BACKGROUND
leishmania  major is one of the agents causing old world cutaneous leishmaniasis  in humans, which, in the case of l.  major, is a rural, zoonotic, vector-borne disease, involving different species of wild rodents as animal reservoir hosts and different species of phlebotomine sand flies as vectors, depending on the geographical location where it occurs  major isolated from many sources over many years have proved to be relatively uniform when studied by most of the classical and more modern methods used for characterizing leishmanial strains.
serological tests like excreted factor  serotyping and the application of leishmania species-specific monoclonal antibodies have shown that antigenic differences exist among different strains of l.  major  <cit> .
multilocus enzyme electrophoresis  exposed some enzyme electrophoretic variation with some isoenzyme variant profiles showing a degree of geographical sub-division within a general enzyme electrophoretic uniformity  <cit> .
the more recent application of various molecular biological methods revealed geographically distributed genetic polymorphism among different strains of l.  major.
analysis of sequence polymorphisms in seven coding, non-coding and anonymous nuclear dna sequences  <cit>  as well as a partial sequence from the kinetoplast dna maxicircle divergent region  <cit>  of strains of l.  major showed that the strains from central asia and the middle east were genetically distinct.
none to very little variation was seen among the central asian strains and somewhat more among the middle eastern ones.
only a few east african strains of l.  major were included in these studies, which tended to be genetically closer to the middle eastern than to the central asian ones.
different patterns in the most variable sequence were attributed to variations in complex microsatellite repeats.
this prompted a search for highly polymorphic microsatellite markers that would allow greater discrimination of otherwise closely related strains of l.  major.
microsatellites are repeated simple motifs of a few nucleotides  flanked by unique sequences.
they are ubiquitous in prokaryotes and eukaryotes and have been shown to exhibit a substantial level of polymorphism in a number of eukaryotic genomes  <cit> .
they are becoming one of the principal genetic marker systems in phylogenetic, population genetic and molecular epidemiological studies.
the leishmanial genome is relatively rich in microsatellite sequences, about  <dig> n loci per haploid genome, with ca being the most abundant dinucleotide repeat as in all vertebrates and fungi investigated so far  <cit> .
microsatellite markers have been used successfully for characterizing and detecting genetic variation in other old world leishmanial strains and species, i. e., l.  donovani and l.  infantum  <cit>  and l.  tropica  <cit> .
this study adds the species l.  major.
ten informative microsatellite loci based on nucleotide sequence information of l.  major obtained from the leishmania genome project were used to determine polymorphism and micro-heterogeneity, and their geographical and epidemiological distribution.
RESULTS
microsatellite analysis
twenty-three sequences located on chromosomes  <dig>   <dig>   <dig>   <dig> and  <dig> that contained nucleotide repeats such as n, n, n, n, and n were selected from the genome sequence of l.  major published by the european bioinformatics institute.
pcr primers were designed for microsatellite loci that contained at least  <dig> repeats in the reference strain l.  major mhom/il/1980/friedlin and were located on different chromosomes or, if located on the same chromosome, were at least distant enough to be considered unlinked .
fifteen of the  <dig> primer pairs yielded a single pcr fragment for the majority of the strains tested.
ten of these  <dig> primer pairs amplified polymorphic fragments of different size in different strains of l.  major .
* in l.  major mhom/il/1980/friedlin
ta annealing temperaturea
only  <dig> out of  <dig> pair-wise combinations  were in significant linkage disequilibrium  when genepop was used.
no linkage was observed using the bonferroni corrections implemented in fstat.
the  <dig> polymorphic loci were used to perform multi-locus microsatellite typing  on  <dig> strains of l.  major collected in  <dig> asian and  <dig> african countries.
four of the  <dig> loci showed only homozygous allele combinations.
however, three gtg loci, two at loci and the gaca locus were heterozygous for one or more of the strains .
in some cases, no pcr product could be obtained in repeated pcr runs, which was treated as missing data for the statistical analyses.
sixty-six differing mlmt profiles comprising the number of repeats in each of the  <dig> markers were revealed among the  <dig> strains .
