Hum Genet (1997) 99 : 443–449
© Springer-Verlag 1997
O R I G I N A L I N V E S T I G AT I O N
D. Comas · F. Calafell · E. Mateu · A. Pérez-Lezaun · E. Bosch · J. Bertranpetit
Mitochondrial DNA variation and the origin of the Europeans
Received: 24 September 1996
Abstract Sequences from the mitochondrial DNA (mtDNA) control region were analyzed in nine European and West Asian populations. They showed low genetic heterogeneity when compared to world populations. However, a Caucasoid population tree displayed a robust eastwest gradient. Within-population diversity (ascertained through various parameters) and mean pairwise differences declined from east to west, in a pattern compatible with ancient population migration and expansion from the Middle East. Estimated expansion times indicate a Paleolithic event with important differences among populations according to their geographical position and thus a slower tempo than previously believed. The replacement of Neanderthals by anatomically modern humans, fully compatible with the present results, may have been a slower and more complex process than cultural change suggests.
Introduction Debates on early human population origins and history take place in interdisciplinary fields where historical sciences (usually archaeology), linguistics, physical anthropology (including paleoanthropology), and genetics play their own and variable roles in unraveling the scenario of the unique human population history (Cavalli-Sforza et al. 1988; Renfrew 1992). This paper analyzes a highly vari-
D. Comas and F. Calafell contributed equally to this paper D. Comas · F. Calafell1 · E. Mateu · A. Pérez-Lezaun · E. Bosch · J. Bertranpetit (Y) Facultat de Biologia (Antropologia), Universitat de Barcelona, Diagonal 645, E-08028 Barcelona, Spain Tel.: +34-3-402 1461; Fax: +34-3-411 0887 e-mail:
[email protected] E. Mateu · J. Bertranpetit Institut de Salut Pública de Catalunya, Barcelona, Spain Present address: of Genetics, School of Medicine, Yale University, USA
1 Department
able genomic region in individuals of several European and West Asian populations in order to reconstruct the origin of Europeans and contrast this genetic evidence with that of other disciplines. There is a wide literature on the usefulness of mitochondrial DNA (mtDNA) variation in the study of human evolution and history, and the sequence analysis of the control region of mtDNA has been shown to be a powerful tool in unraveling the past of human populations both at global (Vigilant et al. 1991; Jorde et al. 1995 and references therein) or at regional (e.g., Ward et al. 1991; Bertranpetit et al. 1995; Mountain et al. 1995) levels. In many papers, the specificities of the mtDNA analysis are discussed; in this introduction we will focus on the evolutionary problem of the origin of Europeans as posed by archaeology and paleoanthropology. Since the initial colonization of Europe by the first hominids (Carbonell et al. 1995) at an uncertain date, but probably more than one million years ago (Gabunia and Vekua 1995), the most significant development of the early human history of Europe is undoubtedly the period centered on 40 000–30 000 years ago (Mellars 1989, 1993), when morphology and culture underwent major, and possibly related, transformations. The typical morphology of the former Europeans (the Neanderthals) seems to vanish and the anatomically modern humans (AMH) emerged, presumably in the Middle East (Stringer 1989; Bar-Yosef and Vandermeersch 1993), replacing, rather than deriving from, Neanderthals. However, debate continues both on the meaning of the relatively recent persistence of Neanderthal morphology (Mercier et al. 1991; Hublin et al. 1996) and on the existence of a morphological, and thus genetic, continuity between both populations (Frayer 1992). Cultural change was dramatic, and is traditionally referred to as the transition (or even the revolution) from the Middle Paleolithic culture (the Mousterian) to the Upper Paleolithic, whose first full-fledged, widespread cultural tradition in Europe and the Middle East was the Aurignacian (Mellars 1989, 1993). The possible chronological overlap of both transitions (or abrupt changes) provides a foundation for a unique in-
444
ing a geographic pattern of variation tracing back ancient population growth (and potential expansion), and relating all these processes to the cultural changes that may have triggered them.
