ST calculations, parsimony, and distance analysis demonstrated relationships among the breeds that largely reflect geographic origins and known breed histories. Low levels of population divergence were observed between breeds that are relatively early on in the process of breed development, and between those with high levels of within-breed diversity, whether due to large population size, ongoing outcrossing, or large within-breed phenotypic diversity. Populations with low within-breed diversity included those which have experienced population bottlenecks, have been under intense selective pressure, or are closed populations with long breed histories. These results provide new insights into the relationships among and the diversity within breeds of horses. In addition these results will facilitate future genome-wide association studies and investigations into genomic targets of selection.Horses were domesticated from the Eurasian steppes 5,000–6,000 years ago. Since then, the use of horses for transportation, warfare, and agriculture, as well as selection for desired traits and fitness, has resulted in diverse populations distributed across the world, many of which have become or are in the process of becoming formally organized into closed, breeding populations (breeds). This report describes the use of a genome-wide set of autosomal SNPs and 814 horses from 36 breeds to provide the first detailed description of equine breed diversity. F With a world-wide population greater than 58 million Prior genetic work aimed at understanding horse domestication has shown that a significant proportion of the diversity observed in modern maternal lineages was present at the time of domestication Diversity in the founding population of the domestic horse has since been exploited to develop a wealth of specialized populations or breeds. While some breeds have been experiencing artificial selection for hundreds of years , in general, most modern horse breeds have been developed recently  and continue to evolve based upon selective pressures for performance and phenotype . Horse bConsidering modern breeds, unlike mtDNA, nuclear markers can discern breed membership The Equine Genetic Diversity Consortium (EGDC), an international collaboration of the equine scientific community, was established in an effort to quantify nuclear diversity and the relationships within and among horse populations on a genome-wide scale. The development of this consortium has facilitated the collection of samples from 36 breeds for genotyping on the Illumina 50K SNP Beadchip. The breeds included in this report represent many of the most popular breeds in the world as well as divergent phenotypic classes, different geographic regions of derivation, and varying histories of breed origin . The staOf the 38 populations sampled, two breeds were represented by geographically distinct populations: the Thoroughbred was sampled in the both the United States (US) and the United Kingdom and Ireland (UK/Ire), and the Standardbred was sampled in the US as well as in Norway. Eight Standardbred horses sampled from the US were noted to be pacing horses as opposed to the Norwegian and remaining US individuals that were classified as trotters. In addition, the International Andalusian and Lusitano Horse Association Registry (IALHA) in the US maintains one stud book but designates whether the individual was derived from Spanish (Pura Raza Española) or Portuguese (Lusitano) bloodlines, or a combination of both. Of the Andalusian horses collected in the US, five were noted to have Portuguese bloodlines.Phenotypic classifications of the horse breeds include those characterized by small stature , breeds characterized by large stature and/or large muscle mass in proportion to size (draft breeds), light horse or riding breeds, gaited breeds, rare breeds, breeds founded in the past 80 years, and populations that are relatively unmanaged (“landrace”). The number of samples, sampling location, region of breed origin, and a list of primary breed characteristics are found in After pruning of individuals for genotyping quality and relationships see , and keeDiversity indices were calculated using 10,536 autosomal SNPs that remained after pruning for minor allele frequency (MAF), genotyping rate, and linkage disequilibrium (LD) across breeds (referred to as the primary SNP set). Diversity indices were also calculated using three other SNP sets, resulting from different levels of LD-based pruning see . Individe), ranged from 0.232 in the Clydesdale, to 0.311 in the Tuva (IS) calculated on the primary SNP set showed significant excess homozygosity in 17 populations, which was greatest in the Andalusian (0.065). Three of the four lowest FIS values were found in the Thoroughbred samples   .Inbreeding coefficients (f) calculated for each individual based upon observed and expected heterozygosity showed several individuals with significant loss of heterozygosity. The highest individual value of f (0.56) was found in an Exmoor pony. Within breeds, average individual estimates of f were greatest in the Clydesdale, Mangalarga Paulista, and Exmoor while the lowest breed means were found in the landrace populations .e), as estimated by LD e were observed in the Eurasian landrace populations, the Mongolian (743) and Tuva (533), and also in the Icelandic (555), Finnhorse (575), and Miniature (521). Breed-specific decay of LD essentially mirrors the results of the Ne calculation given the relationship between the statistics. A plot of LD across 2 Mb in a subset of the breeds that represent the range of Ne estimates is found in Effective population size  also serves to visualize individual relationships within and among breeds. The plot of PC1 vs. PC2 shown in An unrooted neighbor joining (NJ) tree of Nei’s distance Likelihood scores for runs of various K in Structure showed an increase in overall mean ln P(X|K) until K = 35 . A clearThe first breeds to have all individuals assign strongly to one cluster are the Thoroughbred and Clydesdale (with Shire) at K = 2, followed by the Shetland at K = 3; these four breeds do not show signs of admixture at any K value analyzed. Evidence of weak geographic grouping is observed at K  = 4, which consists of: 1, the Middle Eastern and Iberian breeds (pink); 2, the Thoroughbred and breeds to which it continues to be or was historically crossed (yellow); 3, breeds developed in Scandinavia and Northern Europe (orange); and 4, the British Isles draft breeds (blue) .i.e. K <6) the Florida Cracker, Saddlebred, Standardbreds, Morgan, and Tennessee Walking Horse fall into the cluster with the Iberian and Middle Eastern breeds with breed mean q >0.5. At K = 29, each of these breeds is assigned with q >0.72 to an individual cluster with the exception of the Lusitano and Andalusian, which remaining clustered together.As also observed in the NJ tree, clustering of the Iberian and Middle Eastern breeds with the Mangalarga Paulista, Peruvian Paso, and Puerto Rican Paso Fino (q >0.5) is observed until K  = 8, after which point the Mangalarga Paulista assigns with q  = 0.93 to another cluster. The remaining breeds cluster together until K = 12, at which time the Middle Eastern breeds  are assigned to their own cluster, leaving the Iberian breeds clustered with the Peruvian Paso and Puerto Rican Paso Fino. At low values of K  through K  = 5. However, unlike the NJ tree, at K  = 4, the highest value of assignment places the Belgian and Percheron into this cluster although with q <0.5 . The relationship remains until K  = 6, at which time the Miniature, Icelandic, and Shetland fall into a different cluster. At K  = 10, the Icelandic clusters again with the North Swedish Horse and Norwegian Fjord. The Norwegian and United States Standardbred populations, which at K  = 4 assign with q >0.5 to the cluster containing the Scandinavian breeds, separate from the Scandinavian breeds at K  = 5. At K  = 31, substructure appears in the Standardbred samples, which correlates to those individuals identified as pacers and that fall into an individual clade in the parsimony tree . At K  =The Clydesdale and Shire cluster together, and apart from the other breeds beginning at K  = 3. In addition, the Fell Pony, which is placed within the same clade in the NJ and parsimony trees, and proximal to the Clydesdale and Shire in PCA, shows moderate assignment to this cluster (0.29< q <0.41) for several values of K from 4 to 14. At K  = 29, the Shire assigns to the same cluster as the Clydesdale with q  = 0.69. The individual outliers from the Shire breed also noted in parsimony analysis are evident beginning at K  = 3. Excluding these outliers, at K  = 29, the proportion of assignment for the Shires to the cluster with the Clydesdale increases to 0.74.ST values calculated between the 37 populations (excluding the Florida Cracker) were significant as tested by 20,000 permutations (ST  = 0.002), while the greatest divergence was observed between the Clydesdale and Mangalarga Paulista (FST = 0.254). The two Thoroughbred populations had an FST value of 0.004, while the two Standardbred populations had 10-fold greater divergence (FST  = 0.020) than the minimum observed value in this dataset; this value is similar to that observed between the Lusitano and Andalusian (0.021). An FST value of 0.006 was identified between the Tuva and Mongolian populations. The global FST value was 0.100. AMOVA computed on the set of 37 samples (excluding the outliers identified in Structure and the Florida Cracker) showed that 10.03% of the variance was accounted for among populations (p = <0.001), 0.53% of the variance was among individuals within populations (p = 0.19), and 89.44% of the variation was within individuals (p<0.001).All pairwise Futations . The lowThese data are gathered from populations that represent tremendous diversity in phenotype and breed specialization. With breeds sampled across four continents, the resulting relationships observed largely reflect similarities of geographic origin, documented breed histories, and shared phenotypes. In general the highest within breed diversity was observed in breeds that are recently derived, continue to allow introgression of other populations, those that have a large census population size, and landrace populations that experience a lesser degree of controlled breeding. Not surprisingly, low diversity is observed in breeds with small census size, relatively old breeds with closed populations, and those with documented founder effects, whether due to population bottlenecks or selective breeding.A total of seven individuals were identified by parsimony and cluster analysis as outliers with respect to the breed to which they were assigned. The pedigrees of these individuals were unknown. Because it is possible these horses were unknowingly crossbred or subject to mishandling in the field or laboratory, they were excluded from the within-breed analyses to avoid potential bias in indices