BACKGROUND
the x-chromosome has features that make it a good source of information for population genetic studies.
the x-chromosome is present in a single copy in males, which makes it possible to determine the x-chromosome haplotypes in men.
compared with autosomes, the x-chromosome has lower recombination rate, lower mutation rate and smaller effective population size resulting in a faster genetic drift.
in consequence, both linkage disequilibrium  and population structure in the x chromosome are expected to be stronger than those in autosomes  <cit> .
two-thirds of the x-chromosome history has been spent in females.
thus, x chromosome polymorphisms mainly reflect the history of females.
due to recombination, x-chromosome markers in females provide a multilocus system, while the mtdna and y-chromosome are linked haplotypes.
thus, x-chromosome markers are valuable for population genetic studies  <cit> .
due to its history with constant migration movements, the mediterranean area constitutes an challenging region for population genetic studies, both considering the whole area  <cit>  and particular populations  <cit> .
only very little genetic structure has been found among populations living on the northern and eastern shores of the mediterranean sea by analyses of y-chromosome strs  <cit>  and mtdna  <cit> .
the pattern may be a consequence of the neolithic demic diffusion in this region  and/or a high level of gene flow in the area.
the presence of a strong genetic boundary between the northern and southern mediterranean populations, especially in the western side of the sea has been suggested  <cit> .
studies focused on smaller areas have also given interesting results, especially those regarding physical and/or cultural isolates such as corsican  <cit>  and sardinian sub-isolates  <cit> , balearic populations  <cit>  and calabrian populations  <cit> .
in order to further investigate the genetic characteristics of the populations in the mediterranean basin, we analyzed  <dig> snps located on the x-chromosome in eleven populations from the mediterranean area.
in addition, two populations from northern europe and east africa were analyzed.
we carried out a wider analysis using data from the hapmap website on  <dig> out of the  <dig> snps used in this study .
we wanted to use the x-chromosome snps as multilocus markers.
we selected polymorphic x chromosome snps with a physical distance of at least  <dig> kb.
population history such as genetic drift and admixture has an important consequence on the degree of population ld  <cit> .
the level of ld on the x-chromosome is expected to be higher than on autosomes.
we also wanted to use these markers to study the pairwise linkage disequilibrium and multilocus association in order to obtain knowledge of the long-term and background ld in each population, because this information can be useful for studies of population genetics and the genetics of complex diseases.
we studied the informativeness of the markers selected and the sex-biased migration rate and compared the information to that of non-recombinant markers in mtdna and the y-chromosome.
RESULTS
intrapopulation variability
a total of  <dig>  different haplotypes were found in the  <dig> populations analyzed by means of  <dig> x-chromosome snps.
no single haplotype was shared among individuals within or between populations.
all the markers were polymorphic in all the populations studied.
the minor allele frequency varied between a minimum percentage of  <dig> %  and a maximum value of around  <dig> % .
the mean gene diversity  of the  <dig> markers analyzed was very homogeneous in the  <dig> populations sampled.
they ranged from  <dig>  in the somali population to  <dig>  in iraqis and tunisians.
taking into account that the maximum level of expected heterozygosity for biallelic markers is  <dig> , a high degree of gene diversity was found for the  <dig> snps selected in all the populations analyzed.
among the  <dig> pairwise comparisons between the  <dig> snp markers in the populations, between  <dig>  and  <dig> pairwise comparisons  showed significant p-values .
however, after holm-sidak correction, only two pairwise ld values were significant .
nevertheless, when multilocus tests of associations were performed using all the  <dig> markers, the null hypotheses of linkage equilibrium were rejected in two populations: ibiza  and cosenza .
both populations present some peculiarities that may explain the existence of a higher degree of background linkage disequilibrium .
interpopulation differentiation
amova and pairwise fst values
fst:  <dig>
p-value:  <dig>
a degrees of freedom.
b sum of squares deviations among populations.
c sum of squares deviations within populations.
fst :  <dig>
p-value <  <dig>
a degrees of freedom.
b sum of squares deviations among populations.
c sum of squares deviations within populations.
when a locus by locus amova was performed in the mediterranean group, only three out of  <dig> markers showed significant  variations among the mediterranean populations , while  <dig> out of  <dig> markers were important for the differentiation of the  <dig> "worldwide" populations: x <dig>  x <dig>  x <dig>  x <dig>  x <dig>  x <dig>  x <dig>  x <dig>  x <dig>  x <dig>  x <dig>  x <dig>  x <dig>  x <dig>  x <dig>  x <dig> and x <dig>  the markers x <dig>  x <dig>  x <dig> and x <dig> were not included in these analyses due to the lack of information in the populations obtained from the hapmap project.
no correlation  was found between the mean gene diversity and the level of differentiation among populations, as has been suggested by others  <cit> .
