BACKGROUND
arabidopsis thaliana  heynh is an annual weed belonging to the family brassicaceae .
the species is native to europe and central asia, but is now widely distributed in the northern hemisphere ranging from 68°n  to equator   <cit> .
many characteristics, from morphological traits to protein and dna markers, have been used to evaluate natural genetic variation among populations, and to reconstruct an intraspecific phylogeny, for a. thaliana .
it has been found that many nuclear genes comprise two or more major differentiated haplotypes, generally referred to as allelic dimorphism  <cit> .
balancing selection or ancient population subdivision was often invoked to explain the pattern.
the major mechanisms for balancing selection are heterozygote advantage, frequency-dependent selection, or environmental heterogeneity.
it is well known that a. thaliana has an inbreeding mating system.
the estimated outcrossing rate of the species is 1% or less  <cit> .
it seems difficult to imagine that so many loci in a. thaliana have experienced balancing selection via heterozygote advantage  <cit> .
therefore, frequency-dependent selection and/or diversifying selection might be the driving forces for the dimorphism phenomenon, as in the case of pathogen resistance  genes  <cit> .
it is not clear yet if the dimorphism also exists in the chloroplast genome.
the chloroplast genome of a. thaliana is a circular dna composed of  <dig>  bp with a pair of inverted repeats of  <dig>  bp separated by small and large single-copy regions of  <dig>  bp and  <dig>  bp, respectively  <cit> .
the uniparentally inherited chloroplast genome has been utilized in many studies in plant population and evolutionary genetics.
however, studies focused on global patterns of cpdna genetic variation among accessions of a. thaliana are scattered.
in an investigation on the maternal origins of a. suecica,  <dig> cpdna regions were sequenced for  <dig> a. thaliana accessions, which were mainly collected from scandinavia  <cit> .
these authors found considerable variation existed among the non-coding single-copy sequences in the chloroplast genome of a. thaliana.
in another study, the trnl-trnf cpdna intergenic spacer region of  <dig> individuals from  <dig> a. thaliana populations in its native range was sequenced and  <dig> haplotypes were identified  <cit> .
based on the chloroplast and nuclear dna sequence data, beck et al proposed the caucasian area as the possible ancestral area of a. thaliana, and suggested four possibilities for the origin of east asian populations.
they also found that the maternal components of a. suecica shared a high similarity to those in the asian metapopulation of a. thaliana, especially to those from china  <cit> .
in the studies incorporating worldwide sampling of a. thaliana populations, the samples from east asia were very scattered.
he et al conducted a study on the genetic diversity of  <dig> natural arabidopsis thaliana populations in china based on issr and rapd makers, and found that about 42-45% of the total genetic variation existed within populations and there was a significant correlation between geographic distance and genetic distance  <cit> .
however, the phylogenetic relationships of chinese populations with those distributed in other regions of the world, and the history of population dispersal in this region, are not clear.
the goals of the present survey are:  to examine global patterns of cpdna genetic variation in a. thaliana;  to infer phylogenetic relationships among a. thaliana accessions from all over the world based on cpdna sequence data, with particular focus on chinese populations; and  to discuss the possible origin of the chinese populations.
it was found in this study that dimorphism did exist in the chloroplast genome of a. thaliana; the  <dig> accessions studied were grouped into two major clusters; and the chinese populations might have two independent origins.
RESULTS
nucleotide variation in the chloroplast dna sequences
seventy-seven a. thaliana accessions were used in the survey, among them  <dig> accessions were field collected in china .
all sampling locations in china were separated by at least  <dig> km, with most of the locations separated by more than  <dig> km .
no cp dna polymorphism was detected within either accession ahyxx or abd- <dig>  therefore only one individual was chosen for each accession for dna sequence analysis.
about  <dig> nucleotides from the chloroplast genome were amplified and sequenced for each accession, of which  <dig> nucleotides of non-coding fragments were retained for analysis.
*accession no.
begun with 'n' were obtained from the nottingham arabidopsis stock center ; with 'cs' were obtained from the arabidopsis biological resource center ; with "pku" were field collected in china and their detailed information is listed in table  <dig>
the combined data matrix contained  <dig> variable nucleotide sites.
among them,  <dig> were mononucleotide repeat polymorphisms, one was a dinucleotide repeat polymorphism, and four were complicated length variations.
these  <dig> length polymorphisms were excluded in the analyses.
the other  <dig> polymorphic variations comprised  <dig> single nucleotide polymorphisms ,  <dig> insertion/deletion events  and two small fragment inversions.
only one site exhibited three-base polymorphism and the other  <dig> sites showed two-base polymorphism .
the two small fragmental inversions were located at sites 3633- <dig>  and sites 6501- <dig> .
the characteristics of this kind of reversion were:  a central region of  <dig> nt  or  <dig> nt , which could mutate to its reverse complement sequence ; and  two flanking sequences of  <dig> nt  or  <dig> nt , respectively .
the two flanking sequences could be reversely complemented to each other.
it is most likely that each of these two small fragmental inversions could be generated by only one or very few mutation event, but it resulted in multiple snps.
nucleotide diversity  for the entire sequenced regions was  <dig> , but ranged from  <dig>  for the ycf3-trns intergenic spacer  to  <dig>  for psaj-rpl <dig>  .