the mlmt profiles were named lmj, thus indicating that these microsatellite profiles are unique to l.  major, and numbered from  <dig> to  <dig>  figures  <dig> to  <dig> show their genetic inter-relationship.
most profiles were represented by a single strain; eleven were present in more than one strain.
nine microsatellite variant profiles were exposed among the  <dig> strains in the sub-population ca1; six among the  <dig> strains in the sub-population ca2; thirteen among the  <dig> strains in the sub-population me1; fifteen among the  <dig> strains in the sub-population me2; eleven among the  <dig> strains in the sub-population af1; and twelve among the  <dig> strains in the sub-population af <dig> .
key: ph.
= phlebotomus; r. = rhombomys; t. = tatera; ps.
= psammomys; the prefix lmj indicates that these microsatellite profiles are unique to l.  major.
the average sample size , average number of alleles per locus , expected proportion of heterozygotes , observed proportion of heterozygotes   and inbreeding coefficient  were obtained using gda software.
ld was calculated using genepop and fstat software.
population structure
the population structure was investigated, using the bayesian-model based clustering approach implemented in the software structure  <cit> .
the most probable number of populations in this data set by calculating Δk was three: central asia , comprising strains from israel, the palestinian authority, egypt, saudi arabia, kuwait and eastern turkey; and africa , comprising strains from north, east and west africa, western turkey, iran and iraq .
structure analyses were re-done with each of the three main populations, ca, me and af, to expose further sub-division; and each main population did separate into two sub-populations: ca <dig> and ca2; me <dig> and me2; af <dig> and af <dig> .
although calculations of Δk do not allow for differentiation between one or two groups in the dataset, the strains' assignment  was most congruent with the existence of two sub-populations.
the distribution of the different sub-populations is shown in figure  <dig>
on using a predefined clustering approach, the total area from which the strains of l.  major were collected was divided into five geographical sectors: central asia , the middle east , north, west and east africa with the last three being referred to collectively as africa .
the boundaries between these sectors are mainly significant geographical barriers like seas and deserts.
each strain was assigned to one of the sectors, according to its place of collection without regard to underlying genetic relationships .
the central asian cluster was highly homogenous, genetically and according to predefined geographical placement, with all the strains having a very high membership coefficient of  <dig> .
the middle east cluster was also genetically well defined, except for the exclusion of the two western turkish and sole iraqi and iranian strains, which grouped with the african strains.
the entire african cluster was very heterogeneous regarding mlmt profiles and did not separate into distinct north, west and east african regional clusters as envisaged by the predefined clustering approach.
increasing values of k  were used to identify ancestral populations.
at k =  <dig>  the first split separated the population ca from other strains.
at k =  <dig>  three main populations, ca, me and af were exposed.
at k =  <dig>  the african strains split into two clusters.
at k =  <dig>  the middle eastern cluster separated into two distinct sub-clusters, me <dig> and me <dig> while at k =  <dig> the population ca split into the sub-clusters ca <dig> and ca <dig>
construction of distance trees
for the phylogenetic analyses of these strains of l.  major, the dataset was clone-corrected and presented  <dig> microsatellite genotypes.
nj and upmga trees were constructed from the different distance matrices obtained.
all phylograms displayed the same five clusters, albeit with different bootstrap support values, as shown, for example, in the unrooted nj tree  that was based on a dps distance matrix and has: i) a central asian cluster; ii) two clusters of strains from the middle east, corresponding to the sub-populations me <dig> and me <dig> as determined by structure, and iii) two clusters of strains from africa, which correlate perfectly with the sub-populations af <dig> and af <dig> as determined by structure.
the sub-division seen in the central asian cluster is largely congruent with the two sub-populations ca <dig> and ca <dig> identified by structure.
as in the structure analysis, the two western turkish strains  grouped with the strains in the sub-population af <dig> while the eastern turkish one  belonged to the sub-population me  <dig>  the two strains from saudi arabia formed a unique branch in the unrooted nj tree, closer to the central asian cluster, however with only low bootstrap values.
