Calonectris diomedea) is a pelagic seabird that has recently established three new colonies in Galicia (NE Atlantic) thus expanding its distribution range 500 km northwards. This study aimed to describe the establishment and early progress of the new Galician populations and to determine the genetic and morphometric characteristics of the individuals participating in these foundation events. Using 10 microsatellite loci, we tested the predictions supported by different seabird colonization models. Possibly three groups of non-breeders, adding up to around 200 birds, started visiting the Galician colonies in the mid 2000’s and some of them eventually laid eggs and reproduced, thus establishing new breeding colonies. The Galician populations showed a high genetic diversity and a frequency of private alleles similar to or even higher than some of the large historical populations. Most individuals were assigned to several Atlantic populations and a few (if any) to Mediterranean colonies. Our study suggests that a large and admixed population is settling in Galicia, in agreement with predictions from island metapopulation models of colonization. Multiple source colonies imply that some birds colonizing Galicia were dispersing from very distant colonies (> 1500 km). Long-distance colonizations undertaken by relatively large and admixed groups of colonizers can help to explain the low levels of genetic structure over vast areas that are characteristic of most oceanic seabird species.Seabirds are colonial vertebrates that despite their great potential for long-range dispersal and colonization are reluctant to establish in novel locations, often recruiting close to their natal colony. The foundation of colonies is therefore a rare event in most seabird species and little is known about the colonization process in this group. The Cory’s shearwater ( Colonization, defined as the process leading to the establishment of a population in a novel location, is essential for the persistence of species, especially in a changing world  where raAn analysis of historical species introductions has suggested that in birds the probability of establishment increases with the number of founders . Importasensu Forbes and Kaiser [The great majority of seabird species breed colonially . Hence d Kaiser ) and havd Kaiser –31. Thusd Kaiser –34. The Phalacrocorax carbo over Denmark and the Netherlands [Fulmarus glacialis across Britain [Phoebastria immutabilis in the North Pacific Ocean [Rissa tridactyla in Eastern Canada [Several models have been proposed in the seabird literature to explain the formation of new colonies : (i) Thrherlands . (ii) Th Britain , 37. , is the largest of procellariids in the North Atlantic  [C. d. borealis and C. d. diomedea) [Our study model, the Cory´s shearwater  , 43 or siomedea)  is curreiomedea) . Severaliomedea) , but viriomedea) , thus exIn this study we aimed at: (i) describing the establishment and early progress of the new Cory´s shearwater colonies in Galicia; (ii) analyzing the genetic diversity and the number of alleles private to these new populations using microsatellites as genetic markers; (iii) determining the most likely sources (geographic origin) of the individuals participating in these founding events by drawing evidence from genetic markers and morphological characteristics. We were particularly interested in testing which of the three models of colony formation best fit our observations, as they are expected to produce contrasting patterns in the genetic diversity and the number of private alleles in the new populations .We made 168 captures of 128 different individuals, including 9 chicks. A bird observed inside a burrow in Cíes in May 2007 is the first known record indicative of the breeding of Cory´s shearwaters in NW Iberia. Later in that year a few birds were reported prospecting in Coelleira and by 2011, three small colonies and a few scattered burrows were active in three insular localities: Cíes, Sisargas and Coelleira . A totalFelis catus) in 2010. In Cíes, American mink (Neovison vison) predated on European shags nesting close to the area where the shearwaters first established in 2009 and 2010.In any given year, the occupation of burrows varied from full occupation (successful breeders) to sporadic occupation by a single prospector. Overall, the number of AOB´s increased from 24 to 61, albeit in the first years only a few burrows were occupied by laying pairs. The proportion of active burrows occupied by breeders and the number of chicks produced increased over the study period both in Coelleira and Cíes . Thus, i2,14 = 14.75; P < 0.001) and did not vary significantly across years  or months .The number of birds assembling in rafts near the colony fluctuated somewhat from year to year in the three colonies but without a clear trend . The larThe genetic statistics showed that the diversity of the new Galician populations was high and within the range of values of potential source populations . NotablyThe estimated number of private alleles per locus  as evaluated by the rarefaction method implemented in the computer program ADZE indicated that Selvagens possessed the highest number of private alleles, and that Azores showed the smallest values . The valFST values ranged from 0.015 to 0.246  with an average FST = 0.109 (CV = 1.42). These values represent moderate levels of population differentiation  or without  the Galician colonies; the correlation