in order to obtain a graphical representation of the genetic structure of the populations studied, a principal coordinate analysis  was made from the pairwise reynold's fst matrix obtained using  <dig> or  <dig> x-chromosome snps in the mediterranean  and the  <dig> populations , respectively.
in figure 1a, the first two axes accounted for 39% of the total variability .
thus, the mediterranean populations are quite homogeneous.
equivalently to the results of the amova analysis, the moroccan population was considerably displaced in the first axis, and the majorcans were placed in the opposite direction.
in the second axis, the differentiation was less, but it is interesting that the cosenza and ibiza populations were located opposite to their neighbouring populations .
figure 1b shows the relative position of the mediterranean populations in relation to a wider geographical area.
the first two axes accounted for 51% of the global variability .
the first axis displayed east asian populations  opposite to the others .
in the second axis, the mediterranean populations plus the ceu population  formed a group between the north-european and african populations.
only the moroccans showed a slight displacement from the others showing significant fst values in most of the pairwise comparisons inside the mediterranean group.
the ceu population showed an intermediate position between the north of europe, represented by the danish population, and the mediterranean group.
none of the pairwise comparisons between the ceu population and the mediterranean or danish populations showed a significant fst value.
for the grouping proposed in figure 1b,  <dig> % of the variation could be explained by differences between groups .
a more detailed hierarchical amova analysis showed that  <dig> % of the global variation could be explained by the east asian populations ,  <dig> % of the variation was explained by somalis and sub-saharans .
the mediterranean group  contributed to  <dig> % of the global variation, a value that went down to  <dig> % when the moroccan population was excluded from the group .
finally, the north-west european populations  only explained  <dig> % of the global variation .
structure analysis
additionally to the amova analysis, a cluster analysis was performed using the structure  <cit>  software in order to assign individuals to a number of k populations allowing admixture.
we estimated the posterior probability , where x represents the genotypes) for k between  <dig> and  <dig> for  <dig> populations  using  <dig> x-chromosome snps.
we observed that the proportions of the individuals assigned to each k subpopulation were balanced in all cases indicating that the x-chromosome snps did not identify to which subpopulation an individual belongs.
according to the authors of the programme  <cit> , this could be interpreted as a lack of population structure as a consequence of a high level of admixture.
due to the statistical model implemented in structure, it is possible to find significant fst values without finding significant differences using structure when the populations analyzed are closely related  <cit> .
this is not the case of some of the populations included in the analysis.
as it was discussed by other authors  <cit> , it could be that the high migration rate of females could have resulted in a small genetic structure in x-linked loci.
on the other hand, a lack of resolution of the investigated snp markers could also be the cause of the low level of genetic structure observed.
isolation by distance
we did not find any significant correlation between genetic differentiation ) and the natural logarithm of the geographical distance  in the mediterranean area based on the study of  <dig> x-chromosome snps .
according to rousset's isolation-by-distance model  <cit> , some demographic information can be obtained from the model drawn .
an indirect estimation of 4d μσ <dig>  also known as the "neighbourhood size" , could be calculated from the slope value .
considering the  <dig> biallelic markers analyzed in the present work, the estimated value of 4d μσ <dig> would be around  <dig> individuals, which seems to be an underestimated value according to the same author.
this data can be interpreted as the result of a high number of migration events in the recent history of the human populations in the mediterranean basin.
the number of estimated migrants was dramatically reduced when a much wider area was considered ,  <dig> asian populations  and  <dig> european populations .
the analyses of populations belonging to an extended area are more prone to reflect more ancient demographic processes  <cit> .
if the neolithic transition occurred around  <dig>  ybp, and human dispersal was close to  <dig> km per generation, pre-agricultural migration patterns could be observed at distances over  <dig>  km  <cit> .
taking this information into account and the fact that the minimum geographic distance between two populations in figure 2b was  <dig> km, pre-agricultural migration events should be reflected there.
on the basis of  <dig> x-chromosome snps, an indirect estimate of  <dig> migrants between subpopulations was calculated from the regression slope .
in this study, isolation by distance operated better at large geographic scales than at short distances.
x-chromosome snps vs. non-recombinant systems
mantel test
we performed two mantel tests for each of the three sets of markers:  <dig> x-chromosome snps, hvri mtdna and  <dig> y-chromosome snps .
first, we analyzed  <dig> of the  <dig> populations in this work for which results of all the markers were available.
then, only the mediterranean populations were analyzed .
mantel correlations were corrected for geographical distances by computing partial correlations between genetic distances at constant geographic distance .
all tests were significant  except the test between x-chromosome and mtdna in the mediterranean area, where the correlation index was low  but still positive.
the lack of significance was most likely due to the low degree of genetic differentiation of both x-chromosomes and mtdna in the area.
all x-chromosome and mtdna markers showed a significant correlation  with y-chromosome snps both when  <dig> or  <dig> populations were analyzed.
