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

Abstract

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.

Figure 1

Sequences overlapping HVRI positions carrying diagnostic differences between Neandertal and extant humans. All 43 sequences overlapping the three diagnostic positions are shown.

Figure 2

Distribution of pairwise mtDNA sequence differences among 53 humans (green), between humans and the Neandertal mtDNA (red), and between humans and chimpanzee (blue) in (A) the complete mtDNA; (B) the HVRI (Neandertal pos. 16,044–16,411); and (C) the HVRII (pos. 57–372).

Figure 3

Phylogenetic tree and divergence time estimate of mtDNA sequences. (A) Bayesian phylogenetic tree of complete mtDNA sequences of the Neandertal, 10 extant humans, one chimpanzee, and one bonobo. Identical topologies for the Neandertal and chimpanzee/bonobo split are produced by each tree-building methods. The Bayesian posterior probability and the bootstrap support values are shown for two internal nodes. (B) Posterior distribution of divergence times at each internal node using a 6–8 Mya for the ape/hominid divergence (blue node). The extant human divergences are shown in black, the Neandertal/human divergence in red, the chimpanzee/bonobo divergence in yellow, and the ape/hominid in blue.

Figure 4

COX2 protein sequence differences between Neandertal and modern humans in structural context. The amino acid positions of the four differences are shown in red. The copper center of COX2 (Cu_A) is also shown. The amino acid at each position in some primate and the cow sequences (from which the structure (PDB identifier:2EIK) is derived) are indicated.

Figure 5

Sequence coverage and base composition of the Neandertal mtDNA.(A) The expected (grey) and observed (red) distribution of sequence depths in the Neandertal mtDNA assembly at 34.9 fold over-all coverage. (B) The length distribution of sequences (yellow) and, for each length bin, the mean and standard deviation of GC content (blue). (C) G+C content within a sliding window 30 bp 5’ and 3’ of each position (blue) and the observed coverage (red) at each position.