Materials and methods Population samples
Fig. 1 Location of the analyzed samples. (BAS Basques, BRI British, SAR Sardinians, TUS Tuscans, SWI Swiss, BUL Bulgarians, TUR Turks, MEA Middle Easterners)
terpretation in which the AMH spread the Upper Paleolithic cultures in Europe with both a cultural and biological replacement process. This simple model has been a matter of much debate (Mellars and Stringer 1989; Nitecki and Nitecki 1994) and many aspects remain to be explained, including: (1) the first fully AMH are very recent, mainly in western Europe; the few older remains do not seem to have a clear AMH anatomy and might have some Neanderthal traits (Frayer 1992); (2) the spread of Aurignacian is too rapid for a complete demographic replacement (Straus 1989, 1994); (3) large regions present transition cultures, such as Castelperonian, which is associated with Neanderthal remains (like Saint-Césaire); and (4) recent findings in Vindija Cave (Croatia) of 33 000year-old Neanderthal remains in stratigraphic association with Aurignacian stone and bone artifacts (Karavanic 1995). It is likely that by the Last Glacial Maximum (Soffer and Gamble 1990), 18 000 years ago, the direct ancestors of contemporary Europeans were already the settlers of Europe but there is insufficient early skeletal evidence to reconstruct the pace of the transition. The possible genetic impact of the Neolithic transition has been ascertained through the analysis of gene frequencies (Ammerman and Cavalli-Sforza 1984; Sokal et al. 1991), tracing the spread from the Middle East through a genetic footprint in many parts of Europe. Nonetheless, gene frequency studies do not exclude the persistence of an older gene pool in most of Europe. In the present paper we analyze, through multiple techniques, the geographical sequence variation of the 360 bp of segment I of the control region of mtDNA in nine European and West Asian populations (Fig. 1), comprising 483 individuals, and we relate these results to the archaeological and paleoanthropological debate. The geographical distribution of the samples comprises the places involved in the main ancient archaeological events relevant to the origin of Europeans (Champion et al. 1984; Cunliffe 1994). The aspects of population history, which genetic analysis may elucidate, include understanding of the time depth necessary for the observed variation, unravel-
Populations included published samples: 45 Basque individuals (Bertranpetit et al. 1995), 100 British (Piercy et al. 1993), 69 Sardinians (Di Rienzo and Wilson 1991), 74 Swiss (Pult et al. 1994), 49 Tuscans (central Italy, Francalacci et al. 1996), 30 Bulgarians (Calafell et al. 1996), 29 Turks (Calafell et al. 1996), a different sample of 45 Turks (Comas et al. 1996), and 42 Middle Easterners (Di Rienzo and Wilson 1991). All the individuals included in these samples possessed autochthonous (European or West Asian) ancestry. The use of either sample of Turks or of both combined produced the same basic results and were kept separate to check the consistency of the results. Several external populations were used for comparison. These are Havik and Mukri from India (Mountain et al. 1995), Asians, Papuans and Bantu, Hadza, Pygmies and !Kung from Africa (Vigilant et al. 1991), Polynesians (Lum et al. 1994); from the Americas, Nuu-Chah-Nulth (Ward et al. 1991) and Huétar (Santos et al. 1994). Recent publications (Sajantila et al. 1995; Richards et al. 1996) provide data for partially overlapping population sets. These, with many other unpublished sequences, will be jointly analyzed in a forthcoming paper. The analysis encompasses 360 bp, from nucleotide positions 16 024 to 16 383, according to the reference sequence (Anderson et al. 1981). For the 483 European and West Asian individuals, 279 different sequences were found, in which 139 nucleotide positions were variable. Genetic distances and trees Intermatch-mismatch distances (Horai and Hayasaka 1990) were computed, using the equation (d = dij – (di + dj/2), where dij is the average number of nucleotide differences between populations i and j, and di and dj are, respectively, the average pairwise differences within populations i and j. When negative distances were found, a small constant was added to the entire matrix to make it positive; this procedure does not alter the genetic relationships contained in the tree, and enabled us to use tree-building algorithms. Neighbor-joining trees (Saitou and Nei 1987) were built with the PHYLIP 3.5c package (Felsenstein 1989). Their robustness was probed through 1000 bootstrap iterations (Efron 1982; Felsenstein 1985). Internal genetic diversity Several parameters were used to measure genetic diversity within populations: 1. Shannon’s information (or diversity) index, defined as H = –∑ pi log2 pi (where pi is the sample frequency of the ith sequence), a simple and elegant measure much used in ecology. Nonetheless, its dependence on sample size recommends the use of the standardized version, H′ (Magurran 1988), which is simply the division of H by its highest possible value, –log2(l/n), where n is the sample size. A value of H′ = 1 would imply that all the individuals in a sample bore different sequences. 2. The mean number of nucleotide pairwise differences. 3. The rate of the minimum number of steps in a most parsimonious tree to the number of sequences included in the tree. One hundred putatively most parsimonious trees were generated for each population with the DNAPARS program in the PHYLIP 3.5c
