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Abstract The genetic prehistory of human populations in Central America is largely unexplored leaving an important gap in our knowledge of the global expansion of humans. We report genome-wide ancient DNA data for a transect of twenty individuals from two Belize rock-shelters dating between 9,600-3,700 calibrated radiocarbon years before present (cal. BP). The oldest individuals (9,600-7,300 cal. BP) descend from an Early Holocene Native American lineage with only distant relatedness to present-day Mesoamericans, including Mayan-speaking populations. After ~5,600 cal. BP a previously unknown human dispersal from the south made a major demographic impact on the region, contributing more than 50% of the ancestry of all later individuals. This new ancestry derived from a source related to present-day Chibchan speakers living from Costa Rica to Colombia. Its arrival corresponds to the first clear evidence for forest clearing and maize horticulture in what later became the Maya region. 

Maize is a cultigen of global economic importance, but when it first became a staple grain in the Americas, was unknown and contested. Here, we report direct isotopic dietary evidence from 52 radiocarbon-dated human skeletons from two remarkably well-preserved rock-shelter contexts in the Maya Mountains of Belize spanning the past 10,000 years. Individuals dating before ~4700 calendar years before present (cal B.P.) show no clear evidence for the consumption of maize. Evidence for substantial maize consumption (~30% of total diet) appears in some individuals between 4700 and 4000 cal B.P. Isotopic evidence after 4000 cal B.P. indicates that maize became a persistently used staple grain comparable in dietary significance to later maize agriculturalists in the region (>70% of total diet). These data provide the earliest definitive evidence for maize as a staple grain in the Americas.