Ancient DNA Analysis of 8000 B.C. Near Eastern Farmers Supports an Early Neolithic Pioneer Maritime Colonization of Mainland Europe through Cyprus and the Aegean Islands Eva Ferna ´ ndez 1,2 *, Alejandro Pe ´ rez-Pe ´ rez 3 , Cristina Gamba 2 , Eva Prats 4 , Pedro Cuesta 5 , Josep Anfruns 6 , Miquel Molist 6 , Eduardo Arroyo-Pardo 2 , Daniel Turbo ´n 3 1 Research Centre in Evolutionary Anthropology and Paleoecology, Liverpool John Moores University, Liverpool, United Kingdom, 2 Laboratorio de Gene ´tica Forense y Gene ´tica de Poblaciones, Dpto. Toxicologı ´a y Legislacio ´ n Sanitaria, Facultad de Medicina, Universidad Complutense de Madrid, Madrid, Spain, 3 Dpto. Biologı ´a Animal- Unidad de Antropologı ´a, Facultad de Biologı ´a, Universitat de Barcelona, Barcelona, Spain, 4 Centro de Investigacio ´ n y Desarrollo, Consejo Superior de Investigaciones Cientı ´ficas, Barcelona, Spain, 5 Dpto. de Apoyo a la Investigacio ´ n, Servicios informa ´ticos de la Universidad Complutense de Madrid, Madrid, Spain, 6 Dep. Prehistoria, Facultad de Filosofı ´a y Letras, Universitat Auto ´ noma de Barcelona, Bellaterra, Barcelona, Spain Abstract The genetic impact associated to the Neolithic spread in Europe has been widely debated over the last 20 years. Within this context, ancient DNA studies have provided a more reliable picture by directly analyzing the protagonist populations at different regions in Europe. However, the lack of available data from the original Near Eastern farmers has limited the achieved conclusions, preventing the formulation of continental models of Neolithic expansion. Here we address this issue by presenting mitochondrial DNA data of the original Near-Eastern Neolithic communities with the aim of providing the adequate background for the interpretation of Neolithic genetic data from European samples. Sixty-three skeletons from the Pre Pottery Neolithic B (PPNB) sites of Tell Halula, Tell Ramad and Dja’de El Mughara dating between 8,700–6,600 cal. B.C. were analyzed, and 15 validated mitochondrial DNA profiles were recovered. In order to estimate the demographic contribution of the first farmers to both Central European and Western Mediterranean Neolithic cultures, haplotype and haplogroup diversities in the PPNB sample were compared using phylogeographic and population genetic analyses to available ancient DNA data from human remains belonging to the Linearbandkeramik-Alfo ¨ldi Vonaldiszes Kera ´mia and Cardial/Epicardial cultures. We also searched for possible signatures of the original Neolithic expansion over the modern Near Eastern and South European genetic pools, and tried to infer possible routes of expansion by comparing the obtained results to a database of 60 modern populations from both regions. Comparisons performed among the 3 ancient datasets allowed us to identify K and N-derived mitochondrial DNA haplogroups as potential markers of the Neolithic expansion, whose genetic signature would have reached both the Iberian coasts and the Central European plain. Moreover, the observed genetic affinities between the PPNB samples and the modern populations of Cyprus and Crete seem to suggest that the Neolithic was first introduced into Europe through pioneer seafaring colonization. Citation: Ferna ´ ndez E, Pe ´ rez-Pe ´ rez A, Gamba C, Prats E, Cuesta P, et al. (2014) Ancient DNA Analysis of 8000 B.C. Near Eastern Farmers Supports an Early Neolithic Pioneer Maritime Colonization of Mainland Europe through Cyprus and the Aegean Islands. PLoS Genet 10(6): e1004401. doi:10.1371/journal.pgen.1004401 Editor: Scott M. Williams, Dartmouth College, United States of America Received September 5, 2013; Accepted April 9, 2014; Published June 5, 2014 Copyright: ß 2014 Ferna ´ndez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work presented here was funded with the following research projects: BMC2002-2741, CGL2006-07828 and CGL2009-07959 (Ministerio de Ciencia e Innovacio ´ n, Spanish Government), CCG08-UCM/BIO-3938 (Comunidad Auto ´ noma de Madrid), INCO-MED-ICA3-CT-2002-10022 (European Commission) and I&D- PTDC/HAH/64548/2006 (Fundac ¸a ˜ o para a Cie ˆncia e a Tecnologia, Portugal). This work was supported by a Post-Doctoral fellowship from Fundac ¸a ˜ o para a Cie ˆncia e a Tecnologia (Portugal) (Ref. SFRH/BPD/69426/2010) and a research contract Juan de la Cierva from the Spanish Government and the European Social Fund (Ref. JCI-2007-56-261) to EF and with a Pre-Doctoral FPU grant (Ref. AP2006-01586) from the Spanish Government to CG. Excavations at the archaeological site of Tell Halula were partially funded with the project HUM2010-18612 (Ministerio de Ciencia e Innovacio ´ n, Spanish Government). Liverpool John Moores University, Universitat the Barcelona, Universidad Complutense de Madrid and Universitat Autonoma de Barcelona have jointly funded the open access publication costs of this article. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: E.FernandezDominguez@ljmu.ac.uk Introduction The term ‘‘Neolithic’’ refers to the profound cultural and technical changes that accompanied the transition from a hunter- gatherer subsistence economy to an agro-pastoral producing system [1]. The first Neolithic societies originated 12 to 10 thousand years ago in a region of the Near East traditionally known as the ‘‘Fertile Crescent’’ [2]. From this region the Neolithic technology rapidly expanded to Anatolia reaching the rest of Europe in less than 3,000 years by following two main routes linked to different archaeological cultural complexes. The Danubian route, associated to the Linearbandkeramic (LBK) cultural complex, brought the Neolithic to the central European plains and from there to the British Islands and Scandinavia (Funnel Beaker Cultural Complex) while the Mediterranean one, associated to the Cardial-Impressa cultural complex, spread it along the Mediterranean coast up to the Atlantic fac ¸ade of Iberia [3]. PLOS Genetics | www.plosgenetics.org 1 June 2014 | Volume 10 | Issue 6 | e1004401