Reconstructing the genetic history of late Neanderthals.

Although it has previously been shown that Neanderthals contributed DNA to modern humans, not much is known about the genetic diversity of Neanderthals or the relationship between late Neanderthal populations at the time at which their last interactions with early modern humans occurred and before they eventually disappeared. Our ability to retrieve DNA from a larger number of Neanderthal individuals has been limited by poor preservation of endogenous DNA and contamination of Neanderthal skeletal remains by large amounts of microbial and present-day human DNA. Here we use hypochlorite treatment of as little as 9 mg of bone or tooth powder to generate between 1- and 2.7-fold genomic coverage of five Neanderthals who lived around 39,000 to 47,000 years ago (that is, late Neanderthals), thereby doubling the number of Neanderthals for which genome sequences are available. Genetic similarity among late Neanderthals is well predicted by their geographical location, and comparison to the genome of an older Neanderthal from the Caucasus indicates that a population turnover is likely to have occurred, either in the Caucasus or throughout Europe, towards the end of Neanderthal history. We find that the bulk of Neanderthal gene flow into early modern humans originated from one or more source populations that diverged from the Neanderthals that were studied here at least 70,000 years ago, but after they split from a previously sequenced Neanderthal from Siberia around 150,000 years ago. Although four of the Neanderthals studied here post-date the putative arrival of early modern humans into Europe, we do not detect any recent gene flow from early modern humans in their ancestry.

A high-coverage Neandertal genome from Vindija Cave in Croatia.

To date, the only Neandertal genome that has been sequenced to high quality is from an individual found in Southern Siberia. We sequenced the genome of a female Neandertal from ~50,000 years ago from Vindija Cave, Croatia, to ~30-fold genomic coverage. She carried 1.6 differences per 10,000 base pairs between the two copies of her genome, fewer than present-day humans, suggesting that Neandertal populations were of small size. Our analyses indicate that she was more closely related to the Neandertals that mixed with the ancestors of present-day humans living outside of sub-Saharan Africa than the previously sequenced Neandertal from Siberia, allowing 10 to 20% more Neandertal DNA to be identified in present-day humans, including variants involved in low-density lipoprotein cholesterol concentrations, schizophrenia, and other diseases.

Neandertal and Denisovan DNA from Pleistocene sediments.

Although a rich record of Pleistocene human-associated archaeological assemblages exists, the scarcity of hominin fossils often impedes the understanding of which hominins occupied a site. Using targeted enrichment of mitochondrial DNA, we show that cave sediments represent a rich source of ancient mammalian DNA that often includes traces of hominin DNA, even at sites and in layers where no hominin remains have been discovered. By automation-assisted screening of numerous sediment samples, we detected Neandertal DNA in eight archaeological layers from four caves in Eurasia. In Denisova Cave, we retrieved Denisovan DNA in a Middle Pleistocene layer near the bottom of the stratigraphy. Our work opens the possibility of detecting the presence of hominin groups at sites and in areas where no skeletal remains are found.

Ancient gene flow from early modern humans into Eastern Neanderthals.

It has been shown that Neanderthals contributed genetically to modern humans outside Africa 47,000-65,000 years ago. Here we analyse 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 early modern humans met and interbred, possibly in the Near East, many thousands of years earlier than previously thought.

Patterns of coding variation in the complete exomes of three Neandertals.

We present the DNA sequence of 17,367 protein-coding genes in two Neandertals from Spain and Croatia and analyze them together with the genome sequence recently determined from a Neandertal from southern Siberia. Comparisons with present-day humans from Africa, Europe, and Asia reveal that genetic diversity among Neandertals was remarkably low, and that they carried a higher proportion of amino acid-changing (nonsynonymous) alleles inferred to alter protein structure or function than present-day humans. Thus, Neandertals across Eurasia had a smaller long-term effective population than present-day humans. We also identify amino acid substitutions in Neandertals and present-day humans that may underlie phenotypic differences between the two groups. We find that genes involved in skeletal morphology have changed more in the lineage leading to Neandertals than in the ancestral lineage common to archaic and modern humans, whereas genes involved in behavior and pigmentation have changed more on the modern human lineage.

A draft sequence of the Neandertal genome.

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.

Targeted retrieval and analysis of five Neandertal mtDNA genomes.

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.

A complete Neandertal mitochondrial genome sequence determined by high-throughput sequencing.

A complete mitochondrial (mt) genome sequence was reconstructed from a 38,000 year-old Neandertal individual with 8341 mtDNA sequences identified among 4.8 Gb of DNA generated from approximately 0.3 g of bone. Analysis of the assembled sequence unequivocally establishes that the Neandertal mtDNA falls outside the variation of extant human mtDNAs, and allows an estimate of the divergence date between the two mtDNA lineages of 660,000 +/- 140,000 years. Of the 13 proteins encoded in the mtDNA, subunit 2 of cytochrome c oxidase of the mitochondrial electron transport chain has experienced the largest number of amino acid substitutions in human ancestors since the separation from Neandertals. There is evidence that purifying selection in the Neandertal mtDNA was reduced compared with other primate lineages, suggesting that the effective population size of Neandertals was small.