except in particular cases , male and female movements in the mediterranean have apparently had the same overall tendency.
an important difference in the intensity of the migration events of both sexes could also be seen in the level of significance of the different mantel tests.
a populations: den, ibz, irq, maj, mor, rca, sic, som, tun, trk and val.
bpopulations: ibz, irq, maj, mor, rca, sic, tun, trk and val.
for abbreviators see figure  <dig>
isolation-by-distance analyses by using different genetic systems
as expected from previous studies  <cit> , no significant correlation between genetic and geographic distances was found in any case.
from a demographic point of view, the estimated number of migrants based on x-chromosome results would be approximately the same as calculated above , and around  <dig> were calculated from y-chromosome snp data.
surprisingly, a negative slope was obtained in the case of the mtdna, reflecting a major effect of isolation and genetic drift versus migration in certain populations of the mediterranean area.
when x-chromosome snps and mtdna were analyzed in a wider geographic area , a greater female migration rate compared to the male migration rate was still observed.
as it has been commented above, the minimum geographic distance between two populations was approximately  <dig> km and mainly pre-agricultural migration events were then represented in this case.
discussion
a considerable amount of information based on classical genetic markers  <cit> , autosomal strs  <cit> , mtdna  <cit>  and y-chromosome haplotypes  <cit>  has previously been reported concerning the genetics of populations in the mediterranean region.
a low level of genetic structure has been described due to the high degree of migration in the mediterranean area  <cit> .
apparently, the surrounding lands of the mediterranean sea were settled by people from the middle east approximately  <dig>  years ago  <cit> .
the migration events increased during the neolithic era  with the transition to agriculture.
the increase in migration events during the history constitutes the main cause of the genetic homogeneity in the area.
several authors  <cit>  have suggested the existence of a north-south genetic "barrier" in the western part, and no consensus on the current genetic landscape has been established  <cit> .
some isolated populations with deviating genetic differentiation have been described  <cit> .
based on the study of  <dig> x-chromosome snps, we found a low value of population differentiation  in  <dig> mediterranean populations.
the fixation index was significant  but the p-value increased to p =  <dig>  when the moroccan population was excluded from the analysis.
in contrast, a  <dig> times higher fst value  was observed when  <dig> populations from three continents  were analyzed.
not surprisingly, the strongest genetic differentiation was found among the  <dig> african populations.
it is well known that african populations have a higher level of genetic heterogeneity compared to european or asian populations  <cit> .
a certain bias of the results obtained could be due to the fact that the markers initially were selected for forensic purposes with a high level of polymorphism in different ethnic groups.
a high level of intrapopulation variability will to some degree be associated with a low interpopulation variability  <cit> .
nevertheless, the significant fst value found in the mediterranean populations and the highly significant fixation index observed when distant populations were compared documents the suitability of these markers for population genetic studies.
geographic "barrier", genetic isolate and population admixture
the analysis of the mediterranean populations in comparison to the "worldwide" populations showed a clear intermediate position between african and north-european populations with morocco somewhat closer to the african populations than the other mediterranean populations.
when only the mediterranean populations were analyzed in a principal coordinate plot, opposite positions of moroccans and majorcans were observed .
this supports the existence of a north-south gene flow "barrier" in the western part of the mediterranean area  <cit> .
it could be argued that the genetic differentiation of the moroccan population was a result of the arab-berber sub-structure of the individuals sampled in this work, but this hypothesis does not seem likely.
several authors  <cit>  reported a high genetic homogeneity between berbers and arabs in nw africa, so they suggested that the arabisation of this area was probably a cultural phenomenon, which did not imply a replacement of the ancestry population.
our results give support the hypothesis of an early settlement of nw africa  <cit> .
the original berber population seem to have received a low genetic influx from the surrounding areas.
different hypothesis have been suggested to explain the genetic differentiation of the moroccan population.
an initial genetic drift  <cit>  could have caused differences in allele frequency distribution that have not been re-established due to a certain level of geographic isolation.
the strait of gibraltar has been described by several authors  <cit>  as an important genetic barrier.
even a certain level of genetic exchange probably occurred between nw africa and the south of the iberian peninsula  <cit> , sharp frequency changes have been described in this area  <cit> .
also the sahara desert has been suggested as responsible of the genetic isolation of nw african populations from sub-saharan populations  <cit> .
there is no consensus about the impact of the neolithic demic diffusion in the mediterranean area  <cit> .
according to our results, a low impact of the neolithic expansions and/or later migration events on nw african populations would have occurred.
nevertheless, the high genetic homogeneity observed and especially the low level of genetic pairwise differentiations between the iraqi and the western mediterranean populations  supports the impact of several migration events  <cit>  during the history in the genetic constitution of the mediterranean area, where sailing at the mediterranean sea have been an important connection between various geographical areas and populations  <cit> .
