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

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

also among Schizothorax represented in Bhutan (S1 Appendix). Again, the BA-tree differs in placement of the YLTR and Bhutan-1 lineages, with the YLTR-West-A and YLTR West-B groups separated by the Bhutan-1A group, whereas Bhutan-1B and Bhutan-1C groups collapse and YLTR-East placing as sister to it (S1 Fig). Eastern QTP and Southeast Asia The E-QTP/ SEA-A species split into three significantly different clades (Fig 4, S3 Table), encompassing five species from the Irrawaddy/ Salween rivers and reflecting the deepest TIME- TREE estimate at 5.6 Ma (CI = 9.7–3.0). The E-QTP/ SEA-B diverged at 1.7 Ma (CI = 3.5–0.8) and consists of seven species from the Yangtze River, whereas the E-QTP/ SEA-C involves seven species from the Mekong/ Salween drainages. The Mekong divergence was seemingly driven by late Pliocene orogenesis [77], an estimate congruent with our timeline (2.4 Ma; CI = 5.0–1.2). Three species within the upper Salween (i.e., S. gongshanensis, S. lissolabiatus, S. nukiangensis) display low genetic divergence and frequent haplotype sharing, a potential by- product of Quaternary glaciations [79]. However, several E-QTP/ SEA-B species (such as S. davidi, S. koslovi, S. chongli, S. gongshanensis) have yet to be fully described or documented, other than in general terms [80]. Dispersal into Bhutan, Nepal, and western Himalaya The YLTR originates on the QTP, flows eastward then abruptly south through the Namche Barwa Syntaxis [5], with subsequent capture by the Brahmaputra River [6]. It represents an

important freshwater dispersal route from the QTP and its drainage history correlates with an hypothesized evolutionary transition between species on the QTP (sensu stricto) versus more recent divergences via the Lower Brahmaputra. In our analyses, samples from Nepal split into distinct clades, each representing separate drainages that are significantly different from Southeast Asian clades. Interestingly, a set of samples from Bhutan (Bhutan-2; N = 8) form a distinct clade sister to the easternmost Nepali drainage (Koshi River Basin; N = 10, S3 Table). This is consistent with an hypothesized east- to-west dispersal via the lower Brahmaputra. Species in the Koshi River group are tentatively referred to as S. richardsonii, S. esocinus, and S. progastus, whereas samples from Bhutan-2 are currently unidentified but link in the network with a haplotype of S. progastus from the Koshi River (Fig 5). The morphological similarities between S. richardsonii and S. progastus in the Koshi, Kar- nali, and Gandaki rivers are such that their discrimination can be problematic in the field [83], particularly at early life history stages. Despite morphological differences [29], the two species are genetically more similar within than among basins, which could be driven by ecological diversification along elevational gradients, allowing for emergence of similar phenotypes in each basin (i.e., homoplasy). This perspective stems from mtDNA data, but a recent study using ddRADseq indicated substantial admixture between the two in each drainage [84]. Our

study does not (and cannot) revise taxonomy, but it does identify regions (e.g., Nepal, Bhutan) where more focused research is needed to assess genetic and phenotypic diversity, and thus clarify taxonomy of Schizothorax in the Central and Eastern Himalaya. Schizothorax in the Western Himalaya are genetically less well defined and their clustering reflects a mosaic of historic and contemporary drainage arrangements (S1 Appendix). Samples from the Gandaki and the Karnali rivers cluster into distinct groups, with the Gandaki River haplotypes (N = 20) most distinct (separated by 26 mutations, Fig 5) and partitioned into nested clusters of S. richardsonii (three lineages), S. progastus (two lineages), and one repre- senting a mixture of the two (Fig 4). Samples from the Karnali River Basin (far western Nepal, PLOS ONE Himalayan uplift and aquatic evolution PLOS ONE | https://doi.org/10.1371/journal.pone.0289736 October 24, 2023 18 / 27

