BACKGROUND
the yakuts  from the republic of sakha are of particular interest because they contrast with other populations from siberia in many respects such as in their specific funeral practices.
before christianization, part of the population was buried, which is exceptional in this region of the world as most bodies were put in aerial graves on platforms or on trees.
at the same time, the yakuts are considered as the most remarkable example of northward expansion into siberia  <cit>  and especially as these semi-nomadic herders contrast so strikingly with surrounding siberian populations on the basis of their turkic language and their horse- and cattle-breeding economy.
the geographic specificities of central yakutia, favorable to the development of a pastoralist economy and reflecting the environmental conditions of the south, certainly initiated the development of the yakut culture in this region.
although several hypotheses regarding the geographic origin of this atypical population have been proposed from results of the first archaeological investigations of ap okladnikov  <cit>  to the latest population genetic studies  <cit> , the precise origins of the yakuts and their admixture with the indigenous tribes of siberia, especially the tungus, have been debated.
nevertheless, archaeological data currently agree with the appearance of the yakut culture around the 14th century a.d.  <cit> .
recent population genetics data tend to support a dual origin for the maternal lineages of the yakuts and admixture with the tungus, and demonstrate that the paternal lines observed today result from a strong bottleneck that led to the restriction of these lineages in the yakut population  <cit> .
this does not, however, address the question of the number of settlers and the precise origin of the paternal lineages, or the admixture rate with indigenous populations  <cit> .
furthermore, the demographic changes occurring in the yakut population since the russian colonization make it difficult to reconstruct the genetic history using present day samples.
russians came into contact with the yakuts in  <dig>  christianization began around  <dig> and by the beginning of the 19th century, a christian ritual was performed in the majority of burials.
from the 17th to the 19th century smallpox and measles epidemics, caused by contact with russians settlers, decimated the yakut people and their neighbors, and during the second world war a large proportion of young yakut males was killed.
therefore ancient dna , which allows the direct study of an ancient population without their descendants, represents a major advantage in order to avoid the possible bias represented by recent genetic events  <cit> .
this study adopts a population genetics approach to an ancient sample composed of  <dig> individuals from central yakutia in order to address the questions detailed above.
human adna studies' outcome quality is closely related to the preservation of dna and to the absence of potential contaminations .
the environmental conditions present in yakutia, one of the coldest parts of the world, are generally favorable to a good preservation of nucleic acids.
indeed, the exceptional preservation state of most of the excavated graves enabled us to successfully analyze several samples collected from  <dig> frozen bodies or skeletons from the last six centuries .
RESULTS
nuclear dna quantification
the results of the nuclear dna quantification are summarized in table  <dig>  the purification protocol was efficient in removing inhibitors in all except six bone extracts.
in order to avoid the effect of pcr inhibitors, the samples yaka <dig>   <dig>   <dig>   <dig> and  <dig> were diluted by a factor of 1/ <dig> to 1/ <dig> and successful amplifications were subsequently obtained.
for the sample yaka <dig> it was not possible to remove the inhibitors and therefore no results were obtained for further amplifications.
no inhibitors were detected in the teeth or hair extracts.
moreover, the dna extracts obtained from teeth showed the highest mean concentration of nuclear dna and the highest frequency of samples yielding a concentration superior to  <dig> pg.μl- <dig>
y-chromosomal analyses
the multiplex amplification of the y-chromosomal strs was successful for  <dig> of the  <dig>  male samples, where full profiles or profiles with only one missing marker were obtained .
the lowest amplification success rate  was observed for the dys <dig> locus that is the largest marker included in the str panel.
all loci with a length over  <dig> bp showed more than 20% amplification failures.
this molecular behavior is a well known phenomenon and has been described as an effective way to ensure the authenticity of results  <cit> .
twelve different haplotypes were observed.
nine were unique, one was common to two individuals  and two haplotypes were widely shared among our ancient sample set.
these two haplotypes were present in eight  and seven individuals , and differed from each other by only one mutation step at the loci dys389ii and dys <dig>  therefore, ht <dig> and ht <dig> could share a recent common ancestor, since two mutation events in a single father/son transmission have been previously described  <cit> .
besides, ht <dig> and ht <dig> were the most frequent lineages in our present day sample set  as well as in the data published by pakendorf et al  <cit>  .
the genetic diversity indices  show equivalent values for the ancient and the present day sample sets of our study.
surprisingly, the diversity observed in our central yakut sample set is higher than the one presented by pakendorf et al  <cit>  although the samples were collected in the same area.
<dig> present day yakuts, 2number of samples, 3number of haplotypes, 4mpd: mean pairwise differences, 5standard error.
central yakuts  <cit>
sixty one percent  of the haplotypes  were affiliated to the n1c  haplogroup on the basis of the snp analyses.
this haplogroup is considered as the most frequent in the yakut population, and its frequency varies across studies from 75%  <cit>  to 100%  <cit> .
sample yaka <dig> was affiliated to haplogroups k. the snp typing was inconclusive for  <dig> individuals ; nevertheless the affiliation to n1c was excluded on the basis of the absence of the tat-c mutation.
although all the y-str haplotypes have been transmitted to the present day yakuts, only a few of them are shared with other populations.
a comparison to the yhrd database and other literature data showed that only ht <dig> and the haplotype of the yaka <dig> individual matched with evenk male lineages; confirming the specificity of the n1c lineages.
in this case, one can assume that n1c comes from yakut ancestors considering the low n1c frequency in evenks  <cit> .
moreover, the ethnical affiliation of one individual to either the yakut or the evenk population could be difficult nowadays, considering that these two ethnic groups share a similar lifestyle or language in some areas of yakutia.