was however, significant for the subset of historical Atlantic colonies .Pairwise ntiation . After tThe first axis of the factorial correspondence analysis (42% of variance) clearly split Atlantic and Mediterranean populations, whereas the second axis (17% of variance) separate Selvagens from the rest . The oldK = 2) was considerably better than alternate models. These two clusters broadly corresponded to the main geographical areas, Atlantic and Mediterranean. Mean membership coefficients (Q) for all populations into one of these two demes were high , with Selvagens (0.76) and Chafarinas (0.68) as lowest . Four Galician individuals showed mixed or Mediterranean origin (Atlantic Q range: 0.33–0.66), but similar patterns of admixture were found in other Atlantic populations (The Bayesian estimates of population structure corroborated the separation between Mediterranean and Atlantic populations . The mods lowest . Mean meulations .n = 8) and Mediterranean (n = 2) populations. Note that up to four individuals may be misclassified according to the error rate in the DAPC with the reference areas (see above). The three Galician colonies and the two sexes showed similar discriminant loadings (P>0.25).A discriminant analysis of principal components (DAPC) using all source populations had low discriminant power as only 65% of individuals were correctly reclassified. Interestingly, all the individuals from Selvagens classified correctly, but in the other populations the proportion of correct reassignments was low (62%). However, when we used a DAPC with three main reference areas , 96% of individuals were correctly assigned to their actual main breeding area . AccordieneClass2 to assign the origin of Galician founders . However the PC1 scores of one male and two females were considerably smaller than the rest  and dependent on sex; Sex: F1,82 = 100.15 P = 0.001). The relationship was stronger in females, mainly due to the two females assigned to the Mediterranean genetic cluster  neither was the interaction between DF2 and sex (P>0.54).We explored the degree of agreement between morphological and genetic assignments by means of the correlation between the PC1 scores and the first genetic discriminant function DF1 of the Discriminant Analysis of Principal Components (DAPC) on microsatellite data, and found that it was significant  from four colonies targeted the waters offshore Galicia . To reacCory’s shearwaters foraged off Galicia both during incubation  and chick-rearing (Berlenga and Porto Santo). All tracked individuals were successful breeders and usually visited the colony surroundings of other populations of the species. We didn’t notice any specific trend over the years on the number and provenance of the birds visiting Galician waters, though the sample size of tracked individuals (N = 10) is rather small to be conclusive on this topic.The recent colonization of Galicia by Cory´s shearwaters was undertaken by a relatively large and admixed group of prospectors. Several lines of evidence add up to the idea that the new Cory´s shearwater populations in Galicia are composed by individuals from different sources; hence, they do not support the predictions of a dispersal hypothesis from a single colony but rather agree with predictions from island metapopulation models . MoreoveDuring our study, more than two hundred Cory’s shearwaters were visiting the Galician colonies as indicated by the maximum number of individuals in the evening rafts assembling close to the colonies (229 birds in 2013). Both the number of apparently occupied burrows and breeding pairs increased from 2009 to 2013. We recaptured 22% of the banded adults and none of these moved to another island, which suggests that each locality may hold a distinct population of founders. Based on the size of the largest raft, founder populations may comprise around 50–70 (Cíes and Coelleira) and 150 (Sisargas) individuals, which implies that the number of active burrows grossly underestimated the size of the founder populations. This is likely an indication that during colony establishment a relatively large proportion of the birds joining the rafts do not hold a burrow. Interestingly, the same pattern was observed during the early years of the first American colony of Manx shearwaters [C. d. diomedea) in Arcachon (France) in the mid 2000’s [Most Cory’s shearwaters breed for first time at age of six to nine years , 54, butd 2000’s  but it ad 2000’s . Thus, td 2000’s .Galician populations show a high genetic diversity, similar or even higher than other historical and large populations. Moreover, the mean number of private alleles per locus in the three Galician colonies exceeds that from other historical Atlantic colonies, including Berlengas, the nearest known breeding colony. Private alleles  are useful for elucidating genetic diversity and relationships among populations. Assessments of the number of alleles private to different combinations of populations were also consistent with the results for individual populations. Indeed, if we exclude Selvagens, Galicia had more private alleles than the pool of sampled Atlantic populations. The private allelic richness in Galicia-Atlantic and Galicia-Selvagens pairwise combinations was higher than the pair Selvagens-Atlantic, suggesting that an admixture pool of individuals is probably participating in the colonization of Galicia. Ancestral alleles or large historical population size may