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

then to the Bay of Bengal; and lastly, an earlier capture by the Ganges of four western Himalaya rivers (i.e., Jhelum, Chenab, Ravi, Sutlej) which are now incorporated within the contemporary Indus Basin. The latter hypothesis is supported by isotopic and seismic data extracted from the detrital fan of the Indus River, as deposited for >30 Ma in the Arabian Sea. These data document a major shift at ~5 Ma, with earlier Himalayan deposition superseding those from the Indus suture zone to the north [91]. This documents the capture of several large Punjabi rivers (i.e., Jhelum, Chenab, Ravi, Sutlej) by the Indus, as represented in the current basin topography. However, this interpretation is not without controversy. A subsequent comparison of sedi- ments from northern Pakistan with those of the Indus Fan suggests the transition was a poten- tial response to differential erosion rates in the source region (the Indus Basin) rather than drainage rearrangements [90]. The east-west divergence among Himalayan clades provides a framework by which the geo- morphic evolution of the drainage system can be interpreted. In this sense, our phylogeo- graphic results can be inferred as biologically corroborating the capture by the Indus of those drainages previously allocated to the Ganges Basin. For example, Schizothorax from the Lower Ganges River diverged from a lineage representing the Upper Ganges and the Indus River. Similar patterns have been noted for Indus and Gangetic dolphin [92]. These data also suggest

Schizothorax in the western Himalaya (i.e., Koshi, Gandaki, and Karnali rivers) were isolated prior to the capture of the Jhelum by the Indus River. Overall, our analyses reflect these unre- solved questions, as indicated by placement of individual haplotypes apart from others with the same geographic origin (Figs 4 and 5, S1 Fig). A more detailed study is needed as our anal- yses only include a limited number of samples representing this biogeographic region. Taxonomic uncertainty in Schizothorax and its implications Several species in our analyses were polyphyletic, with individuals forming biogeographically defined clusters that differed significantly one from another. In addition, multiple cases also emerged where samples within the same region failed to cluster with conspecifics (e.g., Central Asia; Eastern QTP/Southeast Asia; Koshi; Gandaki; Karnali; Ganges). Incongruence between molecular and morphological phylogenies represents a challenge for the current study, as reflected by morphologically-identified species from different drainages failing to cluster together. In contrast, the converse is true for different species in the same drainage [16, 78]. This can reflect incomplete lineage sorting [21, 77]; hybridization (interge- neric: [5, 93], intrageneric: [94]); rapid diversification [5]; and/or convergent evolution (e.g., juxtaposition of trophic and spatial niches across species; [95, 96]). None can be resolved using mtDNA [22]. Unfortunately, these issues are amplified within online databases by an incorrect association of DNA sequences with taxonomy [97].

Conclusions Schizothorax in the Himalaya is a monophyletic genus hypothesized as originating in Central Asia and subsequently dispersing southward into Bhutan via down-cutting rivers, as well as westward across the Eastern QTP and into Southeast Asia. The subsequent capture of the YLTR by the Brahmaputra allowed for an east-to-west dispersal south of the Himalaya, facili- tating sequential movements northward into Himalaya drainages, first in Bhutan, then Nepal, and eventually into the western Himalaya. Our molecular data corroborate western drainages as being previously affiliated with the Ganges River but subsequently captured by the Indus River. PLOS ONE Himalayan uplift and aquatic evolution PLOS ONE | https://doi.org/10.1371/journal.pone.0289736 October 24, 2023 20 / 27

Many morphologically-recognized species have also diverged substantially across basins, suggesting the potential for cryptic speciation and/or undescribed, relictual biodiversity. More detailed molecular studies are needed to test this hypothesis and thus promote conservation planning at the regional scale. This will require the analysis of populations now geographically isolated across the Himalaya, with cooperation/ collaboration needed among neighboring countries for comprehensive, large-scale studies [98, 99]. This is particularly imperative given extant (as well as predicted) impacts of climate change on the Third Pole in general, and Schi- zothhorax in particular [26]. Supporting information S1 Appendix. Divergence intervals (TIMETREE, MEGA X [30]) for Snowtrout (Cyprini- nae: Schizothorax), as associated with geomorphic and climatic drivers in the Himalaya. Confidence intervals for divergence estimates are provided as parentheticals. (PDF) S1 Table. Schizothorax samples from Nepal (N = 53) by site and species. Site = sampling site (N1-N9); Species = Schizothorax sp. identification; River = basin; Location = collection area within basin (confl = confluence); Lat = Latitude; Long = Longitude; KU-T = Univ. Kansas Tissue number (KU:KUIT:#); KU-V = Univ. Kansas Voucher number (KU:KUIT:#); Haplotype = Univ. Arkansas Sequence number; Accession = GenBank Accession number. Sites depicted topographically in Fig 2A. (PDF) S2 Table. Schizothorax samples from Bhutan (N = 19) by site and species. Site = sampling

