Abstract
The expansion of premodern humans into western and eastern Europe 
40,000
years before the present led to the eventual replacement of the Neanderthals
by modern humans 28,000 years ago1. Here we report the
second mitochondrial DNA (mtDNA) analysis of a Neanderthal, and the first
such analysis on clearly dated Neanderthal remains. The specimen is from one
of the eastern-most Neanderthal populations, recovered from Mezmaiskaya Cave
in the northern Caucasus2. Radiocarbon dating estimated the
specimen to be 29,000 years old and therefore from one of the latest
living Neanderthals3. The sequence shows 3.48% divergence from
the Feldhofer Neanderthal4. Phylogenetic analysis places the
two Neanderthals from the Caucasus and western Germany together in a clade
that is distinct from modern humans, suggesting that their mtDNA types have
not contributed to the modern human mtDNA pool. Comparison with modern populations
provides no evidence for the multiregional hypothesis of modern human evolution.
The first successful extraction and sequencing of the mtDNA hypervariable regions (I and II (HVRI & HVRII)) was performed on the Neanderthal-type specimen from Feldhofer Cave, the Neander valley, Germany4, 5. Phylogenetic analysis of the sequence placed the Neanderthal mtDNA outside the mtDNA pool of modern humans. This was regarded as a breakthrough in the study of modern human evolution, providing molecular evidence that Neanderthals did not contribute mtDNA to modern humans. From this sequence the divergence of Neanderthals and modern humans was estimated to have occurred between 317,000 and 741,000 years ago4, 5. However, these estimates were based on the molecular analysis of a single specimen. The shortage of potentially well preserved Neanderthal material6 and limited access to Neanderthal remains for destructive analysis have hindered the analysis of additional specimens, but genetic characterization of additional Neanderthals is essential to understand their molecular diversity and the relationship between different Neanderthal populations, and to assess their relationship to modern humans further.
The Caucasus, which is located on the southeastern boundary between Europe
and Asia, is one of the areas through which pre-modern humans and anatomically
modern Homo sapiens may have entered Europe from the Near East and
Africa. Neanderthals invaded the region at an unknown point in time7, 8
and may have occupied the region alongside modern humans from 40,000
years before the present (B.P.). During the excavation of the Mezmaiskaya
Cave2, which is located in the northern Caucasus (
Fig. 1), a fragmentary skeleton of an infant was found that contained
a set of morphological characteristics which indicated clear affinities to
the Neanderthals of western and central Europe2. Mitochondrial
DNA analysis was undertaken using one of this Neanderthal's ribs.
Figure 1: The area within which Neanderthal remains have been found (dotted line).
The Mezmaiskaya Cave is situated 1,310 m above sea level within the northern Caucasus at 44° 10' N 40° 00' E on the bank of the Sukhoy Kurdzhips river. The location of the Feldhofer Cave in Germany is also shown.
High resolution image and legend (12K)The preservation of collagen-type debris was used as an indicator of macromolecule
preservation in the bone. The amount of collagen-type debris extracted9 from 130 mg of the Mezmaiskaya Neanderthal rib fragment was
22% of the average level extracted from modern bones, and the extracted collagen
contained 41.6% carbon and 14.7% nitrogen. This is within the values recovered
from prehistoric samples displaying good preservation10. These
data suggested that there were low levels of diagenetic modification. The
high collagen yield made it possible to date the Neanderthal infant to
29,195 
965 (Ua-14512) years B.P. by using a radiocarbon accelerator.
This date does not agree with the previously published dates of more than
45,000 (Le-3841) and 40,660 1,600 (Le-3599) years for the
Mousterian layers in the Mezmaiskaya Cave2 with which the skeleton
was associated. The most likely reason for this discrepancy is the incorrect
identification of the poorly defined layers in this area of the cave. The
value obtained from the bone itself rather than from associated material gives
the most reliable date for this individual.