population genetic data
fst, as a measure of genetic differentiation between populations, calculated in a pair-wise manner for the main populations, revealed high genetic differentiation between the populations ca and me, and ca and af.
genetic isolation was less pronounced between the populations me and af.
when fst was calculated at a sub-population level, moderate , high  and very high  genetic differentiation was observed for the pairs af1/af <dig>  me1/me <dig> and ca1/ca <dig>  respectively .
genetic flow or migration rate, nm, refers to the movement of strains between populations and sub-populations and sets a limit as to how much genetic divergence can occur.
at the sub-population level, the genetic flow was the highest between the two african sub-populations and the least between the two central asian sub-populations .
at the pre-defined population classification, there was clear genetic flow  between the african regions .
the numbers of alleles encompassed by geographical groupings
to compare the number of alleles falling within the three main geographical groupings, the mlmt profiles were grouped according to their geographical origin as specified by their who codes: central asia ; the middle east ; and africa .
in order to compare groups of equal size, a re-sampling procedure was used  <cit> .
the african group had the highest number of alleles  followed by middle eastern group  and then the central asian group .
discussion
elfari et al.
<cit>  showed that genetic and biological variation among strains of l.  major tended to correspond with their geographical origin.
among the several analytical techniques and methods developed in that study, only two employed genetic markers that were polymorphic enough to distinguish between closely related strains.
for population genetic studies of l.  major like this one, highly discriminative markers had to be developed.
of the microsatellite markers designed, ten, all based on sequences published by the leishmania genome project  <cit> , have proved to be suitable for multilocus microsatellite typing .
microsatellite analysis of the  <dig> strains of l.  major revealed  <dig> different microsatellite profiles.
these were unique to this species of leishmania and demonstrated substantial genetic micro-heterogeneity with regard to microsatellite profiles.
in many cases in this study, particular microsatellite profiles were found in single strains.
had many more strains of l.  major been available, these profiles would, probably, have been associated with groups of strains as was seen with the strains isolated at mubarak and termez, uzbekistan, .
the model- and distance-based analyses of the microsatellite data exposed three main populations of l.  major, corresponding to three separate geographical regions, central asia, the middle east and africa.
when the data were analyzed by structure with an increase in the number of populations from  <dig> to  <dig> in order to identify the ancestral source population, the first split separated the central asian strains of l.  major from all the others, suggesting, possibly, their older origin.
the bayesian algorithm implemented in structure was more appropriate for characterizing population structure because it identified distinct sub-populations based on patterns of allele frequencies and, also, determined fractions of the genotype that belong specifically to each sub-population.
structure has been shown to accurately infer individual ancestries  <cit>  and provide information on population relationships and history  <cit> .
the presence of these three populations was supported by the detection of genetic isolation among them, particularly between population ca and the two populations af and me that were separate but genetically closer to one another.
this may reflect the geographical distance and barriers between the three main clusters .
both types of analysis led to sub-division of the main clusters.
congruence of the results supported tripartite clustering despite the absence of strong bootstrap values on the main branches of the distance tree .
the two sub-groups in each of the three main populations were also supported by f-statistics, although the genetic differentiation was less pronounced, especially between the sub-populations af <dig> and af <dig>  and between the sub-populations me <dig> and me <dig>  wright  maintained that even slight genetic differentiation among sub-populations is significant.
within the context of this study, it seems that the populations af and me were more genetically diversified than the population ca.
this might be due to different sampling procedures in the three areas.
the  <dig> strains from africa were collected between  <dig> and  <dig> in  <dig> separated locations.
in contrast to that, the  <dig> me strains were collected between  <dig> and  <dig> from different foci encompassed by the eastern mediterranean region and arabian peninsula, and the  <dig> strains from central asia between  <dig> and  <dig> in only  <dig> countries.
to correct, at least partially, for this sampling bias mean number of alleles have been calculated based on the mlmt profiles present in the three areas, e.g.
by excluding profiles shared by more than one strain.