site (B1-N9); Species = Schizothorax sp. identification; River = basin; Location = collection area within basin (Chhu = river); Lat = Latitude; Long = Longitude; KU-T = Univ. Kansas Tis- sue number (KU:KUIT:#); KU-V = Univ. Kansas Voucher number (KU:KUIT:#); Haplotype = Univ. Arkansas Sequence number; Accession = GenBank Accession number. Sites depicted topographically in Fig 2B. (PDF) S3 Table. Samples of Schizothorax (N = 132) and outgroup species (N = 8) evaluated. Spe- cies Name = Scientific name (A = Acrossocheilus; Pe = Pethia; Pu = Puntius; T = Tor; N = Neo- lissochilus; S = Schizothorax); Haplotype = Univ. Arkansas sequence identifier [i.e., from GenBank = letters and numbers; Univ. of Arkansas = starting with ’58’ (Univ. of Kansas tissue vouchers referenced in S1 Table)]; Location = Country, geographic region or river basin; Group = Regions; Group abbreviations are as follows: QTP = Qinghai-Tibetan Plateau; SEA = Southeastern Asia; YLTR = Yarlung-Tsangpo River; * = indicates samples that cluster with a group outside their sampling region. (PDF) S1 Fig. Phylogenetic tree derived using Bayesian methodology (BEAST v2.61). Phylogenetic relationships among snowtrout (Schizothorax; Cyprinidae) and outgroup taxa derived from sequence analysis of the cytochrome-b mitochondrial gene (1,140 bp, 140 haplotypes). Individ- uals are coalesced into geographic regions, with abbreviations defined as: E-QTP/ SEA = Eastern Qinghai-Tibetan Plateau/ Southeast Asia, with three subclades indicated by A,

B, and C; YLTR = Yarlung-Tsangpo River, with subclades indicated by West-A, West-B, and East. Numbers at nodes represent Bayesian posterior probabilities. Sample metadata in S3 Table. (PDF) PLOS ONE Himalayan uplift and aquatic evolution PLOS ONE | https://doi.org/10.1371/journal.pone.0289736 October 24, 2023 21 / 27

Acknowledgments Nepali samples were collected by DRE under permits issued by Nepali Department of National Parks and Wildlife [1984–86 (permit 7066) and 1996 (permit 802)], with a subset of specimens placed in the University of Kansas Biodiversity Institute (https://biodiversity.ku.edu.htm). We thank Curator Leo Smith and Collection Manager Andrew Bentley for their prompt response and support. We also acknowledge the National Research Centre for Riverine and Lake Fisher- ies (NRCR&LF, Haa, Bhutan) for providing logistic support and personnel with which to sam- ple fishes. Analytical resources were provided by the Arkansas Economic Development Commission (Arkansas Settlement Proceeds Act of 2000) and the Arkansas High Performance Computing Center (AHPCC). This research was made possible through generous endow- ments to the University of Arkansas: The Bruker Professorship in Life Sciences (MRD), the 21st Century Chair in Global Change Biology (MED), Distinguished Doctoral Fellowship award (ZDZ), and Graduate Teaching Assistantships (KW, ZDZ). Analytical assistance was provided by Tyler K. Chafin. Author Contributions Conceptualization: Binod Regmi, Marlis R. Douglas, Karma Wangchuk, Michael E. Douglas. Data curation: Binod Regmi, Zachery D. Zbinden. Formal analysis: Binod Regmi, Michael E. Douglas. Funding acquisition: Marlis R. Douglas, Singye Tshering, Michael E. Douglas. Investigation: Marlis R. Douglas, Karma Wangchuk, Zachery D. Zbinden, David R. Edds, Sin- gye Tshering, Michael E. Douglas. Methodology: Marlis R. Douglas, Karma Wangchuk, David R. Edds.

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