Two sections of one rib (90 mg and 123 mg) were used for DNA extraction in two independent laboratories. In the Glasgow laboratory, a total of 345 base pairs (bp) of HVRI was determined from two overlapping polymerase chain reaction (PCR) fragments with lengths of 232 and 256 bp. Forty PCR amplification cycles produced sufficient product to enable direct sequencing. Products from independent PCR amplifications were also cloned into a TA vector and sequenced (Fig. 2).
Figure 2: Variable sites in the DNA sequences of the PCR fragments obtained by direct sequencing (Direct 1 and 2) and cloned PCR products derived from the Neanderthal from Mezmaiskaya Cave.
The human reference sequence11 and the sequence of the Neanderthal from Feldhofer Cave4 are included for comparison. A full stop indicates that the sequence is the same as the reference. The sequence that could be duplicated in the Stockholm laboratory is shown in bold within the compiled Mezmaiskaya sequence.
High resolution image and legend (8K)The authenticity of the DNA sequence obtained in the Glasgow laboratory is supported by a number of factors. First, a section of the mtDNA was isolated and sequenced with congruent results in the Stockholm laboratory. Second, the PCR products were generated using Neanderthal-specific primer pairs that, under the amplification conditions, failed to amplify any fragments using modern DNA controls from individuals of different ethnic origins. Third, the retrieval of the sequence was not dependent on the primers used. Fourth, the low level of diagenetic modification indicated that the sample could theoretically contain amplifiable DNA. Last, and most convincingly, the sequence is similar to, and after phylogenetic analysis clusters strongly with, the previously analysed Neanderthal sequence4.
Comparison of the 345-bp fragment of HVRI with the Anderson reference sequence11 and the Neanderthal from Feldhofer Cave4 revealed 22 differences (17 transitions, 4 transversions and 1 insertion) and 12 differences (11 transitions and 1 transversion), respectively. The Feldhofer Neanderthal HVRI contained 27 differences to Anderson reference sequence11 (over the equivalent 345 bp4). The two Neanderthals share 19 substitutions relative to the reference sequence. The cloned PCR products contained all the substitutions that were detected by direct sequencing; six other non-reproducible substitutions occurred in seven different clones. No modern sequences were found in the Glasgow laboratory either by direct sequencing or by sequencing cloned PCR products. The Stockholm laboratory experienced problems with contamination: most of the cloned PCR products that they analysed contained sequences that are found in the modern human mtDNA pool, with two haplotypes predominant. However, three clones contained DNA that was the same as the sequence determined in Glasgow (two of these contained non-reproducible substitutions).
The preservation of 256-bp DNA fragments in bone that is 29,000 years
old, that has not been preserved in permafrost and that contained sufficient
DNA to enable direct DNA sequencing after amplification is unprecedented and
may be attributed to specific features of the microenvironment of the limestone
cavern2. The retrieval of mtDNA showed a positive correlation
to the preservation of collagen content and the skeletal morphology.
Phylogenetic analysis using both distance and parsimony optimizations places the two Neanderthal sequences together, in a distinct clade, basal to modern humans. Neighbour-joining analysis supports this separation ( Fig. 3a). Parsimony analysis, which makes minimal assumptions about the model of evolution and optimizes the fit between the tree and data, produced similar results (Fig. 3b).
Figure 3: Phylogenetic relationship of the two Neanderthals and modern humans.
a, A neighbour-joining tree computed using a total of 1,897 haplotypes derived from 5,846 modern humans19. b, A maximum parsimony branch and bound search with the two Neanderthal sequences along with the sequences of one !Kung, three other Africans, three Asians and three Europeans, all randomly selected19. This result is congruent with four additional data sets that were analysed. In both analyses, three chimpanzee sequences13 were used as an outgroup. The numbers in both diagrams refer to the bootstrap frequencies (%) obtained from 1,000 replicates.