when corrected for central asia, the smallest group, the lowest number of alleles was still found in central asia followed by the middle east and then africa.
the sampling bias might still account for the high heterogeneity of the african strains, but the differences in diversity for ca and me strains cannot be explained only by differences in sampling.
most of the me strains were collected in israeli and palestinian foci from a territory much smaller than the central asian area.
the area from where the central asian strains of l.  major studied here came is a landlocked generally arid plateau.
the greater variation of habitats and biotopes within middle eastern environments seems to translate into a greater variety of animal hosts and sand fly vectors compared to central asia.
where groups of strains with the same microsatellite profile were available, it was possible to make some assessment with regard to time and spatial distribution.
for example, the strains in the groups of strains with the microsatellite profiles lmj  <dig>  lmj  <dig> and lmj  <dig> were isolated in a given area for each group at the same time or within short periods of time.
however, strains with the microsatellite profile lmj  <dig>  lmj  <dig> and lmj  <dig> were isolated in several locations at different times.
this, in addition to the observed low heterozygosity at microsatellite loci, seems to provide evidence against extensive and frequent genetic exchange within l.  major.
that microsatellite variants can persist for long periods of time and are restricted or widely spread in their distribution, supposedly, results from the effect of biotopic conditions on the animal hosts and vectors of leishmanial parasites and much less so in the case of the humans, who do alter the biotope by construction, agriculture and many of their other activities.
most of the strains in the population me came from israel and the palestinian authority and their separation into the sub-populations me <dig>  jordan valley-dead sea area, and me <dig>  the negev-sinai area, did seem to parallel geographical distribution but not entirely so.
most 'misplaced' strains were from human cases and could be explained by travel and migration between these areas.
several strains have membership coefficients for both sub-populations, indicating gene flow between them.
the two african sub-populations did not correlate well with the geographical origin of the strains that fell into them.
their analysis was hampered owing to the small number of strains available and their very wide geographical dispersal.
unlike dps-based phenetic analysis , which is not affected by population size  <cit> , structure improves with increasing sample size for each population  <cit> .
in this study, the results obtained using structure were consistent with those of the phenetic analysis.
nevertheless, more strains from different african foci need to be analysed to really determine the population structure of african strains of l.  major.
preferably, one should use strains isolated from sand fly vectors and rodent hosts to avoid the effect of human migration.
small sample size was also a problem in the case of the two single strains from iran and iraq and the two from western turkey that grouped together with the african strains and the eastern turkish strain that grouped with the middle eastern strains .
waki et al.
<cit>  hypothesized that adaptation to the micro-environment in the macrophages of the mammalian hosts, the place of long-term residence of all leishmanial parasites, including l.  major, is most significant in the evolution of the genus leishmania.
they considered adaptation to the sand fly vectors' gut, the place of short-term residence, to be of lesser evolutionary importance.
that the three main populations of l.  major identified by this study exist in endemic foci that each have different rodent species serving as the reservoir hosts  <cit>  seems to support this hypothesis.
variation among the strains of l.  major from the same endemic area leading to different sub-populations could be attributed to differences in sand fly vector populations.
for instance, analysis of mitochondrial cytb haplotypes exposed two genetically distinct populations of ph.
papatasi in the middle east  <cit> .
CONCLUSIONS
multilocus microsatellite typing  based on  <dig> different microsatellite markers and using a bayesian model-based analysis as well as phylogenetic reconstructions based on genetic distances, identified three main populations, corresponding to three separate geographical regions: central asia ; the middle east ; and africa .
each of them separated into two sub-populations which might reflect the existence of different species of rodent host in these areas.
the african and middle eastern populations seemed to be more genetically diversified than the central asian population.
sampling bias might account for the high heterogeneity of the african strains but cannot explain the differences in diversity for central asian and middle eastern strains.