the high genetic homogeneity of x-chromosome markers contrasts with the genetic structure of y-chromosome snps  <cit> .
this highlights the differences between male and female migration rates, and stresses the importance of combining results obtained from different types of genetic markers.
tunisians did not show a significant level of differentiation with northern populations as mentioned by others  <cit> .
a discordant result may be due to the different nature of the markers used in the different works .
also the heterogeneity described in tunisian berbers  <cit>  could explain that studies carried out using different sets of tunisian samples may end up showing different results.
more recent demographic events that took place in some particular populations in the area showed interesting exceptions to the general homogeneity.
this was the case of the cosenza and ibiza populations that were placed relatively "far" from their neighbouring populations in the various statistical analyses performed.
tagarelli et al  <cit>  described the calabria province as a collection of many "human genetic isolates".
in contrast to catanzaro and reggio di calabria, cosenza did not suffer destructive telluric events that would have modified its population structure.
so, the population heterogeneity inside cosenza is more patent than in any of the other two populations.
this is reflected by the heterogenic distribution of various genetic markers at the coast compared to the internal areas  <cit> .
the results obtained with the  <dig> x-chromosome markers were in agreement with the previous studies.
cosenza showed not just a relatively high genetic differentiation compared to the populations from catanzaro and reggio di calabria but also a certain displacement in relation to other mediterranean populations.
this was also the case of the ibiza population.
ibiza, one of the three major islands of the balearic archipelago, was reproductively isolated for centuries, their population effective size was reduced by infectious diseases and a high number of consanguineous marriages were reported in the 15th-17th centuries  <cit> .
in 1970s, the tourist influx considerably increased the population of ibiza  <cit> .
former studies have emphasized the genetic differentiation of this population as a consequence of the stochastic events that happened during its history  <cit> .
moreover, when a parametric multilocus association analysis was performed, significant values of associations were only observed in the ibiza and cosenza populations.
in most human populations, high levels of linkage disequilibrium do not exist between markers separated > <dig> kb  <cit> .
taking the distance  between the markers selected into consideration, we did not expect to find significantly increased levels of ld in any of the populations studied.
on the other hand, it is well known that different demographic scenarios could result in unexpected levels of ld over large genomic regions  <cit> .
sex-biased migration rate: a comparative study among different parts of the genome.
sex-biased migration rates vary between populations as well as through population history.
matrilocality results in a mtdna geographic structure while patrilocality results in a genetic structure in the non-recombinant region of the y-chromosome  <cit> .
the x-chromosome, which has spent 2/ <dig> of its history in females, reflects matrilocality to a higher degree than patrilocality.
in the light of our findings it can be said that the migration rate was higher in women than in man, both when our results were analyzed with the mantel test and under rousset's isolation-by-distance model.
the regression slope obtained from mtdna  and x-chromosome markers were smaller than those obtained from y-chromosome snps.
the negative slope obtained from mtdna data can be explained by the fact that populations geographically close to each other showed a relatively high genetic distance.
in accordance with the findings of other authors  <cit> , the female migration rate was greater than that for males .
when a much wider area was included , the female migration rate was still larger than the male migration rate .
thus, we did not find any indication of a change from matrilocality to patrilocality from pre- to post-agricultural societies as it has been suggested by others  <cit> .
CONCLUSIONS
in summary, a general genetic homogeneity of the x-chromosome snps was observed in a number of populations in the mediterranean area.
the genetic distance between populations in the middle east and the western part of the mediterranean area was very low, most likely reflecting the effect of the neolithic wave and recent migration events.
only the moroccan population showed a significant genetic distance from the remaining mediterranean populations including populations that are geographically close to it, showing the importance of the strait of gibraltar as a geographic barrier and supporting the idea of a low impact of the neolithic demic diffusion and more recent migrations in north-west africa.
in ibiza and cosenza populations, interesting differentiations from their neighbouring populations and significant multilocus associations were observed when all the  <dig> x-chromosome snps were analyzed reflecting the particular demographic histories of these populations.
a deeper study of ld in these two populations could reveal interesting results for disease association studies.
a higher migration rate was observed in females than in males in both the mediterranean area and in a wider geographic area showing a greater genetic flow mediated by females than males probably both in pre-agricultural and post-agricultural societies.
as it was suggested by other authors  <cit> , patrilocality seems to have been common in post-agricultural populations.
according to our results, patrilocality should have also been important in pre-agricultural societies.
the negative slope obtained in rousset's isolation-by-distance model using mtdna data in the mediterranean area emphasizes the effect of genetic drift and genetic isolation observed in females in some of the populations in the western part of the mediterranean sea.