explain the higher number of private alleles found in the Selvagens sample. These results argue against a founder effect resulting in loss of genetic diversity, as expected if the new populations were started by a few members of one original population .All analyses on population structure , revealed two main genetic demes, which broadly corresponded to the Atlantic and the Mediterranean range of the species. The differentiation between these two ocean basins has previously been reported based on mtDNA , 60 and Bayesian assignment and discriminant analysis of principal components showed that only a few individuals could be confidently assigned to single source populations suggesting that birds settling in the Galician colonies probably originated from a genetic pool larger than the one we sampled. Most individuals assigned with a high probability to Atlantic populations, were mainly assigned to Desertas, Azores and Berlengas, but interestingly some were assigned to Selvagens and a few to Mediterranean colonies. Admixture in founder populations was further supported by the clear assignment of some of the individuals captured in Galicia to Selvagens, the only divergent clade within the Atlantic (see above).The genetic assignment of a few individuals to the Mediterranean clade may be questionable. First, our analyses of population structure revealed a weak Mediterranean genetic signature in all Atlantic colonies, thus, the Mediterranean assignment may simply reflect this genetic background. Second, the few individuals assigned to the Mediterranean cluster fall within the error rate of our discriminant analyses, precluding robust conclusions. However, a remarkable result is that three of the smaller Galician birds  were those genetically assigned to the Mediterranean clade. Two clear geographic gradients in size have been detected within the Cory´s shearwaters range . In the In conclusion, the picture that emerges from the array of genetic and morphometric analyses performed suggests that the Galician colonies have originated from an admixture of birds from several source colonies in the Atlantic, whereas the presence of Mediterranean birds cannot be entirely discarded.apoikia, the establishment of a colony far from the source populations, is likely a relatively common phenomenon in Cory´s shearwaters and other procellariids.Our results suggest that a large and admixed population is settling in Galicia, in agreement with predictions from island metapopulation models. Possibly three groups of non-breeders, adding up to around 200 birds, started visiting the Galician colonies in the mid 2000’s and some of them eventually laid eggs and reproduced, thus establishing new breeding colonies. Our results do not support Berlengas, (the closest colony ca. 400 km apart) or any other of the Atlantic colonies as the single main source for the Galician founders. Indeed, several lines of evidence suggest admixture, which implies that some birds colonizing Galicia were dispersing from very distant colonies > 1500 km). Previous studies have suggested that the species performs rare but recurrent long-distance dispersal events . Interes00 km. PrLarge and admixed founder groups are likely to be much more efficient colonizers of new environments than are small single-source ones . The benThis study was carried out with appropriate licensing and permissions for scientific work in restricted access natural areas and bird handling and ringing in Galicia, issued by Xunta de Galicia and the National Park of the Atlantic islands of Galicia. All sampling procedures were specifically approved as part of obtaining the field permit. The issuers of the permit did not require approval of field sampling methods by an animal ethics committee. We made all possible efforts to reduce handling times and to keep disturbance to the birds to a minimum.This study was conducted in the three breeding areas that the Cory´s shearwaters have recently established in Galicia, at the NW of the Iberian peninsula, in the Northeast Atlantic : the islTo assign possible source populations, we genetically analyzed 174 blood samples of individuals from five populations representing the entire range of the Cory´s shearwater in the Atlantic: Berlengas , Tenerife (Canarias), Selvagen Grande (Selvagens), Deserta Grande (Desertas) and Corvo (Azores); and three from the Western Mediterranean: illa de l’Aire (Menorca), Pantaleu  and Habibas (Tunisia) see ; we addiPuffinus mauretanicus [2 (depending on the primer) and 1–2 μl of template DNA. The thermocycling conditions were 94°C for 2 min, followed by 34 cycles of 95°C for 30 seconds 50°C–60°C for 30 seconds and 72°C for 30 seconds, with a final extension of 72°C for 5 min. Reactions were loaded together and the length of the DNA fragments were analyzed directly from PCR product using an ABI 3100 automated sequencer  and software ABI GeneMapper v. 3.7 and finally rechecked by eye.Total DNA was isolated from blood samples by an overnight incubation at 55°C in SET buffer with 30 μl SDS 10% and 2.5 units/ml of proteinase K followed by a standard phenol/chloroform protocol . DNA wasetanicus  and polyetanicus  and 4 spetanicus . AmplifiWe also assigned sex to sampled individuals by PCR amplification of the CHD genes , 83. Blois) and tested for deviations from the Hardy-Weinberg equilibrium. Using permutations (>1000) we tested for the occurrence of non-random associations of pairs of loci (linkage