of diversity. In addition, the potential impact of SNP ascertainment bias on diversity calculations must be acknowledged. The reference genome is from a Thoroughbred mare 2>0.2 as well as MAF of 0.05 or above. Even though additional markers could have been used for analysis, many population-level statistics assume independence of loci. The stringent pruning for LD was therefore undertaken to eliminate bias in the test statistics that may result from substantial breed-specific differences in LD 2 = 0.2 and r2 = 0.4 (using pairwise correlation), and one replicate setting the threshold to R2<0.1 (using the variance inflation factor), to examine the effect of allowing for varying levels of LD and therefore varying numbers of loci .Using LD-based calculations, the estimated NST of all breeds in the study. While these results could be due to geographic distance between this and other breeds, and/or genetic drift, unfortunately these horses were all sampled from only two farms and likely do not represent the entirety of the diversity present in the breed; therefore we cannot rule out sampling error which would inflate the estimated level of divergence between these individuals and the other breeds and result in a decrease in He. However, a lack of diversity in sampling of the breed would not have an effect on estimates of individual inbreeding coefficients, which were among the highest of the entire data set.The Clydesdale and Exmoor, in addition to having high individual estimated coefficients of inbreeding, also show the lowest within-breed diversity observed in the dataset. A lack of diversity in the Clydesdale and another British draft breed, the Shire, is likely a result of a severe population bottleneck observed in most draft breeds with the onset of industrialization and after the conclusion of World War II (WWII) as well as selection for size and color e and Ne, accompanied by low estimates of inbreeding (f and FIS), are observed in the Mongolian, Tuva, and New Forest Pony. The Mongolian and Tuva are unique in that they represent landrace populations that are less managed than the popular breeds of Western Europe and North America; they occupy a diverse range of habitat, have been selected for meat and milk in addition to use in transportation, and originate in the region where domestication was likely to have occurred. The population of Mongolian horses is large and individuals are phenotypically diverse Converse to the above examples, high levels of diversity as measured by both HOther population characteristics are likely the cause of the diversity observed in the Finnhorse, Icelandic, and Miniature. In the case of the Icelandic, the high level of diversity was possibly maintained by a large census population size despite isolation for almost a thousand years and several population bottlenecks due to natural disasters e for these breeds account for only a fraction of the census size, demonstrating non-random mating and selection. Outcrossing is also continued in the Swiss Warmblood and Hanoverian breeds, which show similar trends in diversity measures as the Quarter Horse and Paint. The relatively low Ne in these breeds, accompanied by moderate He may partially be due to significant crossing with the Thoroughbred, which would contribute long blocks of LD e.All of these three factors, large population size, phenotypic diversity within the breed, and a diversity of founding stock, also lead to the relatively high levels of diversity observed in the Paint and Quarter Horse; in addition, these breeds both allow continued outcrossing between themselves and with the Thoroughbred and have experienced a tremendous population expansion since the formal foundation of the breeds within the past 45–75 years. Due to the relative infancy of these populations, it could be argued that the Paint, Quarter Horse, and other, newly-derived breeds, have not yet had time to undergo the evolutionary processes necessary to be genetically distinct populations as is observed in breeds with longer histories and closed studbooks. However, even with high within-breed diversity and large census population sizes , Ne, and low to moderate estimates of f, FIS values in each breed are significantly positive. Significant FIS was also previously observed in the Iberian breeds using microsatellite markers IS in some of these breeds, another instance in which FIS may be significantly positive is in the presence of subpopulation structure within the sample. Evidence of this in the Lusitano and Andalusian is present in parsimony analysis where individuals of the two breeds fall into one clade, but within that clade are two highly supported branches represented by a subset of each breed. In addition, when forcing high values of K in Structure, such as observed at K  = 35, Andalusian and Lusitano individuals fall into one of two clusters with q-value >0.5 in a nonbreed-specific manner (data not shown). These results support IS). Unlike Standardbreds in Europe, which are raced at a trot, those in the US are divergently selected for racing at either the pace or the trot, creating structure within the breed Of note in breeds such as the Quarter Horse, Lusitano, and Andalusian, is that despite moderate to high relative levels of He, He, and estimates of f, given its rarity. After rediscovery of the breed, which historically was believed to represent a type of landrace population, Finally, several rare populations are included in this dataset. The Caspian is one of the oldest breeds in the Middle East and was thought to be extinct until its recent rediscovery in 1965. The Florida Cracker, a now rare breed, was developed in the United States from feral stock of Iberian descent ST values, parsimony, PCA, and NJ dendograms.The expectation of homogeneity within breeds due to closed populations and selection is supported by the results of AMOVA, which show significant variation present among populations, but a non-significant proportion of variance within. However, the variation among samples lends information about current and historic relationships. Observed trends include patterning based upon geographic origin and/or phenotypic similarities, and relatively low divergence observed in comparisons that include breeds with high within-breed diversity. In Structure analysis, K  = 29 was chosen as the most likely value of K; however no single value stood out as the “best” number of clusters to describe these data. Regardless, patterns observed in clustering were also supported by pairwise FST value of 0.006. In addition, with the exception of six Tuva individuals that fell into a clade with the Caspians, both parsimony and NJ analyses place the Tuva and Mongolian into the same clade of each tree. Examining all comparisons, low FST values were observed between the Mongolian and Tuva compared to the other breeds in this study, supporting the potential role of Eurasian horses of similar type in founding modern stocks. This also aligns with high microsatellite diversity observed in Eastern Eurasian “non-breed” (landrace) populations in ST, while those with low diversity show higher values of FST. Low divergence in breeds with high diversity is expected as variation within a breed may indicate outcrossing with other populations, and high variation also makes these breeds more likely to share variation with others by chance. In contrast, if a breed has little within-breed variation, it is less likely to share genetic variation with another breed by chance, especially with another breed that is relatively homogeneous itself. As demonstrated in human literature, source populations are expected to contain greater diversity than those populations which they found ST values of Tuvas and Mongolians supports their role in founding modern breeds, the same argument could be made for the Quarter Horse, which also shows low levels of pairwise divergence; however that argument would be unreasonable as the Quarter Horse was developed in only the past century. Therefore, the relative values of FST are informative, but these FST values and data, which represent modern breeds generally derived from limited founding stock, and subjected to intense artificial selective pressures, cannot be used independently to elucidate the evolution of the modern horse. Also, as is the case for other analyses, while the relationships observed can shed light on the history of breeds, they cannot distinguish between recent admixture and shared ancestry.The Mongolian and Tuva populations are believed to have been influential in the spread of horses across Asia and Europe th and 17th centuries, as well as the general use of the term “Arab” for horses of Middle Eastern descent, it is likely other “Arabian” horses with influence on the Thoroughbred breed also had Turkoman, and Barb bloodlines ST values between the Thoroughbred and the Arabian do not suggest any less divergence than observed between the Thoroughbred and a majority of the other breeds. In addition, at K = 29 the Arabian assigns to the Thoroughbred cluster at only 2.3%. If the Arabian did have significant influence on the Thoroughbred breed, there are several possible explanations for why the supposed Arabian influence is not more apparent. The first is related to SNP ascertainment and the bias of SNPs toward modern variation in Thoroughbred. It is possible that the genes derived from the Arabian are at or near fixation in the Thoroughbred, which would reduce the chance that these SNPs, and the variation described within them are present in the dataset. Another possibility is that the current Arabian sample, taken from the United States, may not reflect the Arabian lineage(s) that were influential in the founding of the Thoroughbred. Finally, as noted above and also suggested elsewhere The Thoroughbred is believed to have founding sires of Arabian, Turk, and Barb ancestry ST of 0.004, similar to that observed between the Hanoverian and Swiss Warmblood (0.008) and Mongolian and Tuva (0.006), but larger than the minimally observed value seen between the Paint and the Quarter Horse (0.002). Although artificial insemination is prohibited in the Thoroughbred and would be anticipated to limit gene flow to some extent, the founder effect in the original European Thoroughbred by few high-impact sires and dams, accompanied by shared selective pressures, relatively recent importation of the breed to the United States, and ongoing shipment of horses between continents are likely contributing to the lack of geographic population structure identified by parsimony and cluster analyses.Within the Thoroughbred itself, divergence between the US and European samples had a significant FST values as well as greater than 30% assignment of the Paint and Quarter Horse to the Thoroughbred cluster. As each of these breeds experience continued gene flow from the Thoroughbred, and had Thoroughbred founding stock, these results are not unexpected. While outcrossing of these breeds is