 the x-chromosome has features that make it a good source of information for population genetic studies. the x-chromosome is present in a single copy in males, which makes it possible to determine the x-chromosome haplotypes in men. compared with autosomes, the x-chromosome has lower recombination rate, lower mutation rate and smaller effective population size resulting in a faster genetic drift. in consequence, both linkage disequilibrium and population structure in the x chromosome are expected to be stronger than those in autosomes. two-thirds of the x-chromosome history has been spent in females. thus, x chromosome polymorphisms mainly reflect the history of females. due to recombination, x-chromosome markers in females provide a multilocus system, while the mtdna and y-chromosome are linked haplotypes. thus, x-chromosome markers are valuable for population genetic studies. due to its history with constant migration movements, the mediterranean area constitutes an challenging region for population genetic studies, both considering the whole area and particular populations. only very little genetic structure has been found among populations living on the northern and eastern shores of the mediterranean sea by analyses of y-chromosome strs and mtdna. the pattern may be a consequence of the neolithic demic diffusion in this region and/or a high level of gene flow in the area. the presence of a strong genetic boundary between the northern and southern mediterranean populations, especially in the western side of the sea has been suggested. studies focused on smaller areas have also given interesting results, especially those regarding physical and/or cultural isolates such as corsican and sardinian sub-isolates, balearic populations and calabrian populations. in order to further investigate the genetic characteristics of the populations in the mediterranean basin, we analyzed snps located on the x-chromosome in eleven populations from the mediterranean area. in addition, two populations from northern europe and east africa were analyzed. we carried out a wider analysis using data from the hapmap website on out of the snps used in this study. we wanted to use the x-chromosome snps as multilocus markers. we selected polymorphic x chromosome snps with a physical distance of at least kb. population history such as genetic drift and admixture has an important consequence on the degree of population ld. the level of ld on the x-chromosome is expected to be higher than on autosomes. we also wanted to use these markers to study the pairwise linkage disequilibrium and multilocus association in order to obtain knowledge of the long-term and background ld in each population, because this information can be useful for studies of population genetics and the genetics of complex diseases. we studied the informativeness of the markers selected and the sex-biased migration rate and compared the information to that of non-recombinant markers in mtdna and the y-chromosome.  intrapopulation variability a total of different haplotypes were found in the populations analyzed by means of x-chromosome snps. no single haplotype was shared among individuals within or between populations. all the markers were polymorphic in all the populations studied. the minor allele frequency varied between a minimum percentage of % and a maximum value of around %. the mean gene diversity of the markers analyzed was very homogeneous in the populations sampled. they ranged from in the somali population to in iraqis and tunisians. taking into account that the maximum level of expected heterozygosity for biallelic markers is, a high degree of gene diversity was found for the snps selected in all the populations analyzed. among the pairwise comparisons between the snp markers in the populations, between and pairwise comparisons showed significant p-values. however, after holm-sidak correction, only two pairwise ld values were significant. nevertheless, when multilocus tests of associations were performed using all the markers, the null hypotheses of linkage equilibrium were rejected in two populations: ibiza and cosenza. both populations present some peculiarities that may explain the existence of a higher degree of background linkage disequilibrium. interpopulation differentiation amova and pairwise fst values fst: p-value: a degrees of freedom. b sum of squares deviations among populations. c sum of squares deviations within populations. fst: p-value < a degrees of freedom. b sum of squares deviations among populations. c sum of squares deviations within populations. when a locus by locus amova was performed in the mediterranean group, only three out of markers showed significant variations among the mediterranean populations, while out of markers were important for the differentiation of the "worldwide" populations: x x x x x x x x x x x x x x x x and x the markers x x x and x were not included in these analyses due to the lack of information in the populations obtained from the hapmap project. no correlation was found between the mean gene diversity and the level of differentiation among populations, as has been suggested by others. in order to obtain a graphical representation of the genetic structure of the populations studied, a principal coordinate analysis was made from the pairwise reynold's fst matrix obtained using or x-chromosome snps in the mediterranean and the populations, respectively. in figure 1a, the first two axes accounted for 39% of the total variability. thus, the mediterranean populations are quite homogeneous. equivalently to the results of the amova analysis, the moroccan population was considerably displaced in the first axis, and the majorcans were placed in the opposite direction. in the second axis, the differentiation was less, but it is interesting that the cosenza and ibiza populations were located opposite to their neighbouring populations. figure 1b shows the relative position of the mediterranean populations in relation to a wider geographical area. the first two axes accounted for 51% of the global variability. the first axis displayed east asian populations opposite to the others. in the second axis, the mediterranean populations plus the ceu population formed a group between the north-european and african populations. only the moroccans showed a slight displacement from the others showing significant fst values in most of the pairwise comparisons inside the mediterranean group. the ceu population showed an intermediate position between the north of europe, represented by the danish population, and the mediterranean group. none of the pairwise comparisons between the ceu population and the mediterranean or danish populations showed a significant fst