ARTICLES NATURE Vol 444 16 November 2006 These positions are likely to have changed on the human lineage after of such errors in the Neanderthal sequences are quantified and the divergence from Neanderthal. Finally, a total of 51 positions removed(see Supplementary Methods),7.9% of the sequence contain different bases in all three groups. hanges along the human lineage are estimated to have occurred after Because the 454 sequencing technology allows the base in a base divergence from the Neanderthal. If the human-chimpanzee diver pair from which a sequence is derived to be determined, the relative gence time is set to 6,500,000 years(refs 40, 41, 44), this implies an frequencies of each of the 12 possible categories of base changes can average human-Neanderthal DNA sequence divergence time of be estimated for each evolutionary lineage. As seen in Fig. 5, the 516,000 years. A 95% confidence interval generated by bootstrap atterns of the zee-specific and human-specific changes re-sampling of the alignment data gives a range of 465,000 to are similar to each other in that the eight transversional changes 569,000 years. Obviously, these divergence estimates are dependent are of approximately equal frequency and about fourfold less fre- on the human-chimpanzee divergence time, which is a much larger quent than each of the four transitional changes, yielding a transition source of uncertainty to transversion ratio of 2.04, typical of closely related mammalian We analysed the DNA sequences generated from a contemporary genomes. For the Neanderthal-specific changes the pattern is very human using the same sequencing protocol as was used for the different in that mismatches are dominated by C to T and G to A Neanderthal. Although ancient DNA is degraded and damaged, this differences. Thus, the pattern of change seen among the Neanderthal- comparison controls for many of the aspects of the analysis including pecific alignment mismatches is typical of the nucleotide substi- sequencing and alignment methodology. In this case, -7.1% of ution pattern observed in PCR of ancient DNA the divergence along the human lineage is assigned to the time Consistent with this, modern human sequences determined by subsequent to the divergence of the two human sequences. The aver 454 sequencing show no excess amount ofC to T or G to A differences age divergence time between alleles within humans is thus (Supplementary Fig. 2), indicating that lesions in the ancient DNA,000 years with a 95% confidence interval between 419,000 rather than sequencing errors account for the majority of the errors in and 498,000 years. As expected, this estimate of the average human the Neanderthal sequences. Assuming that the evolutionary rate of diversity is less than the divergence seen between the human and the DNA change was the same on the Neanderthal and human lineages, Neanderthal sequences, but constitutes a large fraction of it because the majority of observed differences specific to the Neanderthal lin- much of the human sequence diversity is expected to predate the eage are artefacts. All Neanderthal-specific changes were therefore human-Neanderthal split. Neanderthal genetic differences to disregarded in the subsequent analyses and the Neanderthal humans must therefore be interpreted within the context of human equences were used solely to assign changes to the human or chim- diversity panzee lineage where the human and chimpanzee genome sequences differ and the Neanderthal sequence carries either the human or the Ancestral population size Humans differ from apes in that their effective population size is of the order of 10,000 while those of chimpanzees, gorillas and orang Genomic divergence between Neanderthals and humans utans are two to four times larger. Furthermore, the population Assuming that the rates of DNA sequence change along the chim- size of the ancestor of humans and chimpanzees was found to be panzee lineage and the human lineage were similar, it can be esti- similar to those of apes, rather than to humans". The mated that 8.2% of the DNA sequence changes that have occurred on Neanderthal sequence data now allow us to ask if the effective size age since the divergence m the chimpanzee lineage of the population ancestral to humans and Neanderthals was large, as occurred after the divergence of the Neanderthal lineage. However, is the case for apes and the human-chimpanzee ancestor, or small, as although the Neanderthal-specific changes that are heavily influ- for present-day humans. enced by errors are not used for this analy he errors in the We applied a method"that co-estimates the ancestral effective single-pass sequencing reads from the Neanderthal extract will create population size and the split time between Neanderthal and human positions where the Neanderthal is identical either to human or populations( Fig 6a; see Supplementary Methods). As seen in Fig. 6b, himpanzee sequences, and thus affect the estimates of sequence we recover a line describing combinations of population sizes and change on the human and chimpanzee lineages. When the effects split times compatible with the data and lack power to be more Chimpanzee Human pmbF68百后 Human different Figure 5 Schematic tree illustrating the number of nucleotide changes to base-damage and base-calling inferred to have occurred on hominoid lineages. In blue is the distribution human lineage. The distributions of types of changes in each of all aligned positions that did not change on any lineage. In brown are the tegory,corrected changes that occurred either on the chimpanzee lineage() or on the rrors in the Neanderthal sequence(see Supplementary hominid lineage(h)before the human and Neanderthal lineages diverged In red are the changes that are unique to the Neanderthal lineage(n), including 334 E2006 Nature Publishing GroupThese positions are likely to have changed on the human lineage after the divergence from Neanderthal. Finally, a total of 51 positions contain different bases in all three groups. Because the 454 sequencing technology allows the base in a base pair from which a sequence is derived to be determined, the relative frequencies of each of the 12 possible categories of base changes can be estimated