 leishmania major is one of the agents causing old world cutaneous leishmaniasis in humans, which, in the case of l. major, is a rural, zoonotic, vector-borne disease, involving different species of wild rodents as animal reservoir hosts and different species of phlebotomine sand flies as vectors, depending on the geographical location where it occurs major isolated from many sources over many years have proved to be relatively uniform when studied by most of the classical and more modern methods used for characterizing leishmanial strains. serological tests like excreted factor serotyping and the application of leishmania species-specific monoclonal antibodies have shown that antigenic differences exist among different strains of l. major. multilocus enzyme electrophoresis exposed some enzyme electrophoretic variation with some isoenzyme variant profiles showing a degree of geographical sub-division within a general enzyme electrophoretic uniformity. the more recent application of various molecular biological methods revealed geographically distributed genetic polymorphism among different strains of l. major. analysis of sequence polymorphisms in seven coding, non-coding and anonymous nuclear dna sequences as well as a partial sequence from the kinetoplast dna maxicircle divergent region of strains of l. major showed that the strains from central asia and the middle east were genetically distinct. none to very little variation was seen among the central asian strains and somewhat more among the middle eastern ones. only a few east african strains of l. major were included in these studies, which tended to be genetically closer to the middle eastern than to the central asian ones. different patterns in the most variable sequence were attributed to variations in complex microsatellite repeats. this prompted a search for highly polymorphic microsatellite markers that would allow greater discrimination of otherwise closely related strains of l. major. microsatellites are repeated simple motifs of a few nucleotides flanked by unique sequences. they are ubiquitous in prokaryotes and eukaryotes and have been shown to exhibit a substantial level of polymorphism in a number of eukaryotic genomes. they are becoming one of the principal genetic marker systems in phylogenetic, population genetic and molecular epidemiological studies. the leishmanial genome is relatively rich in microsatellite sequences, about n loci per haploid genome, with ca being the most abundant dinucleotide repeat as in all vertebrates and fungi investigated so far. microsatellite markers have been used successfully for characterizing and detecting genetic variation in other old world leishmanial strains and species, i. e., l. donovani and l. infantum and l. tropica. this study adds the species l. major. ten informative microsatellite loci based on nucleotide sequence information of l. major obtained from the leishmania genome project were used to determine polymorphism and micro-heterogeneity, and their geographical and epidemiological distribution.  microsatellite analysis twenty-three sequences located on chromosomes and that contained nucleotide repeats such as n, n, n, n, and n were selected from the genome sequence of l. major published by the european bioinformatics institute. pcr primers were designed for microsatellite loci that contained at least repeats in the reference strain l. major mhom/il/1980/friedlin and were located on different chromosomes or, if located on the same chromosome, were at least distant enough to be considered unlinked. fifteen of the primer pairs yielded a single pcr fragment for the majority of the strains tested. ten of these primer pairs amplified polymorphic fragments of different size in different strains of l. major. * in l. major mhom/il/1980/friedlin ta annealing temperaturea only out of pair-wise combinations were in significant linkage disequilibrium when genepop was used. no linkage was observed using the bonferroni corrections implemented in fstat. the polymorphic loci were used to perform multi-locus microsatellite typing on strains of l. major collected in asian and african countries. four of the loci showed only homozygous allele combinations. however, three gtg loci, two at loci and the gaca locus were heterozygous for one or more of the strains. in some cases, no pcr product could be obtained in repeated pcr runs, which was treated as missing data for the statistical analyses. sixty-six differing mlmt profiles comprising the number of repeats in each of the markers were revealed among the strains. the mlmt profiles were named lmj, thus indicating that these microsatellite profiles are unique to l. major, and numbered from to figures to show their genetic inter-relationship. most profiles were represented by a single strain; eleven were present in more than one strain. nine microsatellite variant profiles were exposed among the strains in the sub-population ca1; six among the strains in the sub-population ca2; thirteen among the strains in the sub-population me1; fifteen among the strains in the sub-population me2; eleven among the strains in the sub-population af1; and twelve among the strains in the sub-population af. key: ph. = phlebotomus; r. = rhombomys; t. = tatera; ps. = psammomys; the prefix lmj indicates that these microsatellite profiles are unique to l. major. the average sample size, average number of alleles per locus, expected proportion of heterozygotes, observed proportion of heterozygotes and inbreeding coefficient were obtained using gda software. ld