value. for the grouping proposed in figure 1b, % of the variation could be explained by differences between groups. a more detailed hierarchical amova analysis showed that % of the global variation could be explained by the east asian populations, % of the variation was explained by somalis and sub-saharans. the mediterranean group contributed to % of the global variation, a value that went down to % when the moroccan population was excluded from the group. finally, the north-west european populations only explained % of the global variation. structure analysis additionally to the amova analysis, a cluster analysis was performed using the structure software in order to assign individuals to a number of k populations allowing admixture. we estimated the posterior probability, where x represents the genotypes) for k between and for populations using x-chromosome snps. we observed that the proportions of the individuals assigned to each k subpopulation were balanced in all cases indicating that the x-chromosome snps did not identify to which subpopulation an individual belongs. according to the authors of the programme, this could be interpreted as a lack of population structure as a consequence of a high level of admixture. due to the statistical model implemented in structure, it is possible to find significant fst values without finding significant differences using structure when the populations analyzed are closely related. this is not the case of some of the populations included in the analysis. as it was discussed by other authors, it could be that the high migration rate of females could have resulted in a small genetic structure in x-linked loci. on the other hand, a lack of resolution of the investigated snp markers could also be the cause of the low level of genetic structure observed. isolation by distance we did not find any significant correlation between genetic differentiation ) and the natural logarithm of the geographical distance in the mediterranean area based on the study of x-chromosome snps. according to rousset's isolation-by-distance model, some demographic information can be obtained from the model drawn. an indirect estimation of 4d μσ also known as the "neighbourhood size", could be calculated from the slope value. considering the biallelic markers analyzed in the present work, the estimated value of 4d μσ would be around individuals, which seems to be an underestimated value according to the same author. this data can be interpreted as the result of a high number of migration events in the recent history of the human populations in the mediterranean basin. the number of estimated migrants was dramatically reduced when a much wider area was considered, asian populations and european populations. the analyses of populations belonging to an extended area are more prone to reflect more ancient demographic processes. if the neolithic transition occurred around ybp, and human dispersal was close to km per generation, pre-agricultural migration patterns could be observed at distances over km. taking this information into account and the fact that the minimum geographic distance between two populations in figure 2b was km, pre-agricultural migration events should be reflected there. on the basis of x-chromosome snps, an indirect estimate of migrants between subpopulations was calculated from the regression slope. in this study, isolation by distance operated better at large geographic scales than at short distances. x-chromosome snps vs. non-recombinant systems mantel test we performed two mantel tests for each of the three sets of markers: x-chromosome snps, hvri mtdna and y-chromosome snps. first, we analyzed of the populations in this work for which results of all the markers were available. then, only the mediterranean populations were analyzed. mantel correlations were corrected for geographical distances by computing partial correlations between genetic distances at constant geographic distance. all tests were significant except the test between x-chromosome and mtdna in the mediterranean area, where the correlation index was low but still positive. the lack of significance was most likely due to the low degree of genetic differentiation of both x-chromosomes and mtdna in the area. all x-chromosome and mtdna markers showed a significant correlation with y-chromosome snps both when or populations were analyzed. except in particular cases, male and female movements in the mediterranean have apparently had the same overall tendency. an important difference in the intensity of the migration events of both sexes could also be seen in the level of significance of the different mantel tests. a populations: den, ibz, irq, maj, mor, rca, sic, som, tun, trk and val. bpopulations: ibz, irq, maj, mor, rca, sic, tun, trk and val. for abbreviators see figure isolation-by-distance analyses by using different genetic systems as expected from previous studies, no significant correlation between genetic and geographic distances was found in any case. from a demographic point of view, the estimated number of migrants based on x-chromosome results would be approximately the same as calculated above, and around were calculated from y-chromosome snp data. surprisingly, a negative slope was obtained in the case of the mtdna, reflecting a major effect of isolation and genetic drift versus migration in certain populations of the mediterranean area. when x-chromosome snps and mtdna were analyzed in a wider geographic area, a greater female migration rate compared to the male migration rate was still observed. as it has been commented above, the minimum geographic distance between two populations was approximately km and mainly pre-agricultural migration events were then represented in this case. discussion a considerable amount of information based on classical genetic markers, autosomal strs, mtdna and y-chromosome haplotypes has previously been reported concerning the genetics of populations in the mediterranean region. a low level of genetic structure has been described due to the high degree of migration in the mediterranean area. apparently, the surrounding lands of the mediterranean sea were settled by people from the middle east approximately years ago. the migration events increased during the neolithic era with the transition to agriculture. the increase in migration events during the history constitutes the main cause of the genetic homogeneity in the area. several authors have suggested the existence of a north-south genetic "barrier" in the western part, and no consensus on the current genetic landscape has been established. some isolated populations with deviating genetic differentiation have been described. based on the study of x-chromosome