the y-str haplotypes of yaka <dig> and yaka <dig> were found in present day mongols, buryats, kalmyks and central asians.
the mds plot based on pairwise fst values clearly shows the separation of the yakuts from all the other populations, since the ancient and present day yakuts are clearly isolated .
indeed, the fst distance computations revealed significant differences for each comparison excepted for the two yakut sample sets.
the median network based on the y-str haplotypes belonging to the n1c haplogroup is undoubtedly affected by sample sizes.
nevertheless, the separation between the yakuts and the other populations is visible .
indeed, both ancient and present day yakuts are grouped in one isolated cluster together with the evenks.
the different haplotypes of this cluster are separated by one mutation step , indicating that these closely related paternal lineages have emerged from a reduced number of individuals in a short period of time.
this observation is consistent with earlier studies  <cit> .
mitochondrial dna analysis
the hvs- <dig> sequences were unambiguously validated for  <dig> individuals .
five subjects were excluded from the analysis: yaka <dig>   <dig>   <dig>   <dig> and  <dig>  no amplification was obtained for the sample yaka <dig> due to the presence of inhibitors in the dna extracts.
amplification failures were also observed for the sample yaka <dig> and only one of the two sub-regions of hvs- <dig> was successfully amplified for sample yaka <dig>  finally, multiple heteroplasmies were observed in the electropherograms of yaka <dig> and  <dig>  the fact that these two samples were collected in museums, increasing the risk of pre-laboratory contamination, led us to exclude these two subjects from our study.
two heteroplasmic positions were observed for three distinct samples.
one t/c heteroplasmy at position  <dig> was observed in the samples yaka <dig> and yaka <dig> in teeth and hair dna extracts only.
this heteroplasmic position is observed in the present day yakut population  and is also well known as a hotspot of post-mortem damage  <cit> .
the second heteroplasmy was observed at position  <dig> in the sample yaka <dig>  this position has been reported as a potential post-mortem mutation  <cit> .
therefore, these heteroplasmic sites were treated as missing data for these specific samples in all the subsequent analyses.
gene diversity in our ancient sample was calculated using the arlequin  <dig>  program and then compared with different populations.
both ancient and present day populations from central yakutia showed a gene diversity of  <dig>  .
this value is in agreement with the data from  <cit> .
we observed  <dig> different haplotypes among the  <dig> studied individuals, and  <dig> were found only once.
twenty nine haplotypes were assigned to different haplogroups with the near matching method.
the subject yaka <dig>  plausibly a young shaman from the early 18th century, was affiliated to haplogroup h <dig> due to the typing of coding region snps  <cit>  since the assignment on the basis of the hvs- <dig> haplotype was inconclusive.
the overall haplogroup distribution  in our ancient sample set corresponds with earlier studies focused on the present day yakuts  <cit> .
as can be seen in table  <dig>  the ancient yakuts present high frequencies of haplogroups c and d. haplogroup c and specifically the lineage c <dig> represents the most abundant haplogroup in our ancient sample set with a frequency of 30%.
the highest frequency of this lineage is observed in the evenks, but c <dig> is also prevalent in south-western siberian populations such as tuvans, buryats and tofalars  <cit> .
among haplogroup d, the d <dig> lineage is present in seven out of  <dig> individuals and represents  <dig> % of the ancient sample set .
this sub-haplogroup is usually present at low frequency in siberian populations  <cit>  and in central asian or mongolian populations  <cit> .
interestingly, the two most frequent d <dig> haplotypes seem to share a recent common ancestor as they differ only by one transition at position  <dig>  this high frequency of subhaplogroup d <dig> has been regarded as specific to the yakut maternal lineages by pakendorf et al.
<cit> .
the buryat ascendance of the d <dig> lineages demonstrated in their analyses was not clearly supported by our ancient dna data.
sub-haplogroup frequencies have been merged under the name of the basal haplogroup to simplify the comparison.
frequencies are given in percent.
the sequence of yaka <dig> belonged to haplogroup a4b, that is present at low frequencies in western and middle siberian populations such as the mansis, tuvans and evenks  <cit> .
yaka <dig> was assigned to the novel haplogroup a <dig>  found in the kalmyks and buryats  <cit> .
two haplotypes were assigned to haplogroup b: yaka <dig> to subhaplogroup b <dig>  which is dispersed throughout mongolia and china  <cit>  and the sequences of yaka <dig> and yaka <dig> to subhaplogroup b <dig> due to the presence of transitions at positions  <dig>   <dig>   <dig> and  <dig>  also present in one buryat studied by derenko et al  <cit> .
two sequences belonged to the f1b lineage , widespread in populations from south siberia  <cit> .
the haplotype of yaka <dig> was affiliated to the m13a subhaplogroup present in buryats, khamnigans  <cit>  and mongols  <cit> .
the proportion of the sequences affiliated to the west eurasian haplogroups  was low in our ancient sample set, as the merged frequencies corresponded to  <dig> % .
the sequence of yaka <dig> was affiliated to subhaplogroup h <dig> which is characteristic for central asians  <cit>  and less frequent in western europe.
these sequences were observed in samples distributed in the second and third period, and no increase in the frequency of the west eurasian lineages could be noted for the period following the russian colonization.
besides, these haplogroups are observed at similar frequencies in various siberian populations  <cit> .
the size of the sample set should be considered as a potential source of bias in the overall haplogroup distribution.
nevertheless, we observed the same distribution as was found in the present day population.
this statement has been verified by the calculation of the fst distances since there are no significant distances between the yakuts from the pre-colonization era and the ones from the christian and present day periods.
in an attempt to trace the origins of the ancient mtdna lineages, each haplotype was compared to data from the literature .