ns = not significant and p >  <dig>
nsa = not significant but  <dig>  > p >  <dig>
* p <  <dig>
** p <  <dig>
less frequent nucleotide polymorphisms  were in excess for the sequenced regions.
singletons were found at a very high frequency:  <dig> among the  <dig> snps and  <dig> among the  <dig> indels were singletons .
the excess of low-frequency polymorphisms resulted in negative tajima's d, fu and li's d* and f* values for most of the sequenced segments; for example,  <dig> out of  <dig> tajima's d values, nine out of  <dig> fu and li's d* values and  <dig> out of  <dig> fu and li's f* values were negative .
the values for the combined data matrix were - <dig>  , - <dig>   and - <dig>  .
when fu and li's d and f tests were conducted using the a. arenosa ortholog as the reference sequence, similar results were obtained: eight out of  <dig> fu and li's d values and nine out of  <dig> fu and li's f values were negative and for the combined data matrix; both the values were negative .
in total, we identified three types of nucleotide variations among the aligned sequences: snps, length polymorphisms , and two small fragmental inversions.
phylogenetic relationships among the accessions
because the single base changes in the two short inverted regions were not independent events, they were excluded from the phylogenetic analysis.
two distinct clusters with high bootstrap values were retrieved in the nj tree.
one cluster included  <dig> accessions and the other included  <dig> accessions .
although the topology of the mp tree differed to that of the nj tree, one branch with  <dig> accessions corresponded to one of the two clusters in the nj tree .
in general, no significant correlation was detected between geographical origins and clusterings in the phylogenetic trees.
accessions from the same country, such as four accessions from italy  and five accessions from usa  failed to cluster together, but were scattered on different branches.
this lack of phylogeographic structure conforms to the hypothesis of a rapid recent expansion of the species with strong involvement of human-mediated migrations  <cit> .
although there was incongruence between the two phylogenies, the topological relationship was relatively stable among a large number of accessions.
we identified four stable branches in both trees .
the only major difference was in branch c. it was placed within one of the two clusters in the nj tree, but formed a deep polytonous branch in the mp tree, containing the same accessions except cvi- <dig>  an accession from cape verde island.
the branches a-e comprised  <dig> out of the  <dig> accessions.
discrimination of two major differentiated haplotypes among a. thaliana accessions
when only the parsimony-informative sites were considered, the nucleotide variation of the  <dig> accessions was structured into two major different haplotype classes .
the mdhc-i and mdhc-ii classes were composed of  <dig> and  <dig> accessions, respectively, and they corresponded well to the two clusters in the nj tree.
the mdhc-i and mdhc-ii classes differed at five nucleotide sites , and these sites were within a fragment about  <dig> kb long from trnl to rpl <dig> in the chloroplast genome.
for interspecific comparison, the homologous sequences of three related species, olimarabidopsis cabulica, a. arenosa and a. suecica, were aligned with the  <dig> a. thaliana accessions.
they were identical to those in mdhc-ii of a. thaliana at all five nucleotide sites where the two mdhcs could be distinguished from each other .
all the sites except the inverted length variants were used to form a binary data set for haplotype network analysis.
forty-nine haplotypes were identified in the  <dig> accessions of a. thaliana.
the  <dig> haplotypes were also bifurcated to form two haplogroups .
haplogroup  <dig>  and haplogroup  <dig>  differed at the same five sites  where mdhc-i and -ii differed, and the accessions in haplogroup  <dig> and haplogroup  <dig> were identical to those in mdhc-i and -ii, respectively.
estimated divergence time for mdhc-i and mdhc-ii, and demographic expansion of a monophyletic group of accessions in asia
the k value between a. arenosa and  <dig> a. thaliana accessions was  <dig>  ±  <dig> .
using equation  <dig>  the substitution rate per nucleotide site per year for the sequenced chloroplast regions was  <dig>  × 10- <dig>  the k value between mdhc-i and mdhc-ii was  <dig>  ±  <dig> .
therefore, the estimated divergence time for mdhc-i and mdhc-ii was estimated  to be about  <dig>  ±  <dig>  mya.
although no significant correlation was detected between geographic origin and genetic distance, the  <dig> accessions collected along the yangtze river, china, were always clustered together with kas- <dig>  an accession from kashmir  in both nj and mp trees .
in the network analysis, they congregated closely and formed a distinct cluster  in haplogroup  <dig> .
the level of nucleotide polymorphism of the  <dig> accessions was very low , only six haplotypes  were detected  and haplotype diversity  was  <dig> .
in comparison, the values of π, h and hd for the  <dig> accessions were  <dig> ,  <dig> and  <dig> , respectively.
to test the model of demographic population growth in the region from which the  <dig> accessions were sampled, especially for the  <dig> accessions along the yangtze river, a mismatch distribution analysis was conducted.
each small fragment inversion was treated as a snp in the analysis.
the ssd between the observed and expected mismatch distribution was  <dig>   and hrag was  <dig>  .
there was an only marginally significant difference in ssd between the observed and the predicted pairwise difference distribution under the sudden-expansion model.
this result provided evidence of rapid population expansion along the yangtze river.
