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...

How and when the Americas were populated remains contentious. Using ancient and modern genome-wide data, we find that the ancestors of all present-day Native Americans, including Athabascans and Amerindians, entered the Americas as a single migration wave from Siberia no earlier than 23 thousand years ago (KYA), and after no more than 8,000-year isolation period in Beringia. Following their arrival to the Americas, ancestral Native Americans diversified into two basal genetic branches around 13 KYA, one that is now dispersed across North and South America and the other is restricted to North America. Subsequent gene flow resulted in some Native Americans sharing ancestry with present-day East Asians (including Siberians) and, more distantly, Australo-Melanesians. Putative ‘Paleoamerican’ relict populations, including the historical Mexican Pericúes and South American Fuego-Patagonians, are not directly related to modern Australo-Melanesians as suggested by the Paleoamerican Model.