High resolution image and legend (15K)The level of pairwise difference found between the two Neanderthals was
higher than the average values found in random samples of 300 Caucasoids (
5.28 2.24) and Mongoloids (6.27 2.29)—less
than 1% of Caucasoid and Mongoloid pairs differ at 12 or more positions—but
comparable to a random sample of 300 Africans (8.36 3.2),
where 37% of pairs differed at 12 or more positions. When analysing ancient
DNA there is the possibility of misincorporating nucleotides in the early
stages of PCR, especially when the target DNA is possibly damaged and present
in low copy number12. As both Neanderthals were analysed in
replicate and the results were consistent, however, errors of this type can
be discounted.
The Feldhofer and Mezmaiskaya Neanderthals were separated geographically by over 2,500 km. Given that these two individuals contained closely related mtDNA, which is phylogenetically distinct from modern humans, and displays only a moderate level of sequence diversity compared with some primates13, these data provide further support for the hypothesis of a very low gene flow between the Neanderthals and modern humans. In particular, these data reduce the likelihood that Neanderthals contained enough mtDNA sequence diversity to encompass modern human diversity.
The 'out-of-Africa' hypothesis for the origin of modern humans
predicts equal distances between the Neanderthal sequences and all modern
sequences. We observed this in our analysis—the average pairwise differences
between the Neanderthals and 300 randomly selected Africans, Mongoloids and
Caucasoids were calculated to be 23.09 2.86, 23.27
4.06 and 25.45 3.27, respectively.
We estimated the age of the most recent common ancestor (MRCA) of the mtDNA of the eastern and western Neanderthals to be 151,000–352,000 years. This coincides with the time of emergence of the Neanderthal lineage in the palaeontological records14. The divergence of modern human and Neanderthal mtDNA was estimated to be between 365,000 and 853,000 years. Using the same model, we estimated the age of the earliest modern human divergences in mtDNA to be between 106,000 and 246,000 B.P.
The results obtained from this specimen suggest that some other Neanderthal samples may be amenable to molecular analysis. To obtain a more complete picture of the relationship of Neanderthals to modern humans, additional Neanderthals and early modern humans must be analysed, especially from the regions where they may have co-existed. The excellent preservation of this specimen leads to the potential of analysing the entire Neanderthal mitochondrial genome.
Methods
DNA extraction, PCR, cloning and sequencing
The DNA extraction methods used in Glasgow4 and Stockholm15 have been described. We took precautions to prevent contamination from modern DNA4. The Neanderthal-specific primer pairs, NL16,055 and NH16,262, and NL16,209 and NH16,400 (5'-TGATTTCAC GGAGGATGGTGA-3') were used in Glasgow and the primers L16,212 (5'-ATGCTTAC AAGCAAGCACA-3') and H16,332 (5'-TTGACTGTAATGTGCTATG-3') were used in Stockholm. The annealing temperatures for the primer pairs NL16,055–NH16,262, NL16,209–NH16,400 and L16,212–H16,332 were 50 °C, 60 °C and 50 °C respectively; 40 cycles were used for the first two pairs, 55 cycles for the third. AmpliTaq Gold (Perkin Elmer Cetus) was used in all PCRs. PCR products were purified using the QIAquick Gel Extraction kit (Qiagen) before direct sequencing using the Dye Terminator sequencing kit (Perkin Elmer) or cloning into the TA vector (Invitrogen) before sequencing with the same kit using the M13 and T7 primers.
Sequence analyses
The neighbour-joining and the maximum parsimony branch and bound trees were both constructed using PAUP* 4.0 (ref. 16). For the neighbour-joining analysis, the Tamura-Nei DNA substitution model17 was used with a gamma distribution of 0.4 (ref. 18), for all other parameters the defaults provided by PAUP* 4.0 were used. The MRCA was calculated using the described methods and assumptions5. PAUP* 4.0 was used to calculate pairwise differences between sequences: the data sets used for this were constructed by randomly selecting appropriate samples from a published data set19.