was calculated using genepop and fstat software. population structure the population structure was investigated, using the bayesian-model based clustering approach implemented in the software structure. the most probable number of populations in this data set by calculating Δk was three: central asia, comprising strains from israel, the palestinian authority, egypt, saudi arabia, kuwait and eastern turkey; and africa, comprising strains from north, east and west africa, western turkey, iran and iraq. structure analyses were re-done with each of the three main populations, ca, me and af, to expose further sub-division; and each main population did separate into two sub-populations: ca and ca2; me and me2; af and af. although calculations of Δk do not allow for differentiation between one or two groups in the dataset, the strains' assignment was most congruent with the existence of two sub-populations. the distribution of the different sub-populations is shown in figure on using a predefined clustering approach, the total area from which the strains of l. major were collected was divided into five geographical sectors: central asia, the middle east, north, west and east africa with the last three being referred to collectively as africa. the boundaries between these sectors are mainly significant geographical barriers like seas and deserts. each strain was assigned to one of the sectors, according to its place of collection without regard to underlying genetic relationships. the central asian cluster was highly homogenous, genetically and according to predefined geographical placement, with all the strains having a very high membership coefficient of. the middle east cluster was also genetically well defined, except for the exclusion of the two western turkish and sole iraqi and iranian strains, which grouped with the african strains. the entire african cluster was very heterogeneous regarding mlmt profiles and did not separate into distinct north, west and east african regional clusters as envisaged by the predefined clustering approach. increasing values of k were used to identify ancestral populations. at k = the first split separated the population ca from other strains. at k = three main populations, ca, me and af were exposed. at k = the african strains split into two clusters. at k = the middle eastern cluster separated into two distinct sub-clusters, me and me while at k = the population ca split into the sub-clusters ca and ca construction of distance trees for the phylogenetic analyses of these strains of l. major, the dataset was clone-corrected and presented microsatellite genotypes. nj and upmga trees were constructed from the different distance matrices obtained. all phylograms displayed the same five clusters, albeit with different bootstrap support values, as shown, for example, in the unrooted nj tree that was based on a dps distance matrix and has: i) a central asian cluster; ii) two clusters of strains from the middle east, corresponding to the sub-populations me and me as determined by structure, and iii) two clusters of strains from africa, which correlate perfectly with the sub-populations af and af as determined by structure. the sub-division seen in the central asian cluster is largely congruent with the two sub-populations ca and ca identified by structure. as in the structure analysis, the two western turkish strains grouped with the strains in the sub-population af while the eastern turkish one belonged to the sub-population me the two strains from saudi arabia formed a unique branch in the unrooted nj tree, closer to the central asian cluster, however with only low bootstrap values. population genetic data fst, as a measure of genetic differentiation between populations, calculated in a pair-wise manner for the main populations, revealed high genetic differentiation between the populations ca and me, and ca and af. genetic isolation was less pronounced between the populations me and af. when fst was calculated at a sub-population level, moderate, high and very high genetic differentiation was observed for the pairs af1/af me1/me and ca1/ca respectively. genetic flow or migration rate, nm, refers to the movement of strains between populations and sub-populations and sets a limit as to how much genetic divergence can occur. at the sub-population level, the genetic flow was the highest between the two african sub-populations and the least between the two central asian sub-populations. at the pre-defined population classification, there was clear genetic flow between the african regions. the numbers of alleles encompassed by geographical groupings to compare the number of alleles falling within the three main geographical groupings, the mlmt profiles were grouped according to their geographical origin as specified by their who codes: central asia; the middle east; and africa. in order to compare groups of equal size, a re-sampling procedure was used. the african group had the highest number of alleles followed by middle eastern group and then the central asian group. discussion elfari et al. showed that genetic and biological variation among strains of l. major tended to correspond with their geographical origin. among the several analytical techniques and methods developed in that study, only two employed genetic markers that were polymorphic enough to distinguish between closely related strains. for population genetic studies of l. major like this one, highly discriminative markers had to be developed. of the microsatellite markers designed, ten, all based on