this comparison showed that more that 73% of the haplotypes found in our ancient samples have been transmitted to the present day yakut population.
among the nine haplotypes absent in the present day yakuts, four were not found in any of the populations included in the database due to their unique combination of polymorphisms or private mutations.
the haplotypes belonging to haplogroup c are shared with various populations but most specifically with buryats , evenks  and tuvans .
on the contrary the sequences assigned to haplogroup d appear to be more restricted to the yakut population.
this specificity is even higher for the d <dig> lineages since they are shared only with two yakut speaking evenks, one buryat, one mongol and one daur.
to evaluate if this comparison could bring some information on the different migration waves which led to the formation of the yakuts, we took the chronological repartition of the samples into consideration.
unfortunately, this approach failed to reveal any clear pattern, since individuals from different periods were distributed among all the haplogroups and matched to populations without any identifiable preference.
the mds plot based on fst distances  revealed, as for the y-chromosomal strs, a high stability through time of the maternal lineages.
indeed, the distances between the chronological groups were not significant.
besides, the fst values confirm the previous statements based on the comparison of individuals since non-significant distances were observed between ancient yakuts, evenks and tuvans.
in order to clarify the previously obtained results, migration rates were computed with the migrate software.
the populations used for the analyses were selected taking the previous results into consideration.
to avoid a potential bias related to the sample size, ancient yakuts from different periods were merged into one group.
as mentioned earlier, this approximation should not be deleterious since the two groups showed non-significant genetic distances.
the results are presented in table  <dig> and indicate that the major genetic contribution to the maternal lineages of the yakuts came from the evenks.
this observation could be explained by the fact that new settlers were more susceptible to admixture than the autochthonous population due to their reduced number and to their cultural practices .
this assumption is also corroborated by the higher genetic diversity of the ancient yakuts compared to the evenks and by the important number of mtdna lineages shared with this population.
first presence of the yakutian ancestors and demographic data
the first yakut graves discovered are dated from the 15th and 16th century.
from this period until  <dig>  only male graves with horse riding equipment  and weapons from the steppe area  are found.
the funerary ritual was influenced more by turkish than by mongolian culture .
it is unlikely that these graves are the first graves of the new settlers, and it is more probable that this period corresponds to the first time with a relatively high population density.
during this early period, y-chromosomal ht <dig> and ht <dig> are already present.
simulations  suggest two possibilities: either a very small group arrived around the 10th century, or a more important one arrived between the disintegration of the mongol empire  and the discovery of the first graves.
<dig>  people is an historical estimation for  <dig> ad and we have made estimation for different steps: the beginning of the 9th century, which is a period of an important mongolo-turkish expansion with some contacts between these populations and yakutia  <cit> ,  <dig> ad which corresponds to the disintegration of the mongol empire with the dispersion of many nomad groups, and  <dig> ad, a date prior to the most ancient yakut graves excavated.
authenticity of sequences and profiles
the possibility that our data arose from contaminated dna was considered highly unlikely, since morphological and molecular typing results for sex determination were always in accordance with each other.
moreover, no contamination was observed in the extraction or the pcr blanks.
reproducible pcr results were obtained from multiple extractions and amplifications of the same samples performed at different times.
the choice of autosomal str markers as a first approach for analyzing the ancient yakut remains was based on their ability to detect degraded and/or contaminated dna.
<dig> out of the  <dig> human samples yielded amplifiable dna for more than two markers, but only  <dig> of the genetic profiles were complete .
the loci d18s <dig>  csfp1o, d7s <dig> and d2s <dig> were often not amplified, probably because they are expressed in the higher molecular weight range.
such an inverse dependence of the amplification efficiency on the size of the dna fragment to be amplified is typical of dna retrieved from ancient remains and results from the degradation of the dna  <cit> .
various factors are thought to affect the success of dna analysis from ancient tissues:  the time from excavation  <cit> , as post-excavation storage conditions are less favorable for dna preservation than pre-excavation condition,  the cortical thickness of long bones which favors good dna preservation  and  the age-at-death of the individual, as it is more difficult to obtain profiles from newborn compared to adult bone.
thus, as time passes after excavation, one would expect a reduction in the number of endogenous dna and an increase in the number of exogenous contaminant template dna molecules extracted from a tissue sample, with additional difficulties for newborn and young children compared to adult samples.
of the ten human samples that yielded no str profiles, four came from the museum and six from frozen burials including four newborn samples.
discussion
during the last few years, several studies have focused on the yakuts using genetic analyses to trace their origins  <cit> .
however, to date the use of ancient dna has been limited due to small numbers of individuals sampled  <cit> , in spite of the exceptional taphonomic conditions of siberia and the possibility of retrieving high quality dna from remains buried in the permafrost  <cit> .
therefore, this study represents the first attempt to trace the origins of the yakut population by ancient dna analyses.
out of the  <dig> ancient individuals analyzed in this study,  <dig> complete y-chromosomal str haplotypes were obtained from  <dig> male individuals, and mtdna sequences were validated for  <dig> individuals.
the quality of the results obtained in our study may be explained by the presence of permafrost which favors the preservation of dna  <cit> .
the excavation strategy and the absence of any post-excavation treatment prior to laboratory analyses might have played an important role in the quality of dna and confirms the statements of pruvost et al.