the average τ-value was  <dig>  .
the initial time when the populations expanded along the yangtze river were calculated using equation  <dig> to obtain u , and then using equation  <dig> to obtain t .
therefore, the initial time of expansion was estimated to be about  <dig>  years ago.
discussion
the level and pattern of nucleotide variation in the sequenced chloroplast regions
the π value of the sequenced chloroplast regions among global samples of a. thaliana accessions was  <dig> , which is about one-quarter of that of the mean nucleotide diversity of the nuclear genes in a. thaliana  <cit> , but double that in another study by sall et al  <cit> , in which  <dig> non-coding single-copy cpdna regions were sequenced for  <dig> a. thaliana accessions .
the differences may be due to the different sampling strategies.
the  <dig> a. thaliana accessions in the latter study were mainly collected from scandinavia, whereas the  <dig> accessions in the present study were sampled worldwide.
for a highly self-fertilizing species, geographical structure may play an important role on a smaller scale in the level of polymorphism, at least for the uniparentally inherited chloroplast genome.
for example, the π value reduced to  <dig>  if only  <dig> accessions in branch a  were considered.
inversions in the chloroplast genome exist in monocotyledonous plants and the asteraceae.
the length of these inversions range from  <dig>  to  <dig> kb, and all have phylogenetic implications  <cit> .
the length of the inversions found in the present study were much shorter, only about 18- <dig> bp.
the accessions with inversions were found mostly scattered on branches b, d, e in the nj and mp trees.
the exception is in branch a, where all accessions had inversion  <dig>  the mechanism responsible for these inversions is not known, but they might have originated several times during the population expansion process.
therefore, it is advisable not to consider them for phylogenetic analysis.
dimorphism in the chloroplast dna of a. thaliana
two significantly differentiated haplotype classes could be identified in the sequenced chloroplast dna regions, just as in the allelic dimorphism found in some nuclear dna sequences of a. thaliana.
at least three different interpretations have been proposed to explain the nuclear dimorphism phenomenon.
first, balanced polymorphisms were usually the mutations maintained in populations by natural selection through heterozygotic advantage  <cit> .
the chloroplast genome is maternally inherited in a. thaliana, and the dna regions selected for analysis in this study are intergenic regions.
therefore, the dimorphism found in the chloroplast may not be caused by balancing selection via heterozygotic advantage.
furthermore, in our investigation, the value of the tajima's d-value was negative.
a negative tajima's d value is a general feature of the arabidopsis thaliana genome  <cit> , and is correlated to demographic factors, such as population growth  <cit> , rather than non-neutral forces such as selection  <cit> .
a second explanation for the nuclear dimorphism is that introgression might result in the allelic dimorphism.
chloroplast dna introgression has been widely reported .
in this study, we found that two related species, olimarabidopsis cabulica and a. arenosa, had all five identical nucleotide site variations with mdhc-ii of a. thaliana, which were the 'markers' to separate mdhc-ii from mdhc-i.
however, the k values between o. cabulica and the  <dig> accessions of a. thaliana, and between a. arenosa and the  <dig> a. thaliana accessions, are  <dig>  and  <dig> , respectively, whereas that between the two mdhcs of a. thaliana is only  <dig> .
the interspecific genetic distance is at least one order of magnitude higher than intraspecific genetic distances.
the estimated divergence time between o. cabulica and a. thaliana is about 10~ <dig> mya, and that between a. arenosa and a. thaliana is about  <dig> ~ <dig>  mya  <cit> .
the genetic distances based on cpdna between these species pairs correlate to their nuclear gene-based estimated divergent time.
these results indicated that the dimorphism in cpdna found in this study was not the result of recent introgressive hybridization events, but we cannot rule out the possibility that the dimorphism might be the result of ancient introgression events.
hybridization between a. thaliana and its closely related species does occur in nature.
for example, several studies confirmed the allotetraploid species, a. suecica, resulted from a hybridization event between a. thaliana and a. arenosa about  <dig>  to  <dig>  years ago .
the third explanation for genetic dimorphism is demographic factors, such as founder effects.
being a small annual weed, a. thaliana is a poor competitor in dense vegetation whereas the highly self-fertilizing characteristic makes it capable of founding a population even from a single seed.
as a result, this species has a tendency for rapid colonization and extinction cycles  <cit> .
founder effects might have occurred repeatedly in the evolutionary history of a. thaliana.
the founder event could enable some rare alleles to spread into additional populations when the founder population expanded rapidly if the unoccupied ecological niches were favourable.
the divergence time between mdhc-i and mdhc-ii was estimated to be about  <dig>  mya based on our cpdna data.
this is earlier than the estimated time of demographic expansion during the eemian interglacial .
therefore, another possible explanation for the cpdna dimorphism might be a founder effect followed by limited gene flow during late pleistocene glaciations and interglacial periods.
as the accessions in mdhc-ii share five specific variable sites with a. arenosa, the chloroplast genomes in mdhc-ii might represent more ancient types than those in mdhc-i.
it is also supported by the fact that more haplotypes are found in mdhc-ii  than in mdhc-i .
origin of chinese populations
the  <dig> accessions of a. thaliana from asia included in this study are scattered compared to those collected from europe.
