BACKGROUND
the cold periods of the pleistocene have had a dramatic impact on most organisms in temperate regions.
whereas the artic ice sheet began to grow about  <dig>  myr ago, more severe climatic oscillations occurred mainly during the last  <dig>  <dig> year  <cit>  with the consequence that the european biota has experienced repeatedly some drastic climate changes.
tree species responded through migration to regions where environmental conditions allowed them to survive  <cit> .
phylogeography investigates on the spatio-temporal dynamics of populations.
it relies on inference from two main sources of information, i) macrofossils and pollen in sediment profiles, ii) population structure in dna markers ideally inferred at the sequence level.
cytoplasmic markers, which are uniparently inherited in most plant species, are especially well suited for phylogeographic studies.
they helped infer the number of genetically distinct ancestral lineages, their location during the last glacial maximum, and the postglacial migration routes for several tree and plant species in europe .
these studies and others based on fossil evidence suggested that most plant and tree refuges were located in southern european peninsulas and the balkans  <cit> .
glacial refugia and recolonization routes were found to coincide often for a number of plant and animal species .
in north america, common patterns are also starting to emerge and much knowledge has been rapidly gained from the analysis of species with wide distributions .
however, there is now increasing evidence that some european species were established much further north and east than previously assumed during glacial time  <cit> .
similar inferences have also been made in north america, where genetic and palynological evidence is accumulating for the presence of glacial populations much closer to the ice fronts as once thought .
phylogeographic studies in europe also indicate asymmetric contributions of glacial refugia to holocene colonization: some ancestral lineages remained endemic, because of geographic barriers limiting migration, while others contributed considerably to the postglacial colonization of more northern latitudes .
scots pine  is a long-lived evergreen monoecious forest tree species growing in a large variety of ecological conditions from western europe to asia.
of all pine species, it has the largest distribution  <cit> .
its natural range extends northwards from spain to northern fennoscandia, and westwards from longitudes 5°w in spain and scotland to 135°e in siberia.
while most of its natural range is contiguous, there are also large populations of scots pine disconnected from the main natural range such as in scotland, spain, france, turkey, and former yugoslavia.
extensive variations in phenotypic traits as well as geographic races and types have been reported  <cit> .
much of this variation has been usually attributed to adaptation to edaphic and climatic factors  <cit> .
studies carried out over the last  <dig> years using isozymes, terpenes and flavonoid markers have also revealed the existence of distinct evolutionary units in scots pine, which might be indicative of isolation in different glacial refugia.
although these studies were not entirely congruent and not aimed at deciphering effects related to glacial and postglacial history, they helped define biogeographical hypotheses for the western and northwestern european range of scots pine  <cit> .
because of the regional scope of many of these studies and the different phenotypic or genetic attributes used, much biogeographical knowledge on the glacial and postglacial history of eurasia remains to be gained from analysing the broad geographic distribution of scots pine and by using homologous gene markers with stronger historical imprints.
in the pinaceae, mtdna is maternally inherited and dispersed through seeds only  <cit> .
this mode of dispersion helps avoid the problems with inferring history from present-day distributions of nuclear or cpdna markers because the latter are dispersed by both seeds and pollen in the pinaceae, which produce a more rapid breakdown of the historical genetic signatures induced by glacial vicariance.
however, one of the major difficulties one faces with plant mtdna is the low level of variation in their exons and introns  <cit> , which limits the detection of variation between and within species at the sequence level .
an indel has been discovered in the intron b/c of the mitochondrial gene nad <dig> in scots pine, which allowed identifying a genetically distinct ancestral lineage in the iberian peninsula  <cit> .
restriction fragment length polymorphisms  of a region encompassing the mitochondrial gene cox <dig> have also been found in populations of scots pine from western europe  <cit> , which helped identifying two genetically distinct groups of populations each distributed on a large latitudinal range.
the possibility for a refuge at northern latitudes was suggested in a previous study  <cit> .
given the little diversity observed in the mtdna regions surveyed and the population sampling limited mostly to western europe, it is likely that the pan-eurasian scots pine possesses more genetically distinct ancestral lineages.
such evidence would contribute towards a more complete understanding of the historical factors and processes leading to the current distribution of species genetic diversity throughout eurasia.
in an effort to discern from a phylogeographic perspective the possible existence and contribution of mid-northern refugia to the glacial and postglacial history of eurasian biota, we investigated the extant genetic structure of scots pine with a set of populations covering much of the natural range in europe and also in asia.
we also report on a novel polymorphic mtdna region in scots pine, the intron  <dig> of the gene nad <dig>  which is indicative of unknown genetically distinct maternal lineages.
the location of one of these lineages in northern europe supports the view that quaternary refugia located outside european peninsulas at mid-northern latitudes might have played a significant role in the holocene recolonization of europe  <cit> .
RESULTS
mtdna polymorphisms and population differentiation
out of  <dig> mtdna regions surveyed , intraspecific polymorphism was detected for only two loci in scots pine.
polymorphism was detected de novo for the intron  <dig> of nad <dig> with three length variants.
they were  <dig>   <dig> and  <dig> bp long and are hereafter called haplotypes a, b, and c, respectively.
the size differences were caused by two single indels: one of  <dig> bp exclusive to haplotype c and one of  <dig> bp exclusive to haplotype b .
as previously reported from a more limited sampling  <cit> , two size variants were detected for the intron b/c of nad <dig> among the  <dig> samples analysed herein.
these fragments differed by a  <dig> bp indel and are hereafter called haplotypes a  and b .
a third variant restricted to italy was recently reported for this gene  <cit> .
it was not detected in our sampled trees which were all from outside the italian peninsula.
primers for these two mtdna regions were also tested on  <dig> to  <dig> individuals for each of five other pines of the subgenus diploxylon  and positive amplifications were obtained .
the first four species belong to the section pinus, but the first three are members of subsection pinus  and pinus heldreichii is member of subsection pinaster.
as for p. contorta, it belongs to section trifoliae, subsection contortae.
fragment length polymorphism was detected for both nad <dig> intron b/c and nad <dig> intron  <dig> in p. heldreichii.
for p. nigra, only nad <dig> intron b/c showed polymorphism.
no polymorphism was detected in p. mugo and p. resinosa.
for p. contorta, polymorphism was only observed for nad <dig> intron  <dig> involving the same minisatellite-like marker as previously reported for this species and the closely related p. banksiana  <cit> .