allowed (with restrictions), it is likely that even in cases of breeds with closed stud books, some outcrossing, intentional, unintentional, and/or undocumented, has occurred; it has been demonstrated that historical pedigrees, while helpful, are not always accurate While within-breed diversity of the Thoroughbred was relatively low, and a notable relationship with the Arabian was not observed, among-breed analysis shows a clear influence of the Thoroughbred on many other breeds. Placed with the Thoroughbred in parsimony analysis are the Hanoverian, Maremmano, and Swiss Warmblood. The Maremmano, an Italian breed, shows a q-value of assignment to the Thoroughbred clade of 0.26 at K  = 29. This is not surprising given reports that Thoroughbreds contributed over 13% of the maternal lineages to the stallion lines within the stud book ST value of 0.020, five-fold greater than that observed between the two Thoroughbred samples. This comparison may reflect geographic structure, the influence of French Trotter bloodlines in the European sample, and selection for pacing horses in the US population. Within the horses included in this study, eight were noted to be pacers. These pacers all fall within one clade of the parsimony tree, supported by a bootstrap value of 98%. The limited sample size does not allow a thorough comparison of the pacing vs. trotting Standardbreds, however significant genetic differentiation between horses of the two racing groups has been reported Finally, the Standardbred samples, which also represent two continents, had a significant pairwise FThe Middle Eastern breeds, Arabian, Akhal Teke, and Caspian, were placed into a single clade of the NJ tree, with the Arabian and Akhal Teke in their own, highly supported clade. This relationship was supported by low values of K in Structure, which placed the Iberian and Middle Eastern breeds into the same cluster until K  = 12. However, parsimony analysis did not support this relationship between the Middle Eastern and Iberian breeds as the Arabian and Akhal Teke individuals were placed into one clade, apart from the Iberian samples and from the Caspians.ST value observed in either breed, 0.021. The parsimony tree and PCA also shows that individuals cannot necessarily be distinguished from one another regardless of whether they were sampled in the US or Portugal. Of the two clades that appear to suggest population substructure, one includes only Portuguese Lusitanos while the other includes only US samples, although two are of Portuguese ancestry.In Europe, the Iberian breeds  have only recently been distinguished from one another depending upon the region in which they are bred and divergent selective pressures. In the US, horses of each breed are occasionally interbred A horse that is considered to be “gaited” naturally moves in a means other than the traditional walk, trot, canter, and gallop. Alternative gaits in horses are distinguished from traditional gaits by their unique footfall pattern and/or rhythm. The genetic basis of gait has recently been investigated and suggests that all modern gaited breeds share a common ancestor as supported by a shared, extended haplotype spanning a variant significantly associated with the ability to pace ST value of 0.037. The Fell Pony, a British breed, falls out as sister taxa to the British draft horses, the Shire and Clydesdales. However FST values show that divergence between the Fell Pony and either the Clydesdale or Shire is not significantly less than seen with most other populations. The other branch of the “draft” clade of the NJ tree contains the breeds from the European mainland, the Belgian, Percheron, and Franches-Montagnes; each of these breeds shows monophyly in parsimony analysis. In addition, similarities among draft and light draft breeds are reflected in cluster analyses at K  = 6, which show the populations from the European mainland and Scandinavian Peninsula  assign to one cluster with q >0.5. The grouping of the Scandinavian breeds is similar to that previously reported The Shire and Clydesdale populations share assignment to the same cluster throughout Structure runs. The similarity between these breeds is also seen in a lack of monophyly in the parsimony tree, the sharing of a branch of the NJ tree, positioning in PCA, and a pairwise FThis data set resulting from a large international collaboration represents the first study in the horse to provide an extensive overview of nuclear genetic diversity within, and relationships among a diverse sample of breeds and landrace populations. These data are now available for use in subsequent studies of population-level relationships and provide a baseline for monitoring changes in breed diversity. With high mtDNA diversity but limited paternal input during domestication, this increased understanding of nuclear diversity within the horse will allow for the identification of genomic regions of importance to breed derivation and will be instrumental in guiding across-breed gene discovery projects.DNA sampling was limited to the collection of blood by jugular venipuncture performed by a licensed veterinarian or from hairs pulled from the mane or tail by the horse owner or researcher. All animal work was conducted in accordance with and approval from the international and national governing bodies at the institutions in which samples were collected ; the University of Kentucky IACUC; the University College Dublin, Animal Research Ethics Committee; Swiss Law on Animal Protection and Welfare; the Ethical Board of the University of Helsinki; the Animal Health Trust Clinical Research Ethics Committee; Norwegian Animal Research Authority; UK Home Office License; and the Lower Saxon state veterinary office).Tissue samples and previously collected genotypes from 1,060 horses were obtained from members of the EGDC or were obtained by our laboratory. 814 samples representing the 38 populations included in this study were selected from the EDGC sample collection with the goal of obtaining as random of a sample as possible and to minimize close relationships among individuals. In some cases, genotypes were available from breeds collected for genome-wide association studies (GWAS). In all cases, when pedigree information was available, no relationships were allowed at or more recent to the grandsire/dam level. If no pedigrees were available, once genotyping was performed, individuals were removed from the analyses to reduce genome sharing as measured by autosomal estimates of identity by descent (pi hat) values in PLINK 2<0.1 in PLINK 2<0.1 in Plink , which is similar to the above method but instead of analyzing pairwise relationships of SNPs as in the former method, uses a multiple regression approach upon the SNPs in the analysis window. Files were converted for usage between analyses programs using PLINK, perl script, CONVERT SNP discovery was conducted using horses from seven breeds  as well as the reference genome of a Thoroughbred mare e) and AMOVA were calculated in Arlequin3.5 IS was calculated and significance tested on the primary data set, with 10,000 permutations of the data in Genetix Expected heterozygosity  cladogram was built using breed allele frequencies calculated from the primary SNP set using the packages seqboot, gendist, neighbor, and consense and Nei’s genetic distance 2<0.2 (–indep-pairwise). The domestic ass was included as an outgroup for traditional and new-technology searches in TNT A parsimony cladogram was constructed using 10,066 SNP markers pruned from the original data set using the MAF and genotyping rate criteria as above and allowing for Rhttp://cran.r-project.org) on the full SNP set consisting of all 814 individuals and 38,755 autosomal SNPs (pruned only for MAF and genotyping rate).Principal component analysis (PCA) was conducted in snpStats in R (Clustering of breeds into genetic groups was examined using the program Structure 2.3.3 e), the full set of 54,602 SNP markers was pruned within each population to remove those with MAF <0.01 and genotyping rate of <0.05. Pairwise r2 values between remaining SNPs were calculated in Haploview e were calculated using the method of To estimate effective population size  for the purpose of reconstructing the analyses. The only exception is the data collected from the Tennessee Walking Horse, which, under agreement from the granting agency  and the Tennessee Walking Horse Foundation (TWHF)), is only available under a Material Transfer Agreement (MTA) between interested individuals and the University of Minnesota.Figure S1Example of LD decay over 2 Mb in 9 breeds of horse. Decay of linkage disequilibrium over 2Mb for 9 of the 36 breeds. Landrace populations such as the Mongolian, and large and/or diverse breeds such as the Finnhorse and Quarter Horse, show more rapid decay than those with small population sizes and less diversity .(TIFF)Click here for additional data file.Figure S2Parsimony relationship among Lusitano and Andalusian individuals. Portion of the parsimony clade shown in (TIFF)Click here for additional data file.Figure S3Principal component 1 vs. 2 as determined from 814 horses from 38 populations. Principal components 1 and 2 as determined from 38,755 SNPs (pruned for MAF and genotyping rate). All 814 individuals are included in the plot.(TIFF)Click here for additional data file.Figure S4Mean of estimated ln P(X|K) for each run in Structure. Mean of estimated ln P(X|K) for each of the three runs for K = 1−45 in Structure.(TIFF)Click here for additional data file.Figure S5Bayesian clustering output for additional values of K in 814 horses of 38 populations. Structure output for additional values of K. Each individual is represented by one vertical line with the proportion of assignment to each cluster shown on the y axis and colored by cluster.(TIF)Click here for additional data file.Figure S6Parsimony relationship among Thoroughbred horses from the US and UK/Ire. The branch of the parsimony clade shown in (TIF)Click here for additional data file.Figure S7Parsimony relationship among Standardbreds from the US and Norway. The branch of the parsimony clade shown in (TIFF)Click here for additional data file.Table S1Cluster to which each population maximally assigns and corresponding q-value for K = 2 to 45. Highest breed q-value of assignment and cluster identity (#) for each value of K examined in Structure. The cluster ID # is not carried through across values of K.(PDF)Click here for additional data file.Table S2Proportion of assignment for 38 horse populations to each of K = 29 clusters. Proportion of assignment to each of K = 29 clusters as determined in Structure. The largest proportion of assignment for each population is outlined and shown in bold; those with 30–50% assignment are shown in italic. The top row notes the breed(s) with >50% of assignment to each of the 29 clusters. This analysis was performed without removal of outlier individuals.(PDF)Click here for additional data file.