six of them belong to mdhc-ii and  <dig> belong to mdhc-i.
of the mdhc-ii group, two collections from china  and two from kazakhstan  are within or very close to the altai mountains.
although these four accessions were not clustered in the same clade in the phylogenies, the two accessions from china were always on the same branch.
one of the kazakhstan accessions  was clustered with xjalt and xjqhx together with a russian accession  in the nj tree .
xjalt and xjqhx are unique in that they share six specific variable sites , the most number of specific variable sites in this study.
the provenances of these two accessions are about  <dig> km apart and located in the middle of the altai mountains range.
based on the cpdna data, the populations on the altai mountain range may have dispersed there during one of the late pleistocene glaciations, and some local habitats along the southern slopes of the altai mountains might have served as refugia.
in contrast to some refugia in europe, where a. thaliana populations had contributed the postglacial colonization of western and northern europe  <cit> , some populations in the asian refugia, such as xjalt and xjqhx, became relatively isolated genetically from other populations after glaciers retreated.
therefore, some fixed mutations were accumulated specifically in these populations.
it is also noticed by beck et al  <cit>  that "differentiation is particularly strong between the central asian refugia and all others, suggesting that either small historical population sizes in central asia, relatively limited gene flow between central asia and other areas, or a combination of the two have produced relatively strong genetic drift in this region".
the other two asian accessions in the mdhc-ii group are from japan  and india .
although tsu- <dig> was clustered with alc- <dig> from spain, the bootstrap support is poor in both nj and mp trees .
tsu- <dig> has two specific variable sites, but only shares one specific site with alc- <dig>  its phylogenetic relationships with other accessions are unclear and more samples are needed to clarify its origin.
kas- <dig> from india shares four specific variable sites with four accessions from europe ; this accession will be discussed later.
in mdhc-i,  <dig> accessions collected along the yangtze river, china, were clustered together with an accession from india .
all data suggested that the chloroplast genomes of these  <dig> accessions originated from a single common ancestor.
the initial time of expansion of the populations along the yangtze river was estimated to be about  <dig>  years ago based on the cpdna sequences.
at least we can rule out the possibility that these populations were introduced by recent human activities, like the populations found in the usa.
both highlands of the western himalayas and caucasus have been proposed as possible ancestral areas of a. thaliana  <cit> .
the populations along the yangtze river could have dispersed eastwards from the ancestral area to the present-day locations via the himalayas or kunlun mountains, which have an east-west trend.
kas- <dig> was collected in kashmir, india , on the southern slopes of the himalayas.
it shares a most frequent haplotype with seven chinese accessions, but it can be distinguished from them only by one specific variable site .
there are at least two explanations for the observed distribution pattern.
the first is that kas- <dig> represents the type that is most similar to the ancestor of the yangtze river populations, and the expansion of the latter was immediately or very shortly after the dispersal event.
however, in the phylogenies, kas- <dig> is not basal among the  <dig> accessions.
the second explanation is that kas- <dig> might have been introduced to kashmir from china.
it is uncertain which explanation is more likely.
more samples from the western part of the himalayas and kunlun mountains are definitely needed for identification of the possible ancestral haplotype of the yangtze river populations.
the other three accessions in mdhc-i are h tsukaya from japan, sorbo from tajikistan, and kas- <dig> from india.
the former shares one haplotype with four accessions from europe and one accession from the usa.
it is apparent that it represents a recent introduction to japan, possibly by human activity.
the accession from tajikistan has a specific haplotype that is characterized by four unique snps , and it shares a haplotype with one accession from sweden, italy and libya, respectively.
kas- <dig> shares the exact haplotype with two accessions from russia  and one from austria .
although kas- <dig> and kas- <dig> were collected at the same locality , as stated on the abrc seed stock centre website, their genetic distinctness has been noted previously  <cit> .
re-sampling from their original locality might help in clarifying the confusion.
the possible maternal parent of arabidopsis suecica
arabidopsis suecica is an allotetraploid species whose maternal parent is a. thaliana.
the species originated more than  <dig>  years ago from an allotetraploid hybrid between a. thaliana and a. arenosa  <cit> .
several studies indicate that the ancestral area of a. suecica is in europe .
however, a recent study by beck et al  <cit>  revealed a genetic similarity between a. suecica and chinese accessions of a. thaliana.
in the present study, the cpdna sequences of a. suecica were more similar to those of some european accessions, especially chi- <dig> from russia .
based on the comparison of cpdna in this study, it is most likely that the maternal parent of a. suecica was from europe.
CONCLUSIONS
elucidating the dispersal of a. thaliana within asia is a very complicated issue.
temperature fluctuations during glaciations and interglacial periods in the late pleistocene, the complex mountain ranges in the central and central-east parts of asia, and human activities have all contributed to the present-day distribution patterns.
it is clear from this study that some populations in east asia, such as those along the yangtze river, were dispersed there along the himalaya or kunlun mountain ranges and underwent rapid expansion about  <dig>  years ago.
altai mountains may have provided refugia for a. thaliana during pleistocene glaciations.
however, more samples from the altai, kunlun, and western part of the himalaya mountain ranges are needed to elucidate more fully the dispersal history of a. thaliana populations in asia.