1targeted regions, annealing temperatures, expected size of pcr products and restriction enzymes tested for primer pairs used to amplify  <dig> mtdna regions of scots pine.
2nad <dig> intronb/c have been amplified  and tested with different restriction enzymes, only internal primers h and i of soranzo et al  <cit>  have shown polymorphism.
3intergenic region.
4multiple fragments.
1polymorphisms detected in the intron  <dig> of the mitochondrial gene nad <dig> and in the intron b/c of the mitochondrial gene nad <dig> in scots pine, and nomenclature used for multi-locus haplotypes .
for scots pine, the nomenclature used for mitochondrial multi-locus haplotypes  followed a two-letter code after the haplotypes observed for nad <dig> intron  <dig>  and nad <dig> intron b/c , respectively .
among the four observed mitotypes, the most abundant one was aa , followed by ba , ca , and ab  .
the minimum spanning tree indicates that the mitotypes were more or less arranged as a star phylogeny with little sequence variation among them and no recombination .
the observed number of mitotypes per population  was  <dig> or  <dig> without exception, with an average of  <dig>  .
the mitotype diversity index per population  was low to moderate  and ranged from  <dig> to  <dig>  .
population differentiation was high among the  <dig> populations, with gst and nst values of  <dig>  and  <dig> , respectively .
when excluding the six more diverse populations from a presumed zone of contact in central and northeastern europe between two distinct mtdna lineages , the gst and nst values increased to  <dig>  and  <dig> , respectively, among the remaining populations.
however, the presence of a formal phylogeographic structure was not supported, as nst was not significantly higher than gst.
lack of test power is likely the reason, given the small genetic divergence observed among the four mtdna variants detected.
1at, austria; bg, bulgaria; by, belarus; cn, china; cz, czech republic; es, spain; fi, finland; fr, france; lt, lithuania; lv, latvia; ro, romania; ru, russian federation; se, sweden; tr, turkey; ua, ukraine; uk, united kingdom.
2the "w" indicates the longitude west of greenwich meridian.
3the first letter of multi-locus haplotype  refers to the haplotype observed at locus nad <dig> intron <dig> and the second letter refers to the haplotype observed at locus nad <dig> intron b/c following nomenclature in table  <dig>
4number of distinct mitotypes per population.
5mitotype diversity.
1estimates for groups of populations of scots pine delineated by samova and maximizing geographic structure.
2sample size.
3number of distinct multi-locus haplotypes  observed in each group.
4average number of mitotypes per population in each group.
5average mitotype diversity per population in each group.
6differentiation among populations in each group.
7fixation index using permut software  <cit> .
8differentiation among the four groups.
geographic structure
the geographic distribution of mitotypes appeared to be essentially non-random, with clusters of mitotypes geographically quite well delineated .
the mediterranean region showed the highest diversity with three of the four mitotypes revealed in the study.
the cosmopolitan haplotype aa was largely distributed and found in most of the populations sampled.
geographic structuring was evident for other mitotypes.
mitotype ca was present only in asia minor, in the pontide mountains of turkey .
mitotype ab was found fixed in some high mountain populations of the iberian peninsula   and was observed as a rare variant in some populations of the balkans .
the distribution of mitotype ba was quite unexpected: it was largely distributed in the lowland region of middle to northern latitudes in eastern europe.
it was prevalent in the baltic region and quite frequent in russia, west of the ural mountains.
but it was also detected in siberia .
a large zone composed of populations with variable mixed genetic background  was observed in central and northeastern europe.
it includes most if not all populations bearing mitotype ba.
the zone extends from the southeast of russia to the baltic states .
the within-population mitotype diversity of these populations  was much higher than elsewhere.
samova was conducted repeatedly by increasing the value of the number of population groups  until the fct value reached a maximum of  <dig> , which was obtained for k =  <dig> .
for k =  <dig>  a maximum fct value of  <dig>  was obtained.
the four groups confirmed trends from visual inspection and corresponded to:  the majority of populations from europe and asia,  northeastern europe,  asia minor and  the high mountains of the iberian peninsula.
group i was the most widespread geographically, including  <dig> populations .
it contained mostly the cosmopolitan mitotype aa and, at a lower frequency, mitotype ba and ab.
differentiation among the populations of the group was moderate.
population differentiation within each of the three other groups  was smaller.
group ii from northeastern europe was largely dominated by mitotype ba.
the two remaining groups iii and iv were highly homogenous.
group iii regrouped populations from turkey fixed or nearly fixed for mitotype ca, and group iv regrouped populations from the iberian peninsula fixed for mitotype ab.
discussion
mtdna diversity and population differentiation
dna sequence variation is low in the mtdna of conifers .
accordingly, little intraspecific variation has been found in the mtdna of p. sylvestris in western europe, including four rflp variants for the cox <dig> region  <cit>  and three size variants at the locus nad <dig> intron b/c, of which two were found endemic to the iberian and italian peninsulas  <cit> .
the three new size variants reported herein for the locus nad <dig> intron  <dig> indicate new region-specific variants for northeastern europe and asia minor.
no other length polymorphisms or substitutions were detected in scots pine in spite of the screening of  <dig> distinct mtdna regions.
highly informative minisatellite-like motifs such as those rarely detected in some spruce  <cit>  and pine taxa  <cit>  could not be detected in scots pine.
the overall level of population differentiation estimated in this study from mitotype variation at the nad <dig> intron b/c and nad <dig> intron  <dig> loci was rather high and similar to that reported in earlier studies restricted to scots pine populations from western europe based on rflps of the cox <dig> region  or indels of the nad <dig> intron b/c .
