BACKGROUND
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  <cit>  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  <cit> .
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  <cit> .
it has been proposed, on the basis of their geographic distributions, that several mitochondrial lineages as v  <cit> , and the h <dig> and h <dig> subgroups  <cit>  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  <cit> , but it could also be possible that this uniformity is due to a lack of resolution of the basque mtdna genealogies  <cit> .
to deal with this possibility, we analyzed a sample of  <dig> unrelated basques using the hypervariable segment of the mtdna control region  and diagnostic rflp analysis, and sequenced the complete mitochondrial dna of the rare lineages.
RESULTS
the analysis of the basque sample showed three haplotypes  that by their mutated positions in the control region had uncertain subhaplogroup adscription, but that by diagnostic rflps  belonged to haplogroup u/k.
also, we found one individual  that belongs to the scarce u8a subhaplogroup.
complete sequencing of the four rare u haplotypes and their inclusion in a phylogenetic tree  <cit>  with all published u complete sequences  allowed their correct subhaplogroup affiliation.
a more schematic tree  shows that one of the lineages  belongs to subhaplogroup k1a <dig>  <cit> , being a back-mutation of the diagnostic position  <dig> its main peculiarity.
its most related k1a <dig> complete sequences are the eleven found by herrnstadt et al.
<cit>  and finn  <dig> in finnilä et al.
<cit> .
applying a mutation-rate of  <dig>  × 10- <dig>  <cit>  to their average sequence divergence  <cit>  a radiation upper bound of  <dig> ±  <dig> ky is obtained for this group.
the other three lineages clustered into the rare subhaplogroup u8a  <cit> .
this subhaplogroup can be rflp diagnosed as - <dig> alu i. what is outstanding of these sequences is their great genetic diversity that extends the range of all known u8a european sequences .
this basque diversity specially contrasts with the lack of variation in the finn sequences.
furthermore, the phylogenetic radiation of their u8a lineages  is characteristic of an old population without recent exponential growth  <cit> .
in fact, the most ancestral sequence  indicates that u8a lineages could have been in the basque country since  <dig> ±  <dig> ky, and that the other basque lineages, belonging to the u8a <dig> subgroup , participated in a more recent european expansion around  <dig> ±  <dig> ky, similar to that estimated for k1a, and congruent with a re-expansion from an iberian refuge when glaciers retreated in europe proposed for other mtdna clades  <cit> .
although all the u8a complete sequences belong to europeans, the ancestral radiation of haplogroup u most probably occurred in western asia shortly after the out of africa episode  <cit> , with early branch expansions to india , europe  and north africa .
u <dig> may be considered another main branch with a broad geographic range.
its first split separated u8a from u8b/k around  <dig> ±  <dig> ky. relatively short in time a new subdivision gave the sister clades u8b and k  <cit> .
until now there was only one completely sequenced u8b subject  <cit> .
the addition of our jordan  <dig> sequence to the tree  gives a branching point for u8b, defined by transitions  <dig>   <dig> and  <dig>  two of them,  <dig> and  <dig>  can be mnli-rflp-detected.
curiously, the similar number of substitutions to the coalescence point of the three u <dig> branches u8a , u8b, k, suggests that all of them radiated at a similar age, supporting the hypothesis that, most probably, global climatic changes favored human expansions simultaneously at a continental scale  <cit> .
when only rflps and/or partial sequence data are available, u8a haplotypes in general can be identified by the - <dig> alu i rflp or the  <dig>   <dig> hvsii motif, and the majority of u8a <dig> derivates in particular by the <dig>   <dig> hvsi motif.
in turn, u8b can be classified by the  <dig>   <dig> hvsi motif.
from a total of  <dig>  sequences studied  <dig>  could be, unequivocally, u8a analyzed  and referenced .
for the remaining , in general, only their u8a <dig> assignation was possible.
analysis of  <dig>  eurasian, and  <dig>  north african published and unpublished rflp/hvsi/hvsii sequences showed a scattered but widespread u8a/ <dig> distribution that is restricted to europe.
its frequency  ranges from  <dig> in the majority of samples to 8%  in the region of var in southeast france  <cit> .
in spite of its moderate sample size an important characteristic of this region is its high u8a polymorphism as all the three lineages detected are different.
the u8a eastern boundaries seem to be in the volga region near the urals  <cit> .
u8b is also a quantitatively minor clade that partially overlaps with u8a in europe.
