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.

Isotopic and molecular analysis on human, fauna and pottery remains can provide valuable new insights into the diets and subsistence practices of prehistoric populations. These are crucial to elucidate the resilience of social-ecological systems to cultural and environmental change. Bulk collagen carbon and nitrogen isotopic analysis of 82 human individuals from mid to late Holocene Brazilian archaeological sites (∼6,700 to ∼1,000 cal BP) reveal an adequate protein incorporation and, on the coast, the continuation in subsistence strategies based on the exploitation of aquatic resources despite the introduction of pottery and domesticated plant foods. These results are supported by carbon isotope analysis of single amino acid extracted from bone collagen. Chemical and isotopic analysis also shows that pottery technology was used to process marine foods and therefore assimilated into the existing subsistence strategy. Our multidisciplinary results demonstrate the resilient character of the coastal economy to cultural change during the late Holocene in southern Brazil.

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Small‐scale fisheries provide food and livelihoods for thousands of people along the Brazilian coastline. However, considerable uncertainties still surround the extent to which artisanal and subsistence fisheries contribute to the total of national landings and their historical ecological significance. Fisheries monitoring is deficient in Brazil, and historical records are limited to irregular accounts spanning the last few decades, while this coastline has supported human populations for at least 6,000 years. Here, we estimate pre‐Columbian subsistence catches for a large subtropical estuary in southern Brazil. Our results suggest that prehistoric populations may have extracted volumes of fish biomass higher than or comparable with historical subsistence fisheries in the region, and that the latter is likely underestimated. If a long‐term perspective is required to evaluate the current economic value and status of fisheries in subtropical and tropical South America, this should go beyond the historical time interval and integrate the contribution of pre‐Columbian archaeology.