Platycladus orientalis (Cupressaceae) is an old-growth tree species which distributed in the imperial parks and ancient temples in Beijing, China. We aim to (1) examine the genetic diversity and reproductive traits of old-growth and young populations of P. orientalis to ascertain whether the older populations contain a higher genetic diversity, more private alleles and a higher reproductive output compared with younger populations; (2) determine the relationships between the age of the population and the genetic diversity and reproductive traits; and (3) determine whether the imperial parks and ancient temples played an important role in maintaining the reproductive capacity and genetic diversity of Platycladus orientalis.Samples from seven young (younger than 100 yrs.) and nine old-growth (older than 300 yrs.) artificial populations were collected. For comparison, three young and two old-growth natural populations were also sampled. Nine microsatellite loci were used to analyze genetic diversity parameters. These parameters were calculated using FSTAT version 2.9.3 and GenAlex v 6.41.P. orientalis have significantly higher genetic diversity than younger artificial populations and similar levels to those in extant natural populations. The imperial parks and ancient temples, which have protected these old-growth trees for centuries, have played an important role in maintaining the genetic diversity and reproductive capacity of this tree species.The old-growth artificial populations of  Picea rubens) have shown positive relationships among the age, stem diameter, height and genetic and reproductive status of old-growth and older stands of P. rubens, and old-growth forests may serve as reservoirs of genetic diversity and seed sources to maintain populations under the pressures of climate change and population fragmentation Old-growth tree populations are assumed to be potential resources of genetic diversity and high reproductive output; however, there is little relevant research and empirical evidence for this assertion Ginkgo biloba, Chinese jujube (Ziziphus jujuba), Sophora japonica, Pinus tabulaeformis, and Pinus bungeana. The largest number of these trees (53.8%) consists of Platycladus orientalis (Cupressaceae), an evergreen tree species that originated in China, and that can live for centuries. Natural populations of Platycladus orientalis are rare and restricted to Beijing, Hebei, Shanxi and Shaanxi provinces P. orientalis is an afforestation tree species which is widespread and naturalized throughout China and as an introduced species in both North Korea and eastern Russia P. orientalis have focused on its chemical components P. orientalis populations can help to determine the differences between these populations and provide empirical evidence for the assertion that the old-growth populations are genetically diverse and potential sources for obtaining seeds for conservation and reintroduction purposes.A great number of imperial parks, tombs and ancient temples were established in Beijing, an old city with a long history as the capital of China. These sites provide a long-term stable environment (hundreds of years) for long-lived trees and protect the genetic diversity of these trees. Beijing City has the largest number of long-lived trees in the world. In a survey of ancient and famous trees in 2007, 28 families, 47 genera, 66 species and 39408 old-growth trees were found, 6122 of which were older than 300 years P. orientalis to investigate the levels of genetic diversity and reproductive traits in old and younger artificial populations and compare these levels to those observed in five natural populations. We hypothesize that older populations will harbor higher genetic diversity than younger populations. Through this study, we will better understand the importance of protecting old-growth trees, both for their cultural significance as a biological archive P. orientalis and to (2) determine the relationships between the age of the population and the genetic diversity and reproductive traits.Here we use 2  =  10.047; p  =  0.002). There was no significant relationship between the average value of the DBH of a population and the percentage of individuals with cones in the artificial populations.Of the 241 individuals (52.28%) of the old-growth populations, 126 produced cones, whereas only 69 of the 187 individuals (36.90%) of the 100-yr. age group displayed cones. In the 300-yr. age group, the proportion of individuals with cones was significantly higher than the 100-yr. age group . Seed length  and weight  for these populations were positively correlated with the average values of the DBH (There were no significant differences in the seed weight between the eight sampled populations (data not shown), except for the TMA population, which was significantly longer  and heavier  than the other seven populations ( the DBH : Older pThe genetic diversity parameters of these sampled populations are shown in p > 0.1). The allele range of Group 2 was significantly higher than that of the other three groups (p < 0.05). The Ho, HE, NA, NE, I and AR values for Group 1 were significantly lower than those of the other groups (p < 0.05), but no difference was detected in the allele range between Group 1 and natural Groups 3 and 4. The private alleles of these groups did not differ significantly.There was no difference between the old-growth artificial tree populations (Group 2) and natural populations (Groups 3 and 4) for the Ho, He, Na, Ne, I and AR parameters , Shannon’s information index , allele richness  and allele range    were not as large as the ancient artificial individuals of   0.015) . There w  0.006) .P. orientalis. According to our results for seed trait measurements and microsatellite experiments, the 300-yr. artificial age group exhibited the highest reproductive traits and can represent most of the genetic diversity of this species. In addition, the 300-yr. artificial age group and the natural groups showed much higher levels of genetic diversity than the artificial 100-yr. age group.Old-growth tree populations can provide long-term stable environments for other species, such as animals and other plants, and forests can play an important role in the soil and water retention Pinus tabulaeformis) and Robinia pseudoacacia forests and some forest shrub, with some of the P. orientalis trees even growing on rocks. Therefore, their growth status, as based on the diameter at breast height and crown, and reproductive output, as based on the cone set, were lower than the artificial trees in the parks and other urban locations, which have adequate space, water and nutrients and were very well protected. Although the fruit-setting percentage of tree populations may have a positive relationship with the DBH Factors affecting reproductive success include fruit yield, seed weight and size, and seed quality directly determining the perpetuation of seed plant species P. orientalis.Seed weight and size have a significant impact on seed germination, whereby the larger or heavier the seed is, the earlier and faster it germinates, which can be associated with higher reproductive fitness E, AR, I, allele range and Ho; P. orientalis population growth and genetic diversity. The higher genetic diversity in the artificial 300-yr. age group showed that the resources of individuals in these populations may be quite extensive In long-living forests, the genetic diversity parameters could be significantly correlated with the tree growth traits, such as the DBH and height P. orientalis. In contrast, the genetic diversity parameters for the artificial young populations were significantly lower than other populations, indicating that, although the number of artificial young P. orientalis was the largest, in terms of the genetic diversity, this group was much less important than the old-growth and natural populations.Almost all of the mean values for the genetic diversity parameters of the old-growth artificial populations were similar to those in the natural populations , indicating that these ancient artificial populations contain similar levels of the genetic diversity of natural populations of P. orientalisNone of the genetic estimates obtained for the natural 100-yr. and 300-yr. age groups were notably different, and there were no relationships found between these estimates and the population average height or DBH, which may have resulted from the small number of sampled natural individuals. In Beijing, the amount of natural populations were limited, despite the large numbers of individuals of As the number of people living in large cities increases with the modern-day urbanization process, the restoration, protection and enhancement of the biodiversity in urban areas become more important The potential negative influences of artificially cultivated plants on the gene pool of the species also require attention: the genetic contamination of natural populations may produce negative effects on the fitness of the natural plants as a result of outbreeding depression P. orientalis populations in the urban areas can serve as reservoirs for genetic diversity and seed sources for this species None of the genetic diversity parameters obtained from the old-growth artificial populations sampled in our study differed from those of the natural populations, and some of them showed an even higher level of genetic diversity than the populations in nature. Moreover, the artificial 300-yr. age group had a greater reproductive capacity than the young artificial and natural populations. These results suggest that the old-growth As garden plants, the artificial populations maintained alleles similar to the nearby natural populations and may hold a higher level of genetic diversity Platycladus orientalis.These royal gardens, imperial tombs and ancient temples are in a solemn atmosphere and thus they protected these old trees which have been given extraordinary significance from deforestation, and indirectly protected their valuable genes. These urban areas play an important role in maintaining seed availability and genetic diversity of According to the records from the parks and temples P. orientalis with a Plant Genomic DNA Kit . Nine microsatellite loci and the same PCR condition as in reference R) and allele range. These parameters were calculated using FSTAT version 2.9.3 Total genomic DNA was extracted from silica gel–dried leaves from individuals’ specimen of The average numbers of the population trait - DBH were calculated. To determine whether older populations harbor higher genetic diversity, we estimated the correlation between DBH, a proxy of age in this species, and genetic diversity parameters using bivariate correlations in SPSS version 16.0. The relationships among the population average DBH and seed size were also analyzed by bivariate correlations. The differences in genetic diversity parameters between the age groups were analyzed using a one-way ANOVA, followed by post-hoc pairwise comparisons between groups using a Bonferroni correction. The differences among groups for reproductive capacity were examined using the Chi-square test.