<cit> .
besides, this limited handling of the ancient remains represents a key factor in the reduction of contamination risk by modern dna.
the combination of the precautions against contamination and the validation strategy  validate the data presented here, even if not all the traditional criteria  <cit>  could be met due to the large number of samples.
the y-chromosome results obtained from our ancient sample are consistent with earlier studies  <cit> .
the specificity of the male lineages was confirmed by statistical analyses  and by individual comparison of the y-str haplotypes with the literature data since only three haplotypes were shared with other populations.
the high frequency of the haplogroup n1c, which is present from the most ancient graves, and the non-significant fst values between ancient and present day yakuts, indicates their stability through time.
thus, this genetic result corroborates the assumption that the yakuts are a highly homogenous population not only geographically  <cit>  but also from a chronological standpoint.
the origin of the most frequent y-chromosomal haplotypes  was difficult to establish on the basis of genetic information.
indeed, these two lineages belonging to haplogroup n1c seem to be restricted to yakut populations, and were probably present since the period they were first located in central yakutia.
interestingly, the comparison with archaeological data revealed that the male individuals  at the beginning of the 18th century, identified as clan chiefs  on the basis of their grave goods , belonged to these two haplotypes.
therefore, archaeological data could bring interesting information in tracing back the origin of these enigmatic male lineages.
indeed, the grave goods of the 15th/17th centuries  and the construction of coffins with an empty trunk from the 18th century are similar to the burial customs of the cis-baïkal area  <cit>  and of the egyin gol necropolis during the 3rd century bc  <cit> .
this suggests that the male ancestors of the yakuts were probably formed of a small group of horse-riders originating from northern mongolia or the baïkal lake.
later, the patrilineal clan organization of the yakuts would have facilitated the diffusion of male lineages borne by the individuals of high social rank.
this hypothesis is supported by the fact that the y-chromosomal haplotypes identified by the warriors of the 15th/17th centuries were transmitted to the tojons of the 18th century.
after russian colonization, the influence of the tojons was strengthened by the decrees made by the empress anna ivanovna  <cit> , and this official reinforcement of their power might have increased the spread of a limited number of y-chromosomal lineages for a second time.
therefore, the early founder effect combined with the yakutian traditions and the russian influence would have led to the present day genetic pattern observed for the paternal lineages.
conversely, the results obtained from the mtdna analyses revealed a more important diversity and varying origins of the maternal lineages.
indeed, the gene diversity observed in our ancient sample is intermediate compared with the diversity found in small and isolated siberian groups such as the chukchi and mongolian  <cit> , buryatian  <cit>  or central asian  <cit>  populations.
the haplogroup distribution observed in our sample matched that found in present day yakuts and is constituted by distant haplogroups found in siberian, mongolian and central asian populations.
the major haplogroup, c <dig>  indicates more specific affinities with the evenks  <cit> .
nevertheless, the individual comparison of maternal lineages with data from literature allowed us to precise that four out of the six lineages belonging to the c <dig> haplogroup were present in the buryat population.
therefore, the buryat origin of some of the d <dig> sub-haplogroups found in the yakuts as demonstrated by pakendorf et al.
<cit>  could be expanded to the c <dig> haplogroup.
the influence of both buryat and evenk populations is clearly visible in the mtdna lineages of the ancient yakuts.
concerning the contribution of the evenks to the yakut's mtdna gene pool, the admixture might have mostly occurred between yakut men and evenk women  according to the high frequency of the c <dig> haplogroup in these two groups, and since the y-chromosomal lineages are highly specific to the yakuts and the genetic diversity indices are lower for y chromosome than for mtdna.
moreover, the number of first settlers who arrived in central yakutia was certainly limited and the patrilocal exogamy practiced by the traditional yakut society  <cit>  corroborates the inclusion of autochthonous women.
this assumption is further confirmed by linguistic data that reveals intermarriages between the yakuts and evenks  <cit> .
finally, the stability through time demonstrated by the y chromosome is also observed in the maternal lineages.
indeed, most of the sequences present in our ancient sample  have been transmitted to the present day population.
however, this could signal a small loss in variation over the two last centuries, which could be associated with stochastic processes linked to demographic changes undergone by the yakut population  or other phenomena  <cit> .
besides, the yakuts from all chronological periods are grouped together in the mds plot and the fst calculation revealed the absence of significant distances between them.
thus, even if the russian colonization had an important impact on the expansion of the yakuts throughout yakutia  <cit> , its genetic influence appears to be relatively low.
based on the analyses of the maternal and paternal lineages of ancient yakuts, we were able to demonstrate that the formation of this population started before the 15th century, with a small group of settlers composed of horse-riders from the cis-baïkal region and a small number of women from different south siberian origins.
these assumptions are in agreement with archaeological data  <cit> , the founder effect dates calculated by pakendorf et al.
<cit>  and with the recent coalescent simulations of yakut mtdna variation by zlojutro et al.
<cit> .
the early expansion of the yakuts was accompanied by intermarriages between yakut men and evenk women as evidenced by the migration rates.
our data also suggest that the genetic characteristics of the yakuts were already well established in the central yakutian population during the 15th century and have remained stable until the present day.
CONCLUSIONS
there are few regions in the world where the microevolution of a population over several centuries, based on y-chromosomal and mitochondrial dna analyses, can be understood.
it is the case, however, in siberia, notably in yakutia, where frozen bodies facilitate the exceptional preservation of biological samples.
in this article we compared  <dig> individuals from central yakutia:  <dig> frozen bodies from the 15th to the late 18th century which were excavated over three years, and seven bodies of a later period from more ancient excavations, with  <dig> individuals from a well-defined contemporary population.
data authenticity from the frozen bodies was determined by the concordance of morphological and molecular typing for sex determination, reproducible pcr results from multiple extractions, and the amplification and analysis of autosomal str markers based on their ability to detect degraded and/or contaminated dna.
if the time from excavation and post-excavation storage conditions were less favourable for dna preservation, age at death also plays a role, as it is more difficult to obtain profiles from newborn compared to adult bones.
we were able to demonstrate that the yakutian population formed before the 15th century, from a small group of settlers from the cis-baïkal region and a small number of women from different south siberian origins.
the genetic characteristics of the yakuts were well established in the central yakutian population during the 15th century, even if there was a small loss in genetic variation during the last two centuries associated with stochastic processes or other phenomena.