disequilibrium). We evaluated the mean number of private alleles per locus in individual populations and in various sets of populations grouped according to major breeding areas [We measured the mean number of alleles per locus, as well as observed and unbiased expected heterozygosity  using thng areas . Since tng areas . Under aST with sample size correction as the basis for generating a neighbor-joining (NJ) tree based on population allele frequencies using the program Poptreew [ST values were taken as representative of genetic distances between populations and the significance of the correlation coefficient between genetic and geographic distances was estimated using a Mantel test implemented in Poptools [Genetix v. 4.05 . Conventionally, the first axis has a larger contribution to total inertia and usually displays the differentiation between species or subspecies.We calculated FPoptreew . The trePoptools . To visuStructure 2.3.4 [F model of correlation in allele frequencies across clusters [Structure Harvester v 0.6.1 [lumpp v. 1.1.1 using FullSearch algorithm [We assessed population differentiation among colonies by Bayesian assignment techniques using the software re 2.3.4 . We idenclusters  and alloclusters  calculat v 0.6.1 . Thus, flgorithm .eneclass2 [We assigned Galician samples to the most likely genetic source location using the Bayesian assignment test method in Gneclass2  and the neclass2 . We firsneclass2 .adegenet package in R [We additionally assigned Galician shearwaters to reference populations by means of a Discriminant Analysis of Principal Components  using tage in R . This meBayesAss analysis was conducted with the microsatellite data set using the default delta values for allelic frequency, migration rate, and inbreeding. Subsequent analyses incorporated different delta values to ensure that proposed changes between chains at the end of the run were between 20% and 60% of the total chain length [Since Galician colonies were grouped within an Atlantic genetic deme, and to help disentangling connectivity patterns, we explored the contemporary gene flow (i.e. during the past one-to-three generations) between the Atlantic colonies using the Bayesian assignment algorithm implemented in BayesAss . A prelin length . Once thThe morphological analyses of the Galician birds were conducted on a total of 86 sexed adults  from the three colonies: Coelleira (N = 43), Sisargas (N = 22) and Cíes (N = 21). We used a principal components analysis (PCA) for each sex separately on the correlation matrix with four variables  standardized to zero mean and unit variance. The normality of distributions was assessed graphically by plotting the cumulative frequency distribution of the samples. In both sexes the first two components explained more than 65% of the observed variability . The PC1 values were used as indicating overall size. Morphometric data from colonies elsewhere was taken from  Table 1Table 1 aWe used telemetry data (compass- and GPS-loggers and PTT transmitters) to assess the geographic origin of the birds that visit the coastal and offshore waters of Galicia during the breeding season. The telemetry data corresponds to 480 individual Cory’s shearwaters tracked in 13 breeding locations throughout the Atlantic and the Mediterranean (Chafarinas), and was mainly obtained from the literature see .


The Chinese Hui population, as the second largest minority ethnic group in China, may have a different genetic background from Han people because of its unique demographic history. In this study, we aimed to identify genetic differences between Han and Hui Chinese from the Ningxia region of China by comparing eighteen single nucleotide polymorphisms in cancer-related genes.2 tests, and FST was used to measure the degree of population differentiation.DNA samples were collected from 99 Hui and 145 Han people from the Ningxia Hui Autonomous Region in China, and SNPs were detected using an improved multiplex ligase detection reaction method. Genotyping data from six 1000 Genomes Project population samples (99 Utah residents with northern and western European ancestry (CEU), 107 Toscani in Italy (TSI), 108 Yoruba in Ibadan (YRI), 61 of African ancestry in the southwestern US (ASW), 103 Han Chinese in Beijing (CHB), and 104 Japanese in Tokyo (JPT)) were also included in this study. Differences in the distribution of alleles among the populations were assessed using χST values (FST>0.000) between the Hui and Han populations.We found that the genetic diversity of many SNPs in cancer-related genes in the Hui Chinese in Ningxia was different from that in the Han Chinese in Ningxia. For example, the allele frequencies of four SNPs  showed different genetic distributions (p<0.05) between Chinese Ningxia Han and Chinese Ningxia Hui. Five SNPs  had different FThese results suggest that some SNPs associated with cancer-related genes vary among different Chinese ethnic groups. We suggest that population differences should be carefully considered in evaluating cancer risk and prognosis as well as the efficacy of cancer therapy. Genetic studies have revealed that different populations have different genetic structures because of their complex demographic histories . TherefoThere are 56 ethnic groups in China. Han is the largest ethnic population, comprising 98% of the total population in China. The populations of the other 55 minority groups vary from thousands to millions, and some of the minority