Sus scrofa) is one of the most widely distributed mammals in Europe. Its demography was affected by various events in the past and today populations are increasing throughout Europe. We examined genetic diversity, structure and population dynamics of wild boar in Central and Eastern Europe. MtDNA control region (664 bp) was sequenced in 254 wild boar from six countries . We detected 16 haplotypes, all known from previous studies in Europe; 14 of them belonged to European 1 (E1) clade, including 13 haplotypes from E1-C and one from E1-A lineages. Two haplotypes belonged respectively to the East Asian and the Near Eastern clade. Both haplotypes were found in Russia and most probably originated from the documented translocations of wild boar. The studied populations showed moderate haplotype (0.714±0.023) and low nucleotide diversity (0.003±0.002). SAMOVA grouped the genetic structuring of Central and Eastern European wild boar into three subpopulations, comprising of: (1) north-eastern Belarus and the European part of Russia, (2) Poland, Ukraine, Moldova and most of Belarus, and (3) Hungary. The multimodal mismatch distribution, Fu's Fs index, Bayesian skyline plot and the high occurrence of shared haplotypes among populations did not suggest strong demographic fluctuations in wild boar numbers in the Holocene and pre-Holocene times. This study showed relatively weak genetic diversity and structure in Central and Eastern European wild boar populations and underlined gaps in our knowledge on the role of southern refugia and demographic processes shaping genetic diversity of wild boar in this part of Europe.The wild boar ( Sus scrofa originated in South-East Asia, where the genus Sus differentiated about 3 million years ago and from where S. scrofa spread throughout Asia, Europe and North Africa The wild boar It is, however, not clear which of these sources of refuge contributed the most to the re-establishment of the current population in Europe, especially in its eastern part. Continental Europe is populated by wild boar belonging to two major haplogroups; clade E1 is widespread throughout the entire continent and clade E2 is restricted to the Italian Peninsula, Sardinia and Croatia However, the genetic affinity of wild boar populations from east and northwest part of Europe has not yet been thoroughly studied. th–19th centuries when the combined effects of climate cooling (Little Ice Age) and overexploitation by humans reduced the population numbers and distribution in many regions th century, re-colonization from populations in Poland, Hungary, Slovakia and possibly the Ukraine started. After World War II, the density and geographical distribution of wild boars increased throughout most of Europe and by the middle of the 20th century wild boar populations were restored In addition to the impact of past glaciation, the contemporary phylogeographic profile of wild boar in Europe could have been affected by more recent events. In Eastern Europe, demographic decline in wild boar occurred in the 17In the last 50 years, a rapid increase in wild boar numbers was observed across Europe The most important event in shaping the pattern of genetic diversity of Western and Central European wild boar was the last glaciation, which was followed by a sudden demographic and spatial expansion of the populations The objective of our study was to characterize genetic diversity, structure, and phylogenetic relationships among Central and Eastern European populations of wild boar using the mitochondrial (mtDNA) control region. Specifically, we aimed at: (1) describing mtDNA variability with a reference to haplotype diversity observed in the rest of Europe and Asia, (2) determining the genetic structure of the populations and (3) understanding if currently observed genetic diversity and structure have a signature of past, post-glacial demographic expansion. We used wide-range geographic sampling over six countries of Central and Eastern Europe to give the first comprehensive genetic characteristics of the wild boar populations from this part of Europe.In total, 254 tissue samples were collected in 2007–2010 in six Central and East European countries  using two primers: forward Ss.L-Dloop: Haplotype and nucleotide diversities, and the number of polymorphic sites were calculated with DnaSP 5.00 jModeltest 2.1.4 Sus barbatus as outgroups (Genbank accession numbers AJ314540 and GQ338953). The best model resulted the HKY model The best-fit model of nucleotide substitution was selected across 288 candidate models using For spatial analyses of the population structure, we divided our sampling area into 8 regions: (1) northern Poland, (2) southern Poland, (3) the Polish part of the Białowieża Forest, (4) the Belarusian part of the Białowieża Forest, (5) most of Belarus; (6) eastern Belarus and western Russia, (7) Ukraine and Moldova, and (8) Hungary . SamplesWe assessed population structure of mtDNA using spatial analysis of molecular variance – SAMOVA −8 mutation rate, 1,5 years generation time was used in BEAST 1.6.1. We used Arlequin 3.1 to test the hypothesis of a past population expansion by calculating Fu's and Tajima's statistics Our alignment (664 bp) of 254 wild boars from Central and Eastern Europe yielded 43 polymorphic sites . They reWe identified 16 haplotypes, and called them H1-H16 . The secCT for all structuring scenarios, except for K = 2, genetic differentiation among populations within groups ΦSC was the lowest and significant for K = 3 . All other pairwise comparisons were insignificant. This is an effect of the low number of shared haplotypes between S1 and other groups and the dominance of H2 within group S1. Optimal spatial structure of the analysed sequences consisted of three subpopulations within the sampling region . Althougor K = 3 . Group SA Bayesian phylogenetic tree and median-joining network were constructed using our sequences and 598 wild boar and domestic pig sequences from GenBank  and 4. TOverall, we obtained negative and non-significant Fu's Fs value and negative non-significant Tajima's D value . For allAnalysis of the prehistorical population size dynamics in Central and Eastern Europe showed slowly declining population number and a sudden recent increase . The absWe present the first comprehensive data on mtDNA diversity and genetic structure of the wild boar populations in Central and Eastern Europe. In our sample of 254 wild boar from six countries, we detected a total of 16 haplotypes. Other studies revealed 16 haplotypes in 98 Italian wild boar and only 7 haplotypes in 47 other European countries  The haplotype diversity for our studied populations (0.714) was lower than those reported for two European wild boar studies Based on data from 38 sampling locations in Europe (Russia excluded), spatial variation in haplotype diversity of wild boar was analysed In our study, we found one individual belonging to Near East haplogroup and one from East Asian haplogroup (both in Russia). Most probably, these two are signs of past translocations. Twenty-two individuals from northern Caucasus  wider sampling, especially to cover the critical areas of Dinaric – northern Balkan region and southern parts of Ukraine and European Russia, which are candidate regions for the LGM refugia of the contemporary Eastern European wild boars, and (2) applying additional genetic tools such as microsatellite markers and genome sequencing to study wild boar populations at pan-European scaleCentral and Eastern wild boar mainly belong to the European E1-C lineage  and only 5% of individuals represented the E1-A lineage. Two animals from Russia had haplotypes typical of East Asian and Near East lineages, and they most likely were traces of past translocations.SAMOVA suggested three genetic subpopulations of wild boar, comprising: (1) north-eastern Belarus and the European part of Russia, (2) Poland, Ukraine, Moldova and most of Belarus, and (3) Hungary.The multimodal mismatch distribution, Fu's Fs index, and Bayesian skyline plot and the occurrence of many shared haplotypes among the populations did not show evidence for strong demographic fluctuations in wild boar numbers in the Holocene and pre-Holocene times.To fully understand the history and to determine the LGM refugia of the extant populations of wild boar Central and Eastern European, it is essential to sample the Dinaric – northern Balkan region, southern Ukraine, and southern portions of Russian Plains, and to study the genetic profile of wild boar by means of microsatellite loci and genome sequencing.Table S1List of the wild boar and domestic pig mtDNA sequences used in this study. Sequences were downloaded from GenBank or obtained by the authors of this study. Clades: A - East Asia, E1 - European 1, E2 - European 2, NE and ME - Eastern . In European 1 (E1) clade, two sides are distinguished: (A) and (C).(PDF)Click here for additional data file.Table S2The haplotypes of wild boar Sus scrofa detected in this study and variable nucleotide positions in relation to a reference sequence from GenBank, accession no. AJ002189.(DOC)Click here for additional data file.Table S3CT: fixation index among groups; ΦSC: fixation index among populations within groups; ΦST: fixation index within populations.Results of SAMOVA, Φ(DOC)Click here for additional data file.


Rhizophora mangle and Avicennia germinans, along the Pacific and Atlantic coasts of Mexico, we set out to test whether concordant genetic signals could elucidate recent evolution of the ecosystem. Genetic diversity of both mangrove species showed a decreasing trend toward northern latitudes along the Pacific coast. The lowest levels of genetic diversity were found at the range limits around the Gulf of California and the outer Baja California peninsula. Lack of a strong spatial genetic structure in this area and recent northern gene flow in A. germinans suggest recent colonization by this species. On the other hand, lack of a signal of recent northern dispersal in R. mangle, despite the higher dispersal capability of this species, indicates a longer presence of populations, at least in the southern Gulf of California. We suggest that the longer history, together with higher genetic diversity of R. mangle at the range limits, likely provides a gene pool better able to colonize northwards under climate change than A. germinans.Mangrove forests in the Gulf of California, Mexico represent the northernmost populations along the Pacific coast and thus they are likely to be source populations for colonization at higher latitudes as climate becomes more favorable. Today, these populations are relatively small and fragmented and prior research has indicated that they are poor in genetic diversity. Here we set out to investigate whether the low diversity in this region was a result of recent colonization, or fragmentation and genetic drift of once more extensive mangroves due to climatic changes in the recent past. By sampling the two major mangrove species,  Cyclical climatic oscillations during the last few million years have had a great effect on biota, causing isolation, genetic subdivision and speciation in some cases and/or hybridization and homogenization in others Populations are dynamic in space and time, and peripheral populations are often of special conservation concern as they are predicted to experience local extinctions at a greater rate, and to have reduced recolonization potential due to smaller population sizes and more limited dispersal Mangroves are highly productive tropical ecosystems that support numerous food chains in the coastal zone and neighboring ecosystems Rhizophora mangle L. experienced range limit oscillations during glacial – interglacial cycles, to the extent that modern ranges are still constrained by post-glacial re-colonization along the Brazilian coast The global distribution of mangrove marsh is mainly influenced by temperature, restricting species to warm tropical and subtropical latitudes R. mangle in northern Mexico are a result of recent colonization or fragmentation and genetic drift of once more extensive mangroves due to climatic changes in the recent past. Recently colonized populations are expected to harbor decreased levels of genetic diversity Mexican mangroves cover 770,057 ha Recent colonization, or fragmentation of older populations could have important consequences as to the likelihood of these northernmost populations providing well-adapted seed sources for colonization at higher latitudes as climate changes in the future.A. germinans and R. mangle) to interpret demographic and biogeographic processes along the Pacific and Atlantic coasts of Mexico.Therefore, we hypothesize that Gulf of California populations of Mexican mangroves are recent colonizations following climatic restriction southwards during the last glacial. Propagule sources for northward advance along the northern Pacific coast would most likely be dominated by leading edge populations in this narrow linear system. Assuming a contiguous dispersal model  over non-contiguous long distance dispersal, founder populations should exhibit low genetic diversity and in this stepping stone system low genetic divergence Permission to collect samples in each location was obtained from General Directorate for Wildlife .A. germinans and 600 of R. mangle was collected from mangrove forests along the Pacific and the Atlantic coasts of