Significance Since prehistory, the Himalayan mountain range has presented a formidable barrier to population migration, whereas at the same time its transverse valleys have long served as conduits for trade and exchange. Yet, despite the economic and cultural importance of Himalayan trade routes, little is known about the region’s peopling and early population history. In this study, we conduct to our knowledge the first ancient DNA investigation of the Himalayan arc and generate genome data for eight individuals ranging in time from the earliest known human settlements to the establishment of the Tibetan Empire. We demonstrate that the region was colonized by East Asians of likely high-altitude origin, followed by millennia of genetic continuity despite marked changes in material culture and mortuary behavior.

Abstract The high-altitude transverse valleys [>3,000 m above sea level (masl)] of the Himalayan arc from Arunachal Pradesh to Ladahk were among the last habitable places permanently colonized by prehistoric humans due to the challenges of resource scarcity, cold stress, and hypoxia. The modern populations of these valleys, who share cultural and linguistic affinities with peoples found today on the Tibetan plateau, are commonly assumed to be the descendants of the earliest inhabitants of the Himalayan arc. However, this assumption has been challenged by archaeological and osteological evidence suggesting that these valleys may have been originally populated from areas other than the Tibetan plateau, including those at low elevation. To investigate the peopling and early population history of this dynamic high-altitude contact zone, we sequenced the genomes (0.04×–7.25×, mean 2.16×) and mitochondrial genomes (20.8×–1,311.0×, mean 482.1×) of eight individuals dating to three periods with distinct material culture in the Annapurna Conservation Area (ACA) of Nepal, spanning 3,150–1,250 y before present (yBP). We demonstrate that the region is characterized by long-term stability of the population genetic make-up despite marked changes in material culture. The ancient genomes, uniparental haplotypes, and high-altitude adaptive alleles suggest a high-altitude East Asian origin for prehistoric Himalayan populations.

The world’s high plateaus and great mountain ranges were among the last places colonized by humans in prehistory (1⇓–3). The challenges of rough terrain, cold stress, hypoxia, and the relative scarcity of resources in these high places significantly slowed the pace at which permanent occupation took place. The Himalayan mountain range and the Tibetan plateau are among the highest places on earth. The Himalayas include 9 of the 10 tallest mountains in the world, and, at an average elevation of 5,000 m above sea level (masl), the Tibetan Plateau is ∼25% higher than the Peruvian altiplano, the next highest plateau in the world (1, 4, 5). Genome-wide studies of the geographic structure of modern populations clearly point to the Himalayas as a barrier to gene flow between East Asians and Western Eurasians (6). However, there is also extensive evidence of cultural and linguistic diversity across the Himalayan arc, which hints at a long history of cross-regional contact (7, 8).

Currently available archaeological and osteological data and genetic data from modern-day populations have been used to support contrasting hypotheses of South Asian (9⇓–11), Central Asian (12), lowland Southeast Asian (13), and high-altitude East Asian (7, 8) origins for the earliest Himalayan inhabitants, and there is likewise little agreement regarding subsequent regional population history (12, 14). Successful permanent habitation of high-altitude environments requires numerous physiological adaptations, and recent genetic studies have identified robust signals of positive natural selection underlying adaptations to hypoxia in Tibetans (15⇓–17) and in the Sherpa (18), an ethnic group that migrated from the eastern Tibetan plateau to Nepal 400–600 y ago (ya) (19). Tibetans and Sherpa are the only two present-day high-altitude East Asian ethnic groups that have been studied to date, using genome-wide markers. Although the Tibetan plateau has been the subject of intense study regarding its population history and high-altitude adaptation (15, 20⇓–22), far less is known about the much later colonization of the surrounding high transverse valleys along the Himalayan arc. Elucidating this history is important because these valleys have long served as natural corridors and trade routes connecting the Tibetan plateau to the Indian subcontinent. Moreover, the role of adaptation to high-altitude hypoxia in the initial colonization of these valleys and in the subsequent gene flow through them is entirely unexplored.

The Annapurna Conservation Area (ACA) of Upper Mustang, Nepal (Fig. 1) is a major high-elevation corridor (2,800–4,500 masl) that includes the earliest known archaeological sites containing preserved human remains in a Himalayan transverse valley (23). Foodstuffs within ACA prehistoric funerary contexts include domesticates of both West Asian (e.g., barley, buckwheat, lentils, peas, sheep, and goats) and East Asian (e.g., rice) origin (24). In addition to locally made utilitarian wares, prestige objects include copper ornaments and vessels, carnelian beads, marine shell pendants, and faience suggestive of a strong South Asian connection, as well as bamboo baskets, mats, and cups and wooden furniture and design motifs suggesting contact with Central Asia and Xinjiang (12, 25). Later periods after ca. 1,750 y before present (yBP) also include Chinese silk and glass beads from Sassania (modern-day Iran) and central and far southern India, as well as gold and silver masks that resemble those found in western Tibet, Ladahk, and Kyrgyzstan (14). Finally, local mortuary practices initially resemble those observed in northern Xinjiang, but after ca. 1,500 yBP include defleshing, a practice that may have multiple origins but is primarily associated with Western Asian cultures (23). Therefore, there is evidence that early populations in the Himalayan transverse valleys were exposed to influences from a remarkably wide geographic extent, from Iran to eastern China.