445 package (Felsenstein 1989), The minimum values obtained did not change after shuffling the data several times to avoid bias due to the original order of the sequences. Dating of expansions Pairwise difference distributions were least-square fitted to the Rogers and Harpending (1992) model. The τ parameter in the Rogers and Harpending model was used to estimate expansion time according to the equation τ = 2 µlt, where µ is the mutation rate per nucleotide and generation, l is the sequence length and t is the time in generations. Several estimations for the mutation rate have been proposed in the literature and those used here cover the range of most of those available: 2.16 × 10–6 and 1.44 × 10–6 (Vigilant et al. 1991), and 4.14 × 10–6 (Ward et al. 1991) were used. Horai et al. (1995) obtained an estimate of 2.06 × 10–6, which closely agrees with one of those reported by Vigilant et al. (1991). Thus, independent evidence seems to favor estimates toward the middle of the range given. A generation time of 20 years was assumed. Distances between sequences were computed with the DNADIST program in the PHYLIP 3.5c package (Felsenstein 1989) according to Kimura’s two-parameter model (Kimura 1980), with the ratio of transitions to transversions set to 15 : 1 according to the proposal of Tamura and Nei (1993); other values of this parameter produced nearly identical trees. A neighbor-joining tree (Saitou and Nei 1987) was built from the distance matrix and was used to estimate a gene tree.
Results and discussion Classical polymorphic markers (i.e., blood groups, protein electromorphs, and HLA antigens) had revealed that Europe is a genetically homogenous continent, with few outliers (such as Saami, Sardinians, Icelanders, and Basques) among the small and clinal geographical variation (Cavalli-Sforza et al. 1993, 1994; Piazza 1993). The present analysis of mtDNA sequence also showed a high degree of homogeneity among European populations, which can be seen by: (1) allele sharing; unlike other continents, especially Africa, there are many sequences shared by more than one individual, often from different populations; and (2) small genetic distances (Pult et al. 1994; Bertranpetit et al. 1995); when European sequences were compared to published sequences from the rest of the world, genetic distances between European populations were found to be much smaller than between populations from other continents, especially Africa (results not shown). In a neighbor-joining tree of several world populations, European populations clustered in a tiny compact group [assembled under (1) in Fig. 2] close to Turkey and the Middle East, while other populations were connected to each other with much longer branches, a sign of a higher heterogeneity among populations. This genetic homogeneity is compatible either with a recent origin of the European population or with a migration rate throughout human history higher than in other continents; the second hypothesis is less likely even if the relatively small geographic distances among European populations are taken into account, as ancient human movements have been dependent on climatic and ecological conditions, not unique or special in Europe.
Fig. 2 Mitochondrial DNA (mtDNA) neighbor-joining tree linking 20 world populations. Genetic distances are intermatch-mismatch distances. Besides the nine European and West Asian populations analyzed, non-caucasoid populations have been included
Fig. 3 mtDNA neighbor-joining tree of European and West Asian populations. Numbers at the nodes are bootstrap percentages after 1000 resamplings
Although the mtDNA variability in Europe is small, it shows a robust pattern of differentiation. An unrooted genetic tree built for nine European and West Asian populations (Fig. 3) shows an approximately east-west gradient, with the Middle East at one extreme, followed by the two Turkish samples, most European populations, and, at the other end, the Basque sample. The tree obtained was also the bootstrap consensus tree (Felsenstein 1985), and most branches show high bootstrap supports, which strengthen the consistency of the topology presented. When two African populations (!Kung and Pygmies, Vigilant et al. 1991) were added as outgroups to the tree, they connected to the Middle East with a long branch. In 1000 bootstrap replicates, the African populations always clustered together and joined the Middle East in 52.8% of the itera-
446 Table 1 Samples used in this study. [N Number of individuals in the sample, k number of different sequences found, a number of variable nucleotides, b mean nucleotide pairwise differences, d mean number of steps per sequence in a maximum parsimony tree, H′ ratio of H (sequence diversity, estimated through the Shannon index) to Hmax (maximum H for a given sample size)]
Basques British Sardinians Tuscans Swiss Bulgarians Turks-1 Turks-2 Middle Easterners
N
k
a
b
d
H′
45 100 69 49 74 30 45 29 42
27 72 46 40 43 24 40 27 38
32 68 52 55 42 38 56 55 59
3.15 4.35 4.22 5.03 3.63 4.55 5.38 6.59 7.08
1.481 1.528 1.826 2.025 1.465 1.875 1.900 2.704 2.211
0.912 0.930 0.899 0.938 0.908 0.978 0.987 0.993 0.991
tions and either Turkish sample in 40% of the bootstrapped trees. We have found a direction to the spatial pattern of this gradient, which can be related to the direction of the main population expansion. As genetic distances alone cannot show a geometric sense in the genetic gradient (east to west or west to east), we resorted to the comparison of within-population parameters of genetic variation and complexity (Table 1): 1. The diversity of sequences within each population measured by H′ (see Materials and methods) presented its higher values in the east, where sequence diversity was richest, and genetic diversity declined markedly to the west, with a Spearman correlation coefficient (Sokal and Rohlf 1995) of –0.802 (P = 0.0082) with geographical distance from the Middle East.
