The mitochondrial lineage U8a reveals a Paleolithic settlement in the Basque country
© González et al; licensee BioMed Central Ltd. 2006
Received: 16 November 2005
Accepted: 23 May 2006
Published: 23 May 2006
It is customary, in population genetics studies, to consider Basques as the direct descendants of the Paleolithic Europeans. However, until now there has been no irrefutable genetic proof to support this supposition. Even studies based on mitochondrial DNA (mtDNA), an ideal molecule for constructing datable maternal genealogies, have failed to achieve this. It could be that incoming gene flow has replaced the Basque ancient lineages but it could also be that these lineages have not been detected due to a lack of resolution of the Basque mtDNA genealogies. To assess this possibility we analyzed here the mtDNA of a large sample of autochthonous Basques using mtDNA genomic sequencing for those lineages that could not be unequivocally classified by diagnostic RFLP analysis and control region (HVSI and HVSII) sequencing.
We show that Basques have the most ancestral phylogeny in Europe for the rare mitochondrial subhaplogroup U8a. Divergence times situate the Basque origin of this lineage in the Upper Palaeolithic. Most probably, their primitive founders came from West Asia. The lack of U8a lineages in Africa points to an European and not a North African route of entrance. Phylogeographic analysis suggest that U8a had two expansion periods in Europe, the first, from a south-western area including the Iberian peninsula and Mediterranean France before 30,000 years ago, and the second, from Central Europe around 15,000–10,000 years ago.
It has been demonstrated, for the first time, that Basques show the oldest lineages in Europe for subhaplogroup U8a. Coalescence times for these lineages suggest their presence in the Basque country since the Upper Paleolithic. The European U8 phylogeography is congruent with the supposition that Basques could have participated in demographic re-expansions to repopulate central Europe in the last interglacial periods.
Considering Basques as the direct descendants of Paleolithic Europeans has become a multidisciplinary premise. However, there is no irrefutable evidence for this supposition. The Basque country has a well represented archaeological record in Paleolithic and Mesolithic periods  but Archaeology can seldom differentiate an "in situ" cultural evolution from successive waves of new incomers. Basques speak a non Indo-European language with no close affinities with any other extant language but, even if their roots could be found, they would not reach the Paleolithic deepness due to the fast rate of change of languages. Classical population genetic studies, showed the Basques as one of the major outliers in Europe . Nevertheless, these results can be explained by genetic drift which implies isolation but not necessarily an old history for that population. Lack of recombination and the fast mutation rate made mtDNA the ideal molecule to construct maternal genealogies, which frame in time and space the evolution and dispersion of human populations. However, until now, mtDNA studies on the Basques have only confirmed its low genetic diversity in a common Western Europe background [3–5]. It has been proposed, on the basis of their geographic distributions, that several mitochondrial lineages as V [6, 7], and the H1 and H3 subgroups [4, 5, 8] are markers of a Paleolithic human dispersal from southwestern Europe, including the Basque country, to Northeast Europe. However, diversities for these lineages are not higher in Basques than in Central Europeans. It could be possible that this lack of distinctness in Basques is real, in fact, even small levels of gene flow during enough time might have replaced the majority of their ancient lineages , but it could also be possible that this uniformity is due to a lack of resolution of the Basque mtDNA genealogies .
To deal with this possibility, we analyzed a sample of 211 unrelated Basques using the hypervariable segment of the mtDNA control region (HVSI-II) and diagnostic RFLP analysis, and sequenced the complete mitochondrial DNA of the rare lineages.
Results and discussion
Gene diversity (p) and frequency of U8a/1 in different European macro-regions.
p ± σ
2.0 ± 1.7
2.8 ± 1.9
1.3 ± 1.4
0.0 ± 0.0
0.7 ± 0.8
2.2 ± 1.6
1.6 ± 1.1
0.0 ± 0.0
0.0 ± 0.0
In summary, the analysis of U8a lineages supports the idea that Basques have lived in their country since the Paleolithic, and that they could have participated in demographic re-expansions to repopulate central Europe in the last interglacial periods. Furthermore, these primitive U8a founders most probably reached the Basque area from the East through Europe and not through North Africa. However, the fact that we can trace some Basque lineages back to the Paleolithic does not support the generalized supposition that the present day Basque population is the best representative of Paleolithic Europeans. First of all, U8a haplotypes only represent 1% of the present day Basque maternal pool, therefore, a complex set of different mtDNA lineages with possible different histories are left unstudied. In addition, there is empiric evidence that Basques have received recent male gene flow from adjacent areas , and even possible maternal North African influences predating the Muslim Iberian invasion . Furthermore, ancient DNA studies on Basque historic and prehistoric samples  have detected important mtDNA haplogroup frequency fluctuations along different periods. Definitively, like other European populations, Basques have also suffered migration and genetic drift effects throughout its long history.
DNA isolated from bucal swabs or blood samples from 211 autochthonous, unrelated Basques from the Iberian provinces were analyzed. Appropriate informed consent to anonymously use their data was obtained from all the individuals sampled.
HVSI-II and RFLPs
Total DNA was PCR amplified as in Pinto et al , and directly sequenced for both complementary strands as detailed in Rando et al . A sequence of 978 bp of the HVSI-II of the mtDNA control region, from position 15997 to 00408  was determined and sorted into defined haplogroups . To confirm this HVS-based haplogroup classification, all individuals assigned to a specific haplogroup were additionally tested by restriction analysis of the diagnostic coding region mutations proposed to unambiguously classify sequences into haplogroups .
Complete mtDNA sequences
Four Basques (three U8a, one K1) and one Jordan (U8b) rare lineages belonging to the U/K haplogroup were fully sequenced. The complete mitochondrial DNAs (mtDNA) were amplified by PCR using primer pairs already described . Amplified products were sequenced for both complementary strands with the Big Dye Terminator Cycle sequencing kit (Applied Biosystems). Sequencing reactions were analyzed on an Applied Biosystems 3100 DNA analyzer.
Genetic diversity (p ± σ) was estimated as the average number of nucleotide differences between two sequences , using the HVSI region in the range from 16070 to 16365 nucleotide positions.
Phylogenetic relationships among complete mtDNA sequences, and among control region mtDNA sequences, were established using the reduced median network algorithm . In addition to our five sequences, seven lineages were added: Dutch (Dut86; GenBank: DQ112821) [ and P. Shen, pers. comm], Spanish (Spa, GenBank: AY882392) and Italian (Ita, GenBank: AY882393) , 3 Finns (Fin; GenBank: AY339551, AY339552 and AY339553)  and British-Australian (BriT7; [T. Obayashi, M. Tanaka, pers. comm.]).
The presence of U8a was tested in published sequences by the 16146 16342 HVSI motif and/or the 73 282 HVSII motif, and U8b by the 16189 16234 motif. 19,133 Eurasian and 1,430 North-African published and unpublished HVSI/HVSII sequences were analyzed.
Time estimations, based on control region mtDNA, were calculated as the mean divergence ρ  from inferred ancestral sequence types and converted into time by assuming that one transition within np 16090–16365 corresponds to 20,180 years .
The five complete mitochondrial DNA sequences are registered in [GenBank: DQ200801, DQ200802, DQ200803, DQ200804, and DQ200805].
We thank Dr. P. Shen and Drs. T. Obayashi and M. Tanaka, for providing us information about the Dutch (Dut86) and British-Australian (BriT7) sequences, respectively.
This study was supported by grants BMC2001-3511 from Ministerio de Ciencia y Tecnología and COF2002-015 from Gobierno de Canarias to V.M.C
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