00 MONTH 2016 | VOL 000 | NATURE | 1 LETTER doi:10.1038/nature17405 Nuclear DNA sequences from the Middle Pleistocene Sima de los Huesos hominins Matthias Meyer 1 , Juan-Luis Arsuaga 2,3 , Cesare de Filippo 1 , Sarah Nagel 1 , Ayinuer Aximu-Petri 1 , Birgit Nickel 1 , Ignacio Martínez 2,4 , Ana Gracia 2,4 , José María Bermúdez de Castro 5,6 , Eudald Carbonell 7,8 , Bence Viola 9 , Janet Kelso 1 , Kay Prüfer 1 & Svante Pääbo 1 A unique assemblage of 28 hominin individuals, found in Sima de los Huesos in the Sierra de Atapuerca in Spain, has recently been dated to approximately 430,000 years ago 1 . An interesting question is how these Middle Pleistocene hominins were related to those who lived in the Late Pleistocene epoch, in particular to Neanderthals in western Eurasia and to Denisovans, a sister group of Neanderthals so far known only from southern Siberia. While the Sima de los Huesos hominins share some derived morphological features with Neanderthals, the mitochondrial genome retrieved from one individual from Sima de los Huesos is more closely related to the mitochondrial DNA of Denisovans than to that of Neanderthals 2 . However, since the mitochondrial DNA does not reveal the full picture of relationships among populations, we have investigated DNA preservation in several individuals found at Sima de los Huesos. Here we recover nuclear DNA sequences from two specimens, which show that the Sima de los Huesos hominins were related to Neanderthals rather than to Denisovans, indicating that the population divergence between Neanderthals and Denisovans predates 430,000 years ago. A mitochondrial DNA recovered from one of the specimens shares the previously described relationship to Denisovan mitochondrial DNAs, suggesting, among other possibilities, that the mitochondrial DNA gene pool of Neanderthals turned over later in their history. When modern humans spread out of Africa and the Near East some 75,000–50,000 years ago, at least two archaic hominin groups, Neanderthals and Denisovans, inhabited Eurasia. While Neanderthals are known from an abundant fossil record in Europe and western and central Asia, Denisovan remains are currently only known from the Altai Mountains in southern Siberia 3,4 . However, Denisovan ancestry is detected in present-day human populations from Oceania, main- land Asia and in Native Americans 5 , suggesting that they were once more widespread. High-quality genome sequences recovered from one Neanderthal and one Denisovan show that they were more closely related to each other than to modern humans 6,7 and that they diverged from a common ancestral population between 381,000 and 473,000 years ago 7 if a mutation rate of 0.5 × 10 -9 per site per year is used. The Middle Pleistocene fossils from Sima de los Huesos (SH) are relevant for the question of when and where the ancestral populations of Neanderthals and Denisovans lived, but their relationship to these later archaic groups is unclear. They share some derived dental and cranial features with Late Pleistocene Neanderthals, for example, a midfacial prognathism and some aspects of the supraorbital torus, the occipital bone and the glenoid cavity 1,8 . In apparent contrast to this, the mitochondrial (mt)DNA determined from one SH individual is more similar to an mtDNA ancestral to Denisovan than to Neanderthal mtDNAs 2 . However, the mtDNA is inherited as a single unit from mothers to offspring and does not necessarily reflect the overall relationship of individuals and populations. To clarify the relationships of the SH hominins to Neanderthals and Denisovans, we therefore set out to retrieve nuclear DNA from SH hominins. However, DNA pres- ervation in these fossils is poor owing to their great age. Femur XIII, from which the SH mtDNA genome was sequenced, contains only small amounts of highly degraded endogenous DNA (30–45 base pairs (bp)) in a large excess of microbial DNA. To reconstruct its mtDNA genome, almost 2 g of bone had to be used to produce DNA libraries from which mtDNA fragments were isolated by hybridization capture. Furthermore, because of the presence of modern human DNA contam- ination, putatively endogenous sequences had to be identified on the basis of the presence of C to T substitutions that accumulate at the ends of DNA fragments over time owing to cytosine deamination 9 , which are largely absent in recent human DNA that contaminates fossils 10,11 . To retrieve nuclear DNA sequences from femur XIII, we generated approximately 2.6 billion sequence reads from the library with the highest frequency of terminal C to T substitutions (library A2021 (ref. 2)). In addition, between 600 million and 900 million reads were col- lected from each of four new specimens that were recovered from the site for molecular analyses (Extended Data Table 1). These were an incisor (AT-5482), a femur fragment (AT-5431), a molar (AT-5444) and a scapula (AT-6672). In addition to sequencing random fragments from these specimens, we also isolated mtDNA fragments from the four new specimens by hybridization capture. Between 1,419 and 3,742 unique mtDNA frag- ments of 30 bp or longer were retrieved (Extended Data Table 2). To investigate whether they represented endogenous DNA or present-day human contamination, we determined the frequency of C to T sub- stitutions relative to the human mitochondrial genome at each posi- tion in the fragments. The fragments from femur AT-5431 carry 44% C to T substitutions at the 5′ ends and 41% at the 3′ ends, compati- ble with the presence of endogenous ancient mtDNA. The other three specimens do not show discernible evidence of deamination-induced substitutions. Because there were too few DNA fragments to reconstruct the complete mtDNA genome of femur AT-5431, we restricted further analyses to ‘diagnostic’ positions in the mtDNA genome where each lineage in the mtDNA tree differs from the other hominin lineages and from the chimpanzee. At positions where modern humans differ from Neanderthals, Denisovans, SH femur XIII and the chimpanzee, 41% (17 out of 41) of the mtDNA fragments share the modern human state, indicating that they are derived from present-day human contamination 1 Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, 04103 Leipzig, Germany. 2 Centro de Investigación Sobre la Evolución y Comportamiento Humanos, Universidad Complutense de Madrid–Instituto de Salud Carlos III, 28029 Madrid, Spain. 3 Departamento de Paleontología, Facultad de Ciencias Geológicas, Universidad Complutense de Madrid, 28040 Madrid, Spain. 4 Área de Paleontología, Departamento de Geografía y Geología, Universidad de Alcalá, Alcalá de Henares, 28871 Madrid, Spain. 5 Centro Nacional de Investigación sobre la Evolución Humana, Paseo Sierra de Atapuerca, 09002 Burgos, Spain. 6 Department of Anthropology, University College London, 14 Taviton Street, London WC1H 0BW, UK. 7 Institut Català de Paleoecologia Humana i Evolució Social, C/Marcel·lí Domingo s/n (Edifici W3), Campus Sescelades, 43007 Tarragona, Spain. 8 Àrea de Prehistòria, Departament d’Història i Història de l’Art, Universitat Rovira i Virgili, Facultat de Lletres, Avinguda de Catalunya, 35, 43002 Tarragona, Spain. 9 Department of Anthropology, University of Toronto, 19 Russell Street, Toronto, Ontario M5S 2S2, Canada.