The human occupation history of Southeast Asia (SEA) remains heavily debated. Current evidence suggests that SEA was occupied by Hòabìnhian hunter-gatherers until ~4000 years ago, when farming economies developed and expanded, restricting foraging groups to remote habitats. Some argue that agricultural development was indigenous; others favor the "two-layer" hypothesis that posits a southward expansion of farmers giving rise to present-day Southeast Asian genetic diversity. By sequencing 26 ancient human genomes (25 from SEA, 1 Japanese Jōmon), we show that neither interpretation fits the complexity of Southeast Asian history: Both Hòabìnhian hunter-gatherers and East Asian farmers contributed to current Southeast Asian diversity, with further migrations affecting island SEA and Vietnam. Our results help resolve one of the long-standing controversies in Southeast Asian prehistory.

Gigantopithecus blacki was a giant hominid that inhabited densely forested environments of Southeast Asia during the Pleistocene 1. Its evolutionary relationships to other great ape species, and their divergence during the Middle and Late Miocene (16-5.3 Mya), remains disputed 2,3. Hypotheses regarding relationships between Gigantopithecus and extinct and extant hominids are difficult to substantiate because of its highly derived dentognathic morphology and the absence of cranial and post-cranial remains 1,3-6. Therefore, proposed hypotheses on the phylogenetic position of Gigantopithecus among hominids have been wide-ranging, but none have received independent molecular validation. We retrieved dental enamel proteome sequences from a 1.9 million years (Mya) old Gigantopithecus blacki molar found in Chuifeng Cave, China 7,8. The thermal age of these protein sequences is approximately five times older than any previously published mammalian proteome or genome. We demonstrate that Gigantopithecus is a sister clade to orangutans (genus Pongo) with a common ancestor about 10-12 Mya, implying that the Gigantopithecus divergence from Pongo is part of the Miocene radiation of great apes. Additionally, we hypothesize that the expression of alpha-2-HS-glycoprotein (AHSG), which has not been observed in enamel proteomes previously, had a role in the biomineralization of the thick enamel crowns that characterize the large molars in the genus 9,10. The survival of an Early Pleistocene dental enamel proteome in the subtropics further expands the scope of palaeoproteomic analysis into geographic areas and time periods previously considered incompatible with genetic preservation. Gigantopithecus blacki is an extinct, potentially giant hominid species that once inhabited Asia. It was first discovered and identified by von Koenigswald in 1935 when he described an isolated tooth that he found in a Hong Kong drugstore 11. The entire Gigantopithecus blacki fossil record, dated between the Early Pleistocene (~2.0 Mya) and the late Middle Pleistocene (~0.3 Mya 12), includes thousands of teeth and four partial mandibles from subtropical Southeast Asia 1,13,14. All the known Gigantopithecus blacki localities are situated in southern China, stretching from Longgupo Cave, just south of the Yangtze River, to the Xinchong Cave on Hainan Island, and, possibly, into northern Vietnam and Thailand 15,16. To address the evolutionary relationships between Gigantopithecus and extant hominoids, we performed protein extractions on dentine and enamel samples of a single molar (CF-B-16) found in Chuifeng Cave, China, that is morphologically assigned to Gigantopithecus blacki 7,8. The site is dated using multiple approaches to 1.9±0.2 Mya (Extended Data Figs. 1, 2). Enamel and dentine samples were processed using recently established digestion-free protocols optimized for extremely degraded ancient proteomes 17 (Methods). Enamel demineralization was replicated using two different acids, trifluoroacetic acid (TFA) and hydrochloric acid (HCl). Welker et ...