groups differ substantially from Han Chinese in terms of morphological and genetic characteristics. For example, the Uyghur (UIG) population, the fifth largest minority group in China, differs significantly from the Han Chinese in terms of facial features and has an approximately 55% European genetic component . Hui ChiAll subjects were from the Ningxia Hui Autonomous region in China. A total of 99 Hui individuals were selected from the physical examination center of a county hospital located in the Ningxia Haiyuan region of China. A total of 145 Han individuals were selected from the physical examination center of the General Hospital of Ningxia Medical University. The inclusion criteria were: (1) All subjects were from Ningxia Han or Hui residents whose ancestral native living places were Ningxia Hui Autonomous Region and at least three generations of their families were also the same ethnic people. (2) All subjects were proved to be physically healthy by their history, physical examination, and clinical examination when their samples were collected. (3) All subjects were proved to be free of benign or malignant tumors, both previously and at present, by their history, physical examination, and clinical examination. Demographic data including age, gender, and alcohol and tobacco consumption were obtained using a survey. Blood samples were collected from all subjects for DNA isolation and genotyping of the eighteen SNPs. Signed informed conset was obtained from each participant. All procedures were approved by the Medical Ethics Review Committee of Ningxia Medical University .TP63 gene (rs4488809 at 3q28), TERT-CLPTM1 L gene (rs465498 at 5p15), MIPEP-TNFRSF19 gene (rs753955 at 13q12), and MTMR3-HORMAD2-LIF gene (rs17728461 at 22q12). Two SNPs related to esophageal cancer are located in the PLCE1 gene (rs2274223 at 10q23) and C20orf54 gene (rs13042395 at 20p13). Two SNPs related to breast cancer are located in the TAB2 gene (rs9485372 at 6q25) and LOC100506172 (rs9934948 at chromosome 16). Two SNPs with independent effects and significant gastric cancer associations are located in the PRKAA1 (rs13361707 at 5p13) and ZBTB20 genes (rs9841504 at 3q13).Eighteen SNPs were selected for the analysis. Among all the SNPs, ten including rs13042395, rs465498, rs753955, rs17728461, rs2274223, rs13361707, rs9841504, rs9485372, rs4488809, and rs9934948 were reported by GWAS to be associated with cancer risk. For example, four SNPs associated with lung cancer are located in the CDKN1A, both of which play a critical role in carcinogenesis in the p53 pathway. For example, only one SNP, rs1042522, is located in the p53 gene (at 17p13), which represents one of the most studied tumor suppressor genes in cancer biology. A great number of genetic association studies have reported that rs1042522 is a risk factor for human malignancies [CDKN1A gene , six of which have been analyzed for their associations with longevity, esophageal squamous cell carcinoma, or lung cancer except rs3176320 [Eight other SNPs, including rs1042522, rs3176320, rs3829964, rs762624, rs4135234, rs730506, rs3829963, and rs2395655, are in p53 or s3176320 ,15,16,17Genomic DNA was extracted using Qiagen genomic DNA extraction kits . SNP genotyping was performed using an improved multiplex ligase detection reaction method  as previously described . The priwww.1000genomes.org) as controls [Genotyping data from six 1000 Genomes Project population samples (99 Utah residents with northern and western European ancestry (CEU), 107 Toscani in Italy (TSI), 108 Yoruba in Ibadan (YRI), 61 persons of African ancestry in the southwestern United States (ASW), 103 Han Chinese in Beijing (CHB), and 104 Japanese in Tokyo (JPT)) were included in this study. According to two references by Xu et al. and Hu et al, we downloaded the genotype data of individuals from six populations from the 1000 Genomes Project web site  version 17.0 statistical software was used for the statistical analyses. The FST value, originally defined by Wright, was introduced as the correlation between gametes chosen randomly from within the same subpopulation relative to the entire population. FST can be thought of either as the proportion of genetic diversity due to allele frequency differences among populations or as the correlations between alleles within populations relative to the entire population [ST calculations were performed using Arlequin 3.5. The FST of SNPs were calculated following Weir and Cockerham [ST of an SNP was two-tailed and were considered statistically significant at p<0.05.Genotype and allele frequencies were obtained by direct counting. Differences in the distribution of alleles among the populations were assessed using the χpulation ,23,24. FWe investigated a total of 244 subjects, including 99 Hui and 145 Han subjects. ST value, a measure of genetic differentiation, was calculated for each SNP to quantify the differences among the different populations. As shown in ST values among the eight populations, varying from 0.013 to 0.192, and all p values were less than 0.05. This finding suggests that the cancer-related genes differ substantially among the studied populations.To identify cancer-related genes that are highly differentiated with respect to allele frequency among the six 1000 Genomes Project populations and the two populations from the Ningxia region of China, an FST value between each pair of subpopulations for the eighteen SNP sites was also