Although an inverse relationship is expected in ancient DNA samples between the number of surviving DNA fragments and their length, ancient DNA sequencing libraries are strikingly deficient in molecules shorter than 40 bp. We find that a loss of short molecules can occur during DNA extraction and present an improved silica-based extraction protocol that enables their efficient retrieval. In combination with single-stranded DNA library preparation, this method enabled us to reconstruct the mitochondrial genome sequence from a Middle Pleistocene cave bear (Ursus deningeri) bone excavated at Sima de los Huesos in the Sierra de Atapuerca, Spain. Phylogenetic reconstructions indicate that the U. deningeri sequence forms an early diverging sister lineage to all Western European Late Pleistocene cave bears. Our results prove that authentic ancient DNA can be preserved for hundreds of thousand years outside of permafrost. Moreover, the techniques presented enable the retrieval of phylogenetically informative sequences from samples in which virtually all DNA is diminished to fragments shorter than 50 bp.T race amounts of DNA can occasionally survive the decomposition of organic matter for long periods of time after the death of an organism. However, the retrieval of these ancient DNA molecules is severely impeded by their small size. DNA fragmentation is at least partly driven by depurination (1, 2), a continually occurring process. It is thus predicted that the degree of DNA fragmentation increases with sample age. This correlation has, in fact, been established in a recent study that analyzed samples of different ages from the same archeological sites (3), but the correlation vanishes in comparisons across different sites (4). The important role of environmental conditions, especially temperature, in DNA preservation is well recognized and reflected-for example, in the concept of thermal age (5). Unsurprisingly, permafrost environments have yielded the oldest credible records of DNA survival, including short stretches of plant and invertebrate DNA with an estimated age of up to 800,000 y that were amplified by PCR from Artic ice cores (6, 7) and the genome sequence of a 700,000-y-old horse published recently (8). More temperate environments have yielded many DNA sequences from the Holocene and the Late Pleistocene, some as old as ∼100,000 (9) or ∼120,000 y (10), but only a single study has convincingly raised the possibility of DNA survival extending far into the Middle Pleistocene outside of permafrost (11). In this study, short PCR products of ∼50 bp were retrieved from several bone samples of Middle Pleistocene cave bears from European caves, the oldest coming from the site of Sima de los Huesos (Atapuerca, Spain) and estimated to be >300,000 y old.It is important to note that direct PCR amplification provides limited power to reconstruct sequences from short DNA fragments, because only fragments that are long enough to allow for the hybridization of two PCR primers around a stretch of informative sequence are amena...

The earliest hominin occupation of Europe is one of the most debated topics in palaeoanthropology. However, the purportedly oldest of the Early Pleistocene sites in Eurasia lack precise age control and contain stone tools rather than human fossil remains. Here we report the discovery of a human mandible associated with an assemblage of Mode 1 lithic tools and faunal remains bearing traces of hominin processing, in stratigraphic level TE9 at the site of the Sima del Elefante, Atapuerca, Spain. Level TE9 has been dated to the Early Pleistocene (approximately 1.2-1.1 Myr), based on a combination of palaeomagnetism, cosmogenic nuclides and biostratigraphy. The Sima del Elefante site thus emerges as the oldest, most accurately dated record of human occupation in Europe, to our knowledge. The study of the human mandible suggests that the first settlement of Western Europe could be related to an early demographic expansion out of Africa. The new evidence, with previous findings in other Atapuerca sites (level TD6 from Gran Dolina), also suggests that a speciation event occurred in this extreme area of the Eurasian continent during the Early Pleistocene, initiating the hominin lineage represented by the TE9 and TD6 hominins.

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