Mexico. Sampling data of R. mangle from the Baja California peninsula and the Gulf of California have already been reported in a previous work Leaf tissue from 448 individuals of A. germinansR. manglea priori, to be neutral with respect to natural selection. The forward primers were fluorescent-labeled with FAM, VIC, PET and NED (Applied Biosystems Inc). All amplifications were performed on a MyCycler BIORAD thermal cycler in 20 μL PCR reactions containing 1×Buffer , 2.0 mM MgCl2, 200 μM of each dNTP, 0.15 μM of each primer, 1 unit Taq DNA polymerase (SIGMA) and 20 ng of genomic DNA. With the exception of some small variations on annealing temperatures, all microsatellite loci were amplified with a similar thermocycler profile. The profile consisted of an initial denaturation step at 95°C for 5 min, followed by 35 amplification cycles as follows: 95°C for 30 s, annealing temperature , for 30 s and 45 s at 72°C, ending with an extension cycle at 72°C for 30 min. To ensure reproducibility and consistency in PCR amplification, approximately 5% of samples were re-amplified. In addition, a negative control was run for each set of PCR reactions and genotyped to check for contamination. Amplified products were run on an ABI 310 automated DNA sequencer, and microsatellite alleles were visualized and scored in the program GeneMarker 1.97 (Softgenetics).Individuals were genotyped at seven loci  previously designed for E) and observed heterozygosity (HO) for each locus across all localities using GDA 1.1 To determine spatial patterns in genetic diversity that might confirm latitudinal trends consistent with recent colonization northwards, we estimated the number of alleles (A), unbiased expected heterozygosity  ST) pRST) . This analysis compares the observed RST value (before randomization) with the distribution of pRST values obtained for all possible configurations of allele size permutations. If observed RST is within the upper 5% of the distribution of pRST the contribution of mutations to population differentiation is non-negligible compared with genetic drift and migration Genetic differentiation among populations was evaluated at each locus and over all loci by calculating the measures of relative genetic differentiation among populations defined under the infinite allele model  following Rousset To test the significance of Isolation By Distance (IBD), the correlation between genetic and geographic distance matrices was tested using a Mantel test with 2000 permutations A. germinans, of which 35.5% were exclusive to the Atlantic, 35.5% to the Pacific and 29% were common to both coasts. For R. mangle, only 34 alleles were detected, of which 11.8% were exclusive to the Atlantic, 38.2% to the Pacific and 50% were shared between both coasts.Microchecker analyses showed no evidence of null alleles nor allelic drop out that might interfere with posterior analysis on genetic diversity. We detected 93 alleles in A. germinans and R. mangle  of genetic diversity (HO) versus latitude as a covariate  was significantly larger  than differentiation based on allele identities (FST = 0.47 and 0.54), indicating that stepwise mutations contributed to overall among-population differentiation. However, when we only analyzed populations from the northwestern coast , in both mangrove species RST was not significantly higher than FST , suggesting that more recent evolutionary processes of migration and genetic drift were responsible for differentiation over this geographic range.In general, overall pairwise values of FTRUCTURE . AccordiA. germinans, isolation by distance was significant over all sampling locations along the Pacific coast and over the more restricted geographic range of the northwestern coast  ranged from 0.83 to 1.81 and from 0.00 to 0.26 (MIGRATE and BAYESASS respectively)  and “Marismas Nacionales” (MN), including Central Pacific (CP) as a control . Evolutictively) , whereas to 0.05 . BayesiaToday, mangroves in the Gulf of California and along the Pacific coasts of the Baja California peninsula form the northern limit of this ecosystem in the eastern Pacific. Populations are relatively small and fragmented and prior research has indicated that they are poor in genetic diversity A. germinans from the Gulf of California and those from “Marismas Nacionales” and Central Pacific, consistent with Gulf of California populations being the product of recent founder events, for example the BC population represented by the red color in the STRUCTURE analysis. For A. germinans, this hypothesis was supported by estimates of recent directional migration inferred using the program BAYESASS, which showed a predominantly northward migration from “Marismas Nacionales” to the Gulf of California.We found that broad patterns of genetic diversity along the Mexican Pacific coast were consistent for both species; a linear decrease in genetic diversity with latitude, becoming impoverished in the Gulf of California. Although patterns of genetic diversity were broadly similar, demographic processes inferred from population structure and directional migration rates revealed a more complex evolution of populations and the ecosystem. For both species, populations from “Marismas Nacionales” and the Central Pacific were highly admixed and levels of admixture declined into the Gulf of California , 4. BaseR. mangle shared very little recent ancestry with those of “Marismas Nacionales” and Central Pacific and we found no detectable recent migration among these populations. This would seem to indicate: 1) A longer presence of R. mangle in the Gulf of California, 2) Confounding due to unsampled source populations, 3) Effects associated with enhanced dispersability of R. mangle propagules. We were careful to sample most populations along the Pacific mainland coast and, although extinct populations could have been the source of Gulf populations, it seems unlikely that they would have been genetically distinct from “Marismas Nacionales” and Central Pacific. Today “Marismas Nacionales” is one of the more extensive areas of mangrove forests along the Mexican Pacific coast. Therefore, we find it unlikely that unsampled source populations will have confounded our results. Our data do indicate that R. mangle propagules are more effectively dispersed than those of A. germinans. Whereas, southern Mexican Pacific populations of A. germinans formed a discrete cluster in the STRUCTURE analysis, those of R. mangle were admixed with “Marismas Nacionales” and Central Pacific populations. The intervening coastline between central and southern populations is not suitable for mangrove establishment, so long-distance dispersal appears to have been more effective for R. mangle. Nevertheless, there is no evidence of shared ancestry between Gulf of California populations of R. mangle and those from the southern Mexican coast to suggest a source of founder propagules for Gulf populations. We find the most likely explanation of the contrasting demographic patterns between A. germinans and R. mangle is that populations of the latter species have been present somewhere in the Gulf of California longer than A. germinans. Interestingly, evolutionary rates of migration estimated from MIGRATE, also yielded contrasting results; no significant rates of migration between the Gulf of California and “Marismas Nacionales” or Central Pacific for A. germinans, but significant southward migration from the Gulf to “Marismas Nacionales” for R. mangle. Taken together, these data support a scenario of glacial restriction followed by Holocene latitudinal advance for A. germinans, and possible persistence of populations of R. mangle somewhere in the Gulf of California during the harsh conditions of the last glacial maximum.Despite comparable latitudinal trends in genetic diversity, we found contrasting demographic signals in the two species. Gulf of California populations of A. germinans along the mainland of the Gulf of Mexico advanced and went extinct during climatic cycles of the Pleistocene. Today, whereas both species are common components of Floridan mangroves, A. germinans extends further north than R. mangle. However, along the Pacific coast, R. mangle has the northernmost distribution A. germinans was about twice as great in the Atlantic compared with the Pacific coast, but that mean genetic diversity was about equal for R. mangle. This is in part, due to greater sharing of alleles between the two coasts in R. mangle; R. mangle shared almost 50% of alleles between Atlantic and Pacific populations, while A. germinans shared around 29% of alleles. This could be an effect of higher mutation rates at the studied loci in A. germinans, or of more extensive early dispersal of R. mangle before closure of the Central American Isthmus (CAI). Genetic structure analysis showed the highest genetic differentiation among populations from the Atlantic and the Pacific coasts, supporting previously reported molecular data Whereas, we detected a clear linear trend of decreasing genetic diversity among populations of both species along the Pacific coast, no trend was detected along the Atlantic coast. Indeed, STRUCTURE assigned all individuals from the Atlantic to single clusters for each species. These opposing trends support our hypothesis that a stronger pattern of decreasing genetic diversity would be expected along the Pacific coast because of the more linear distribution of mangroves and inclusion of the populations from the range limit here. Sherrod and McMillan R. mangle extends further north than A. germinans along the Pacific coast. As discussed earlier, the patterns of genetic diversity that we observed suggest a longer presence of R. mangle in the Gulf of California and the Baja California peninsula. This could be explained by earlier colonization of R. mangle because of its more effective dispersal capabilities R. mangle, however, this would not explain the presence of genetic structure in both mangrove species. Therefore, we prefer an interpretation that populations of R. mangle persisted, perhaps in the lower Gulf, through the last glacial, which could explain the greater genetic diversity observed in R. mangle than A. germinans along the Gulf of California. However, it is difficult to explain why R. mangle has been more successful than A. germinans at the range limit along the north-western coast of Mexico. The answer may lie with shoreline topography, which may propitiate local extinction of mangroves on coastlines with no gradual gradient A. germinans hindsighted onto last glacial maximum climate from the PMIP database, suggests that mangroves could have survived along the Sinaloa coastline and into the lower Gulf of California through the last glacial (Dodd unpublished data). This is also supported by fossil record of prop roots of A. germinans in BC population dating from the Pliocene R. mangle over A. germinans because the former is more tolerant to changes in the hydroperiod, which have occurred periodically through time Today, A. germinans through northward propagule dispersal, perhaps from relictual stands around present-day “Marismas Nacionales”. On the other hand, R. mangle showed no signal of recent northern dispersal despite the greater dispersal capability of this species, suggesting the presence of populations at least in the southern Gulf of California for a longer time. Future climate change could favor colonization at higher latitudes, but the source propagules must be well adapted to the arid and highly saline conditions. Populations with a longer history at a site are likely to be better adapted to local conditions than founders from populations under more benign conditions. Under this scenario, R. mangle would serve as a better adapted gene pool for latitudinal advances under climate change, even though A. germinans showed evidence of more successful northward migration in recent time.In conclusion, our data provide compelling evidence that the Gulf of California and outer Baja California were recently colonized by


