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Indigenous peoples have occupied the island of Puerto Rico since at least 3000 BC. Due to the demographic shifts that occurred after European contact, the origin(s) of these ancient populations, and their genetic relationship to present-day islanders, are unclear. We use ancient DNA to characterize the population history and genetic legacies of precontact Indigenous communities from Puerto Rico. Bone, tooth, and dental calculus samples were collected from 124 individuals from three precontact archaeological sites: Tibes, Punta Candelero, and Paso del Indio. Despite poor DNA preservation, we used target enrichment and high-throughput sequencing to obtain complete mitochondrial genomes (mtDNA) from 45 individuals and autosomal genotypes from two individuals. We found a high proportion of Native American mtDNA haplogroups A2 and C1 in the precontact Puerto Rico sample (40% and 44%, respectively). This distribution, as well as the haplotypes represented, supports a primarily Amazonian South American origin for these populations and mirrors the Native American mtDNA diversity patterns found in present-day islanders. Three mtDNA haplotypes from precontact Puerto Rico persist among Puerto Ricans and other Caribbean islanders, indicating that present-day populations are reservoirs of precontact mtDNA diversity. Lastly, we find similarity in autosomal ancestry patterns between precontact individuals from Puerto Rico and the Bahamas, suggesting a shared component of Indigenous Caribbean ancestry with close affinity to South American populations. Our findings contribute to a more complete reconstruction of precontact Caribbean population history and explore the role of Indigenous peoples in shaping the biocultural diversity of present-day Puerto Ricans and other Caribbean islanders.

The Caribbean was one of the last regions of the Americas to be settled by humans, but where they came from and how and when they reached the islands remain unclear. We generated genome-wide data for 93 ancient Caribbean islanders dating between 3200 and 400 calibrated years before the present and found evidence of at least three separate dispersals into the region, including two early dispersals into the Western Caribbean, one of which seems connected to radiation events in North America. This was followed by a later expansion from South America. We also detected genetic differences between the early settlers and the newcomers from South America, with almost no evidence of admixture. Our results add to our understanding of the initial peopling of the Caribbean and the movements of Archaic Age peoples in the Americas.