Neolithic millet farmers contributed to the permanent settlement of the Tibetan Plateau by adopting barley agriculture.

The permanent human settlement of the Tibetan Plateau (TP) has been suggested to have been facilitated by the introduction of barley agriculture ∼3.6 kilo-years ago (ka). However, how barley agriculture spread onto the TP remains unknown. Given that the lower altitudes in the northeastern TP were occupied by millet cultivators from 5.2 ka, who also adopted barley farming ∼4 ka, it is highly possible that it was millet farmers who brought barley agriculture onto the TP ∼3.6 ka. To test this hypothesis, we analyzed mitochondrial DNA (mtDNA) from 8277 Tibetans and 58 514 individuals from surrounding populations, including 682 newly sequenced whole mitogenomes. Multiple lines of evidence, together with radiocarbon dating of cereal remains at different elevations, supports the scenario that two haplogroups (M9a1a1c1b1a and A11a1a), which are common in contemporary Tibetans (20.9%) and were probably even more common (40-50%) in early Tibetans prior to historical immigrations to the TP, represent the genetic legacy of the Neolithic millet farmers. Both haplogroups originated in northern China between 10.0-6.0 ka and differentiated in the ancestors of modern Tibetans ∼5.2-4.0 ka, matching the dispersal history of millet farming. By showing that substantial genetic components in contemporary Tibetans can trace their ancestry back to the Neolithic millet farmers, our study reveals that millet farmers adopted and brought barley agriculture to the TP ∼3.6-3.3 ka, and made an important contribution to the Tibetan gene pool.