such estimates are in line with averages reported for maternally inherited markers for a number of conifer and angiosperm taxa  <cit> .
the amount of population differentiation determined by nuclear markers such as isozymes was generally much smaller in scots pine with gst values ranging between  <dig>  and  <dig>   <cit> ].
however, these studies were carried out mostly at the regional level.
among-population differentiation was also much higher for mtdna markers than for paternally inherited cpdna markers in a scots pine study covering much of the species range , as usually observed in other conifers .
geographic structure
the present study confirms the existence of a genetically distinct set of populations in the high mountains of the iberian peninsula  <cit>  while variants discovered in the intron  <dig> of nad <dig> signal distinctive evolution in asia minor and northeastern europe.
haplotype b at locus nad <dig> intron b/c was observed mostly in the iberian peninsula in some high elevation populations of the zaragoza/valencia regions where it was fixed or nearly fixed.
it was observed only sparingly west of catalonia.
such a distribution indicates that these catalan populations have been isolated during glaciations and have not contributed to the recolonization of europe .
a distinct dynamics of catalan populations from the rest of the iberian peninsula has also been reported for other species like aleppo pine  <cit> , maritime pine  <cit> , and white oaks  <cit> .
in the present study, haplotype b of nad <dig> intron b/c has also been discovered, albeit at low frequency, in five other populations located in bulgaria, in the balkan peninsula, thus far away from the center of prevalence for this haplotype in the iberian peninsula.
long-distance seed dispersal could be invoked but is unlikely over such long distances, given the heterogenous topography and relative isolation of iberia.
the probability of such long-distance movements would be higher in the northern and more eastern parts of scots pine natural range, given the lack of major physical barriers.
another perhaps more plausible explanation for the rare presence of haplotype b of nad <dig> intron b/c in some balkan populations could be that it is ancient and was present in more than one southern ice refugia.
its high or low frequency, depending on the region considered, could be attributable to stochastic variation, especially if southern refugia populations suffered from severe bottlenecks.
the weak occurrence of this haplotype in the balkans might also result from human activity, as previously suggested for the rare presence of this variant in poland  <cit> .
whereas the hypothesis of seed source transfer cannot be totally ruled out, it is difficult to sustain because of  the lack of hard evidence for such seed source transfer between the balkans and the iberian peninsula in the past;  natural stands in which seeds were collected existed long before forest management activities began in europe; and  the relative conservatism of forest seed transfer guidelines in the balkan region  <cit> .
a third explanation could relate to ancient mtdna capture from hybridizing species in the region  <cit> .
accordingly, phylogeographical studies of other pine species in the region using the same gene locus might help clarify the issue.
the detection and high prevalence of haplotype c of nad <dig> intron  <dig> in asia minor populations only is a strong signal for the presence of a genetically distinct glacial population.
this relictual population would have experienced severe bottlenecks, because populations were all fixed for this haplotype except for one tree harbouring the cosmopolitan haplotype a. after glaciation, it would have remained isolated, eastward by the caucasus mountains, northward by the black sea, and westward by the bosphorus passage.
the cold and arid climate in this region during the holocene might have also impeded the expansion of this population  <cit> , but not its survival as scots pine can survive under very harsh conditions  <cit> .
the high prevalence of haplotype b of nad <dig> intron  <dig> in northeastern europe suggests the presence of a genetically distinct glacial population at more northern latitudes than previously thought, an idea that had already been suggested for scots pine  <cit> .
a recent mutation during the holocene giving rise to haplotype b of nad <dig> intron  <dig> is unlikely, given the widespread distribution of this haplotype.
as well, homoplasy is unlikely, given the low rate of variation of angiosperm and conifer mitochondrial introns  <cit> .
a large number of populations had mixed composition of both haplotypes a and b of nad <dig> intron  <dig> in central and northeastern europe.
their diversity index was highest among all populations surveyed, which could result for the meeting of two genetically distinct glacial lineages , or indicates that the glacial population harboring haplotype b had mixed genetic composition.
the uneven distribution of haplotype b of nad <dig> intron  <dig> among these populations, with a higher prevalence of haplotype b as one moves northward, could result from successive founder events during colonization  <cit> , as recently proposed for european hedgehogs  <cit> .
this is likely, given than that migration from more southern latitudes was necessary to colonize these previously glaciated areas.
implications for glacial and postglacial history of european vegetation
there is some evidence from fossil and palynological data that cold adapted tree species occupied mid-altitude sites in the mountains of southern europe during the last glacial stage  <cit> .
moreover, the southern peninsulas of iberia, italy, the balkans, and greece, which are separated from the rest of europe by major mountain ranges such as the pyrenees, alps and caucasus, were identified as major glacial refugia for many species .
our genetic data indicates that a genetically distinct glacial refuge existed in asia minor as well.
tree pollen, including that of the genus pinus, was recorded in the balkans about  <dig>  <dig> years bp  <cit>  while it was barely present as late as  <dig>  <dig> years bp in southwestern turkey where the climate was cold and dry with steppe vegetation  <cit> .
pine pollen attained its maximum only in early holocene in western anatolia and in mid-holocene in more eastern areas, likely because of similar climatic limitations  <cit> .
thus, it is apparent that tree populations would have been scarce in the region at lgm,  <dig> kyr ago.
the refuge is also unlikely to have contributed much to the postglacial colonization of europe, given the genetic discontinuity detected in scots pine.
the peculiar location of turkey between the black and mediterranean seas would have promoted isolation from more northern areas.
it is likely that other elements of the steppe vegetation in the region also represent relicts from the pre-holocene era and suffered similar effects during glacial periods, as shown recently for field mouse  <cit> .
as such, it is expected that they would show a distinct genetic background, as that detected in scots pine.
the exact location and size of the scots pine glacial population giving rise to the large spread of haplotype b of nad <dig> intron  <dig> in eastern or northeastern europe remain unknown.
