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

(13) a composite western Himalaya clade that included Rara Lake and the Karnali drainage (western Nepal), the upper Ganges (northwest India), and the Indus River Basin (Pakistan). A more detailed perspective is presented in Fig 4, with regional composites listed in S3 Table. Most noteworthy is the partition of the Eastern QTP/ Southeast Asia (E-QTP/ SEA) clade into three distinct geographic clusters: E-QTP/ SEA-A (Salween-Irrawaddy drainages); E-QTP/ SEA-B (upper Yangtze drainage); (9) E-QTP/SEA-C (Mekong-Salween drainages). In the BA tree (S1 Fig) the western Himalaya was additionally partitioned into two subclades: Indus/Upper Ganges versus Karnali/Rara Lake. In the ML tree, samples from Central Asia formed a distinct regional clade that clusters as sister to all other samples, including. S. argentatus, which is sister to all remaining species. These form two distinct composite clades. In the first, samples representing the Central QTP form a group, followed by the YLTR West-A and YLTR West-B, then Bhutan-1A separated from Bhutan-1B, and finally a group representing by YLTR East and Bhutan-1C. The second large ML cluster is subdivided into two distinct sister clades, each partitioned into subclades consistent with geographic regions. The first comprises the Eastern QTP/ Southeast Asia and is represented by three significantly different groups (labeled A, B, C). The second contains the Koshi River (eastern Nepal) as sister to the Bhutan-2 group (representing eight undescribed Bhutanese samples), followed by the Gandaki River as sister to a mixed

western Himalaya clade containing the Karnali River/ Rara Lake, the upper Ganges River, and the Indus River (Figs 3 and 4). Clustering of S. richardsonii and S. progastus in the Koshi, Kar- nali, Ganges, and Indus River drainages is noteworthy because individuals do not cluster sepa- rately by taxonomic designation within each drainage, but instead group as co-mingled components. Major differences between ML (Figs 3 and 4) and BA (S1 Fig) trees include: Position of the Central Asia and E-QTP/ SEA clades, relationship of YLTR and Bhutan-1 subgroups, and position of S. argentatus in relation to the Central Asia group. The regions are the same within the BA tree, but distributed across two large regional clusters, with Central Asia included within the first, rather than being distinct from it. YLTR West is subdivided into groups by the presence of Bhutan-1A, whereas Bhutan-1B is instead subsumed within it rather than being sister to YLTR West/Bhutan-1C. The E-QTP/ SEA-A clade is also further subdivided within the BA tree (a pattern also distinct in the haplotype network, Fig 5). And lastly, the Ganges and Indus rivers appear as distinct western Himalaya clades, whereas they are unresolved in the ML tree. Temporal divergences We employed RELTIME to derive major time-calibrated events, 13 of which are depicted in our ML-based tree (Fig 3; other dates annotated within discussion; See also S1 Appendix for the associations between divergence intervals and geomorphic/ climatic drivers in the Himalaya).

The average divergence time for Central Asia was 11.6 Ma (CI = 15.4–8.7). Two large sub- clades sister to Central Asia then separated at ~7.3 Ma (CI = 11.3–4.7). In the first, the Central QTP diverged at 6.2 Ma (10.2–3.7), followed by the YLTR West (4.2 Ma; CI = 7.4–2.4), then Bhutan-1A (3.3 Ma; CI = 6.7–1.5), with YLTR East and Bhutan-1B at 2.7 Ma (CI = 5.9–1.2). In the second large subclade, the Eastern QTP/ Southeast Asia region separated at 6.6 Ma (CI = 10.7–4.1), followed by a second large composite clade at 4.8 Ma (CI = 8.8–2.6). Here, the grouped within each. Numbers at nodes represent RELTIME TIMETREE mean divergence estimates (in Ma; confidence intervals in text; See also S1 Appendix). https://doi.org/10.1371/journal.pone.0289736.g003 PLOS ONE Himalayan uplift and aquatic evolution PLOS ONE | https://doi.org/10.1371/journal.pone.0289736 October 24, 2023 12 / 27