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

N = 25) fall within a composite that encompasses samples from the Upper Ganges (N = 8) and Indus rivers (N = 4; western Himalaya). These generally group by basin in the BA tree (S1 Fig) yet are weakly separated in the haplotype network (Fig 5), where haplotypes from the Karnali River Basin form a distinct cluster, separated by 11 mutations from the Indus and Ganges (Fig 5). Samples from the Ganges and Indus River basins were designated as S. progastus and S. pla- giostomus, with S. richardsonii also identified in the Upper Ganges, and S. esocinus in the Indus River (S3 Table). The BA tree topology separates the Indus and Ganges and places this group as sister to the distinct Karnali River/ Rara Lake group. These groupings are congruent with contemporary drainage arrangements of the Koshi, Gandaki, and Karnali rivers which flow into the Ganges River of India (Fig 2A) to ultimately drain into the Bay of Bengal. In con- trast, the contemporary Indus River drains westward to the Arabian Sea, but the close associa- tion of haplotypes between Indus, Upper Ganges and Karnali rivers suggest an alternate historic drainage arrangement. Phylogeography of Rara Lake, Nepal Three morphologically differentiated and reproductively isolated Schizothorax (i.e., S. macro- phthalmus, S. nepalensis, S. raraensis) occupy separate trophic niches in Rara Lake (Karnali River Basin, northwestern Nepal: [85]), with S. raraensis and S. nepalensis listed as critically endangered (IUCN, 2017; http://www.iucnredlist.org/details/168564/0/). Of the three, only S.

nepalensis could be differentiated using mtDNA, and was hypothesized [86] as sharing mtDNA haplotypes with S. macrophthalmus and S. raraensis via hybridization. Similarly, two morphologically distinct Snowtrout in the upper Karnali River (i.e., S. richardsonii and S. pro- gastus) could not be discriminated, again hypothesized as interspecific hybrids [86]. Our mito- chondrial marker could not resolve this hypothesis, thus necessitating further nuclear analyses that may unequivocally test the role of hybridization as a source of phylogenetic discordance. Such an examination, however, is again complicated by polyploidy and its variance within Schizothorax. Haplotypes of Rara Lake species are generally distinct, but not clearly separated from each other or from Karnali River samples (Fig 5). The geologic age of Rara Lake is estimated at ~60 kya [87]. Its endemic species do not form a monophyletic clade but cluster instead with samples from tributaries of the Karnali River which drains the lake (Fig 5, S1 Fig). Haplotypes of S. richardsonii and S. progastus do not dif- fer significantly from one another in the Karnali River Basin, but do so from conspecifics in the Koshi and Gandaki rivers [86], a pattern also confirmed in our analyses. Species in the Kar- nali River Basin are poorly resolved with respect to mitochondrial divergence, with a genetic signal likely obscured by the limited timeframe within which ancestral alleles could sort (i.e., incomplete lineage sorting). More refined molecular approaches, such as reduced-representa-

tion genomics [68–70, 88, 89], could potentially provide the necessary resolution, although it will again be compounded by polyploidy, as characterized within Cyprininae. Dispersal into western Himalaya tributaries The southern QTP and western Himalaya are now drained by the contemporary Indus River, which extends south and west from the Karakorum Range rather than from the High Hima- laya. Given its western placement, the geomorphic history of the Indus River has an interesting relationship with the biogeography of Himalayan Schizothorax. Samples from the Indus and the Ganges (immediately to the east) are unsettled, as reflected in our analyses, with several hypotheses proposed [90]. Relative stasis between the two river basins is the most conservative, whereas others involve various stream-capture scenarios, such as: The Ganges initially flowing westward to the Indus, then to the Arabian Sea; The Indus flowing eastward into the Ganges, PLOS ONE Himalayan uplift and aquatic evolution PLOS ONE | https://doi.org/10.1371/journal.pone.0289736 October 24, 2023 19 / 27