SummaryWe report genome-wide ancient DNA from 49 individuals forming four parallel time transects in Belize, Brazil, the Central Andes, and the Southern Cone, each dating to at least ∼9,000 years ago. The common ancestral population radiated rapidly from just one of the two early branches that contributed to Native Americans today. We document two previously unappreciated streams of gene flow between North and South America. One affected the Central Andes by ∼4,200 years ago, while the other explains an affinity between the oldest North American genome associated with the Clovis culture and the oldest Central and South Americans from Chile, Brazil, and Belize. However, this was not the primary source for later South Americans, as the other ancient individuals derive from lineages without specific affinity to the Clovis-associated genome, suggesting a population replacement that began at least 9,000 years ago and was followed by substantial population continuity in multiple regions.

The purpose of this paper is to contribute to dietary studies of Early-Middle Holocene Pampean hunter-gatherers, and to evaluate the intra-group dietary differences. Results from stable carbon isotope analysis of the organic and inorganic fractions of bone from 26 human remains from the sites Arroyo Seco 2 (Interserrana area) and Monte Hermoso 1 (Atlantic coast) are presented and discussed. Isotopic values from archaeofauna -including megafauna and current fauna -are also reported. d 13 C COL isotopic values on human remains from these sites suggest the existence of different diets in Early-Middle Holocene groups, including individuals consuming mainly marine resources, individuals with a terrestrial diet based on C 3 plants and/ or the intake of C 3 -consuming herbivores, and finally, individuals with a mixed diet. d 13 C COL data from human remains are consistent with the isotopic results obtained for the most abundant faunal remains at the sites (guanaco, Pampean deer, and seal). Apatite analysis was carried out on 22 individuals from AS2. The apatite results suggest C 4 plants or marine food consumption or a combination of both.

Summary The Southern Cone of South America (SCSA) is a key region for investigations about the peopling of the Americas. However, little is known about the eastern sector, the Argentinian Pampas. We analyzed 18 mitochondrial genomes—7 of which are novel—from human skeletal remains from 3 Early to Late Holocene archaeological sites. The Pampas present a distinctive genetic makeup compared to other Middle to Late Holocene pre-Columbian SCSA populations. We also report the earliest individuals carrying SCSA-specific mitochondrial haplogroups D1j and D1g from Early and Middle Holocene, respectively. Using these deep calibration time points in Bayesian phylogenetic reconstructions, we suggest that the first settlers of the Pampas were part of a single and rapid dispersal ∼15,600 years ago. Finally, we propose that present-day genetic differences between the Pampas and the rest of the SCSA are due to founder effects, genetic drift, and a partial population replacement ∼9,000 years ago.