sequences published by the leishmania genome project, have proved to be suitable for multilocus microsatellite typing. microsatellite analysis of the strains of l. major revealed different microsatellite profiles. these were unique to this species of leishmania and demonstrated substantial genetic micro-heterogeneity with regard to microsatellite profiles. in many cases in this study, particular microsatellite profiles were found in single strains. had many more strains of l. major been available, these profiles would, probably, have been associated with groups of strains as was seen with the strains isolated at mubarak and termez, uzbekistan,. the model- and distance-based analyses of the microsatellite data exposed three main populations of l. major, corresponding to three separate geographical regions, central asia, the middle east and africa. when the data were analyzed by structure with an increase in the number of populations from to in order to identify the ancestral source population, the first split separated the central asian strains of l. major from all the others, suggesting, possibly, their older origin. the bayesian algorithm implemented in structure was more appropriate for characterizing population structure because it identified distinct sub-populations based on patterns of allele frequencies and, also, determined fractions of the genotype that belong specifically to each sub-population. structure has been shown to accurately infer individual ancestries and provide information on population relationships and history. the presence of these three populations was supported by the detection of genetic isolation among them, particularly between population ca and the two populations af and me that were separate but genetically closer to one another. this may reflect the geographical distance and barriers between the three main clusters. both types of analysis led to sub-division of the main clusters. congruence of the results supported tripartite clustering despite the absence of strong bootstrap values on the main branches of the distance tree. the two sub-groups in each of the three main populations were also supported by f-statistics, although the genetic differentiation was less pronounced, especially between the sub-populations af and af and between the sub-populations me and me wright maintained that even slight genetic differentiation among sub-populations is significant. within the context of this study, it seems that the populations af and me were more genetically diversified than the population ca. this might be due to different sampling procedures in the three areas. the strains from africa were collected between and in separated locations. in contrast to that, the me strains were collected between and from different foci encompassed by the eastern mediterranean region and arabian peninsula, and the strains from central asia between and in only countries. to correct, at least partially, for this sampling bias mean number of alleles have been calculated based on the mlmt profiles present in the three areas, e.g. by excluding profiles shared by more than one strain. when corrected for central asia, the smallest group, the lowest number of alleles was still found in central asia followed by the middle east and then africa. the sampling bias might still account for the high heterogeneity of the african strains, but the differences in diversity for ca and me strains cannot be explained only by differences in sampling. most of the me strains were collected in israeli and palestinian foci from a territory much smaller than the central asian area. the area from where the central asian strains of l. major studied here came is a landlocked generally arid plateau. the greater variation of habitats and biotopes within middle eastern environments seems to translate into a greater variety of animal hosts and sand fly vectors compared to central asia. where groups of strains with the same microsatellite profile were available, it was possible to make some assessment with regard to time and spatial distribution. for example, the strains in the groups of strains with the microsatellite profiles lmj lmj and lmj were isolated in a given area for each group at the same time or within short periods of time. however, strains with the microsatellite profile lmj lmj and lmj were isolated in several locations at different times. this, in addition to the observed low heterozygosity at microsatellite loci, seems to provide evidence against extensive and frequent genetic exchange within l. major. that microsatellite variants can persist for long periods of time and are restricted or widely spread in their distribution, supposedly, results from the effect of biotopic conditions on the animal hosts and vectors of leishmanial parasites and much less so in the case of the humans, who do alter the biotope by construction, agriculture and many of their other activities. most of the strains in the population me came from israel and the palestinian authority and their separation into the sub-populations me jordan valley-dead sea area, and me the negev-sinai area, did seem to parallel geographical distribution but not entirely so. most'misplaced' strains were from human cases and could be explained by travel and migration between these areas. several strains have membership coefficients for both sub-populations, indicating gene flow between them. the two african sub-populations did not correlate well with the geographical origin of the strains that fell into them. their analysis was hampered owing