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

Central Asia Five species from Central Asia form a distinct group, but their relationships within Schi- zothorax differs per ML and BA analyses, as does S. argentatus. In the ML tree (Figs 3 and 4), the Central Asia group clusters as a clade apart from all other Schizothorax, with S. argentatus positioned as sister to all remaining species. In contrast, the BA tree (S1 Fig) places the Eastern QTP/ Southeast Asia as a separate clade outside other species, with the Central Asia group clustering with S. argentatus as sister to the remaining species. These six species represent the same geographic region (Fig 3) and exhibit potential regional and/or taxonomic substructure, with S. pseudoaksaiensis and S. argentatus distributed in Lake Balkhash and its catchment basin (Kazakhstan, central Asia), as well as the Ili River (northwest China; [81]). Similarly, S. biddulphi [80] and S. eurystomus [82] occur in the Tarim River and tributaries (an endorheic basin in the Central Asiatic Desert of northwest China), whereas S. intermedius (represented by two distinct haplotypes) has a more extensive range through Iran, Uzbekistan, Kazakhstan, and Kyrgyzstan. Our TIMETREE estimation indicated the Central Asia group emerged at 11.6 Ma (CI = 14.3–6.2), an estimate comparable with previous results (11.4–10.5 Ma; [5]). Our estimate for S. argentatus (9.4 Ma; CI = 14.3–6.2) is also corroborated by earlier estimates (9.6–8.9 Ma; [5]). Based on the results of our ML tree, it seems reasonable to hypothesize Cen- tral Asia as the schizothoracine center of origin.

Central QTP, YLTR, and ’Early Bhutan’ The next major split, at 7.3 Ma (CI = 11.3–4.7), is again consistent with previous estimates (8.3–7.6 Ma) and defines two large groups (Figs 3 and 4). The first encompasses species of the Central QTP, Bhutan (immediately south), and the YLTR. The second is split into two sister clades: One represents species from eastern QTP/ Southeast Asia, whereas the second incorpo- rates Himalayan species hypothesized to have dispersed westward via the Brahmaputra River. The two Central QTP species (S. molesworthi and S. malacanthus) separated in early Mio- cene (6.2 Ma; CI = 10.2–3.7), whereas the YLTR West diverged late Pliocene (4.2 Ma; CI = 7.3–2.4). The YLTR-West species cluster as two separate groups, with S. macropogon (3.2 Ma; CI = 6.6–1.5, late Pliocene) as sister to S. waltoni (YLTR West-A), which subsequently diverged into western and eastern groups detectable via microsatellite DNA but not cyt-b [76]. These species are sister to a second geographically structured group (YLTR West-B) comprised of S. oconnori and S. wangchiachii, which clusters as sister group to samples from Bhutan and YLTR East. The evolutionary history of S. oconnori (endemic to the YLTR; [81]) underscores a late Miocene divergence driven by QTP uplift. During this process, the development of a precipi- tous canyon [72] separated S. oconnori into distinct western and eastern components [74]. The results of our composite analyses (SH-ALRT, UFBOOT, AMOVA) sustain this hypothesis, with the western group (to include S. wangchiachii) differing significantly from eastern S.

oconnori (YLTR-East), and separated from it by two undescribed Bhutanese lineages (i.e., Bhutan-1A, Bhutan-1B). Interestingly, a third Bhutanese lineage (Bhutan-1C) clusters with YLTR-East. We consider these three as representing ‘Early Bhutan,’ given their divergence from a fourth Bhutanese clade (Bhutan-2) that apparently originages from a more recent dis- persal event (Fig 3). Similarly, separate dispersals seemingly occurred for S. oconnori in the ancestral YLTR. One into western Bhutan (Bhutan-1A), and a subsequent one into central/eastern Bhutan (Bhutan-1B,C). Each is currently unverified as to species but can be tentatively allocated to S. oconnori (i.e., as Schizothorax sp. cf oconnori). It thus appears as if Quaternary climatic oscilla- tions not only yielded distinct evolutionary groupings in both S. waltoni and S. oconnori, but PLOS ONE Himalayan uplift and aquatic evolution PLOS ONE | https://doi.org/10.1371/journal.pone.0289736 October 24, 2023 17 / 27