calculated . However, the average FST value between two different ethnic groups of people among the European groups, African groups, and two East Asian groups varied from 0.07961 to 0.16061, suggesting that there was considerable genetic differentiation. For example, the maximum average FST value was 0.16061 between CEU and YRI, and the minimum value was 0.07961 between ASW and JPT. We found that the average FST value between Chinese Ningxia Han and CEU showed the greatest differentiation (0.10627) between Chinese Ningxia Han and the other populations; furthermore, the average FST value between Chinese Ningxia Han and CHB showed the least differentiation (0.00000), and the value between Chinese Ningxia Han and Hui was between the maximum and minimum (0.00363). Similarly, regarding the differentiation between Chinese Ningxia Hui and the other populations, the average FST value between Chinese Ningxia Hui and YRI indicated the maximum differentiation (0.10129), and the average FST value between Chinese Ningxia Hui and CHB indicated the least differentiation (0.00108).The average Flculated . The FSTST values were also calculated for each SNP to quantify the differences among the CHB, Hui, and Han populations  between the CHB and Chinese Ningxia Han samples. By contrast, the FST values of five SNPs between the Hui and Han populations ranged from 0.000 to 0.050, suggesting the existence of genetic differentiation between the two populations. The other thirteen SNPs had FST values ≤0.000 between the two populations , as shown in The allele frequency and relative physical coordinates of the eighteen SNPs are shown in Molecular genetics studies in the last few decades have provided the basis for ancestral analysis and analysis of the geographic origins of human populations using genetic data. Starting approximately 100,000 years ago, anatomically modern humans migrated out of East Africa and gradually spread to South Asia, Australia, Europe, East Asia, and eventually the Americas. All people living today are direct descendants of these earlier humans. Populations living in different parts of the world today exhibit a small number of genetic differences due to migration, mutation, genetic drift, natural selection, and reproductive isolation .ST to measure the degree of population differentiation [ST results were consistent with the allele frequency comparisons among the different populations.In this study, we sought to investigate the diversity pattern of cancer-related genes between Hui and Han Chinese from the Ningxia region of China to explore the hereditary differences between the two populations. We selected eighteen SNPs from cancer-related genes for the analysis. We first used Fntiation , and ourST values to investigate the diversity pattern of cancer-related genes among eight subpopulations with respect to the eighteen SNP sites. We found that there was little genetic differentiation between any two European, African, or East Asian groups, although there was remarkable genetic differentiation between each pair of the aforementioned ethnic groups.We then used average FWe subsequently investigated the genetic differentiation between CHB and Han Chinese in Ningxia. Our data indicated that the allele frequencies of all eighteen SNPs were very similar between the two populations. The Han Chinese population is generally thought to be naturally divided by the Yangtze River into two groups: the Southern Han and Northern Han groups. A previous study showed that the difference between these two groups of Han Chinese is greater than that between a given subpopulation and ethnic minorities at the same location . BecauseOur study further showed that the allele frequencies of four SNPs differentiated Ningxia Hui Chinese from Ningxia Han Chinese, indicating that there was some genetic differentiation in the distribution of four SNPs from cancer-related genes between Chinese Ningxia Hui and Han. Among four SNPS, SNP rs753955 had been reported to be associated with lung cancer in Chinese Han population by GWAS. SNP rs753955 was also found to be related to non-cardia gastric cancer in Chinese Ningxia Han in our previous study . HoweverOur results showed for the first time that Hui Chinese in Ningxia exhibit differences in certain cancer-related genes compared with Han Chinese in Ningxia. Therefore, we suggest that population differences in cancer susceptibility, the efficacy of cancer therapy, and prognosis should be carefully considered. However, there are some limitations to our study, such as the small sample size, the small number of genetic markers, and the very limited geographic location. In addition, although we have investigated the associations between some of the SNPs and cancer in Ningxia Han population , we coulS1 Table(DOC)Click here for additional data file.