based on fossil data, scots pine glacial populations existed in the balkans and possibly even in central europe .
the large spread of haplotype a of nad <dig> intron  <dig> throughout europe is likely the consequence of an expansion from glacial populations from that region  <cit> .
such spread northwestwards towards germany and france and northeastwards throughout eastern europe and siberia correlates geographic dispersion patterns and colonization routes inferred for a grasshopper  <cit> , for european beech  <cit> , for the oaks  <cit> , and for norway spruce  <cit> .
however, some of these colonization routes are still open to alternative interpretation.
furthermore, such observations do not correlate well with the exceptionally northern location of haplotype b of nad <dig> intron  <dig>
the peculiar northern distribution of haplotype b of nad <dig> intron  <dig> indicates that scots pine has likely survived in a refuge distinct from those usually recognized in the southern peninsulas of europe, perhaps under the form of scattered glacial populations spread over sizeable latitudinal areas reaching european mid-latitudes not far from the ice front.
scots pine is known to survive and grow reasonably well upon permafrost  <cit> .
evidence is also accumulating regarding the existence of glacial populations located at more northern latitudes  and their significant contribution to holocene colonization  <cit> .
in addition, if the mixed zone observed in north-central and northeastern europe between the cosmopolitan aa mitotype and the northern ba mitotype represents truly a zone of secondary contact, the northern location and shape of this zone would not correspond well to the meeting of two ancestral lineages both located in or near southern european peninsulas.
while the existence of such zones with typically higher genetic diversity has been reported for other tree species in europe  as well as in north america  <cit>  where ancestral lineages from southern latitudes would converge at more northern latitudes, this possible zone of contact would more likely correspond to the junction between a lineage moving northwards from the balkans or from even more east , and a lineage of more northern or northeastern origin .
in one rare occurrence of similar observation, taberlet et al  <cit>  recognized a contact zone of migration fronts as far north as in scandinavia for picea abies.
they proposed that the area was presumably colonized from the south and from the northeast by populations originating from different refugia.
while the current areas of maximum abundance of haplotype b of nad <dig> intron <dig>  finland and northwestern russia, were completely glaciated at lgm  <cit> , it is likely that trees colonizing the region would have migrated northward from a region anywhere west of the ural mountains.
there are indications from general circulation models that the climate during the last glacial maximum was warm enough to allow scots pine to survive well as far north as above 50°n in isolated regions of northern ukraine and southwestern russia, for instance  <cit> .
a location more south is also possible if founder events after long-distance dispersal are hypothetized, followed by drift and lineage sorting to account for the high abundance of haplotype b of nad <dig> intron  <dig> as one moves into finland and northwestern russia.
however, this hypothesis becomes unlikely for long-distance dispersal from refuges located in the southern european peninsulas, given the absence of haplotype b of nad <dig> intron  <dig> in southern populations of scots pine.
more mtdna polymorphisms will need to be discovered in order to distinguish scots pine disjunct populations from eastern europe and asia and investigate if significant glacial vicariance factors existed in this vast region.
siberian populations where the cosmopolitan mitotype aa was dominant might have been established from a glacial population located in southwestern siberia, in the upper irtysh river valley  <cit> , or from a more southern location.
as for scots pine populations located in the far-east in china where the cosmopolitan mitotype aa was found fixed, it is not clear if it is derived from a more western refuge located in southwestern siberia  or if a refuge existed in northeastern china.
palynological evidence suggests that pine was present at several locations in the region during the last glaciation, but it is unclear if the fossil pollen was representative of scots pine as many pine species are endemic to the region  <cit> .
CONCLUSIONS
the present study suggests a more complex biogeographical history than previously thought for scots pine and presumably, for its associated plant congeners.
the study is congruent with others  about the asymetry of glacial refugia in contributing to the postglacial recolonization of eurasia.
this trend is best exemplified by the prevalent nad <dig> intron  <dig> haplotype found specific to the relictual population from turkey and from nowhere else in eurasia.
it indicates that asia minor is likely to represent a prime area of endemism supporting genetically distinct lineages unlikely to be found in the rest of eurasia.
such a trend highlights the geography of the region as an important vicariance factor limiting migration and gene flow.
similarly as for other areas of endemism in europe , seed source movements from the rest of eurasia to asia minor should be restricted.
reserves could be considered to ensure in situ preservation of relictual genetic diversity and structure, which need to be further documented at the level of the chloroplast and nuclear genomes as well as for other plant and animal species.
the discovery of a genetically distinct north-european lineage suggests that some glacial populations may have survived isolated from refugia in south european peninsulas at more mid- northern latitudes.
given the colonization time lag for southern populations to reach northern latitudes due to distance, topographic barriers  <cit>  and competition from a possible network of resident populations sparingly distributed, such genetically distinct glacial populations have apparently played a major role in the postglacial colonization of parts of northern europe.
a likely consequence of such glacial vicariance is that extant northern populations of nordic plant species might not be as homogenous as assumed by simply considering them as offsets of glacial populations located at southern latitudes.
this observation implies that they might have evolved distinctive genetic adaptations during glacial vicariance, worth evaluating and considering in conservation programs  <cit> .
as well, the extensive mixing of two ancestral lineages in central and northeastern europe provides the opportunity to perhaps identify and preserve unique adaptive diversity.

