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

a surface area of 10.6 km2. All three basins drain south into the east-flowing Ganges River which joins with the Brahmaputra River to terminate in the Bay of Bengal (Figs 1 and 2A). Bhutan has five major north-south river systems (Fig 2B): The Amo Chhu and Wang Chhu descend from the QTP, flow southeasterly through western Bhutan and then into West Bengal (India). The Punatsang Chhu (= Sunkosh) drains the Great Himalaya Range in west-central Bhutan, and flows into Assam (India). The Manas River and its major tributaries drain cen- tral-eastern Bhutan. The Dangme Chhu originates in Arunachal Pradesh (India) and flows southwesterly through Bhutan, whereas the Kuri Chhu (from the QTP), the Chamkhar Chhu, and the Mangde Chhu drain central Bhutan. The eastern-most river, the Nyera Ama Chhu, is the only drainage that originates entirely within Bhutan. All river systems eventually drain into the Brahmaputra River (northern India), which curves south and west through Bangladesh to merge first with the Ganges River before flowing into the Bay of Bengal (Figs 1, 2A and 2B). Amplification and sequencing of mtDNA samples Nepali samples (N = 53; S1 Table, Fig 2A) represent five Schizothorax species. Three [S. macro- phthalmus (N = 3), S. nepalensis (N = 4), and S. raraensis (N = 3); total N = 10] represent a landlocked species-flock within Rara Lake (Karnali River drainage; Fig 2A). Two other riverine species [S. richardsonii (N = 26), and S. progastus (N = 17)] were sampled from (west-to-east): Karnali River (N = 12/N = 3, respectively); Gandaki River (N = 11/9); Koshi River (N = 5/3).

Nepali samples were supplemented with those from Bhutan (N = 19; S2 Table, Fig 2B), col- lected from four west-to-east drainage basins: Wang Chhu (N = 2); Punatsang Chhu (N = 11); Mangde Chhu (N = 3); Dangme Chhu (N = 3). Genomic DNA was extracted using the Qiagen DNeasy kit, per manufacturer’s instruc- tions. We utilized published primers to amplify mtDNA cyt-b [28], with products enzymati- cally purified, sequenced using BIGDYE (ver.3.1) chemistry (Applied Biosystems Inc. [ABI], Forest City, CA), and analyzed on an ABI Prism 3700 Genetic Analyzer (W.M. Keck Center for Comparative and Functional Genomics, University of Illinois, Urbana/Champaign). In addition, we accessed 68 mtDNA cyt-b sequences representing 39 species (N = 60 Schizothorax plus eight outgroups [Acrossocheilus (N = 2); Pethia (N = 2); Puntius (N = 1); Tor (N = 2); Neo- lissochilus (N = 1); S3 Table] (National Institute of Health; https://www.ncbi.nlm.nih.gov/ genbank/). To avoid potential ambiguities with regards to evolutionary trajectories, we chose outgroups from select tribes identified as having originated prior to evolution of the Schi- zothoracini. These include (in sequence): Torini (i.e., Neolissochilus); Smilogastrini (i.e., Pun- tius, Pethia); and Acrossocheilini (i.e., Acrossocheilus, the most immediate outgroup) (S3 Table). Additional details on generating molecular data are provided [29]. Sequences were edited/aligned using SEQUENCHER v.5.3 (Gene Codes Inc., Ann Arbor, MI), with nucleotide composition and uncorrected p-distances derived among groups (MEGA X; [30]).

Polyploidy and choice of marker system The subfamily Cyprininae displays extensive polyploidy (>400 species; [20, 31]), and given the number of polyploid species, different ploidy levels, and the variety of independent polyploidi- zation events identified, it may represent the most complex vertebrate polyploid system cur- rently known [32]. Ploidy is an important consideration when molecular phylogenies are reconstructed in that it significantly impacts statistical approaches, results, and subsequent interpretations. For example, polyploidy complicates the detection of population structure, assignment tests, and principal component analyses [33]. These limitations are particularly problematic given the greater redundancy of nuclear genes in polyploid genomes. All may potentially diverge one from another over evolutionary time, such that results can differ PLOS ONE Himalayan uplift and aquatic evolution PLOS ONE | https://doi.org/10.1371/journal.pone.0289736 October 24, 2023 7 / 27