 arabidopsis thaliana heynh is an annual weed belonging to the family brassicaceae. the species is native to europe and central asia, but is now widely distributed in the northern hemisphere ranging from 68°n to equator. many characteristics, from morphological traits to protein and dna markers, have been used to evaluate natural genetic variation among populations, and to reconstruct an intraspecific phylogeny, for a. thaliana. it has been found that many nuclear genes comprise two or more major differentiated haplotypes, generally referred to as allelic dimorphism. balancing selection or ancient population subdivision was often invoked to explain the pattern. the major mechanisms for balancing selection are heterozygote advantage, frequency-dependent selection, or environmental heterogeneity. it is well known that a. thaliana has an inbreeding mating system. the estimated outcrossing rate of the species is 1% or less. it seems difficult to imagine that so many loci in a. thaliana have experienced balancing selection via heterozygote advantage. therefore, frequency-dependent selection and/or diversifying selection might be the driving forces for the dimorphism phenomenon, as in the case of pathogen resistance genes. it is not clear yet if the dimorphism also exists in the chloroplast genome. the chloroplast genome of a. thaliana is a circular dna composed of bp with a pair of inverted repeats of bp separated by small and large single-copy regions of bp and bp, respectively. the uniparentally inherited chloroplast genome has been utilized in many studies in plant population and evolutionary genetics. however, studies focused on global patterns of cpdna genetic variation among accessions of a. thaliana are scattered. in an investigation on the maternal origins of a. suecica, cpdna regions were sequenced for a. thaliana accessions, which were mainly collected from scandinavia. these authors found considerable variation existed among the non-coding single-copy sequences in the chloroplast genome of a. thaliana. in another study, the trnl-trnf cpdna intergenic spacer region of individuals from a. thaliana populations in its native range was sequenced and haplotypes were identified. based on the chloroplast and nuclear dna sequence data, beck et al proposed the caucasian area as the possible ancestral area of a. thaliana, and suggested four possibilities for the origin of east asian populations. they also found that the maternal components of a. suecica shared a high similarity to those in the asian metapopulation of a. thaliana, especially to those from china. in the studies incorporating worldwide sampling of a. thaliana populations, the samples from east asia were very scattered. he et al conducted a study on the genetic diversity of natural arabidopsis thaliana populations in china based on issr and rapd makers, and found that about 42-45% of the total genetic variation existed within populations and there was a significant correlation between geographic distance and genetic distance. however, the phylogenetic relationships of chinese populations with those distributed in other regions of the world, and the history of population dispersal in this region, are not clear. the goals of the present survey are: to examine global patterns of cpdna genetic variation in a. thaliana; to infer phylogenetic relationships among a. thaliana accessions from all over the world based on cpdna sequence data, with particular focus on chinese populations; and to discuss the possible origin of the chinese populations. it was found in this study that dimorphism did exist in the chloroplast genome of a. thaliana; the accessions studied were grouped into two major clusters; and the chinese populations might have two independent origins.  nucleotide variation in the chloroplast dna sequences seventy-seven a. thaliana accessions were used in the survey, among them accessions were field collected in china. all sampling locations in china were separated by at least km, with most of the locations separated by more than km. no cp dna polymorphism was detected within either accession ahyxx or abd- therefore only one individual was chosen for each accession for dna sequence analysis. about nucleotides from the chloroplast genome were amplified and sequenced for each accession, of which nucleotides of non-coding fragments were retained for analysis. *accession no. begun with'n' were obtained from the nottingham arabidopsis stock center; with'cs' were obtained from the arabidopsis biological resource center; with "pku" were field collected in china and their detailed information is listed in table the combined data matrix contained variable nucleotide sites. among them, were mononucleotide repeat polymorphisms, one was a dinucleotide repeat polymorphism, and four were complicated length variations. these length polymorphisms were excluded in the analyses. the other polymorphic variations comprised single nucleotide polymorphisms, insertion/deletion events and two small fragment inversions. only one site exhibited three-base polymorphism and the other sites showed two-base polymorphism. the two small fragmental inversions were located at sites 3633- and sites 6501-. the characteristics of this kind of reversion were: a central region of nt or nt, which could mutate to its reverse complement sequence; and two flanking sequences of nt or nt, respectively. the two flanking sequences could be reversely complemented to each other. it is most likely that each of these two small fragmental inversions could be generated by only one or very few mutation event, but it resulted in multiple snps. nucleotide diversity for the entire sequenced regions was, but ranged from for the ycf3-trns intergenic spacer to for psaj-rpl. ns = not significant and p > nsa = not significant but > p > * p < ** p < less frequent nucleotide polymorphisms were in excess for the sequenced regions. singletons were found at a very high frequency: among the snps and among the indels were singletons. the excess of low-frequency polymorphisms resulted in negative tajima's d, fu and li's d* and f* values for most of the sequenced segments; for example, out of tajima's d values, nine out of fu and li's d* values and out of fu and li's f* values were negative. the values for the combined data matrix were -, - and -. when fu and li's d and f tests were conducted using the a. arenosa ortholog as the reference sequence, similar results were obtained: eight out of fu and li's d values and nine out of fu and li's f values were negative and for the combined data matrix; both the values were negative. in total, we identified three types of nucleotide variations among the aligned sequences: snps, length