 the yakuts from the republic of sakha are of particular interest because they contrast with other populations from siberia in many respects such as in their specific funeral practices. before christianization, part of the population was buried, which is exceptional in this region of the world as most bodies were put in aerial graves on platforms or on trees. at the same time, the yakuts are considered as the most remarkable example of northward expansion into siberia and especially as these semi-nomadic herders contrast so strikingly with surrounding siberian populations on the basis of their turkic language and their horse- and cattle-breeding economy. the geographic specificities of central yakutia, favorable to the development of a pastoralist economy and reflecting the environmental conditions of the south, certainly initiated the development of the yakut culture in this region. although several hypotheses regarding the geographic origin of this atypical population have been proposed from results of the first archaeological investigations of ap okladnikov to the latest population genetic studies, the precise origins of the yakuts and their admixture with the indigenous tribes of siberia, especially the tungus, have been debated. nevertheless, archaeological data currently agree with the appearance of the yakut culture around the 14th century a.d.. recent population genetics data tend to support a dual origin for the maternal lineages of the yakuts and admixture with the tungus, and demonstrate that the paternal lines observed today result from a strong bottleneck that led to the restriction of these lineages in the yakut population. this does not, however, address the question of the number of settlers and the precise origin of the paternal lineages, or the admixture rate with indigenous populations. furthermore, the demographic changes occurring in the yakut population since the russian colonization make it difficult to reconstruct the genetic history using present day samples. russians came into contact with the yakuts in christianization began around and by the beginning of the 19th century, a christian ritual was performed in the majority of burials. from the 17th to the 19th century smallpox and measles epidemics, caused by contact with russians settlers, decimated the yakut people and their neighbors, and during the second world war a large proportion of young yakut males was killed. therefore ancient dna, which allows the direct study of an ancient population without their descendants, represents a major advantage in order to avoid the possible bias represented by recent genetic events. this study adopts a population genetics approach to an ancient sample composed of individuals from central yakutia in order to address the questions detailed above. human adna studies' outcome quality is closely related to the preservation of dna and to the absence of potential contaminations. the environmental conditions present in yakutia, one of the coldest parts of the world, are generally favorable to a good preservation of nucleic acids. indeed, the exceptional preservation state of most of the excavated graves enabled us to successfully analyze several samples collected from frozen bodies or skeletons from the last six centuries.  nuclear dna quantification the results of the nuclear dna quantification are summarized in table the purification protocol was efficient in removing inhibitors in all except six bone extracts. in order to avoid the effect of pcr inhibitors, the samples yaka and were diluted by a factor of 1/ to 1/ and successful amplifications were subsequently obtained. for the sample yaka it was not possible to remove the inhibitors and therefore no results were obtained for further amplifications. no inhibitors were detected in the teeth or hair extracts. moreover, the dna extracts obtained from teeth showed the highest mean concentration of nuclear dna and the highest frequency of samples yielding a concentration superior to pg.μl- y-chromosomal analyses the multiplex amplification of the y-chromosomal strs was successful for of the male samples, where full profiles or profiles with only one missing marker were obtained. the lowest amplification success rate was observed for the dys locus that is the largest marker included in the str panel. all loci with a length over bp showed more than 20% amplification failures. this molecular behavior is a well known phenomenon and has been described as an effective way to ensure the authenticity of results. twelve different haplotypes were observed. nine were unique, one was common to two individuals and two haplotypes were widely shared among our ancient sample set. these two haplotypes were present in eight and seven individuals, and differed from each other by only one mutation step at the loci dys389ii and dys therefore, ht and ht could share a recent common ancestor, since two mutation events in a single father/son transmission have been previously described. besides, ht and ht were the most frequent lineages in our present day sample set as well as in the data published by pakendorf et al. the genetic diversity indices show equivalent values for the ancient and the present day sample sets of our study. surprisingly, the diversity observed in our central yakut sample set is higher than the one presented by pakendorf et al although the samples were collected in the same area. present day yakuts, 2number of samples, 3number of haplotypes, 4mpd: mean pairwise differences, 5standard error. central yakuts sixty one percent of the haplotypes were affiliated to the n1c haplogroup on the basis of the snp analyses. this haplogroup is considered as the most frequent in the yakut population, and its frequency varies across studies from 75% to 100%. sample yaka was affiliated to haplogroups k. the snp typing was inconclusive for individuals; nevertheless the affiliation to n1c was excluded on the basis of the absence of the tat-c mutation. although all the y-str haplotypes have been transmitted to the present day yakuts, only a few of them are shared with other populations. a comparison to the yhrd database and other literature data showed that only ht and the haplotype of the yaka individual matched with evenk male lineages; confirming the specificity of the n1c lineages. in this case, one can assume that n1c comes from yakut ancestors considering the low n1c frequency in evenks. moreover, the ethnical affiliation of one individual to either the yakut or the evenk population could be difficult nowadays, considering that these two ethnic groups share a similar lifestyle or language in some areas of yakutia. the y-str haplotypes of yaka and yaka were found in present day mongols, buryats, kalmyks and central asians. the mds plot based on pairwise fst values clearly shows the separation of the yakuts from all the other populations, since the ancient and present day yakuts are clearly isolated. indeed, the fst distance computations revealed significant differences for each comparison excepted for the two yakut sample sets. the median network based on the y-str haplotypes belonging to the n1c haplogroup is undoubtedly affected by sample sizes. nevertheless, the separation between the yakuts and the other populations is visible. indeed, both ancient and present day yakuts are grouped in one isolated cluster together with the evenks. the different haplotypes of this cluster are separated by one mutation step, indicating that these closely related paternal lineages have emerged from a reduced