snps, we found a low value of population differentiation in mediterranean populations. the fixation index was significant but the p-value increased to p = when the moroccan population was excluded from the analysis. in contrast, a times higher fst value was observed when populations from three continents were analyzed. not surprisingly, the strongest genetic differentiation was found among the african populations. it is well known that african populations have a higher level of genetic heterogeneity compared to european or asian populations. a certain bias of the results obtained could be due to the fact that the markers initially were selected for forensic purposes with a high level of polymorphism in different ethnic groups. a high level of intrapopulation variability will to some degree be associated with a low interpopulation variability. nevertheless, the significant fst value found in the mediterranean populations and the highly significant fixation index observed when distant populations were compared documents the suitability of these markers for population genetic studies. geographic "barrier", genetic isolate and population admixture the analysis of the mediterranean populations in comparison to the "worldwide" populations showed a clear intermediate position between african and north-european populations with morocco somewhat closer to the african populations than the other mediterranean populations. when only the mediterranean populations were analyzed in a principal coordinate plot, opposite positions of moroccans and majorcans were observed. this supports the existence of a north-south gene flow "barrier" in the western part of the mediterranean area. it could be argued that the genetic differentiation of the moroccan population was a result of the arab-berber sub-structure of the individuals sampled in this work, but this hypothesis does not seem likely. several authors reported a high genetic homogeneity between berbers and arabs in nw africa, so they suggested that the arabisation of this area was probably a cultural phenomenon, which did not imply a replacement of the ancestry population. our results give support the hypothesis of an early settlement of nw africa. the original berber population seem to have received a low genetic influx from the surrounding areas. different hypothesis have been suggested to explain the genetic differentiation of the moroccan population. an initial genetic drift could have caused differences in allele frequency distribution that have not been re-established due to a certain level of geographic isolation. the strait of gibraltar has been described by several authors as an important genetic barrier. even a certain level of genetic exchange probably occurred between nw africa and the south of the iberian peninsula, sharp frequency changes have been described in this area. also the sahara desert has been suggested as responsible of the genetic isolation of nw african populations from sub-saharan populations. there is no consensus about the impact of the neolithic demic diffusion in the mediterranean area. according to our results, a low impact of the neolithic expansions and/or later migration events on nw african populations would have occurred. nevertheless, the high genetic homogeneity observed and especially the low level of genetic pairwise differentiations between the iraqi and the western mediterranean populations supports the impact of several migration events during the history in the genetic constitution of the mediterranean area, where sailing at the mediterranean sea have been an important connection between various geographical areas and populations. the high genetic homogeneity of x-chromosome markers contrasts with the genetic structure of y-chromosome snps. this highlights the differences between male and female migration rates, and stresses the importance of combining results obtained from different types of genetic markers. tunisians did not show a significant level of differentiation with northern populations as mentioned by others. a discordant result may be due to the different nature of the markers used in the different works. also the heterogeneity described in tunisian berbers could explain that studies carried out using different sets of tunisian samples may end up showing different results. more recent demographic events that took place in some particular populations in the area showed interesting exceptions to the general homogeneity. this was the case of the cosenza and ibiza populations that were placed relatively "far" from their neighbouring populations in the various statistical analyses performed. tagarelli et al described the calabria province as a collection of many "human genetic isolates". in contrast to catanzaro and reggio di calabria, cosenza did not suffer destructive telluric events that would have modified its population structure. so, the population heterogeneity inside cosenza is more patent than in any of the other two populations. this is reflected by the heterogenic distribution of various genetic markers at the coast compared to the internal areas. the results obtained with the x-chromosome markers were in agreement with the previous studies. cosenza showed not just a relatively high genetic differentiation compared to the populations from catanzaro and reggio di calabria but also a certain displacement in relation to other mediterranean populations. this was also the case of the ibiza population. ibiza, one of the three major islands of the balearic archipelago, was reproductively isolated for centuries, their population effective size was reduced by infectious diseases and a high number of consanguineous marriages were reported in the 15th-17th centuries. in 1970s, the tourist influx considerably increased the population of ibiza. former studies have emphasized the genetic differentiation of this population as a consequence of the stochastic events that happened during its history. moreover, when a parametric multilocus association analysis was performed, significant values of associations were only observed in the ibiza and cosenza populations. in most human populations, high levels of linkage disequilibrium do not exist between markers separated > kb. taking the distance between the markers selected into consideration, we did not expect to find significantly increased levels of ld in any of the populations studied. on the other hand, it is well known that different demographic scenarios could result in unexpected levels of ld over large genomic regions. sex-biased migration rate: a comparative study among different parts of the genome. sex-biased migration rates vary between populations as well as through population history. matrilocality results