Fig. 1. Map of the ACA and sampling locations. The ACA (dark gray), located in the Upper Mustang of north-central Nepal and bordering Tibet (Inset), is situated between the Annapurna and Dhaulagiri Massifs of the main Himalayan mountain range. The ACA includes 14 mountains in excess of 6,000 masl, and it contains a single major drainage, the Kali Gandaki River, which originates on the Tibetan plateau. Data from ref. 69.

Given the complexity in material culture, currently available archaeological data cannot determine whether population replacement, cultural diffusion, or both are responsible for these diverse influences. Furthermore, interpretation of linguistic and genetic data from present-day populations is complicated by multiple historically documented Tibetan migrations after ca. 1,300 yBP linked to the rise and fall of the Tibetan Empire, extensive warfare, and the establishment of modern nation states (26, 27). For these reasons, the analysis of ancient human genomes provides a unique and direct means for resolving competing hypotheses regarding the population history of the high Himalayas.

To investigate the peopling and early population history of the ACA, we obtained genome-wide sequences and high-coverage mitochondrial sequences from eight individuals dating to three periods with distinct material culture: Chokhopani (3,150–2,400 yBP), Mebrak (2,400–1,850 yBP), and Samdzong (1,750–1,250 yBP) (Table 1). Following initial population affinity analyses, we then further sequenced the genomes of five individuals to >2× coverage to obtain higher-resolution genome data and increase the coverage of two genes associated with high-altitude adaptation, EGLN1 (egl-9 family hypoxia-inducible factor 1) and EPAS1 (endothelial PAS domain protein 1). Our results are consistent with long-term genetic stability in the region; additionally, genome sequences, uniparental haplotypes, and high-altitude adaptive alleles support a high-altitude East Asian origin for these prehistoric Himalayan populations.

Table 1. ACA dental samples investigated in this study

Discussion The role of geography in migration and population structure has been a central topic in population genetics studies of our species and others (43, 44). At a genetic level, the Himalayan arc delineates a sharp genetic barrier between South Asian and East Asian populations, a striking anomaly against a general isolation-by-distance pattern of human population structure across much of Eurasia (6). The asymmetric topography of the Himalayan massif, bordered by a high-elevation plateau to the north and lowland plains to the south, is reflected in the current regional genetic structure of human populations, evidenced by autosomal and Y chromosome STR (short tandem repeat) frequencies in modern Nepalese populations (7, 8). Our results suggest that the Himalayas have long served as a remarkably resistant barrier to northward but not southward gene flow and that this genetic boundary has been stable for at least the last three millennia. The eight ancient ACA individuals analyzed in this study exhibit a strong and consistent genetic affiliation to contemporary East Asian, and especially to high-altitude East Asian (Sherpa and Tibetan), populations. Therefore, previous proposals of a South Asian (9⇓–11), Central Asian (12), or low-elevation Southeast Asian (13) origin of the first inhabitants of this region, as well as speculation regarding subsequent prehistoric population replacement or large-scale admixture with lowland populations (12, 14, 45), are not supported. It is interesting that the high-altitude barrier to migration seems to be more permeable from the northern, as opposed to the southern, side of the Himalayan arc. One can speculate that this disparity may be due to the topographical differences between the northern and southern sides of the arc: the altitudinal gradient is much more gradual in the north than in the south. Thus, ascending populations on the north side may have been able to stay at intermediate altitudes for extended periods of time, allowing for acclimatization and the accumulation of genetic and subsistence adaptations, whereas potential migrants from the south side had no access to such a buffer zone because of the limited availability of sufficient habitable land at intermediate altitudes. This scenario is supported by the archaeological record of the Tibetan plateau. Archaeological data from the northeastern Tibetan plateau indicate an initial occupation ca. 15,000 ya (20, 46), long before the colonization of the high-traverse valleys in the Himalayan arc. Archeological data also support later influences from the East Asian side of the plateau associated with the appearance of agriculture after 5,500 yBP, evidenced by the adoption of Neolithic domesticates, first from East Asia (millets and pigs) and later from West Asia (via Central Asia: barley, sheep, and goats). It has been proposed that these changes enabled populations on the plateau to move to higher and more marginal lands after ca. 4,000 yBP (47), where they may have subsequently served as a source population for the Himalayan transverse valleys. It is beyond the scope of our current study, however, to address whether the spread of agriculture onto the plateau was accompanied by population migration. Genetic adaptation to high altitude also likely facilitated this asymmetric colonization. Accumulation of beneficial mutations is a feature expected for a population gradually adapting to a new environment. Evolution of such beneficial mutations across time provides crucial information for understanding the strength and cause of natural selection. Contemporary high-altitude East Asians on the Tibetan plateau have at least two such genes, EPAS1 and EGLN1, that exhibit strong signatures of positive natural selection as well as functional properties consistent with an adaptive role in high-altitude environments (15, 16, 37, 39, 48). Importantly, we found that the oldest Chokhopani sample (C1) and three later Samdzong individuals (S10, S35, and S41) are most likely homozygous for a derived nonsynonymous allele of the EGLN1 SNP (rs186996510), suggesting that this allele was already common in the founding population. In contrast, derived alleles from the EPAS1 SNPs were observed only in Samdzong individuals, implying an asynchronous evolution of the two genes. However, sequencing of additional ancient samples through time is necessary to reconstruct the adaptive evolution of these and other beneficial mutations in the ACA. Given the unusually high quality of aDNA from the ACA, population-level ancient genome sequencing is likely an achievable goal once additional early archaeological specimens are available. It is tempting to compare this case to archaeogenetic studies in Europe, which suggest that large-scale cultural transitions are frequently associated with massive population movements (49⇓⇓–52). In the Himalayas, we observe two discrete cultural transitions (associated with the Mebrak and Samdzong periods) without evidence of changes in the genetic makeup of the population. One sample from Samdzong (S41) may be an exception in that it is the only one showing some amount of non-East Asian ancestry; however, this proportion is estimated to be small (Fig. 3). Therefore, the predominance of East Asian ancestry in the ACA samples supports our hypothesis that certain topographies, specifically very high altitudes, require a unique set of adaptations, genetic or cultural, that differ from those sufficient for low-altitude migration and colonization. However, because current archaeological data are largely limited to funerary contexts, we caution that the archaeological changes we observe in the ACA may not represent full-scale cultural transitions. In this study, we conducted to our knowledge the first successful ancient DNA investigation of prehistoric Himalayan populations and retrieved high proportions of endogenous aDNA from eight high-altitude ACA individuals dating to three distinct cultural periods spanning 3,150–1,250 yBP. Our population genetic analysis strongly supports the genetic affiliation of prehistoric Himalayan populations with contemporary East Asians and at a subcontinental level suggests a closer affinity with present-day high-altitude East Asians, such as Tibetans and Sherpa, than with low-altitude East Asians. Moreover, this affinity is consistent through time, suggesting that temporal changes in material culture and mortuary behavior largely reflect acculturation or cultural diffusion rather than large-scale gene flow or population replacement from outside East Asia. Finally, we provide to our knowledge the first empirical evidence for differing evolutionary dynamics of selection on the EGLN1 and EPAS1 genes in prehistoric high-altitude populations. Considering the pivotal role of the Himalayan high transverse valleys in connecting far-flung Eurasian populations, as well as the environmental challenges they impose on their inhabitants, our study has deep implications for the understanding of human migration history and adaptation to local environments and for future genetic archaeology studies.