to the small number of strains available and their very wide geographical dispersal. unlike dps-based phenetic analysis, which is not affected by population size, structure improves with increasing sample size for each population. in this study, the results obtained using structure were consistent with those of the phenetic analysis. nevertheless, more strains from different african foci need to be analysed to really determine the population structure of african strains of l. major. preferably, one should use strains isolated from sand fly vectors and rodent hosts to avoid the effect of human migration. small sample size was also a problem in the case of the two single strains from iran and iraq and the two from western turkey that grouped together with the african strains and the eastern turkish strain that grouped with the middle eastern strains. waki et al. hypothesized that adaptation to the micro-environment in the macrophages of the mammalian hosts, the place of long-term residence of all leishmanial parasites, including l. major, is most significant in the evolution of the genus leishmania. they considered adaptation to the sand fly vectors' gut, the place of short-term residence, to be of lesser evolutionary importance. that the three main populations of l. major identified by this study exist in endemic foci that each have different rodent species serving as the reservoir hosts seems to support this hypothesis. variation among the strains of l. major from the same endemic area leading to different sub-populations could be attributed to differences in sand fly vector populations. for instance, analysis of mitochondrial cytb haplotypes exposed two genetically distinct populations of ph. papatasi in the middle east.  multilocus microsatellite typing based on different microsatellite markers and using a bayesian model-based analysis as well as phylogenetic reconstructions based on genetic distances, identified three main populations, corresponding to three separate geographical regions: central asia; the middle east; and africa. each of them separated into two sub-populations which might reflect the existence of different species of rodent host in these areas. the african and middle eastern populations seemed to be more genetically diversified than the central asian population. sampling bias might account for the high heterogeneity of the african strains but cannot explain the differences in diversity for central asian and middle eastern strains. 
multilocus microsatellite typing based on different microsatellite markers was applied to strains of l. major from different regions where it is endemic. on applying a bayesian model-based approach, three main populations were identified, corresponding to three separate geographical regions: central asia; the middle east; and africa. the model- and distance-based analyses of the microsatellite data exposed three main populations of l. major, central asia, the middle east and africa, each of which separated into two sub-populations. the two african sub-populations did not correlate well with strains' geographical origin. leishmania major, one of the agents causing cutaneous leishmaniasis in humans, is widely distributed in the old world where different species of wild rodent and phlebotomine sand fly serve as animal reservoir hosts and vectors, respectively. this was congruent with phylogenetic reconstructions based on genetic distances. to investigate the population structure of the species highly polymorphic microsatellite markers were employed for greater discrimination among it's otherwise closely related strains, an approach applied successfully to other species of leishmania. this probably correlates with the different species of rodent host. strains falling into the sub-populations ca and me did mostly group according to their place of isolation although some anomalies were seen, probably, owing to human migration.
 leishmania major, one of the agents causing cutaneous leishmaniasis in humans, is widely distributed in the old world where different species of wild rodent and phlebotomine sand fly serve as animal reservoir hosts and vectors, respectively. despite this, strains of l. major isolated from many different sources over many years have proved to be relatively uniform. to investigate the population structure of the species highly polymorphic microsatellite markers were employed for greater discrimination among it's otherwise closely related strains, an approach applied successfully to other species of leishmania.  multilocus microsatellite typing based on different microsatellite markers was applied to strains of l. major from different regions where it is endemic. on applying a bayesian model-based approach, three main populations were identified, corresponding to three separate geographical regions: central asia; the middle east; and africa. this was congruent with phylogenetic reconstructions based on genetic distances. re-analysis separated each of the populations into two sub-populations. the two african sub-populations did not correlate well with strains' geographical origin. strains falling into the sub-populations ca and me did mostly group according to their place of isolation although some anomalies were seen, probably, owing to human migration.  the model- and distance-based analyses of the microsatellite data exposed three main populations of l. major, central asia, the middle east and africa, each of which separated into two sub-populations. this probably correlates with the different species of rodent host. 