however, its presence in the caucasus  <cit> , iran  <cit> , the near east  <cit> , and north africa  <cit> , where u8a has not been detected, attests a more southern geographic distribution.
the third sister clade k, is the most widespread and abundant covering the u8a and u8b ranges  <cit>  and even reaching india  <cit> .
a network  <cit>  built with the  <dig> u8a sequences found , could be rooted and resolved attending to the phylogeny of the u <dig> complete sequences.
its most ancestral node is represented by bq <dig> and the only anatolian lineage assignable to haplogroup u8a, both carrying the crs motif in hvsi, and transitions  <dig>   <dig> in hvsii.
this ancient connection might trace the hypothetic route followed by the u8a ancestor from west asia to the basque country.
the absence of u8a in north-africa and its extremely rare presence in the eastern mediterranean area further reinforces this continental route of entrance against a southern alternative.
it is deduced from the network  that a first u8a radiation in europe affected iberia, central europe and reached the baltic.
a second, u8a <dig>  broader expansion further enlarged its range to russia and scotland where the u8a diversities are lower than in the central area .
as the total rooted network does not achieve the star genealogy, to calculate coalescence ages  <cit> , we estimated the average distance and coalescence age to the u8a <dig> founder haplotype  and to the ancestral u8a haplotype  independently, representing, in the last case, the u8a <dig> radiation by only one basic lineage.
time estimations for the younger u8a <dig> expansion was  <dig> ±  <dig> ky and  <dig> ±  <dig> ky for the u8a subset that, if added to first, would give a total time for the u8a coalescence of around  <dig> ±  <dig> ky. notice that both hvsi estimations are higher than, but not significantly different, from those calculated using complete sequences.
to compare the u8a diversity  among regions, we grouped the european populations in different paleolithic areas  <cit> .
the greatest diversity was found in the iberian peninsula when basques are included, followed by the north central area .
these data agree with the primary and secondary origins of expansion proposed on phylogenetic grounds, weakening the possibility that basques would have obtain their total u8a diversity through recent immigrations from other european areas and reinforcing the hypothesis that the first u8a radiation in europe happened in an area in which the basque country was included.
a name region: bq = basque; ip = iberian peninsula; mc = mediterranean centre; me = mediterranean east; an = anatolia; ca = caucasus; sw = southwest europe; al = alpine; se = southeast europe; nw = northwest europe; nc = north central europe; ne = northeast europe; vu = volga-ural; sc = scandinavia
CONCLUSIONS
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  <cit> , and even possible maternal north african influences predating the muslim iberian invasion  <cit> .
furthermore, ancient dna studies on basque historic and prehistoric samples  <cit>  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.

 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. it has been proposed, on the basis of their geographic distributions, that several mitochondrial lineages as v, and the h and h subgroups 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 unrelated basques using the hypervariable segment of the mtdna control region and diagnostic rflp analysis, and sequenced the complete mitochondrial dna of the rare lineages.  the analysis of the basque sample showed three haplotypes that by their mutated positions in the control region had uncertain subhaplogroup adscription, but that by diagnostic rflps belonged to haplogroup u/k. also, we found one individual that belongs to the scarce u8a subhaplogroup. complete sequencing of the four rare u haplotypes and their inclusion in a phylogenetic tree with all published u complete sequences allowed their correct subhaplogroup affiliation. a more schematic tree shows that one of the lineages belongs to subhaplogroup k1a, being a back-mutation of the diagnostic position its main peculiarity. its most related k1a complete sequences are the eleven found by herrnstadt et al. and finn in finnilä et al.. applying a mutation-rate of × 10- to their average sequence divergence a radiation upper bound of ± ky is obtained for this group. the other three lineages clustered into the rare subhaplogroup u8a. this subhaplogroup can be rflp diagnosed as - alu i. what is outstanding of these sequences is their great genetic diversity that extends the range of all known u8a european sequences. this basque diversity specially contrasts with the lack of variation in the finn sequences. furthermore, the phylogenetic radiation of their u8a lineages is characteristic of an old population without recent exponential growth. in fact, the most ancestral sequence indicates that u8a lineages could have been in the basque country since ± ky, and that the other basque lineages, belonging to the u8a subgroup, participated in a more recent european expansion around ± ky, similar to that estimated for k1a, and congruent with a re-expansion from an iberian refuge when glaciers retreated in europe proposed for other mtdna clades. although all the u8a complete sequences belong to europeans, the ancestral radiation of haplogroup u most probably occurred in western asia shortly after the out of africa episode, with early branch expansions to india, europe and north africa. u may be considered another main branch with a broad geographic range. its first split