Fig. 4 Nucleotide pairwise difference distributions in European and West Asian populations. For clarity, the nine populations have been distributed into two separate charts. Note that the distributions are shifted to the right in the West Asian (and presumably older) populations
2. Sequences were also more different from each other in the West Asian samples. Mean pairwise nucleotide differences decreased from 7.08 in the Middle East to 3.15 in the Basques and showed a Spearman correlation coefficient of –0.874 (P = 0.0022) with geographic distance. A comparable gradient was found in the mean number of steps linking each sequence in a putatively most parsimonious tree for each population. Therefore, the complexity of the structure of the sequence pool within a population decreased from east to west. Migration from Asia and Africa could have enriched the Middle Eastern mtDNA sequence pool and contributed to its higher diversity. However, as seen in Fig. 2, the Middle East presents genetic distances to African and Asian populations that are not much shorter than those between Europe and the other continents. Thus, migration from Africa and Asia does not seem to have produced significant inputs to the Middle East. The most likely explanation for such a pattern of decline in genetic diversity is a stepwise migration wave from east to west, with a reduction in population size at every step. Populations at the west may be considered as subsamples of those eastward, and thus migratory events in the opposite direction seem to have had little impact in the making of the main genetic variation features.
The distribution of nucleotide pairwise differences has been shown to reflect ancient population history, particularly expansions (or lack thereof) and their timing (Rogers and Harpending 1992; Harpending et al. 1993). A bellshaped pairwise difference distribution is the hallmark of a population expansion, and the mode of this distribution increases towards higher nucleotide difference values with time after the expansion. To date, all European samples studied (save the Saami, Sajantila et al. 1995) present smooth, bell-shaped pairwise difference distributions (Fig. 4) and their relative position denotes the same pattern of a series of expansions occurring sequentially from east to west. Again, this pattern is consistent with a migration from east to west tied to a population expansion. The dating of a population expansion hinges on precise knowledge of the mutation rate. Several estimates for the mutation rate in the hypervariable segment I of the control region have been published (as discussed in Materials and methods); the highest estimates are three times larger than the lowest. Therefore, only broad ranges for expansion times can be given, although there appears to be stronger support for intermediate rates and times. A least-square fit of the Rogers and Harpending model (Rogers and Harp-
447 Table 2 τ parameter from Rogers and Harpending (1992) model and population expansion times. Different mutation rates (given in mutations per nucleotide and generation) were used to estimate expansion dates. A generation time of 20 years was assumed. Horai et al. (1995) derived an estimate of µ = 2.06 × 10–6
Basques British Sardinians Tuscans Swiss Bulgarians Turks-1 Turks-2 Middle Easterners
τ
µ = 4.14 × µ = 2.16 × µ = 1.44 × 10–6 10–6 10–6
2.138 2.987 4.052 4.800 3.778 3.765 5.100 5.806 7.019
14 467 20 042 27 174 32 206 25 349 25 262 34 219 38 956 47 101
28 074 38 413 52 083 61 728 48 585 48 418 65 586 74 666 90 278
42 619 57 700 78 234 92 593 72 878 72 627 98 516 111 998 135 605
ending 1992; Harpending et al. 1993) has been computed in order to estimate expansion times from pairwise difference distributions. Such estimates are given in Table 2. The population with the oldest expansion is the Middle East; their ancestors seem to have expanded 50 000–135 000 years ago. Not surprisingly, the population whose ancestors expanded most recently (15 000–42 000 years ago), the Basques, are also the most distant from the Middle East. The decrease in the τ parameter from the Middle East shows a significant correlation with geographical distance (Spearman correlation coefficient, r = –0.826, P = 0.0057). Thus, expansion times also showed a significant gradient from east to west, much more gradual than would be expected for known cultural expansions (Champion et al. 1984; Straus 1989, 1994; Cunliffe 1994). A gene tree for all 279 different sequences (not shown) displayed the Cambridge reference sequence (Anderson et al. 1981) close to its center, which raises the possibility that this sequence was ancestral to all modern European sequences. In this case, and assuming that mutations accumulate in a Poisson process (Hudson 1990), the