake (2,990m elevation), a RAMSAR site with PLOS ONE Himalayan uplift and aquatic evolution PLOS ONE | https://doi.org/10.1371/journal.pone.0289736 October 24, 2023 5 / 27