During the evolution and differentiation of human populations, different functional genes and pathways  would have displayed different degrees of genetic diversity or evolutionary conservatism. To query the genetic differences of functional genes or pathways in populations, we have developed the human gene population genetic difference (HGPGD) database. Currently, 11 common population genetic features, 18,158 single human genes, 220 KEGG (Kyoto Encyclopedia of Genes and Genomes) human pathways and 4,639 Gene Ontology (GO) categories  are available in the HGPGD database. The 11 population genetic features are related mainly to three aspects: allele frequency, linkage disequilibrium pattern, and transferability of tagSNPs. By entering a list of Gene IDs, KEGG pathway IDs or GO category IDs and selecting a population genetic feature, users can search the genetic differences between pairwise HapMap populations. We hope that, when the researchers carry out gene-based, KEGG pathway-based or GO category-based research, they can take full account of the genetic differences between populations. The HGPGD database (V1.0) is available at  Any two unrelated individuals share about 99.9% of their genomic DNA sequence. The approximately 0.1% that is different is important in explaining some human phenotypic differences, such as skin color Furthermore, because genes often act in groups to perform a specific biological function or cellular process, one or a few genes may not represent the functional status of an entire system The genetic differences of single genes, KEGG pathways and GO categories were calculated based on the SNP genotype data in the International HapMap project. Currently, 11 sample populations are in the HapMap database. The 11 populations are: African Americans from the American Southwest (ASW), Utah residents with Northern and Western European ancestry from the Centre d'Étude du Polymorphisme Humain (CEPH) collection (CEU), Han Chinese in Beijing, China (CHB), Chinese in Metropolitan Denver, Colorado (CHD), Gujarati Indians in Houston, Texas (GIH), Japanese in Tokyo, Japan (JPT), Luhya in Webuye, Kenya (LWK), Mexican ancestry in Los Angeles, California (MEX), Maasai in Kinyawa, Kenya (MKK), Toscans in Italy (TSI), and Yoruba in Ibadan, Nigeria (YRI). A sample population of 1,117 unrelated individuals was selected from the HapMap populations The human gene position information was obtained from the ‘‘seq_gene’’ file on the NCBI ftp website. In the HGPGD database, we used 18,158 autosomal genes that contained at least two SNPs in all 11 populations to calculate the population genetic differences. A total of 11 common population genetic features are included in the HGPGD database. The 11 features are mainly related to three genetic aspects: allele frequency, linkage disequilibrium (LD) pattern and transferability of tagSNPs (SNPs in a region of the genome with high LD).For each gene region, two features are related to allele frequency. (1) MAF (maf): The average differences of allele frequency for each gene region between pairwise HapMap populations were measured. The minor allele in the ASW population was used as the reference. The minor allele frequencies in the ASW population and the frequencies of the same allele in the other populations were calculated. For each gene region, we defined the allele frequency difference 2 (r2): The LD coefficient r2 between pairwise SNPs was calculated. (2) D prime (D’): The D’ between between pairwise SNPs was calculated. (3) Block number: The Four Gamete Test For each gene region, six features are related to LD pattern. (1) The LD coefficient r2 threshold was 0.8. (2) Captured percent: For any two populations, A and B, if a SNP in population A exhibited a pairwise r2>0.8 with at least one tagSNP selected from population B, then the SNP was defined as a SNP that was captured by population B 2: The average maximum r2 was defined as the average value of the maximum r2 between tagSNPs in population A and SNPs captured by these tagSNPs in population B. The differences in tagSNP transferability were calculated as: For each gene region, three features are related to tagSNP transferability. (1) Tag percent: The tag percent is the percentage of the number of tagSNPs compared to the total number of SNPs. The tagSNPs were identified using the TAGGER panel in HaploView. The rHuman genes are not independent of each other and genes in the same functional pathway often act together to perform specific biological tasks. Under the action of natural selection, different functional pathways have evolved to display different degrees of genetic differences. The HGPGD database provides the option to query genetic differences in the KEGG pathways in the different HapMap populations.The KEGG pathways database has been widely used for the systematic analysis of gene functions that involve networks of molecular interactions in cells The genetic differences for a KEGG pathway were obtained by combining the differences of all the genes in that pathway. In the previous section, we described how the genetic differences of single genes were measured. For pathways, the same weight was assigned to the genes in that pathway and genetic difference scores were calculated separately for each of the 11 features. Genetic difference scores for allele frequency were defined as:The GO database provides a controlled vocabulary of terms to define biological descriptors (GO categories) and to support biologically meaningful annotation of gene products In the HGPGD database, each GO category is seen as a basic functional unit. The same weight was assigned to genes in the same GO category and the genetic difference scores were calculated. The genetic difference scores for the GO categories were calculated as described above for the KEGG pathways.The HGPGD database is a freely available database that focuses on population genetic differences in human genes. The current version (v1.0) of the HGPGD database contains 18,158 single human genes, 220 KEGG human pathways and 4,639 GO categories. For each gene, KEGG pathway or GO category, users can obtain the differences in 11 common genetic features between the 11 HapMap populations. For each of 18,158 single genes in the HGPGD database, by entering a list of Gene IDs, and selecting a population genetic feature, users can obtain the genetic differences between each pairwise HapMap population.When a list of Entrez Gene IDs is entered and “allele frequency” is selected as the query term , the seaThe KEGG pathway search page is similar to the search page for a single gene. When a list of KEGG pathway IDs is entered and a “genetic feature”  is selected as the query terms, the genetic difference results are displayed on a separate page. The search results include detailed information about the pathway (i.e. pathway name and number of genes in the pathway), and by clicking on the link, the matrix of the genetic differences, reference distribution and boxplot are displayed.The GO category search page is similar to search page for the KEGG pathway. When a list of GO IDs is entered and “genetic feature”  is selected, the genetic difference results are displayed on a separate page. The search results include detailed information about the GO category (i.e. GO category name and number of genes in the GO category), and by clicking on the link, the matrix of genetic difference, reference distribution and boxplot are displayed.Human populations have been subjected to a large number of demographic events, such as migration, population expansion and colonization, and, as a result, different populations have been exposed to many different environments. These demographic events together with evolutionary events (such as mutation and selection) have had an effect on the human genome, leading to the population genetic differences that we see today


Schistosoma japonicum still causes severe parasitic disease in mainland China, but mainly in areas along the Yangtze River. However, the genetic diversity in populations of S. japonicum has not been well understood across its geographical distribution, and such data may provide insights into the epidemiology and possible control strategies for schistosomiasis.Oncomelania snails were collected from areas in the middle and lower (ML) reaches of the Yangtze River, including Hubei, Hunan, Anhui, Jiangxi and Jiangsu provinces, and in the upper reaches of the river, including Sichuan and Yunnan provinces in southwest (SW) China. The adult parasites obtained from experimentally infected mice using isolated cercariae were sequenced individually for several fragments of mitochondrial regions, including Cytb-ND4L-ND4, 16S-12S and ND1. Populations in the ML reaches exhibited a relatively high level of diversity in nucleotides and haplotypes, whereas a low level was observed for populations in the SW, using either each single fragment or the combined sequence of the three fragments. Pairwise analyses of F-statistics (Fst) revealed a significant genetic difference between populations in the ML reaches and those in the SW, with limited gene flow and no shared haplotypes in between. It is rather obvious that genetic diversity in the populations of S. japonicum was significantly correlated with the geographical distance, and the geographical separation/isolation was considered to be the major factor accounting for the observed difference between populations in the ML reaches and those in the SW in China.In this study infected S. japonicum in mainland China exhibits a high degree of genetic diversity, with a similar pattern of genetic diversity as observed in the intermediate host snails in the same region in China. Schistosoma japonicum has not been investigated across its wide geographical distribution in China, and such information may provide insight into the disease epidemiology and the development of its control measures. In this study, the adult parasites, obtained through infecting mice with cercariae from snails of the genus Oncomelania collected from a wide range of localities in currently endemic areas of schistosomiasis in the middle and lower (ML) reaches of the Yangtze River, and in Sichuan and Yunnan provinces in the upper reaches of the river in southwest (SW) China, were sequenced individually for mitochondrial genes. In general, a relatively high degree of genetic variation was observed in populations in the ML reaches in terms of nucleotide and haplotype diversity, but a low level was observed in populations in the SW. The significant difference in genetic diversity as revealed by F-statistics, and the existence of no shared haplotypes, were observed between populations in the ML reaches and those in the SW, indicating the effect of geographical separation/isolation upon the schistosomes and probably the parasite-snail system in China.Despite the existing threat of schistosomiasis in some rural areas along the Yangtze River, the genetic diversity of  Schistosoma still infecting more than 200 million people in the world S. japonicum in respect with its genetic diversity, host immune response etc. when compared with other schistosomes Schistosomiasis is one of the most neglected tropical diseases, with six species in the Oncomelania, which is the intermediate host of S. japonicum, was classified into different species and/or subspecies according to their morphology, biogeography and phylogeny Oncomelania which has been verified using various markers S. japonicum is of great interest for research from a co-evolutionary point of view. How diverse the digenean S. japonicum really is in such a large geographical range has not been well assessed especially in mainland China. An accurate measure of its population genetic diversity is certainly needed to clarify our understanding on the epidemiology of schistosomiasis The genus S. japonicum populations. Gasser et al. S. japonicum. Sorensen et al. S. japonicum populations from 6 localities in mainland China using NADH dehydrogenase subunit 1 (ND1) gene, but could not detect variability conclusively at the intrapopulation level. Bøgh et al. S. japonicum in mainland China, but provided very limited information relating to the population genetic diversity of this species. Upon the identification of polymorphic microsatellite loci, Shrivastava et al. S. japonicum populations from 8 geographical locations in 7 endemic provinces across mainland China, and a high level of polymorphism was reported between and within populations. They considered that populations of S. japonicum in mainland China