number of individuals in a short period of time. this observation is consistent with earlier studies. mitochondrial dna analysis the hvs- sequences were unambiguously validated for individuals. five subjects were excluded from the analysis: yaka and no amplification was obtained for the sample yaka due to the presence of inhibitors in the dna extracts. amplification failures were also observed for the sample yaka and only one of the two sub-regions of hvs- was successfully amplified for sample yaka finally, multiple heteroplasmies were observed in the electropherograms of yaka and the fact that these two samples were collected in museums, increasing the risk of pre-laboratory contamination, led us to exclude these two subjects from our study. two heteroplasmic positions were observed for three distinct samples. one t/c heteroplasmy at position was observed in the samples yaka and yaka in teeth and hair dna extracts only. this heteroplasmic position is observed in the present day yakut population and is also well known as a hotspot of post-mortem damage. the second heteroplasmy was observed at position in the sample yaka this position has been reported as a potential post-mortem mutation. therefore, these heteroplasmic sites were treated as missing data for these specific samples in all the subsequent analyses. gene diversity in our ancient sample was calculated using the arlequin program and then compared with different populations. both ancient and present day populations from central yakutia showed a gene diversity of. this value is in agreement with the data from. we observed different haplotypes among the studied individuals, and were found only once. twenty nine haplotypes were assigned to different haplogroups with the near matching method. the subject yaka plausibly a young shaman from the early 18th century, was affiliated to haplogroup h due to the typing of coding region snps since the assignment on the basis of the hvs- haplotype was inconclusive. the overall haplogroup distribution in our ancient sample set corresponds with earlier studies focused on the present day yakuts. as can be seen in table the ancient yakuts present high frequencies of haplogroups c and d. haplogroup c and specifically the lineage c represents the most abundant haplogroup in our ancient sample set with a frequency of 30%. the highest frequency of this lineage is observed in the evenks, but c is also prevalent in south-western siberian populations such as tuvans, buryats and tofalars. among haplogroup d, the d lineage is present in seven out of individuals and represents % of the ancient sample set. this sub-haplogroup is usually present at low frequency in siberian populations and in central asian or mongolian populations. interestingly, the two most frequent d haplotypes seem to share a recent common ancestor as they differ only by one transition at position this high frequency of subhaplogroup d has been regarded as specific to the yakut maternal lineages by pakendorf et al.. the buryat ascendance of the d lineages demonstrated in their analyses was not clearly supported by our ancient dna data. sub-haplogroup frequencies have been merged under the name of the basal haplogroup to simplify the comparison. frequencies are given in percent. the sequence of yaka belonged to haplogroup a4b, that is present at low frequencies in western and middle siberian populations such as the mansis, tuvans and evenks. yaka was assigned to the novel haplogroup a found in the kalmyks and buryats. two haplotypes were assigned to haplogroup b: yaka to subhaplogroup b which is dispersed throughout mongolia and china and the sequences of yaka and yaka to subhaplogroup b due to the presence of transitions at positions and also present in one buryat studied by derenko et al. two sequences belonged to the f1b lineage, widespread in populations from south siberia. the haplotype of yaka was affiliated to the m13a subhaplogroup present in buryats, khamnigans and mongols. the proportion of the sequences affiliated to the west eurasian haplogroups was low in our ancient sample set, as the merged frequencies corresponded to %. the sequence of yaka was affiliated to subhaplogroup h which is characteristic for central asians and less frequent in western europe. these sequences were observed in samples distributed in the second and third period, and no increase in the frequency of the west eurasian lineages could be noted for the period following the russian colonization. besides, these haplogroups are observed at similar frequencies in various siberian populations. the size of the sample set should be considered as a potential source of bias in the overall haplogroup distribution. nevertheless, we observed the same distribution as was found in the present day population. this statement has been verified by the calculation of the fst distances since there are no significant distances between the yakuts from the pre-colonization era and the ones from the christian and present day periods. in an attempt to trace the origins of the ancient mtdna lineages, each haplotype was compared to data from the literature. this comparison showed that more that 73% of the haplotypes found in our ancient samples have been transmitted to the present day yakut population. among the nine haplotypes absent in the present day yakuts, four were not found in any of the populations included in the database due to their unique combination of polymorphisms or private mutations. the haplotypes belonging to haplogroup c are shared with various populations but most specifically with buryats, evenks and tuvans. on the contrary the sequences assigned to haplogroup d appear to be more restricted to the yakut population. this specificity is even higher for the d lineages since they are shared only with two yakut speaking evenks, one buryat, one mongol and one daur. to evaluate if this comparison could bring some information on the different migration waves which led to the formation of the yakuts, we took the chronological repartition of the samples into consideration. unfortunately, this approach failed to reveal any clear pattern, since individuals from different periods were distributed among all the haplogroups and matched to populations without any identifiable preference. the mds plot based on fst distances revealed, as for the y-chromosomal strs, a high stability through time of the maternal lineages. indeed, the distances between the chronological groups were not significant. besides, the fst values confirm the previous statements based on the comparison of individuals since non-significant distances were observed between ancient yakuts, evenks and tuvans. in order to clarify the previously obtained results, migration rates were computed with the migrate software. the populations used for the analyses were selected taking the previous results into consideration. to avoid a potential bias related to the sample size, ancient yakuts from different periods were merged into one group. as mentioned earlier, this approximation should not be deleterious since the two groups showed non-significant genetic distances. the results are presented in table and indicate that the major genetic contribution to the maternal lineages of the yakuts came from the evenks. this observation could be explained by the fact that new settlers were more susceptible to admixture than the autochthonous population due to their reduced number and to their cultural practices. this assumption is also corroborated by the higher genetic diversity