Final Report Summary - TGOFA (Tracking the Genetic Origins of the First Americans) BackgorundIt is generally accepted that the genetic composition of all extant Amerindian populations is of Asian origin. However, there is little consensus across disciplines on the number of migrations and source populations that gave rise to the first inhabitants of the New World and their descendants. The aim of this project was to use ancient DNA techniques to analyse the genetic diversity of ancient inhabitants of America in order to reveal information about the origins and peopling of the New World.Based on the clear craniofacial discontinuity between the Pleistocene (Paleoamerican) and Holocene (Amerindian) populations, it has been suggested that the Americas were populated twice, from different Asian sources. Under this assumption, a first migration wave originating from Southeast Asia gave rise to the Paleoamericans, whereas all modern Amerindian groups would derive from a second wave of migration originating in Northeast Asia.This scenario has been challenged by genetic studies as well as by the presence of Paleoamerican craniofacial traits in Late Holocene groups, such as the Pericues in Baja California, Mexico, and the very southern populations of Patagonia and Tierra del Fuego. The high morphological affinities among the Fuegans and Pericues with Paleoamericans have led to suggestions that these are a temporal extension of the first colonizers of the Americas.In order to test this hypothesis, DNA from skeletal material of the Pericues in Baja California and the Fuegans in the Chilean and Argentinian Patagonia were genetically profiled by shotgun sequencing. Additionally, a set of 10 pre Columbian Peruvian samples was included to test for possible admixture of populations prior to the European arrival into the Americas. Mummified Amerindian remains from northern Mexico were used as an external control.ResultsThe preliminary results show that the Pericues and Fuegans have an Amerindian genetic composition, both at the nuclear and mitochondrial level. In order to rule out a first migration wave with a different genetic pool as the second one, we would need to directly genotype a Paleoamerican population. Not finding a possible differentiated Paleoamerican genetic component among modern Amerindians can be explained by the Paleoamericans not having contributed to the gene pool of later populations or by a genetic continuity despite the morphological differences. For this reason it is of particular interest to sequence the complete genome of the Anzick material in Montana, USA, radiocarbon dated to ca. 11 000 years old, in concordance with the Clovis occupation in North America. This material is ongoing work and some preliminary results have been obtained but much work is required to complete the sequencing of its genome.Although this data cannot discard a possible first migration originating from Southeast Asia, we can determine with high confidence that the populations studied here are not a temporal extension of this hypothesized first migration, as previously suggested. In my data, I do not observe a genetic differentiation of these two groups when compared to Native American populations, which would be expected if these did not share an ancestral origin with Amerindian groups. This is an ongoing project and more data is to be generated, nevertheless, the observed pattern is not expected to change. It is, however, anticipated that there will be a considerable increase in the resolution, which will allow us to characterize the population history of the Pericues and the Fuegans ethnographic groups and identify how they relate to modern Native American populations.Discussion and Conclusions To the extent of our knowledge, there are no published datasets to date derived from NGS of skeletal material of Native Americans from non-permafrost environments. This has represented a challenge and has complicated data generation and analysis. Nonetheless the ~24 Mb of sequence we successfully recovered from samples with endogenous DNA contents ranging between 0.014 and 0.4%, have allowed us to characterize these two controversial groups as Amerindian, discarding the previous idea of these being the direct descendants of the first colonizers of the New World. However, during the last year there has been great advance in the development of new methodologies and technologies, which we are currently incorporating to our sample set and more data is expected in the following months that will help clarify the two-wave migration model into the Americas with statistical confidence. Craniofacial morphological variations observed between Pleistocene and Holocene populations remain to be studied. Discrepancies between these two could be attributed to different factors such as adaptation to extreme climate environments and changes in diet, as well as convergent evolution or random processes such as genetic drift, etc. Genetic characterization of Paleoamerican remains is imperative in order to understand this peculiar discontinuity.This study has demonstrated that aDNA techniques can work on non-permafrost remains from temperate and desert environments. In addition, an extensive network has been created in the Americas involving different Universities and Museums for collaboration in this project as well as many future ones that are already planned. Maintaining the European Union as a world-class center for archaeological and anthropological science and Ancient DNA.As a result of this project I have now one manuscript at final stage of preparation on insights to the peopling of the Americas, and a second one submitted and currently in review on NGS methods applied to fossil material for whole genome capture. A third one based on Clovis material is at its initial stage of preparation. .