could be separated mainly into the populations in Sichuan and Yunnan provinces as being in southwest (SW) China and those in low-lying lake regions along the middle and lower (ML) reaches of Yangtze River. With three partial mitochondrial genes  from 28 individual adult worms, Zhao et al. S. japonicum from a wide range of its geographical distributions, especially in severely endemic areas along the ML reaches of Yangtze River in China.In recent years, several molecular markers have been used to detect the variability of S. japonicum collected from localities in seven provinces of China, where schistosomiasis is geographically endemic. The diversity in nucleotides and haplotypes was analyzed for different populations based on each of the three mitochondrial sequences and their combined sequences. Phylogenetic tree and parsimony network were constructed for observed haplotypes, and the genetic distance was examined against the geographical distance in order to understand the genetic diversity in populations of S. japonicum in mainland China.In this study, mitochondrial DNA sequences including Cytb-ND4L-ND4, 16S-12S and ND1 were examined for The procedures involving animals were carried out in accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). The animal study protocol was approved by the Institutional Animal Care and Use Committee of Wuhan University.Oncomelania hupensis, from 18 localities of 7 schistosomiasis endemic provinces in mainland China, including Hubei, Hunan, Anhui, Jiangxi, Jiangsu provinces in the ML reaches of Yangtze River, and Sichuan and Yunnan provinces which are in the higher reaches of the river in SW China, but separated from the ML reaches by mountain ranges  and Pengze in Jiangxi province (JXpz) where only three and four infected snails were obtained respectively, were exposed to light for 4 hours to stimulate the emergence of cercariae. Five laboratory mice were infected percutaneously with 30 cercariae per mouse for each geographical locality. 6 weeks following the infection, adult worms were retrieved by perfusion from mesenteric veins using 0.9% NaCl, and worms from each mouse infected with cercariae were pooled together, and washed extensively in saline before being preserved in 95% ethanol at 4°C.The intermediate host, n ranges , were coThe total genomic DNA was extracted individually from both male and female schistosomes using a standard sodium dodecyl sulfate-proteinase K procedure 5′- TTGGGGGTTGTCATGCGGAGTA -3′) and the reverse primer ND4R (5′- CAAATACCCAATAGCAACGGAACAC -3′) were used based on available GenBank sequence AF215860. For the ND1 fragment, the forward primer ND1F (5′- TAGAGGGTTTGTTGGTTGTTTTG -3′) and the reverse primer ND1R (5′- ACCATACTTTCATACTACTGCC -3′) were used based on available GenBank sequence AF215860. For the 16–12S fragment, the forward primer 16S-12SF (5′- GATTATTTCTAGTTCCCGAATGG -3′) and the reverse primer 16–12SR (5′- TGTAACGCACAACAACCTATACC -3′) were used based on available GenBank sequence AF215860. The PCR protocols were 94°C for 3 min followed by 30 cycles of 94°C for 30 s, 58°C (for ND1) or 63°C (for Cytb-ND4L-ND4 and 16S-12S) for 30 s, and 72°C for 90 s and then a final elongation step at 72°C for 10 min. The amplified products were purified on 1.0% agarose gel stained with ethidium bromide, using the DNA gel extraction kit (Omega Bio-Tek). The purified PCR products were sequenced using ABI PRISM BigDye Terminators v3.0 Cycle Sequencing (ABI). The DNA sequences were deposited in the GenBank database under accession numbers FJ851893–FJ852573.For each adult worm, three fragments, i.e. Cytb-ND4L-ND4, ND1 and 16S-12S of the mitochondrial genome were sequenced. For the Cytb-ND4L-ND4 fragment, the forward primer ND4F  values. Selective neutrality was tested with Tajima's DF test F-statistics (Fst) based on gene flow (Nm). A Mantel g-test to compare the correlation between pairwise distance and geographical distance among localities was analyzed in Arlequin, with geographic distances (km) for the correlation analysis between geographical distance and genetic distance calculated using the great circle distance between localities.Nucleotide divergences within and between populations were calculated in Arlequin3.11 6 generations with random starting tree, and four Markov chains  sampled every 100 generations. Posterior probability values were estimated by generating a 50% majority rule consensus tree following the discard of first 3000 trees as part of a burn-in procedure. The HKY+I+G model was determined as the best-fit model of sequence evolution by using the hierarchical likelihood ratio tests implemented in Modeltest 3.7 Schistosoma mansoni as outgroup.The phylogenetic analysis for 96 haplotypes generated using combined mitochondrial DNA data was performed with Bayesian inference (BI), which was carried out with MrBayes 3.1 The genetic structure was phylogenetically evaluated by constructing unrooted parsimony network of haplotypes for combined mtDNA data sets using TCS version 1.21 The primary sequence data were obtained by amplifying and sequencing three partial regions of the mitochondrial genome, i.e. Cytb-ND4L-ND4 with 793–794 bp, ND1 with 767 bp, and 16S-12S with 1463–1466 bp. Measures of diversity of haplotypes and nucleotides within populations on the basis of the three mitochondrial regions are presented in Fst) for all 18 populations for Cytb-ND4L-ND4  and 16S-12S , respectively, which indicates that genetic distance increased with the increase in geographical distance (P>0.05) (P>0.05) and 0.061 (P>0.05) for Cytb-ND4L-ND4 and 16S-12S, respectively  . Howeverectively , implyinS. japonicum, as indicated either by Tajima's D or Fu's F test (P>0.05) in each of the three regions.Although some base substitutions were observed, selective neutrality of the observed nucleotide polymorphisms was suggested for Fst of all pairwise analyses varied from 0.482 to 0.870 between populations in the ML reaches and those in the SW (P<0.001). Among the 3 populations in the SW, the Fst between SCxc and two Yunnan populations (YNey and YNhq) showed highly significant differences (P<0.001), whereas no significant difference was observed between YNey and YNhq (P>0.05). Among the 15 populations in the ML reaches, the Fst varied from 0.014 to 0.807 (P<0.05). When all specimens were classified into two populations according to whether they were from above or below the three Gorges region, i.e. population in the ML reaches of the Yangtze River and population in Sichuan and Yunnan provinces of the SW China, the value of genetic distance (Fst) and the gene flow (Nm) between them was 0.381 (P<0.001) and 0.410, respectively.As many studies have shown that longer genes contain generally more variable characters with proportionally more signals, and hence yield accurate phylogenetic estimates than shorter ones n the SW , showingto 0.807 , with moFst) among all 18 populations for combined mtDNA , indicating that genetic distance increased with the increase in geographical distance (P>0.05)  , implyinAs shown in the Bayesian phylogenetic tree , two claThe network constructed by statistical parsimony from 96 haplotypes on the basis of combined mtDNA sequences showed some characters as observed in the phylogenetic tree. The haplotype network was rather complicated, without any obvious lineages for those haplotypes from localities in the ML reaches . HoweverS. japonicum populations was demonstrated in samples collected from currently epidemic areas of schistosomiasis in mainland China, with the use of three mitochondrial fragments, Cytb-ND4L-ND4, ND1 and 16S-12S, respectively, and the combined sequences of these three fragments. The present study contains the mostly widespread and the largest number of S. japonicum populations in any attempts so far to examine the parasite genetic diversity in China. Overall, populations of S. japonicum in mainland China showed a relatively large degree of variation in terms of nucleotide and haplotype diversity. However, it is apparent that across the geographical distribution of schistosomiasis endemic areas in China, the genetic distance was correlated significantly with geographical distance when Cytb-ND4L-ND4, 16S-12S, and combined mtDNA were used, although non-significance was observed for ND1. It is even more obvious that as revealed through analyses of nucleotide and haplotype diversity, populations in Hubei, Hunan, Anhui, Jiangxi and Jiangsu provinces, namely in the ML reaches of Yangtze River showed a much larger degree of genetic variation than those in Sichuan and Yunnan provinces of the SW China in the upper reaches of the river, and no haplotypes were shared between populations in the ML reaches and those in the SW. Significant difference was also observed in genetic distance between populations in the ML reaches and populations in the SW, as revealed in pairwise analyses using individual and/or combined mitochondrial sequences.The difference in genetic diversity of S. japonicum is geographically separated by the gorge area of the river. This apparent geographical separation may account for the observed difference in no-shared haplotypes, and in the genetic distance for S. japonicum between areas in the ML reaches of Yangtze River and areas in the SW China. When populations from the ML reaches and from the SW were further grouped separately, the Fst value (0.381) was greater than 0.25, a value which was considered to be ‘very great’ by Wright S. japonicum, due to at least the geographical separation by the Three Gorges area and mountains. Phylogenetic analyses and haplotype network may support this conclusion, as parasites from Sichuan and Yunnan provinces in the SW were all closely clustered in the phylogenetic tree and the haplotype network. Using different molecular markers, other authors S. japonicum populations in the SW and those in the flood plain of the ML reaches of the Yangtze River.Along the Yangtze River, are the endemic areas of schistosomiasis, and severe epidemic areas are mainly in the ML reaches Fst of pairwise analyses using ND1, 16S-12S, and the combined mtDNA sequences, with the exception of Cytb-ND4L-ND4. Sichuan and Yunnan provinces are both distributed in Hengduan Mountains, and schistosomiasis was reported historically in various localities in these two provinces S. japonicum in this region at a large geographical scale. However, only three populations were included in the present study and efforts to obtain more parasite samples have been unsuccessful, although the intermediate host snails were collected in a much wider range (unpublished data), due possibly to the continuous and extensive practices in either snail control or human chemotherapy in the two provinces. Thus, whether there is an effect of geographical isolation on populations of S. japonicum in this mountainous area will likely remain unknown, and whether the observed low level of genetic variation in these populations resulted from a recent bottleneck effect as a consequence of intensive control practices may also remain to be answered.Despite the finding that the mean nucleotide and haplotype diversity of populations in the SW were rather low when compared with the same parameters in the ML reaches, the genetic distance had some significant difference between the population from Sichuan, SCxc, and the two populations from Yunnan, YNey and YNhq, as revealed by S. japonicum in mainland China S. japonicum might have experienced frequent gene flows in most populations in this region Click here for additional data file.Table S2Within-locality diversity of Schistosoma japonicum from 18 localities in mainland China based on ND1 fragment.(XLS)Click here for additional data file.Table S3Within-locality diversity of Schistosoma japonicum from 18 localities in mainland China based on 16S-12S fragment.(XLS)Click here for additional data file.Table S4Geographical population pairwise genetic distance (Fst) and gene flow (Nm) based on ND1.(XLS)Click here for additional data file.Table S5Geographical population pairwise genetic distance (Fst) and gene flow (Nm) based on 16S-12S.(XLS)Click here for additional data file.Table S6Geographical population pairwise genetic distance (Fst) and gene flow (Nm) based on Cytb-ND4L-ND4.(XLS)Click here for additional data file.