number of mutations relative to the reference sequence would follow a Poisson distribution with a mean λ = µlt, where µ is the mutation rate per nucleotide and generation, l is the sequence length, and t is the number of generations elapsed. European sequences have accumulated a mean λ = 3.30 mutations from their putative ancestor, which yields a time since origin of 45 000–127 000 years ago, according to different µ estimates. As some variation should exist in the first expanding population, this represents an upper boundary in the age of the most recent common ancestor, which is clearly much more recent than the ancestor of Neanderthals and supports a population replacement model. One haplogroup in the sequence tree was found to stem from a sequence absent from 11 geographically dispersed, non-European samples, comprising 376 individuals. Therefore, variation within this group should coalesce after the European and West Asia split from the rest. The mutation common to all sequences in this haplogroup is a C transition in position 16 126 and is the most frequent polymorphism in the set of sequences analyzed.
This mutation has not been found outside Europe except in a single Yoruba sample, making its independent origin likely. Its frequency is clearly clinal, from high values in the Middle East (47.6%) to a minimum in the Basque Country (6.7%). The mean number of substitutions within this group is λ = 3.40, which results in age estimations close to those derived from the whole ensemble of sequences. When considering the few sequences with a large number of substitutions (9–11), found in Sardinia, Turkey, and the Middle East, and closely related to each other, their frequency is fully consistent with a Poisson process as analyzed by the Kolgomorov-Smirnov goodness of fit test. From a phylogenetic point of view, none of the sequences may be considered significantly apart from the rest, making it unlikely that in the 483 individuals surveyed, control region sequences predating the uncovered expansion into Europe could persist. A sequence derived from a Neanderthal would be readily identifiable, as the time depth of their ancestors was much older (Wolpoff 1989) than that estimated from the actual European samples. It is expected that a sequence contributed by the Neanderthal would present a significantly large number of differences with respect to other sequences. As discussed above, no such sequence has been found in our sample of 483 sequences. Manderscheid and Rogers (1996) showed from a theoretical point of view that such an observation would rule out a Neanderthal admixture larger than 1%, given a global female effective population size of 16 000. The present results fully agree with the replacement hypothesis for the origin of AMH in Europe, because they place an upper limit on the date of the most recent common ancestor which is well below the time of the Homo erectus migration out of Africa. Yet our analyses show that the replacement dynamics is much more complex than usually assumed and cannot be understood by a single event such as the Upper Paleolithic expansion in Europe. Our results show that the replacement must have been slow, and that the demographic transition was a smoother process, not strictly tied to the successive Upper Paleolothic cultural changes. Due to the lack of human remains from the early Upper Paleolithic in Europe, it is not known whether concordance could be found with the pace of morphological change, but it is likely that it was not so dramatic as previously believed. Recent archaeological evidence (Hublin et al. 1996) supports a long term coexistence with technocultural interactions between the first modern humans and the last Neanderthals, in agreement with the present genetic results. Thus, the making of the basic modern European gene pool was a relatively recent, complex, and slow process that may have taken many millennia in the successive Upper Paleolithic cultural phases. Acknowledgements We thank M. Crawford, B. Vandermeersch, L. L. Cavalli-Sforza, C. Renfrew, D. Frayer, and C. Stringer for discussion and comments, and S. Iyengar for assistance with statistical packages. Lilianne Meignen provided valuable unpublished information. The meeting, Cultural Change and Human Evolution: the crisis at 40 000 BP, organized by E. Carbonell in Capellades, provided an excellent framework for discussion. Research was supported by Dirección General de Investigación Científica y Técnica (Spain) grant PB92-0722 and PB95-0267-C02-01, by Grup de
448 Recerca Consolidat 1995 SGR00205 from Generalitat de Catalunya and, by the Human Capital and Mobility Program from the European Commission (ERCHRXCT92-0032 and ERBCHRXCT920090) to J. B.
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