in a mtdna geographic structure while patrilocality results in a genetic structure in the non-recombinant region of the y-chromosome. the x-chromosome, which has spent 2/ of its history in females, reflects matrilocality to a higher degree than patrilocality. in the light of our findings it can be said that the migration rate was higher in women than in man, both when our results were analyzed with the mantel test and under rousset's isolation-by-distance model. the regression slope obtained from mtdna and x-chromosome markers were smaller than those obtained from y-chromosome snps. the negative slope obtained from mtdna data can be explained by the fact that populations geographically close to each other showed a relatively high genetic distance. in accordance with the findings of other authors, the female migration rate was greater than that for males. when a much wider area was included, the female migration rate was still larger than the male migration rate. thus, we did not find any indication of a change from matrilocality to patrilocality from pre- to post-agricultural societies as it has been suggested by others.  in summary, a general genetic homogeneity of the x-chromosome snps was observed in a number of populations in the mediterranean area. the genetic distance between populations in the middle east and the western part of the mediterranean area was very low, most likely reflecting the effect of the neolithic wave and recent migration events. only the moroccan population showed a significant genetic distance from the remaining mediterranean populations including populations that are geographically close to it, showing the importance of the strait of gibraltar as a geographic barrier and supporting the idea of a low impact of the neolithic demic diffusion and more recent migrations in north-west africa. in ibiza and cosenza populations, interesting differentiations from their neighbouring populations and significant multilocus associations were observed when all the x-chromosome snps were analyzed reflecting the particular demographic histories of these populations. a deeper study of ld in these two populations could reveal interesting results for disease association studies. a higher migration rate was observed in females than in males in both the mediterranean area and in a wider geographic area showing a greater genetic flow mediated by females than males probably both in pre-agricultural and post-agricultural societies. as it was suggested by other authors, patrilocality seems to have been common in post-agricultural populations. according to our results, patrilocality should have also been important in pre-agricultural societies. the negative slope obtained in rousset's isolation-by-distance model using mtdna data in the mediterranean area emphasizes the effect of genetic drift and genetic isolation observed in females in some of the populations in the western part of the mediterranean sea. 
due to its history, with a high number of migration events, the mediterranean basin represents a challenging area for population genetic studies. our results support both the hypothesis of a reduced impact of the neolithic wave and more recent migration movements in nw-africa, and the importance of the strait of gibraltar as a geographic barrier. a certain level of background linkage disequilibrium in populations in ibiza and cosenza could be attributed to their demographic background. a large number of genetic studies have been carried out in the mediterranean area using different markers but no consensus has been reached on the genetic landscape of the mediterranean populations. a high overall homogeneity was found among the mediterranean populations except for the population from morocco, which seemed to differ genetically from the rest of the populations in the mediterranean area. in contrast, the high genetic homogeneity observed in the mediterranean area could be interpreted as the result of the neolithic wave caused by a large demic diffusion and/or more recent migration events. a differentiated contribution of males and females to the genetic landscape of the mediterranean area was observed with a higher migration rate in females than in males. in order to further investigate the genetics of the human mediterranean populations, we typed individuals from mediterranean populations with single-nucleotide polymorphisms located on the x-chromosome. a very low genetic distance was found between populations in the middle east and most of the western part of the mediterranean sea. multilocus association was observed among the snps on the x-chromosome in the populations from ibiza and cosenza. a higher migration rate in females versus males was observed by comparing data from x-chromosome, mt-dna and y-chromosome snps both in the mediterranean and a wider geographic area.
 due to its history, with a high number of migration events, the mediterranean basin represents a challenging area for population genetic studies. a large number of genetic studies have been carried out in the mediterranean area using different markers but no consensus has been reached on the genetic landscape of the mediterranean populations. in order to further investigate the genetics of the human mediterranean populations, we typed individuals from mediterranean populations with single-nucleotide polymorphisms located on the x-chromosome.  a high overall homogeneity was found among the mediterranean populations except for the population from morocco, which seemed to differ genetically from the rest of the populations in the mediterranean area. a very low genetic distance was found between populations in the middle east and most of the western part of the mediterranean sea. a higher migration rate in females versus males was observed by comparing data from x-chromosome, mt-dna and y-chromosome snps both in the mediterranean and a wider geographic area. multilocus association was observed among the snps on the x-chromosome in the populations from ibiza and cosenza.  our results support both the hypothesis of a reduced impact of the neolithic wave and more recent migration movements in nw-africa, and the importance of the strait of gibraltar as a geographic barrier. in contrast, the high genetic homogeneity observed in the mediterranean area could be interpreted as the result of the neolithic wave caused by a large demic diffusion and/or more recent migration events. a differentiated contribution of males and females to the genetic landscape of the mediterranean area was observed with a higher migration rate in females than in males. a certain level of background linkage disequilibrium in populations in ibiza and cosenza could be attributed to their demographic background. 