Neandertals, the closest evolutionary relatives of present-day humans, lived in large parts of Europe and western Asia before disappearing 30,000 years ago. We present a draft sequence of the Neandertal genome composed of more than 4 billion nucleotides from three individuals. Comparisons of the Neandertal genome to the genomes of five present-day humans from different parts of the world identify a number of genomic regions that may have been affected by positive selection in ancestral modern humans, including genes involved in metabolism and in cognitive and skeletal development. We show that Neandertals shared more genetic variants with present-day humans in Eurasia than with present-day humans in sub-Saharan Africa, suggesting that gene flow from Neandertals into the ancestors of non-Africans occurred before the divergence of Eurasian groups from each other.

Recent genomic data has revealed multiple interactions between Neandertals and humans, but there is currently little genetic evidence about Neandertal behavior, diet, or health. We shotgun sequenced ancient DNA from five Neandertal dental calculus specimens to characterize regional differences in Neandertal ecology. At Spy, Belgium, Neandertal diet was heavily meat based, and included woolly rhinoceros and wild sheep-animals characteristic of a steppe environment. In El Sidrón, Spain, no meat was detected in the dental calculus, but dietary components including mushrooms, pine nuts, and moss reflected forest gathering. Differences in diet were also linked to an overall shift in the oral bacterial community (microbiota) in Neandertals, suggesting that meat consumption contributed to significant variation between Neandertal microbiota. Evidence for self-medication was identified in one El Sidrón Neandertal with a dental abscess, who also likely suffered from a chronic gastrointestinal pathogen (Enterocytozoon bieneusi). Lastly, we characterized a nearly complete genome of the archaeal commensal Methanobrevibacter oralis in Neandertals-the oldest draft microbial genome generated to date at ~48,000 years old (10.2 depth). DNA preserved within dental calculus represents an important new resource of behavioral and health information for ancient hominid specimens, as well as a unique long-term study system for microbial evolution.

Analysis of Neandertal DNA holds great potential for investigating the population history of this group of hominins, but progress has been limited due to the rarity of samples and damaged state of the DNA. We present a method of targeted ancient DNA sequence retrieval that greatly reduces sample destruction and sequencing demands and use this method to reconstruct the complete mitochondrial DNA (mtDNA) genomes of five Neandertals from across their geographic range. We find that mtDNA genetic diversity in Neandertals that lived 38,000 to 70,000 years ago was approximately one-third of that in contemporary modern humans. Together with analyses of mtDNA protein evolution, these data suggest that the long-term effective population size of Neandertals was smaller than that of modern humans and extant great apes.

It has been shown that Neanderthals contributed genetically to modern humans outside Africa 47,000–65,000 years ago. Here, we analyze the genomes of a Neanderthal and a Denisovan from the Altai Mountains in Siberia together with the sequences of chromosome 21 of two Neanderthals from Spain and Croatia. We find that a population that diverged early from other modern humans in Africa contributed genetically to the ancestors of Neanderthals from the Altai Mountains roughly 100,000 years ago. By contrast, we do not detect such a genetic contribution in the Denisovan or the two European Neanderthals. We conclude that in addition to later interbreeding events, the ancestors of Neanderthals from the Altai Mountains and of modern humans met and interbred, possibly in the Near East, many thousands of years earlier than previously reported.