separated u8a from u8b/k around ± ky. relatively short in time a new subdivision gave the sister clades u8b and k. until now there was only one completely sequenced u8b subject. the addition of our jordan sequence to the tree gives a branching point for u8b, defined by transitions and two of them, and can be mnli-rflp-detected. curiously, the similar number of substitutions to the coalescence point of the three u branches u8a, u8b, k, suggests that all of them radiated at a similar age, supporting the hypothesis that, most probably, global climatic changes favored human expansions simultaneously at a continental scale. when only rflps and/or partial sequence data are available, u8a haplotypes in general can be identified by the - alu i rflp or the hvsii motif, and the majority of u8a derivates in particular by the hvsi motif. in turn, u8b can be classified by the hvsi motif. from a total of sequences studied could be, unequivocally, u8a analyzed and referenced. for the remaining, in general, only their u8a assignation was possible. analysis of eurasian, and north african published and unpublished rflp/hvsi/hvsii sequences showed a scattered but widespread u8a/ distribution that is restricted to europe. its frequency ranges from in the majority of samples to 8% in the region of var in southeast france. in spite of its moderate sample size an important characteristic of this region is its high u8a polymorphism as all the three lineages detected are different. the u8a eastern boundaries seem to be in the volga region near the urals. u8b is also a quantitatively minor clade that partially overlaps with u8a in europe. however, its presence in the caucasus, iran, the near east, and north africa, where u8a has not been detected, attests a more southern geographic distribution. the third sister clade k, is the most widespread and abundant covering the u8a and u8b ranges and even reaching india. a network built with the u8a sequences found, could be rooted and resolved attending to the phylogeny of the u complete sequences. its most ancestral node is represented by bq and the only anatolian lineage assignable to haplogroup u8a, both carrying the crs motif in hvsi, and transitions in hvsii. this ancient connection might trace the hypothetic route followed by the u8a ancestor from west asia to the basque country. the absence of u8a in north-africa and its extremely rare presence in the eastern mediterranean area further reinforces this continental route of entrance against a southern alternative. it is deduced from the network that a first u8a radiation in europe affected iberia, central europe and reached the baltic. a second, u8a broader expansion further enlarged its range to russia and scotland where the u8a diversities are lower than in the central area. as the total rooted network does not achieve the star genealogy, to calculate coalescence ages, we estimated the average distance and coalescence age to the u8a founder haplotype and to the ancestral u8a haplotype independently, representing, in the last case, the u8a radiation by only one basic lineage. time estimations for the younger u8a expansion was ± ky and ± ky for the u8a subset that, if added to first, would give a total time for the u8a coalescence of around ± ky. notice that both hvsi estimations are higher than, but not significantly different, from those calculated using complete sequences. to compare the u8a diversity among regions, we grouped the european populations in different paleolithic areas. the greatest diversity was found in the iberian peninsula when basques are included, followed by the north central area. these data agree with the primary and secondary origins of expansion proposed on phylogenetic grounds, weakening the possibility that basques would have obtain their total u8a diversity through recent immigrations from other european areas and reinforcing the hypothesis that the first u8a radiation in europe happened in an area in which the basque country was included. a name region: bq = basque; ip = iberian peninsula; mc = mediterranean centre; me = mediterranean east; an = anatolia; ca = caucasus; sw = southwest europe; al = alpine; se = southeast europe; nw = northwest europe; nc = north central europe; ne = northeast europe; vu = volga-ural; sc = scandinavia  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. 
the european u phylogeography is congruent with the supposition that basques could have participated in demographic re-expansions to repopulate central europe in the last interglacial periods. even studies based on mitochondrial dna, an ideal molecule for constructing datable maternal genealogies, have failed to achieve this. the lack of u8a lineages in africa points to an european and not a north african route of entrance. 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 sequencing. it is customary, in population genetics studies, to consider basques as the direct descendants of the paleolithic europeans. 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 years ago, and the second, from central europe around – years ago. coalescence times for these lineages suggest their presence in the basque country since the upper paleolithic.
 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, 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 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 years ago, and the second, from central europe around – 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 u phylogeography is congruent with the supposition that basques could have participated in demographic re-expansions to repopulate central europe in the last interglacial periods. 