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Neotithic transition in the Iberian Peninsula: reviewing an old question from new technological and computational genome wide approaches

Periodic Reporting for period 1 - NeoGenHeritage (Neotithic transition in the Iberian Peninsula: reviewing an old question from new technological and computational genome wide approaches)

Reporting period: 2015-09-01 to 2017-08-31

In the research project NeoGenHeritage we studied the Neolithic transition in Southern Europe, particularly in the Iberian Peninsula (IP). The Neolithic transition was the most global and revolutionary event in human prehistory, both at the cultural and demographic level, with populations shifting from a subsistence mostly depending on nomadic hunting and gathering to settled communities based on farming. The first archaeological evidence of Neolithic farming is in the Near East around 12000 years before present (yBP) and by 5000 yBP agriculture had spread all over Europe, replacing the Mesolithic lifestyle. Whether this major change was mediated by the diffusion of people or ideas, is a hotly debated question in archaeology, anthropology and population genetics.
In recent years, ancient DNA (aDNA) studies have strongly contributed to shed light on this question by directly describing genetic diversity in past human populations. Thanks to the application of cutting edge technologies such as NGS (next generation sequencing) and DNA hybridization capture, the recovery of whole nuclear genomes from ancient remains has became feasible. The number of ancient human individuals analysed at the nuclear DNA level has grown from a few tens to several hundreds in just the last two years.
However, there are still several problems in paleogenome studies. They are related with the paucity of data from some geographic regions and/or to the low quality/coverage of the genome data available. Also, aDNA studies are now moving from analysing individual mitogenomes and partial nuclear sequences, to the analysis of complete genomes at the population scale. This implies a radical increase in complexity, along with the challenge of incorporating the peculiarities of aDNA (molecular damage, temporal dimension, etc) into the statistical framework of population-genomic analyses.
In our MSCA we have worked to improve the standards in aDNA studies by generating high quality ancient genome data and exploiting the information contained in them by advanced biostatistical inferential methods. We have combined these laboratory and computational approaches to:
- Generate whole nuclear genomes from prehistoric samples in the Iberian Peninsula, covering the time interval between the Mesolithic and Bronze Age. Having been the last region of Europe reached by the Neolithic diffusion, the IP offers a particularly complex (and challenging) opportunity for understanding the relative role of migration and cultural changes at the onset of the Neolithic.
- Test hypotheses about the changes in the genetic landscape of the Iberian populations over time.
- Investigate the age of genetic variants, which have been proposed as target of recent selective events in human evolution.
By the achivement of this research objectives, our MSCA is contributing to a better understanding of human demographic dynamics in southern Europe and of the impact of cultural changes in recent human evolution. A deeper understanding of the origins and evolution of human diversity in Europe is fundamental for designing immigration policies, and for dismissing racist or xenophobic ideas which are on the rise in the public debate. Furthermore, the data that have been generated and the training followed by the researcher, will contribute to reinforce the world leading role of Europe in aDNA research.
A total of 45 ancient human remains were sampled in the Iberian Peninsula for this MSCA project. Their genome analysis involved a wet-lab stage for the generation of the genomic sequences; and an in-silico stage for their analysis.
The wet-lab stage was carried out during a 6-month secondment in the University of Potsdam (Germany). DNA was extracted from petrous bone and teeth, and then built into NGS libraries (Figure1). The libraries with endogenous DNA content > 30% were selected for shotgun sequencing, while those with relatively low endogenous DNA (0.81-19%) were subjected to whole nuclear capture. We developed an optimized capture protocol that yields DNA enrichments of 2-9 fold. Finally, using a combination of shotgun and capture approaches we reconstructed the complete mitogenomes for 23 ancient samples and whole nuclear genomes (3.3 to 6.25x coverage) for 8 individuals, ranging in age from 8500 to 3300 yBP, i.e. from the Mesolithic to the early Bronze age.
At the in-silico stage, the team in the host institution, the University of Ferrara, provided me with the material resources and theoretical training needed to place these genomes in the context of human genome variation. At the same time, I brought to the team my expertise in aDNA analysis and NGS data processing from ancient remains, both at the stage of mapping and quality filtering.
The newly generated genomes were merged and analysed together with: (1) a dataset of 257 ancient individuals from Europe and the Near East, compiled from public databases; (2) a dataset of 1267 modern genomes from all over the world. By PCA and ADMIXTURE two major genome components were found in our ancient samples: a West Hunter-gatherer (WHG) and an Early Neolithic (EN) components. Formal tests for admixture revealed that the Northern samples share more genetic drift with the Iberian Mesolithic genomes, than the contemporary samples from the South do. This may suggest either the existence of a genetic structure in Iberia before the arrival of the Neolithic farmers, or a higher rate of admixture in the South than in the North, diluting the local WHG component. The anlysis of complete mitogenomes revealed in southern Iberia a typically African sequence, never found in ancient samples before. This is the first genetic evidence of movement of people from Africa into Southern Spain and opens new perspectives about prehistoric migration corridors into Europe.
Part of my research during the MSCA has already been published in scientific journals (Gonzalez-Fortes et al 2017. Current Biology) and conferences (EMBO Workshop, Napoli 2017; SIBE 2017, Rome). At the moment I am drafting two manuscripts reporting the main findings described in above. We have also been active at communicating our research to the general public, by taking part in outreach activities at museums, science festivals and secondary schools.
The last two years have seen an amazing increase of aDNA data production. However, most of the paleogenome data that have been published came from capture experiments and have been sequenced at low coverage (0.1-1x coverage). Both factors severely limit the inferential power of the analyses based on such data. Through our MSCA (i) we have generated high quality genomes that will be available to the scientific community for further studies, and (ii) we have designed a nuclear capture protocol performing up to 4 times better in terms of enrichment, than any other capture protocol published so far for aDNA.
Our results, together with those of other recently published aDNA studies, contribute to show that the genetic background of modern Europeans is the result of multiple waves of prehistoric and historic migrations, leading to both cultural and genetic changes. In a time when xenofobic feelings are on the rise in Europe, studies like ours will hopefully contribute to raising awareness on the scientific fact that we are all descended from immigrants, and giving strong factual arguments against racist fallacies that are threatening our peaceful coexistence.