of the ancient yakuts compared to the evenks and by the important number of mtdna lineages shared with this population. first presence of the yakutian ancestors and demographic data the first yakut graves discovered are dated from the 15th and 16th century. from this period until only male graves with horse riding equipment and weapons from the steppe area are found. the funerary ritual was influenced more by turkish than by mongolian culture. it is unlikely that these graves are the first graves of the new settlers, and it is more probable that this period corresponds to the first time with a relatively high population density. during this early period, y-chromosomal ht and ht are already present. simulations suggest two possibilities: either a very small group arrived around the 10th century, or a more important one arrived between the disintegration of the mongol empire and the discovery of the first graves. people is an historical estimation for ad and we have made estimation for different steps: the beginning of the 9th century, which is a period of an important mongolo-turkish expansion with some contacts between these populations and yakutia, ad which corresponds to the disintegration of the mongol empire with the dispersion of many nomad groups, and ad, a date prior to the most ancient yakut graves excavated. authenticity of sequences and profiles the possibility that our data arose from contaminated dna was considered highly unlikely, since morphological and molecular typing results for sex determination were always in accordance with each other. moreover, no contamination was observed in the extraction or the pcr blanks. reproducible pcr results were obtained from multiple extractions and amplifications of the same samples performed at different times. the choice of autosomal str markers as a first approach for analyzing the ancient yakut remains was based on their ability to detect degraded and/or contaminated dna. out of the human samples yielded amplifiable dna for more than two markers, but only of the genetic profiles were complete. the loci d18s csfp1o, d7s and d2s were often not amplified, probably because they are expressed in the higher molecular weight range. such an inverse dependence of the amplification efficiency on the size of the dna fragment to be amplified is typical of dna retrieved from ancient remains and results from the degradation of the dna. various factors are thought to affect the success of dna analysis from ancient tissues: the time from excavation, as post-excavation storage conditions are less favorable for dna preservation than pre-excavation condition, the cortical thickness of long bones which favors good dna preservation and the age-at-death of the individual, as it is more difficult to obtain profiles from newborn compared to adult bone. thus, as time passes after excavation, one would expect a reduction in the number of endogenous dna and an increase in the number of exogenous contaminant template dna molecules extracted from a tissue sample, with additional difficulties for newborn and young children compared to adult samples. of the ten human samples that yielded no str profiles, four came from the museum and six from frozen burials including four newborn samples. discussion during the last few years, several studies have focused on the yakuts using genetic analyses to trace their origins. however, to date the use of ancient dna has been limited due to small numbers of individuals sampled, in spite of the exceptional taphonomic conditions of siberia and the possibility of retrieving high quality dna from remains buried in the permafrost. therefore, this study represents the first attempt to trace the origins of the yakut population by ancient dna analyses. out of the ancient individuals analyzed in this study, complete y-chromosomal str haplotypes were obtained from male individuals, and mtdna sequences were validated for individuals. the quality of the results obtained in our study may be explained by the presence of permafrost which favors the preservation of dna. the excavation strategy and the absence of any post-excavation treatment prior to laboratory analyses might have played an important role in the quality of dna and confirms the statements of pruvost et al.. besides, this limited handling of the ancient remains represents a key factor in the reduction of contamination risk by modern dna. the combination of the precautions against contamination and the validation strategy validate the data presented here, even if not all the traditional criteria could be met due to the large number of samples. the y-chromosome results obtained from our ancient sample are consistent with earlier studies. the specificity of the male lineages was confirmed by statistical analyses and by individual comparison of the y-str haplotypes with the literature data since only three haplotypes were shared with other populations. the high frequency of the haplogroup n1c, which is present from the most ancient graves, and the non-significant fst values between ancient and present day yakuts, indicates their stability through time. thus, this genetic result corroborates the assumption that the yakuts are a highly homogenous population not only geographically but also from a chronological standpoint. the origin of the most frequent y-chromosomal haplotypes was difficult to establish on the basis of genetic information. indeed, these two lineages belonging to haplogroup n1c seem to be restricted to yakut populations, and were probably present since the period they were first located in central yakutia. interestingly, the comparison with archaeological data revealed that the male individuals at the beginning of the 18th century, identified as clan chiefs on the basis of their grave goods, belonged to these two haplotypes. therefore, archaeological data could bring interesting information in tracing back the origin of these enigmatic male lineages. indeed, the grave goods of the 15th/17th centuries and the construction of coffins with an empty trunk from the 18th century are similar to the burial customs of the cis-baïkal area and of the egyin gol necropolis during the 3rd century bc. this suggests that the male ancestors of the yakuts were probably formed of a small group of horse-riders originating from northern mongolia or the baïkal lake. later, the patrilineal clan organization of the yakuts would have facilitated the diffusion of male lineages borne by the individuals of high social rank. this hypothesis is supported by the fact that the y-chromosomal haplotypes identified by the warriors of the 15th/17th centuries were transmitted to the tojons of the 18th century. after russian colonization, the influence of the tojons was strengthened by the decrees made by the empress anna ivanovna, and this official reinforcement of their power might have increased the spread of a limited number of y-chromosomal lineages for a second time. therefore, the early founder effect combined with the yakutian traditions and the russian influence would have led to the present day genetic pattern observed for the paternal lineages. conversely, the results obtained from the mtdna analyses revealed a more important diversity and varying origins of the maternal lineages. indeed, the gene diversity observed in our ancient sample is intermediate compared with the diversity found in small and isolated siberian groups such as the chukchi and mongolian, buryatian or central asian populations. the haplogroup distribution observed in