Capparis spinosa, a xeromorphic Tethyan relict, to discuss its divergence pattern and explore how it responded in a stepwise fashion to Pleistocene geologic and climatic changes. 267 individuals from 31 populations were sampled and 24 haplotypes were identified, based on three cpDNA fragments . SAMOVA clustered the 31 populations into 5 major clades. AMOVA suggests that gene flow between them might be restricted by vicariance. Molecular clock dating indicates that intraspecific divergence began in early Pleistocene, consistent with a time of intense uplift of the Himalaya and Tianshan Mountains, and intensified in mid-Pleistocene. Species distribution modeling suggests range reduction in the high mountains during the Last Glacial Maximum (LGM) as a result of cold climates when glacier advanced, while gorges at midelevations in Tianshan appear to have served as refugia. Populations of low-altitude desert regions, on the other hand, probably experienced only marginal impacts from glaciation, according to the high levels of genetic diversity.Complex geological movements more or less affected or changed floristic structures, while the alternation of glacials and interglacials is presumed to have further shaped the present discontinuous genetic pattern of temperate plants. Here we consider An essential purpose of biogeography is to comprehend the mechanisms resulting in the current distribution patterns of organisms during their evolutionary histories , 2. EvenPhylogeography, a discipline which began more than 20 years ago, has mostly considered the response of geographic distribution and genetic structure of current species to historical events and the cycles of glaciation –16. Chlo Capparis spinosa, a typical xerophyte, to verify the hypothesis of Pleistocene historical influence through phylogeographic analysis and discuss how its spatial genetic structure responded to paleogeologic and paleoclimatic changes. The perennial creeping subshrub is a type member of a relatively large genus of the Capparaceae, with a natural distribution in the Mediterranean, West, and Central Asia [Capparis Tourn. ex L.) with one single species (C. spinosa L.) belonging exclusively to this family [ C. spinosa ranges over the Western Himalayas, Eastern Pamir Plateau, Tianshan Mountains, Turpan-Hami Basin, and the Hexi Corridor. Previous studies on Capparis have paid more attention to autecology, phenology, quantitative morphology, and reproductive ecology [We useral Asia , 32. In s family . C. spin ecology –37. Mole ecology , 39 and  trnL-trnF, rps12-rpl20, and ndhF, to trace historical divergence patterns of C. spinosa. We design to address the following concrete issues. (1) According to phylogenetic relationships yielded by cpDNA sequence variation, what is the genetic pattern of the species? (2) When and how have allopatric divergence and genetic differentiation of this xerophyte been influenced by glacial-interglacial cycles associated with geologic and climatic changes?Here we employed three maternally inherited cpDNA spacers, C. spinosa from 31 natural populations were investigated in the present study , three cpDNA regions, trnL-trnF, rps12-rpl20, and ndhF , were found because they have more sequence variation in individuals among populations. Polymerase chain reaction (PCR) amplification and DNA sequencing were performed using primer pairs for these three regions. PCRs were carried out in 30 μL reaction volumes including 1.5 μL of 10x buffer, 2 μL of 25 mmol/L MgCl2, 2.2 μL of 50 ng/μL each primer , 3 μL of 2.5 mmol/L dNTP, 0.3 μL of 5 U/μL Taq DNA polymerase, and 1.0 μL of 5 ng/μL genomic DNA. PCR amplification was run on a Gene-Amp PCR system 9700 DNA Thermal Cycler  with parameters set as follows: an initial start at 94°C with 4 min; followed by 30 cycles of 94°C with 30 s, then 59°C, 52°C, and 52°C, respectively, for trnL-trnF, rps12-rpl20, and ndhF with 30 s and 72°C with 90 s; plus a subsequent additional extension at 72°C with 10 min. Sizes of PCR products were visualized on 1% TAE-agarose gel electrophoresis. All PCR products were purified with PCR product purification kit  and then sequenced in both directions for trnL-trnF, rps12-rpl20, and ndhF using an ABI Prism 3730 xl automated sequencer in Sangon Biological Engineering Technology & Service Co., Ltd., Shanghai, China.Silica-gel-dried leaf tissues were ground into powder in liquid nitrogen with a Fastprep-24 device . Total genomic DNA was extracted from about 50 mg of powdered tissues using a modified 2x CTAB method . Through trnL-trnF, KU866560–KU866562 and KU940220 for rps12-rpl20, and KU866548–KU866558 and KU940221–KU940223 for ndhF ; and accessions numbers of C. bodinieri as the outgroup are KU940219 for trnL-trnF, KU866563 for rps12-rpl20, and KU866559 for ndhF . Different cpDNA haplotypes were identified by DnaSP 5.0 [http://www.fluxus-engineering.com/sharenet_rn.htm).The chloroplast DNA sequences were edited by SeqMan . All sequences were aligned with Clustal X version 1.81 , then renaSP 5.0 . GeograpnaSP 5.0 , which wK groups) based on genetic differentiation and geographical homogeneity [K values ranged from 2 to 12 by repeated simulated annealing process until largest FCT index was obtained. However, when K ≥ 6, at least one of the groups contained only one population, implying that information on the group structure would have been disappearing [K was set as 2–5.To maximize the differences of haplotype composition between geographical locations, spatial analysis of molecular variance algorithm was conducted using SAMOVA 1.0 procedure. This method divided the sampled populations into inferred partition (ogeneity . We initppearing –48. To mHd) and nucleotide diversity (π), were computed in Arlequin 3.1 program [HS), total gene diversity across all populations (HT), the number of substitution types (NST), and genetic differentiation over populations (GST), were calculated using Permut version 1.0 [http://www.pierroton.inra.fr/genetics/labo/Software/PermutCpSSR/index.html). To test for the existence of phylogeographical structure, the comparison of GST versus