 the cold periods of the pleistocene have had a dramatic impact on most organisms in temperate regions. whereas the artic ice sheet began to grow about myr ago, more severe climatic oscillations occurred mainly during the last year with the consequence that the european biota has experienced repeatedly some drastic climate changes. tree species responded through migration to regions where environmental conditions allowed them to survive. phylogeography investigates on the spatio-temporal dynamics of populations. it relies on inference from two main sources of information, i) macrofossils and pollen in sediment profiles, ii) population structure in dna markers ideally inferred at the sequence level. cytoplasmic markers, which are uniparently inherited in most plant species, are especially well suited for phylogeographic studies. they helped infer the number of genetically distinct ancestral lineages, their location during the last glacial maximum, and the postglacial migration routes for several tree and plant species in europe. these studies and others based on fossil evidence suggested that most plant and tree refuges were located in southern european peninsulas and the balkans. glacial refugia and recolonization routes were found to coincide often for a number of plant and animal species. in north america, common patterns are also starting to emerge and much knowledge has been rapidly gained from the analysis of species with wide distributions. however, there is now increasing evidence that some european species were established much further north and east than previously assumed during glacial time. similar inferences have also been made in north america, where genetic and palynological evidence is accumulating for the presence of glacial populations much closer to the ice fronts as once thought. phylogeographic studies in europe also indicate asymmetric contributions of glacial refugia to holocene colonization: some ancestral lineages remained endemic, because of geographic barriers limiting migration, while others contributed considerably to the postglacial colonization of more northern latitudes. scots pine is a long-lived evergreen monoecious forest tree species growing in a large variety of ecological conditions from western europe to asia. of all pine species, it has the largest distribution. its natural range extends northwards from spain to northern fennoscandia, and westwards from longitudes 5°w in spain and scotland to 135°e in siberia. while most of its natural range is contiguous, there are also large populations of scots pine disconnected from the main natural range such as in scotland, spain, france, turkey, and former yugoslavia. extensive variations in phenotypic traits as well as geographic races and types have been reported. much of this variation has been usually attributed to adaptation to edaphic and climatic factors. studies carried out over the last years using isozymes, terpenes and flavonoid markers have also revealed the existence of distinct evolutionary units in scots pine, which might be indicative of isolation in different glacial refugia. although these studies were not entirely congruent and not aimed at deciphering effects related to glacial and postglacial history, they helped define biogeographical hypotheses for the western and northwestern european range of scots pine. because of the regional scope of many of these studies and the different phenotypic or genetic attributes used, much biogeographical knowledge on the glacial and postglacial history of eurasia remains to be gained from analysing the broad geographic distribution of scots pine and by using homologous gene markers with stronger historical imprints. in the pinaceae, mtdna is maternally inherited and dispersed through seeds only. this mode of dispersion helps avoid the problems with inferring history from present-day distributions of nuclear or cpdna markers because the latter are dispersed by both seeds and pollen in the pinaceae, which produce a more rapid breakdown of the historical genetic signatures induced by glacial vicariance. however, one of the major difficulties one faces with plant mtdna is the low level of variation in their exons and introns, which limits the detection of variation between and within species at the sequence level. an indel has been discovered in the intron b/c of the mitochondrial gene nad in scots pine, which allowed identifying a genetically distinct ancestral lineage in the iberian peninsula. restriction fragment length polymorphisms of a region encompassing the mitochondrial gene cox have also been found in populations of scots pine from western europe, which helped identifying two genetically distinct groups of populations each distributed on a large latitudinal range. the possibility for a refuge at northern latitudes was suggested in a previous study. given the little diversity observed in the mtdna regions surveyed and the population sampling limited mostly to western europe, it is likely that the pan-eurasian scots pine possesses more genetically distinct ancestral lineages. such evidence would contribute towards a more complete understanding of the historical factors and processes leading to the current distribution of species genetic diversity throughout eurasia. in an effort to discern from a phylogeographic perspective the possible existence and contribution of mid-northern refugia to the glacial and postglacial history of eurasian biota, we investigated the extant genetic structure of scots pine with a set of populations covering much of the natural range in europe and also in asia. we also report on a novel polymorphic mtdna region in scots pine, the intron of the gene nad which is indicative of unknown genetically distinct maternal lineages. the location of one of these lineages in northern europe supports the view that quaternary refugia located outside european peninsulas at mid-northern latitudes might have played a significant role in the holocene recolonization of europe.  mtdna polymorphisms and population differentiation out of mtdna regions surveyed, intraspecific polymorphism was detected for only two loci in scots pine. polymorphism was detected de novo for the intron of nad with three length variants. they were and bp long and are hereafter called haplotypes a, b, and c, respectively. the size differences were caused by two single indels: one of bp exclusive to haplotype c and one of bp exclusive to haplotype b. as previously reported from a more limited sampling, two size variants were detected for the intron b/c of nad among the samples analysed herein. these fragments differed by a bp indel and are hereafter called haplotypes a and b. a third variant restricted to italy was recently reported for this gene. it was not detected in our sampled trees which were all from outside the italian peninsula. primers for these two mtdna regions were also tested on to individuals for each of five other pines of the subgenus diploxylon and positive amplifications were obtained. the first four species belong to the section pinus, but the first three are members of subsection pinus and pinus heldreichii is member of subsection pinaster. as for p. contorta, it belongs to section trifoliae, subsection contortae. fragment length polymorphism was detected for both nad intron b/c and nad intron in p. heldreichii. for p. nigra, only nad intron b/c showed polymorphism. no polymorphism was detected in p. mugo and p. resinosa. for p. contorta, polymorphism was only observed for nad intron involving the same minisatellite-like marker as previously reported for this species and the closely related p. banksiana. 1targeted regions, annealing temperatures, expected size of pcr products and restriction enzymes tested for primer pairs used to amplify mtdna regions of scots pine. 2nad intronb/c have been amplified and tested with different restriction enzymes, only internal primers h and i of soranzo et al have shown polymorphism. 3intergenic region. 4multiple fragments. 