Mitochondrial DNA, which is inherited through the maternal line, has been a favored DNA sequence for determining relationships between human populations, and there is a large amount of data on the mitochondrial DNA sequences present in humans of many different ethnic groups from all over the world. In 1997, Krings and colleagues [7
] first amplified and sequenced mitochondrial DNA (mtDNA) from a Neanderthal fossil – in fact, the original Neanderthal specimen. By late 2007, 14 other specimens had yielded mitochondrial sequences that could be compared (Figure and Table ). Several have yielded sequences over 300 bp long from the hypervariable region 1 of the mitochondrial control region (Figure ). Other sequences are shorter but contain informative nucleotide positions. These mtDNA data indicate that all Neanderthal specimens sequenced up to now form a monophyletic lineage that split from the human lineage several hundred thousand years before populations of modern humans began to diverge from each other (Figure ).
Sites of Neanderthal fossils that have provided ancient DNA. Red, mitochondrial sequences only. Green, mitochondrial and nuclear sequences.
Mitochondrial sequences published as of 2007
Neanderthal mtDNA sequences. CRS, Cambridge reference sequence . Only nucleotide positions that vary between Neanderthals and humans, or within Neanderthals, are shown. Numbering based on the CRS. See Table 1 for further details of the sequences.
Nevertheless, some researchers have argued that the absence of Neanderthal-related haplotypes in modern human populations does not necessarily mean that there was no interbreeding between Neanderthals and modern humans 30,000 or more years ago [8
]. Sequencing of mtDNA from anatomically modern human fossils 24,000 years old by Caramelli et al
] strongly suggested that there was no relationship with Neanderthals. But there were questions about the reliability of the DNA techniques and the possibility of contamination by DNA from those who had handled the specimens. To address such problems, Serre et al
] sequenced a series of Neanderthal specimens and contemporaneous early modern human fossils using Neanderthal-specific PCR primers, to avoid detecting any contaminating present-day DNA. The Neanderthal fossils yielded Neanderthal mtDNA haplotypes, but no amplifications were obtained from the well-preserved early modern samples. Serre et al
. interpreted this as a significant lack of evidence of Neanderthal-modern human admixture near the time at which it may have been possible.
More recently, researchers have been successful in isolating and sequencing DNA from the Neanderthal nuclear genome. Ancient DNA entered the genomics age with the publication of around 27,000 bp of Pleistocene cave bear sequence [12
] and more than 13 million bp of woolly mammoth DNA [13
]. These studies used cell-based and emulsion-bead approaches to create metagenomic libraries of fossil DNA extracts [12
]. Such libraries contain both endogenous DNA from the fossils and exogenous microbial DNA from modern contaminants and from microbes that colonized the organism after death or lived in the soil matrix. These approaches were applied to Neanderthals. A 38,000-year-old fossil from Vindija in Croatia (Vindija 80, Figure and Table ) was chosen for analysis because a preliminary PCR and subcloning of the fossil's mtDNA indicated well preserved DNA that was largely free of contamination [15
]. Noonan et al
] obtained 65,250 bp of Neanderthal genomic sequence using a cell-based approach, while Green et al
] obtained more than 1 Mb of genomic sequence using an emulsion-bead based approach.
Both groups made alignments of their sequences with orthologous chimpanzee and human sequences and characterized the substitutions along each lineage. From these, an average sequence divergence time between Neanderthals and modern humans could be calculated. This parameter does not, however, necessarily measure the time that the two populations actually split. To estimate that, the two groups compared their Neanderthal sequence with information on single nucleotide polymorphisms (SNPs) in present-day humans collected by the HapMap project [17
]. If the split between humans and Neanderthals is ancient, Neanderthals should rarely, or almost never, carry the 'derived' variant of a human SNP – that is, a variant that is present in some modern human lineages but not in the ancestral human lineage from which both Neanderthals and modern humans descend. On the other hand, if the split is recent, derived variants will be common in the Neanderthal genome and we should expect alleles to be shared between modern Europeans and Neanderthals.
Although they were working with DNA from the same specimen, the two teams came to very different conclusions. Noonan et al
] arrived at an average divergence time between Neanderthals and humans of 706,000 years and an estimated time for a population split at 370,000 years ago. They found derived human SNP variants at only three sites in the Neanderthal DNA, two of which are only found in sub-Saharan Africans and not in Europeans. They concluded that the Neanderthal contribution to modern genetic diversity was zero. Green and colleagues [15
], on the other hand, calculated the average sequence divergence time between Neanderthals and humans as 516,000 years. To check whether this degree of divergence is comparable to that found within humans, they resequenced a modern human using an identical approach and compared the data to the chimp and human reference genomes. They found the average sequence divergence time between the resequenced human and the reference genome to be 459,000 years. And when Green et al
. compared their Neanderthal sequence with the corresponding HapMap data, they found that around 30% of the SNPs were of the derived human type. They therefore concluded that a single ancient split between Neanderthals and humans is unlikely, and there must have been some level of recent gene flow.
Such conflicting conclusions from the same DNA sample not surprisingly led to a reanalysis of the data. Contaminating modern DNA should be less fragmented than genuine ancient DNA. To check their data for evidence of contamination, Noonan et al
] had compared their long sequence reads to their short sequence reads and confirmed an equal sequence divergence from modern humans across their data, indicating the absence of contamination. Green et al
] had not taken this step. Wall and Kim [18
] reanalyzed Green et al
.'s dataset and found that their long sequence reads showed significantly lower sequence divergence from modern humans than their short sequence reads, and that their short sequence reads showed an indistinguishable level of sequence divergence from Noonan et al
.'s data. Wall and Kim concluded that the sequence used by Green et al
. had been contaminated by human DNA – and, using a maximum likelihood analysis, estimated the contamination to be as high as 78%. We also note that Noonan et al
. found that 1.3% of their metagenomic library was Neanderthal in origin, whereas Green et al
. found 6.2% to be Neanderthal. If this difference is due to contamination, then it is in close agreement with Wall and Kim's likelihood estimates. We believe these findings serve as a cautionary tale that even with extremely stringent protocols, contamination of fossils with modern human DNA will remain a problem.