polymorphisms, and two small fragmental inversions. phylogenetic relationships among the accessions because the single base changes in the two short inverted regions were not independent events, they were excluded from the phylogenetic analysis. two distinct clusters with high bootstrap values were retrieved in the nj tree. one cluster included accessions and the other included accessions. although the topology of the mp tree differed to that of the nj tree, one branch with accessions corresponded to one of the two clusters in the nj tree. in general, no significant correlation was detected between geographical origins and clusterings in the phylogenetic trees. accessions from the same country, such as four accessions from italy and five accessions from usa failed to cluster together, but were scattered on different branches. this lack of phylogeographic structure conforms to the hypothesis of a rapid recent expansion of the species with strong involvement of human-mediated migrations. although there was incongruence between the two phylogenies, the topological relationship was relatively stable among a large number of accessions. we identified four stable branches in both trees. the only major difference was in branch c. it was placed within one of the two clusters in the nj tree, but formed a deep polytonous branch in the mp tree, containing the same accessions except cvi- an accession from cape verde island. the branches a-e comprised out of the accessions. discrimination of two major differentiated haplotypes among a. thaliana accessions when only the parsimony-informative sites were considered, the nucleotide variation of the accessions was structured into two major different haplotype classes. the mdhc-i and mdhc-ii classes were composed of and accessions, respectively, and they corresponded well to the two clusters in the nj tree. the mdhc-i and mdhc-ii classes differed at five nucleotide sites, and these sites were within a fragment about kb long from trnl to rpl in the chloroplast genome. for interspecific comparison, the homologous sequences of three related species, olimarabidopsis cabulica, a. arenosa and a. suecica, were aligned with the a. thaliana accessions. they were identical to those in mdhc-ii of a. thaliana at all five nucleotide sites where the two mdhcs could be distinguished from each other. all the sites except the inverted length variants were used to form a binary data set for haplotype network analysis. forty-nine haplotypes were identified in the accessions of a. thaliana. the haplotypes were also bifurcated to form two haplogroups. haplogroup and haplogroup differed at the same five sites where mdhc-i and -ii differed, and the accessions in haplogroup and haplogroup were identical to those in mdhc-i and -ii, respectively. estimated divergence time for mdhc-i and mdhc-ii, and demographic expansion of a monophyletic group of accessions in asia the k value between a. arenosa and a. thaliana accessions was ±. using equation the substitution rate per nucleotide site per year for the sequenced chloroplast regions was × 10- the k value between mdhc-i and mdhc-ii was ±. therefore, the estimated divergence time for mdhc-i and mdhc-ii was estimated to be about ± mya. although no significant correlation was detected between geographic origin and genetic distance, the accessions collected along the yangtze river, china, were always clustered together with kas- an accession from kashmir in both nj and mp trees. in the network analysis, they congregated closely and formed a distinct cluster in haplogroup. the level of nucleotide polymorphism of the accessions was very low, only six haplotypes were detected and haplotype diversity was. in comparison, the values of π, h and hd for the accessions were, and, respectively. to test the model of demographic population growth in the region from which the accessions were sampled, especially for the accessions along the yangtze river, a mismatch distribution analysis was conducted. each small fragment inversion was treated as a snp in the analysis. the ssd between the observed and expected mismatch distribution was and hrag was. there was an only marginally significant difference in ssd between the observed and the predicted pairwise difference distribution under the sudden-expansion model. this result provided evidence of rapid population expansion along the yangtze river. the average τ-value was. the initial time when the populations expanded along the yangtze river were calculated using equation to obtain u, and then using equation to obtain t. therefore, the initial time of expansion was estimated to be about years ago. discussion the level and pattern of nucleotide variation in the sequenced chloroplast regions the π value of the sequenced chloroplast regions among global samples of a. thaliana accessions was, which is about one-quarter of that of the mean nucleotide diversity of the nuclear genes in a. thaliana, but double that in another study by sall et al, in which non-coding single-copy cpdna regions were sequenced for a. thaliana accessions. the differences may be due to the different sampling strategies. the a. thaliana accessions in the latter study were mainly collected from scandinavia, whereas the accessions in the present study were sampled worldwide. for a highly self-fertilizing species, geographical structure may play an important role on a smaller scale in the level of polymorphism, at least for the uniparentally inherited chloroplast genome. for example, the π value reduced to if only accessions in branch a were considered. inversions in the chloroplast genome exist in monocotyledonous plants and the asteraceae. the length of these inversions range from to kb, and all have phylogenetic implications. the length of the inversions found in the present study were much shorter, only about 18- bp. the accessions with inversions were found mostly scattered on branches b, d, e in the nj and mp trees. the exception is in branch a, where all accessions had inversion the mechanism responsible for these inversions is not known, but they might have originated several times during the population expansion process. therefore, it is advisable not to consider them for phylogenetic analysis. dimorphism in the chloroplast dna of a. thaliana two significantly differentiated haplotype classes could be identified in the sequenced chloroplast dna regions, just as in the allelic dimorphism found in some nuclear dna sequences of a. thaliana. at least three different interpretations have been proposed to explain the nuclear dimorphism phenomenon. first, balanced polymorphisms were usually the mutations maintained in populations by natural selection through