1polymorphisms detected in the intron of the mitochondrial gene nad and in the intron b/c of the mitochondrial gene nad in scots pine, and nomenclature used for multi-locus haplotypes. for scots pine, the nomenclature used for mitochondrial multi-locus haplotypes followed a two-letter code after the haplotypes observed for nad intron and nad intron b/c, respectively. among the four observed mitotypes, the most abundant one was aa, followed by ba, ca, and ab. the minimum spanning tree indicates that the mitotypes were more or less arranged as a star phylogeny with little sequence variation among them and no recombination. the observed number of mitotypes per population was or without exception, with an average of. the mitotype diversity index per population was low to moderate and ranged from to. population differentiation was high among the populations, with gst and nst values of and, respectively. when excluding the six more diverse populations from a presumed zone of contact in central and northeastern europe between two distinct mtdna lineages, the gst and nst values increased to and, respectively, among the remaining populations. however, the presence of a formal phylogeographic structure was not supported, as nst was not significantly higher than gst. lack of test power is likely the reason, given the small genetic divergence observed among the four mtdna variants detected. 1at, austria; bg, bulgaria; by, belarus; cn, china; cz, czech republic; es, spain; fi, finland; fr, france; lt, lithuania; lv, latvia; ro, romania; ru, russian federation; se, sweden; tr, turkey; ua, ukraine; uk, united kingdom. 2the "w" indicates the longitude west of greenwich meridian. 3the first letter of multi-locus haplotype refers to the haplotype observed at locus nad intron and the second letter refers to the haplotype observed at locus nad intron b/c following nomenclature in table 4number of distinct mitotypes per population. 5mitotype diversity. 1estimates for groups of populations of scots pine delineated by samova and maximizing geographic structure. 2sample size. 3number of distinct multi-locus haplotypes observed in each group. 4average number of mitotypes per population in each group. 5average mitotype diversity per population in each group. 6differentiation among populations in each group. 7fixation index using permut software. 8differentiation among the four groups. geographic structure the geographic distribution of mitotypes appeared to be essentially non-random, with clusters of mitotypes geographically quite well delineated. the mediterranean region showed the highest diversity with three of the four mitotypes revealed in the study. the cosmopolitan haplotype aa was largely distributed and found in most of the populations sampled. geographic structuring was evident for other mitotypes. mitotype ca was present only in asia minor, in the pontide mountains of turkey. mitotype ab was found fixed in some high mountain populations of the iberian peninsula and was observed as a rare variant in some populations of the balkans. the distribution of mitotype ba was quite unexpected: it was largely distributed in the lowland region of middle to northern latitudes in eastern europe. it was prevalent in the baltic region and quite frequent in russia, west of the ural mountains. but it was also detected in siberia. a large zone composed of populations with variable mixed genetic background was observed in central and northeastern europe. it includes most if not all populations bearing mitotype ba. the zone extends from the southeast of russia to the baltic states. the within-population mitotype diversity of these populations was much higher than elsewhere. samova was conducted repeatedly by increasing the value of the number of population groups until the fct value reached a maximum of, which was obtained for k =. for k = a maximum fct value of was obtained. the four groups confirmed trends from visual inspection and corresponded to: the majority of populations from europe and asia, northeastern europe, asia minor and the high mountains of the iberian peninsula. group i was the most widespread geographically, including populations. it contained mostly the cosmopolitan mitotype aa and, at a lower frequency, mitotype ba and ab. differentiation among the populations of the group was moderate. population differentiation within each of the three other groups was smaller. group ii from northeastern europe was largely dominated by mitotype ba. the two remaining groups iii and iv were highly homogenous. group iii regrouped populations from turkey fixed or nearly fixed for mitotype ca, and group iv regrouped populations from the iberian peninsula fixed for mitotype ab. discussion mtdna diversity and population differentiation dna sequence variation is low in the mtdna of conifers. accordingly, little intraspecific variation has been found in the mtdna of p. sylvestris in western europe, including four rflp variants for the cox region and three size variants at the locus nad intron b/c, of which two were found endemic to the iberian and italian peninsulas. the three new size variants reported herein for the locus nad intron indicate new region-specific variants for northeastern europe and asia minor. no other length polymorphisms or substitutions were detected in scots pine in spite of the screening of distinct mtdna regions. highly informative minisatellite-like motifs such as those rarely detected in some spruce and pine taxa could not be detected in scots pine. the overall level of population differentiation estimated in this study from mitotype variation at the nad intron b/c and nad intron loci was rather high and similar to that reported in earlier studies restricted to scots pine populations from western europe based on rflps of the cox region or indels of the nad intron b/c. such estimates are in line with averages reported for maternally inherited markers for a number of conifer and angiosperm taxa. the amount of population differentiation determined by nuclear markers such as isozymes was generally much smaller in scots pine with gst values ranging between and ]. however, these studies were carried out mostly at the regional level. among-population differentiation was also much higher for mtdna markers than for paternally inherited cpdna markers in a scots pine study covering much of the species range, as usually observed in other conifers. geographic structure the present study confirms the existence of a genetically distinct set of populations in the high mountains of the iberian peninsula while variants discovered in the intron of nad signal distinctive evolution in asia minor and northeastern europe. haplotype b at locus nad intron b/c was observed mostly in the iberian peninsula in some high elevation populations of the zaragoza/valencia regions where it was fixed or nearly fixed. it was observed only sparingly west of catalonia. such a distribution indicates that these catalan populations have been isolated during glaciations and have not contributed to the recolonization of europe. a distinct dynamics of catalan populations from the rest of the iberian peninsula has also been reported for other species like aleppo pine, maritime pine, and white oaks. in the present study, haplotype b of nad intron b/c has also been discovered, albeit at low frequency, in five other populations located in bulgaria, in the balkan peninsula, thus far away from the center of prevalence for this haplotype in the iberian peninsula. long-distance seed dispersal could be invoked but is unlikely over such long distances, given the heterogenous topography and relative isolation of iberia. the probability of such long-distance movements would be higher in the northern and more eastern parts of scots pine natural range, given the lack of major physical barriers. another perhaps more plausible explanation for the rare presence of haplotype b of nad intron b/c in some balkan populations could be that it is ancient and was present in more than one southern ice refugia. its high or low frequency, depending on the region considered, could be attributable to stochastic variation, especially if southern refugia populations suffered from severe bottlenecks. the weak occurrence of this haplotype in the balkans might also result from human activity, as previously suggested for the rare presence of