our sample matched that found in present day yakuts and is constituted by distant haplogroups found in siberian, mongolian and central asian populations. the major haplogroup, c indicates more specific affinities with the evenks. nevertheless, the individual comparison of maternal lineages with data from literature allowed us to precise that four out of the six lineages belonging to the c haplogroup were present in the buryat population. therefore, the buryat origin of some of the d sub-haplogroups found in the yakuts as demonstrated by pakendorf et al. could be expanded to the c haplogroup. the influence of both buryat and evenk populations is clearly visible in the mtdna lineages of the ancient yakuts. concerning the contribution of the evenks to the yakut's mtdna gene pool, the admixture might have mostly occurred between yakut men and evenk women according to the high frequency of the c haplogroup in these two groups, and since the y-chromosomal lineages are highly specific to the yakuts and the genetic diversity indices are lower for y chromosome than for mtdna. moreover, the number of first settlers who arrived in central yakutia was certainly limited and the patrilocal exogamy practiced by the traditional yakut society corroborates the inclusion of autochthonous women. this assumption is further confirmed by linguistic data that reveals intermarriages between the yakuts and evenks. finally, the stability through time demonstrated by the y chromosome is also observed in the maternal lineages. indeed, most of the sequences present in our ancient sample have been transmitted to the present day population. however, this could signal a small loss in variation over the two last centuries, which could be associated with stochastic processes linked to demographic changes undergone by the yakut population or other phenomena. besides, the yakuts from all chronological periods are grouped together in the mds plot and the fst calculation revealed the absence of significant distances between them. thus, even if the russian colonization had an important impact on the expansion of the yakuts throughout yakutia, its genetic influence appears to be relatively low. based on the analyses of the maternal and paternal lineages of ancient yakuts, we were able to demonstrate that the formation of this population started before the 15th century, with a small group of settlers composed of horse-riders from the cis-baïkal region and a small number of women from different south siberian origins. these assumptions are in agreement with archaeological data, the founder effect dates calculated by pakendorf et al. and with the recent coalescent simulations of yakut mtdna variation by zlojutro et al.. the early expansion of the yakuts was accompanied by intermarriages between yakut men and evenk women as evidenced by the migration rates. our data also suggest that the genetic characteristics of the yakuts were already well established in the central yakutian population during the 15th century and have remained stable until the present day.  there are few regions in the world where the microevolution of a population over several centuries, based on y-chromosomal and mitochondrial dna analyses, can be understood. it is the case, however, in siberia, notably in yakutia, where frozen bodies facilitate the exceptional preservation of biological samples. in this article we compared individuals from central yakutia: frozen bodies from the 15th to the late 18th century which were excavated over three years, and seven bodies of a later period from more ancient excavations, with individuals from a well-defined contemporary population. data authenticity from the frozen bodies was determined by the concordance of morphological and molecular typing for sex determination, reproducible pcr results from multiple extractions, and the amplification and analysis of autosomal str markers based on their ability to detect degraded and/or contaminated dna. if the time from excavation and post-excavation storage conditions were less favourable for dna preservation, age at death also plays a role, as it is more difficult to obtain profiles from newborn compared to adult bones. we were able to demonstrate that the yakutian population formed before the 15th century, from a small group of settlers from the cis-baïkal region and a small number of women from different south siberian origins. the genetic characteristics of the yakuts were well established in the central yakutian population during the 15th century, even if there was a small loss in genetic variation during the last two centuries associated with stochastic processes or other phenomena. 
furthermore, mtdna data showed that intermarriages between the first settlers with evenks women led to the establishment of genetic characteristics during the 15th century that are still observed today. a comparison with the same markers on seven museum specimens excavated to years ago showed significant differences in dna quantity and quality. the yakuts contrast strikingly with other populations from siberia due to their cattle- and horse-breeding economy as well as their turkic language. this study attempts to better understand the origins of the yakuts by performing genetic analyses on mummified frozen bodies dated from the 15th to the 19th century, excavated from yakutia. direct access to ancient genetic data from these molecular markers combined with the archaeological evidence, demographical studies and comparisons with contemporary individuals from the same location as the frozen bodies helped us to clarify the microevolution of this intriguing population. on the basis of ethnological and linguistic criteria as well as population genetic studies, it has been assumed that they originated from south siberian populations. high quality data were obtained for the autosomal strs, y-chromosomal strs and snps and mtdna due to exceptional sample preservation. we were able to trace the origins of the male lineages to a small group of horse-riders from the cis-baïkal area.
 the yakuts contrast strikingly with other populations from siberia due to their cattle- and horse-breeding economy as well as their turkic language. on the basis of ethnological and linguistic criteria as well as population genetic studies, it has been assumed that they originated from south siberian populations. however, many questions regarding the origins of this intriguing population still need to be clarified. this study attempts to better understand the origins of the yakuts by performing genetic analyses on mummified frozen bodies dated from the 15th to the 19th century, excavated from yakutia.  high quality data were obtained for the autosomal strs, y-chromosomal strs and snps and mtdna due to exceptional sample preservation. a comparison with the same markers on seven museum specimens excavated to years ago showed significant differences in dna quantity and quality. direct access to ancient genetic data from these molecular markers combined with the archaeological evidence, demographical studies and comparisons with contemporary individuals from the same location as the frozen bodies helped us to clarify the microevolution of this intriguing population.  we were able to trace the origins of the male lineages to a small group of horse-riders from the cis-baïkal area. furthermore, mtdna data showed that intermarriages between the first settlers with evenks women led to the establishment of genetic characteristics during the 15th century that are still observed today. 