for each evolutionary lineage. As seen in Fig. 5, the patterns of the chimpanzee-specific and human-specific changes are similar to each other in that the eight transversional changes are of approximately equal frequency and about fourfold less fre￾quent than each of the four transitional changes, yielding a transition to transversion ratio of 2.04, typical of closely related mammalian genomes43. For the Neanderthal-specific changes the pattern is very different in that mismatches are dominated by C to T and G to A differences. Thus, the pattern of change seen among the Neanderthal￾specific alignment mismatches is typical of the nucleotide substi￾tution pattern observed in PCR of ancient DNA. Consistent with this, modern human sequences determined by 454 sequencing show no excess amount of C to T or G to A differences (Supplementary Fig. 2), indicating that lesions in the ancient DNA rather than sequencing errors account for the majority of the errors in the Neanderthal sequences. Assuming that the evolutionary rate of DNA change was the same on the Neanderthal and human lineages, the majority of observed differences specific to the Neanderthal lin￾eage are artefacts. All Neanderthal-specific changes were therefore disregarded in the subsequent analyses and the Neanderthal sequences were used solely to assign changes to the human or chim￾panzee lineage where the human and chimpanzee genome sequences differ and the Neanderthal sequence carries either the human or the chimpanzee base. Genomic divergence between Neanderthals and humans Assuming that the rates of DNA sequence change along the chim￾panzee lineage and the human lineage were similar, it can be esti￾mated that 8.2% of the DNA sequence changes that have occurred on the human lineage since the divergence from the chimpanzee lineage occurred after the divergence of the Neanderthal lineage. However, although the Neanderthal-specific changes that are heavily influ￾enced by errors are not used for this analysis, some errors in the single-pass sequencing reads from the Neanderthal extract will create positions where the Neanderthal is identical either to human or chimpanzee sequences, and thus affect the estimates of sequence change on the human and chimpanzee lineages. When the effects of such errors in the Neanderthal sequences are quantified and removed (see Supplementary Methods), ,7.9% of the sequence changes along the human lineage are estimated to have occurred after divergence from the Neanderthal. If the human–chimpanzee diver￾gence time is set to 6,500,000 years (refs 40, 41, 44), this implies an average human–Neanderthal DNA sequence divergence time of ,516,000 years. A 95% confidence interval generated by bootstrap re-sampling of the alignment data gives a range of 465,000 to 569,000 years. Obviously, these divergence estimates are dependent on the human–chimpanzee divergence time, which is a much larger source of uncertainty. We analysed the DNA sequences generated from a contemporary human using the same sequencing protocol as was used for the Neanderthal. Although ancient DNA is degraded and damaged, this comparison controls for many of the aspects of the analysis including sequencing and alignment methodology. In this case, ,7.1% of the divergence along the human lineage is assigned to the time subsequent to the divergence of the two human sequences. The aver￾age divergence time between alleles within humans is thus ,459,000 years with a 95% confidence interval between 419,000 and 498,000 years. As expected, this estimate of the average human diversity is less than the divergence seen between the human and the Neanderthal sequences, but constitutes a large fraction of it because much of the human sequence diversity is expected to predate the human–Neanderthal split25. Neanderthal genetic differences to humans must therefore be interpreted within the context of human diversity. Ancestral population size Humans differ from apes in that their effective population size is of the order of 10,000 while those of chimpanzees, gorillas and orang￾utans are two to four times larger45–47. Furthermore, the population size of the ancestor of humans and chimpanzees was found to be similar to those of apes, rather than to humans42,48. The Neanderthal sequence data now allow us to ask if the effective size of the population ancestral to humans and Neanderthals was large, as is the case for apes and the human–chimpanzee ancestor, or small, as for present-day humans. We applied a method42 that co-estimates the ancestral effective population size and the split time between Neanderthal and human populations (Fig. 6a; see Supplementary Methods). As seen in Fig. 6b, we recover a line describing combinations of population sizes and split times compatible with the data and lack power to be more A C T G C A G T C G G C A T T A A G T C G A C T A C T G C A G T C G G C A T T A A G T C G A C T A C T G C A G T C G G C A T T A A G T C G A C T Human=Neanderthal; Chimpanzee different Neanderthal=Chimpanzee; Human different Human=Chimpanzee; Neanderthal different Human= Neanderthal= Chimpanzee ACG T Neanderthal n s Human h p Neanderthal base Aligned base 0 50,000 100,000 150,000 200,000 250,000 736,941 total (739,966 corrected) Chimpanzee 0 500 1,000 1,500 2,000 0 400 800 1,200 p+h=10,167 (10,208 corrected) s=434 (422 corrected) n=3,447 (422 corrected) 0 20 40 60 80 100 Figure 5 | Schematic tree illustrating the number of nucleotide changes inferred to have occurred on hominoid lineages. In blue is the distribution of all aligned positions that did not change on any lineage. In brown are the changes that occurred either on the chimpanzee lineage (p) or on the hominid lineage (h) before the human and Neanderthal lineages diverged. In red are the changes that are unique to the Neanderthal lineage (n), including all changes due to base-damage and base-calling errors. In yellow are changes unique to the human lineage. The distributions of types of changes in each category are also given. The numbers of changes in each category, corrected for base-calling errors in the Neanderthal sequence (see Supplementary Methods), are shown within parentheses. ARTICLES NATURE| Vol 444| 16 November 2006 334 ©2006 NaturePublishingGroup