Acknowledgments We thank the Department of Archaeology of the Government of Nepal for permission to conduct archaeological research in the ACA and for access to curated samples, Pete Athans for mountaineering assistance, Jacqueline Eng for osteological assistance, Raul Tito for assistance with sample preparation, Arielle Reivant Munters for technical assistance, John Novembre for helpful discussions during data analysis, and the local residents of the ACA who have assisted our research team during many seasons of fieldwork. Computational resources for data analysis were provided by the Beagle supercomputer at the Computation Institute and by the Center for Research Informatics at the University of Chicago. This work was supported by the US National Science Foundation [Grant ATM/HSD-057620 (to M.S.A.) and Grant BCS-1528698 (to M.S.A., C.W., and A.D.R.)], the US National Institutes of Health [Award R01HL119577 (to A.D.R.)], the European Research Council [Starting Grant 311413 (to M.J.)], the University of Chicago Comprehensive Cancer Center Support Grant P30 CA14599 (to A.D.R.) with particular support from the Genomics Core Facility, the National Geographic Society (M.S.A.), the Henry Luce Foundation (M.S.A.), a Samsung Scholarship (to C.J.), The North Face company (Pete Athans), and the Field Museum.

Footnotes Author contributions: C.M.L., M.S.A., A.D.R., and C.W. designed research; A.T.O., H.M., H.E., C.A.H., R.W.H., M.S.A., and C.W. performed research; M.J. and A.D.R. contributed new reagents/analytic tools; C.J., D.B.W., and C.W. analyzed data; and C.J. and C.W. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. L.B.J. is a guest editor invited by the Editorial Board.

Data deposition: Metagenomic DNA sequences have been deposited in the NCBI Short Read Archive (SRA) (project accession no. SRP065070 and sample accession nos. SRR2751055–SRR2751058, SRR2751060–SRR2751063, SRR2751066–SRR2751067, SRR2751070, SRR2751142, SRR2751148, SRR2751152, SRR3222643, SRR3222649, SRR3222655, SRR3222659, SRR3222661, SRR3222664, SRR3222686, SRR3222749, SRR3222758, SRR3222765, and SRR3222772).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1520844113/-/DCSupplemental.