heterozygotic advantage. the chloroplast genome is maternally inherited in a. thaliana, and the dna regions selected for analysis in this study are intergenic regions. therefore, the dimorphism found in the chloroplast may not be caused by balancing selection via heterozygotic advantage. furthermore, in our investigation, the value of the tajima's d-value was negative. a negative tajima's d value is a general feature of the arabidopsis thaliana genome, and is correlated to demographic factors, such as population growth, rather than non-neutral forces such as selection. a second explanation for the nuclear dimorphism is that introgression might result in the allelic dimorphism. chloroplast dna introgression has been widely reported. in this study, we found that two related species, olimarabidopsis cabulica and a. arenosa, had all five identical nucleotide site variations with mdhc-ii of a. thaliana, which were the'markers' to separate mdhc-ii from mdhc-i. however, the k values between o. cabulica and the accessions of a. thaliana, and between a. arenosa and the a. thaliana accessions, are and, respectively, whereas that between the two mdhcs of a. thaliana is only. the interspecific genetic distance is at least one order of magnitude higher than intraspecific genetic distances. the estimated divergence time between o. cabulica and a. thaliana is about 10~ mya, and that between a. arenosa and a. thaliana is about ~ mya. the genetic distances based on cpdna between these species pairs correlate to their nuclear gene-based estimated divergent time. these results indicated that the dimorphism in cpdna found in this study was not the result of recent introgressive hybridization events, but we cannot rule out the possibility that the dimorphism might be the result of ancient introgression events. hybridization between a. thaliana and its closely related species does occur in nature. for example, several studies confirmed the allotetraploid species, a. suecica, resulted from a hybridization event between a. thaliana and a. arenosa about to years ago. the third explanation for genetic dimorphism is demographic factors, such as founder effects. being a small annual weed, a. thaliana is a poor competitor in dense vegetation whereas the highly self-fertilizing characteristic makes it capable of founding a population even from a single seed. as a result, this species has a tendency for rapid colonization and extinction cycles. founder effects might have occurred repeatedly in the evolutionary history of a. thaliana. the founder event could enable some rare alleles to spread into additional populations when the founder population expanded rapidly if the unoccupied ecological niches were favourable. the divergence time between mdhc-i and mdhc-ii was estimated to be about mya based on our cpdna data. this is earlier than the estimated time of demographic expansion during the eemian interglacial. therefore, another possible explanation for the cpdna dimorphism might be a founder effect followed by limited gene flow during late pleistocene glaciations and interglacial periods. as the accessions in mdhc-ii share five specific variable sites with a. arenosa, the chloroplast genomes in mdhc-ii might represent more ancient types than those in mdhc-i. it is also supported by the fact that more haplotypes are found in mdhc-ii than in mdhc-i. origin of chinese populations the accessions of a. thaliana from asia included in this study are scattered compared to those collected from europe. six of them belong to mdhc-ii and belong to mdhc-i. of the mdhc-ii group, two collections from china and two from kazakhstan are within or very close to the altai mountains. although these four accessions were not clustered in the same clade in the phylogenies, the two accessions from china were always on the same branch. one of the kazakhstan accessions was clustered with xjalt and xjqhx together with a russian accession in the nj tree. xjalt and xjqhx are unique in that they share six specific variable sites, the most number of specific variable sites in this study. the provenances of these two accessions are about km apart and located in the middle of the altai mountains range. based on the cpdna data, the populations on the altai mountain range may have dispersed there during one of the late pleistocene glaciations, and some local habitats along the southern slopes of the altai mountains might have served as refugia. in contrast to some refugia in europe, where a. thaliana populations had contributed the postglacial colonization of western and northern europe, some populations in the asian refugia, such as xjalt and xjqhx, became relatively isolated genetically from other populations after glaciers retreated. therefore, some fixed mutations were accumulated specifically in these populations. it is also noticed by beck et al that "differentiation is particularly strong between the central asian refugia and all others, suggesting that either small historical population sizes in central asia, relatively limited gene flow between central asia and other areas, or a combination of the two have produced relatively strong genetic drift in this region". the other two asian accessions in the mdhc-ii group are from japan and india. although tsu- was clustered with alc- from spain, the bootstrap support is poor in both nj and mp trees. tsu- has two specific variable sites, but only shares one specific site with alc- its phylogenetic relationships with other accessions are unclear and more samples are needed to clarify its origin. kas- from india shares four specific variable sites with four accessions from europe; this accession will be discussed later. in mdhc-i, accessions collected along the yangtze river, china, were clustered together with an accession from india. all data suggested that the chloroplast genomes of these accessions originated from a single common ancestor. the initial time of expansion of the populations along the yangtze river was estimated to be about years ago based on the cpdna sequences. at least we can rule out the possibility that these populations were introduced by recent human activities, like the populations found in the usa. both highlands of the western himalayas and caucasus have been proposed as possible ancestral areas of a. thaliana. the populations along the yangtze river could have dispersed eastwards from the ancestral area to the present-day locations via the himalayas or kunlun mountains, which have an east-west trend. kas- was collected in kashmir, india, on the southern slopes of the himalayas. it shares a most frequent haplotype with seven chinese accessions, but it can be distinguished from them only by one specific variable