this variant in poland. whereas the hypothesis of seed source transfer cannot be totally ruled out, it is difficult to sustain because of the lack of hard evidence for such seed source transfer between the balkans and the iberian peninsula in the past; natural stands in which seeds were collected existed long before forest management activities began in europe; and the relative conservatism of forest seed transfer guidelines in the balkan region. a third explanation could relate to ancient mtdna capture from hybridizing species in the region. accordingly, phylogeographical studies of other pine species in the region using the same gene locus might help clarify the issue. the detection and high prevalence of haplotype c of nad intron in asia minor populations only is a strong signal for the presence of a genetically distinct glacial population. this relictual population would have experienced severe bottlenecks, because populations were all fixed for this haplotype except for one tree harbouring the cosmopolitan haplotype a. after glaciation, it would have remained isolated, eastward by the caucasus mountains, northward by the black sea, and westward by the bosphorus passage. the cold and arid climate in this region during the holocene might have also impeded the expansion of this population, but not its survival as scots pine can survive under very harsh conditions. the high prevalence of haplotype b of nad intron in northeastern europe suggests the presence of a genetically distinct glacial population at more northern latitudes than previously thought, an idea that had already been suggested for scots pine. a recent mutation during the holocene giving rise to haplotype b of nad intron is unlikely, given the widespread distribution of this haplotype. as well, homoplasy is unlikely, given the low rate of variation of angiosperm and conifer mitochondrial introns. a large number of populations had mixed composition of both haplotypes a and b of nad intron in central and northeastern europe. their diversity index was highest among all populations surveyed, which could result for the meeting of two genetically distinct glacial lineages, or indicates that the glacial population harboring haplotype b had mixed genetic composition. the uneven distribution of haplotype b of nad intron among these populations, with a higher prevalence of haplotype b as one moves northward, could result from successive founder events during colonization, as recently proposed for european hedgehogs. this is likely, given than that migration from more southern latitudes was necessary to colonize these previously glaciated areas. implications for glacial and postglacial history of european vegetation there is some evidence from fossil and palynological data that cold adapted tree species occupied mid-altitude sites in the mountains of southern europe during the last glacial stage. moreover, the southern peninsulas of iberia, italy, the balkans, and greece, which are separated from the rest of europe by major mountain ranges such as the pyrenees, alps and caucasus, were identified as major glacial refugia for many species. our genetic data indicates that a genetically distinct glacial refuge existed in asia minor as well. tree pollen, including that of the genus pinus, was recorded in the balkans about years bp while it was barely present as late as years bp in southwestern turkey where the climate was cold and dry with steppe vegetation. pine pollen attained its maximum only in early holocene in western anatolia and in mid-holocene in more eastern areas, likely because of similar climatic limitations. thus, it is apparent that tree populations would have been scarce in the region at lgm, kyr ago. the refuge is also unlikely to have contributed much to the postglacial colonization of europe, given the genetic discontinuity detected in scots pine. the peculiar location of turkey between the black and mediterranean seas would have promoted isolation from more northern areas. it is likely that other elements of the steppe vegetation in the region also represent relicts from the pre-holocene era and suffered similar effects during glacial periods, as shown recently for field mouse. as such, it is expected that they would show a distinct genetic background, as that detected in scots pine. the exact location and size of the scots pine glacial population giving rise to the large spread of haplotype b of nad intron in eastern or northeastern europe remain unknown. based on fossil data, scots pine glacial populations existed in the balkans and possibly even in central europe. the large spread of haplotype a of nad intron throughout europe is likely the consequence of an expansion from glacial populations from that region. such spread northwestwards towards germany and france and northeastwards throughout eastern europe and siberia correlates geographic dispersion patterns and colonization routes inferred for a grasshopper, for european beech, for the oaks, and for norway spruce. however, some of these colonization routes are still open to alternative interpretation. furthermore, such observations do not correlate well with the exceptionally northern location of haplotype b of nad intron the peculiar northern distribution of haplotype b of nad intron indicates that scots pine has likely survived in a refuge distinct from those usually recognized in the southern peninsulas of europe, perhaps under the form of scattered glacial populations spread over sizeable latitudinal areas reaching european mid-latitudes not far from the ice front. scots pine is known to survive and grow reasonably well upon permafrost. evidence is also accumulating regarding the existence of glacial populations located at more northern latitudes and their significant contribution to holocene colonization. in addition, if the mixed zone observed in north-central and northeastern europe between the cosmopolitan aa mitotype and the northern ba mitotype represents truly a zone of secondary contact, the northern location and shape of this zone would not correspond well to the meeting of two ancestral lineages both located in or near southern european peninsulas. while the existence of such zones with typically higher genetic diversity has been reported for other tree species in europe as well as in north america where ancestral lineages from southern latitudes would converge at more northern latitudes, this possible zone of contact would more likely correspond to the junction between a lineage moving northwards from the balkans or from even more east, and a lineage of more northern or northeastern origin. in one rare occurrence of similar observation, taberlet et al recognized a contact zone of migration fronts as far north as in scandinavia for picea abies. they proposed that the area was presumably colonized from the south and from the northeast by populations originating from different refugia. while the current areas of maximum abundance of haplotype b of nad intron finland and northwestern russia, were completely glaciated at lgm, it is likely that trees colonizing the region would have migrated northward from a region anywhere west of the ural mountains. there are indications from general circulation models that the climate during the last glacial maximum was warm enough to allow scots pine to survive well as far north as above 50°n in isolated regions of northern ukraine and southwestern russia, for instance. a location more south is also possible if founder events after long-distance dispersal are hypothetized, followed by drift and lineage sorting to account for the high abundance of haplotype b of nad intron as one moves into finland and northwestern russia. however, this hypothesis becomes unlikely for long-distance dispersal from refuges located in the southern european peninsulas, given the absence of haplotype b of nad intron in southern populations of scots pine. more mtdna polymorphisms will need to be discovered in order to distinguish scots pine disjunct populations from eastern europe and asia and investigate if significant glacial vicariance factors existed in this vast region. siberian populations where the cosmopolitan mitotype aa was dominant might have been established from a glacial population located in southwestern siberia, in the upper irtysh river valley, or from a more southern location. as for scots pine populations located in the far-east in china where the cosmopolitan mitotype aa was found fixed, it is not clear if it is derived from a more western refuge located in southwestern siberia or if a refuge existed in northeastern china. palynological evidence suggests that pine was present at several locations in the region during the last glaciation, but it is unclear if the fossil pollen was representative of scots pine as many pine species are endemic to the region.  