site. there are at least two explanations for the observed distribution pattern. the first is that kas- represents the type that is most similar to the ancestor of the yangtze river populations, and the expansion of the latter was immediately or very shortly after the dispersal event. however, in the phylogenies, kas- is not basal among the accessions. the second explanation is that kas- might have been introduced to kashmir from china. it is uncertain which explanation is more likely. more samples from the western part of the himalayas and kunlun mountains are definitely needed for identification of the possible ancestral haplotype of the yangtze river populations. the other three accessions in mdhc-i are h tsukaya from japan, sorbo from tajikistan, and kas- from india. the former shares one haplotype with four accessions from europe and one accession from the usa. it is apparent that it represents a recent introduction to japan, possibly by human activity. the accession from tajikistan has a specific haplotype that is characterized by four unique snps, and it shares a haplotype with one accession from sweden, italy and libya, respectively. kas- shares the exact haplotype with two accessions from russia and one from austria. although kas- and kas- were collected at the same locality, as stated on the abrc seed stock centre website, their genetic distinctness has been noted previously. re-sampling from their original locality might help in clarifying the confusion. the possible maternal parent of arabidopsis suecica arabidopsis suecica is an allotetraploid species whose maternal parent is a. thaliana. the species originated more than years ago from an allotetraploid hybrid between a. thaliana and a. arenosa. several studies indicate that the ancestral area of a. suecica is in europe. however, a recent study by beck et al revealed a genetic similarity between a. suecica and chinese accessions of a. thaliana. in the present study, the cpdna sequences of a. suecica were more similar to those of some european accessions, especially chi- from russia. based on the comparison of cpdna in this study, it is most likely that the maternal parent of a. suecica was from europe.  elucidating the dispersal of a. thaliana within asia is a very complicated issue. temperature fluctuations during glaciations and interglacial periods in the late pleistocene, the complex mountain ranges in the central and central-east parts of asia, and human activities have all contributed to the present-day distribution patterns. it is clear from this study that some populations in east asia, such as those along the yangtze river, were dispersed there along the himalaya or kunlun mountain ranges and underwent rapid expansion about years ago. altai mountains may have provided refugia for a. thaliana during pleistocene glaciations. however, more samples from the altai, kunlun, and western part of the himalaya mountain ranges are needed to elucidate more fully the dispersal history of a. thaliana populations in asia. 
the analysis of the nucleotide sequences showed that the accessions of a. thaliana were partitioned into two major differentiated haplotype classes. the chinese populations along the yangtze river were characterized by five haplotypes, and the two accessions collected from the middle range of the altai mountains in china shared six specific variable sites. in the studies incorporating worldwide sampling of a. thaliana populations, the samples from east asia, especially from china, were very scattered; and the studies focused on global patterns of cpdna genetic variation among accessions of a. thaliana are very few. the chinese populations along the yangtze river may have dispersed eastwards to their present-day locations from the himalayas. the dimorphism in the chloroplast dna could be due to founder effects during late pleistocene glaciations and interglacial periods, although introgression cannot be ruled out. forty-nine haplotypes were detected among the accessions, which exhibited nucleotide diversity of. in this study, chloroplast dna sequence variability was used to infer phylogenetic relationships among arabidopsis thaliana accessions from around the world, with the emphasis on samples from china. two accessions collected from the middle range of the altai mountains in china may have survived in a local refugium during late pleistocene glaciations. these populations originated from a common ancestor, and a rapid demographic expansion began approximately years ago. a data set comprising accessions of a. thaliana, including field-collected chinese accessions together with three related species as the out-group, was compiled. the estimated divergence time of the two mdhcs was about mya. the natural populations from china with specific genetic characteristics enriched the gene pools of global a. thaliana collections.
 in the studies incorporating worldwide sampling of a. thaliana populations, the samples from east asia, especially from china, were very scattered; and the studies focused on global patterns of cpdna genetic variation among accessions of a. thaliana are very few. in this study, chloroplast dna sequence variability was used to infer phylogenetic relationships among arabidopsis thaliana accessions from around the world, with the emphasis on samples from china.  a data set comprising accessions of a. thaliana, including field-collected chinese accessions together with three related species as the out-group, was compiled. the analysis of the nucleotide sequences showed that the accessions of a. thaliana were partitioned into two major differentiated haplotype classes. the estimated divergence time of the two mdhcs was about mya. forty-nine haplotypes were detected among the accessions, which exhibited nucleotide diversity of. the chinese populations along the yangtze river were characterized by five haplotypes, and the two accessions collected from the middle range of the altai mountains in china shared six specific variable sites.  the dimorphism in the chloroplast dna could be due to founder effects during late pleistocene glaciations and interglacial periods, although introgression cannot be ruled out. the chinese populations along the yangtze river may have dispersed eastwards to their present-day locations from the himalayas. these populations originated from a common ancestor, and a rapid demographic expansion began approximately years ago. two accessions collected from the middle range of the altai mountains in china may have survived in a local refugium during late pleistocene glaciations. the natural populations from china with specific genetic characteristics enriched the gene pools of global a. thaliana collections. 