the present study suggests a more complex biogeographical history than previously thought for scots pine and presumably, for its associated plant congeners. the study is congruent with others about the asymetry of glacial refugia in contributing to the postglacial recolonization of eurasia. this trend is best exemplified by the prevalent nad intron haplotype found specific to the relictual population from turkey and from nowhere else in eurasia. it indicates that asia minor is likely to represent a prime area of endemism supporting genetically distinct lineages unlikely to be found in the rest of eurasia. such a trend highlights the geography of the region as an important vicariance factor limiting migration and gene flow. similarly as for other areas of endemism in europe, seed source movements from the rest of eurasia to asia minor should be restricted. reserves could be considered to ensure in situ preservation of relictual genetic diversity and structure, which need to be further documented at the level of the chloroplast and nuclear genomes as well as for other plant and animal species. the discovery of a genetically distinct north-european lineage suggests that some glacial populations may have survived isolated from refugia in south european peninsulas at more mid- northern latitudes. given the colonization time lag for southern populations to reach northern latitudes due to distance, topographic barriers and competition from a possible network of resident populations sparingly distributed, such genetically distinct glacial populations have apparently played a major role in the postglacial colonization of parts of northern europe. a likely consequence of such glacial vicariance is that extant northern populations of nordic plant species might not be as homogenous as assumed by simply considering them as offsets of glacial populations located at southern latitudes. this observation implies that they might have evolved distinctive genetic adaptations during glacial vicariance, worth evaluating and considering in conservation programs. as well, the extensive mixing of two ancestral lineages in central and northeastern europe provides the opportunity to perhaps identify and preserve unique adaptive diversity. 
fractures in its extant genetic structure might be indicative of glacial vicariance and how different refugia contributed to the current distribution at the continental level. the contribution of the various ancestral lineages to the current distribution of scots pine was asymmetric and extant endemism reflected the presence of large geographic barriers to migration. the results suggest a complex biogeographical history with glacial refugia shared with temperate plant species in southern european peninsulas and asia minor, and a genetically distinct glacial population located more north. these results confirm recent observations for cold tolerant species about the possible existence of refugial populations at mid-northern latitudes contributing significantly to the recolonization of northern europe. thus, eurasian populations of nordic plant species might not be as genetically homogenous as assumed by simply considering them as offsets of glacial populations located in southern peninsulas. more or less disjunct refugial populations of plants were dispersed in southern europe, often trapped between mountain ranges and seas. as such, they might have evolved distinctive genetic adaptations during glacial vicariance, worth evaluating and considering for conservation. population differentiation was high and the distribution of the mitotypes was geographically highly structured, suggesting at least four genetically distinct ancestral lineages. scots pine is a nordic conifer with a wide natural range covering much of eurasia. the population structure of scots pine was investigated on much of its eurasian natural range using maternally inherited mitochondrial dna polymorphisms. a new lineage was restricted to more northern latitudes in northeastern europe and the baltic region. from trees distributed among populations, four distinct multi-locus mitochondrial haplotypes were detected based on the three nad intron haplotypes and two previously reported size variants for nad intron b/c. a previously reported lineage limited to the iberian peninsula was confirmed. a novel polymorphic region of the scots pine mitochondrial genome has been identified, the intron of nad with three variants caused by insertions-deletions. genetic and paleobotanical evidences indicate that these populations have contributed much to holocene recolonization of more northern latitudes. less supportive evidence has been found for the existence of glacial populations located closer to the ice margin.
 at the last glacial maximum, fennoscandia was covered by an ice sheet while the tundra occupied most of the rest of northern eurasia. more or less disjunct refugial populations of plants were dispersed in southern europe, often trapped between mountain ranges and seas. genetic and paleobotanical evidences indicate that these populations have contributed much to holocene recolonization of more northern latitudes. less supportive evidence has been found for the existence of glacial populations located closer to the ice margin. scots pine is a nordic conifer with a wide natural range covering much of eurasia. fractures in its extant genetic structure might be indicative of glacial vicariance and how different refugia contributed to the current distribution at the continental level. the population structure of scots pine was investigated on much of its eurasian natural range using maternally inherited mitochondrial dna polymorphisms.  a novel polymorphic region of the scots pine mitochondrial genome has been identified, the intron of nad with three variants caused by insertions-deletions. from trees distributed among populations, four distinct multi-locus mitochondrial haplotypes were detected based on the three nad intron haplotypes and two previously reported size variants for nad intron b/c. population differentiation was high and the distribution of the mitotypes was geographically highly structured, suggesting at least four genetically distinct ancestral lineages. a cosmopolitan lineage was widely distributed in much of europe throughout eastern asia. a previously reported lineage limited to the iberian peninsula was confirmed. a new geographically restricted lineage was found confined to asia minor. a new lineage was restricted to more northern latitudes in northeastern europe and the baltic region.  the contribution of the various ancestral lineages to the current distribution of scots pine was asymmetric and extant endemism reflected the presence of large geographic barriers to migration. the results suggest a complex biogeographical history with glacial refugia shared with temperate plant species in southern european peninsulas and asia minor, and a genetically distinct glacial population located more north. these results confirm recent observations for cold tolerant species about the possible existence of refugial populations at mid-northern latitudes contributing significantly to the recolonization of northern europe. thus, eurasian populations of nordic plant species might not be as genetically homogenous as assumed by simply considering them as offsets of glacial populations located in southern peninsulas. as such, they might have evolved distinctive genetic adaptations during glacial vicariance, worth evaluating and considering for conservation. 