NST was conducted using Permut based on 1,000 random permutations of haplotypes across populations. If NST is significantly higher than GST, then genealogically closely related haplotypes tend to occur together within populations, indicating the presence of phylogeographical structure [P values was examined by the U Test using the Haplonst procedure [Parameters of Nei's diversity, containing haplotype diversity  and a SD of 6.08 × 10−9 for most angiosperm species were adopted as a criterion for the analytical operation [To estimate the divergence times of cpDNA haplotypes, the BEAST version 1.6.1  was carrperation , 53, to peration  to checkTo investigate spatial genetic patterns of geographical distance among populations for each defined regional group, genetic landscape shape analysis was performed using Alleles In Space . PrimariD and Fu's Fs statistics) [D value (not 0) may imply that a population underwent expansion or bottlenecks [Fs value probably indicates a recent population expansion [Possible demographic historical expansions for defined groups were inferred by mismatch distribution analysis using Arlequin with 1,000 parametric bootstrap replicates . Populattistics) , 61 weretistics) , 63. A ptlenecks , while ahttp://www.cvh.org.cn/cms/) and our field survey locations (http://www.worldclim.org/download/). To avoid the potential problems of overfitting [To explore the potential distribution changes of the species between the present-day and the Last Glacial Maximum , the ecological niche modeling was performed in MAXENT version 3.3.1 , followiocations . The currfitting , we remo trnL-trnF, 728 bp for rps12-rpl20, and 603 bp for ndhF ). Based on the total sequences, thirty-six polymorphic sites (thirty nucleotide substitutions and six indels) were obtained (Table S2). A total of 24 haplotypes (H1–H24) were identified from 267 samples among 31 populations across the entire geographic range of C. spinosa  the Western Himalayas Group, (II) the Eastern Pamir Group, (III) the Ili Valley Group, (IV) the north side of the Tianshan Mountains Group, and (V) the south side of the Tianshan Mountains Group . The maiDistribution of the haplotypes showed high geographic structure. Haplotypes H22, H6, H7, H1, H8, H5, and H10 had the most frequent and widespread distributions: H22 only was distributed in the populations of Group I; H6 occurred in all populations of Group II; H7 only occurred in the populations of Group III; H1 and H8 were dominantly distributed in the populations of Group V, while H5 and H10 were mainly present in the populations of Group IV .HT = 0.916, s.e. = 0.0172) was much higher than average gene diversity , which indicated that there was considerable population differentiation across the distributional range. The U Test showed that total NST  was significantly higher than GST  , indicating cpDNA variation with signal phylogeographic structure for the species.Genetic diversity analysis revealed that total genetic diversity  of total variation existed between the five groups (Hd) within the 31 populations ranged from 0 to 0.8485, and nucleotide diversities (π) ranged from 0 to 0.0021. Considering the whole species, high degrees of haplotype diversity (Hd = 0.8925) and nucleotide diversity (π = 0.0037) were found. At the level of population, six populations  in Group V showed considerably high degrees of genetic diversity . Differences among geographical groups were significant, and AMOVA results showed that 71.83% , mean temperature of the coldest quarter (22.97%), precipitation of the coldest quarter (15.48%), mean temperature of the driest quarter (8.25%), and precipitation seasonality (6.37%). The five most contributive bioclimatic variables of the MIROC model in the LGM were annual mean temperature (24.32%), precipitation of the coldest quarter (20.18%), mean temperature of the coldest quarter (13.87%), mean temperature of the driest quarter (12.18%), and precipitation of the wettest month (11.89%). The range differences under the distinct climatic conditions of the two models revealed a diminished amount of suitable habitat for the species during the LGM period. In comparison with the present potential distribution, the MIROC scenario indicated that the high-altitude ranges of the species in the Western Himalayas, eastern Pamir, and western Tianshan Mountains were diminished  was detected. Compared with other species where three intergenic spacers have been sequenced, gene diversity in C. spinosa was higher, for instance, Juniperus sabina (HT = 0.577) [ Gymnocarpos przewalskii (HT = 0.903) [Hd = 0.8925)   and Gymn= 0.903) . The res 0.8925) . High va C. spinosa mainly include anemochory, myrmecochory, and endozoochory by rodent vector [Dramatic genetic variation among the total populations was shown, accounting for a far greater proportion than that within populations . The gent vector . In the The timescales of intraspecific divergences are usually related to historical geologic and climatic events , 12. The C. spinosa during the LGM period were contracted  in the middle Tianshan Mountains as potential refugia during glacial epochs, in accordance with the high levels of genetic diversity within these populations.According to previous glaciological research, maximum glaciation was likely to have occurred in the early phase of the mid-Pleistocene (0.8–0.6 Ma) in Eastern Central Asia , 77. Durountains , as a reulations , 82. Refulations , 22 and ulations . A numbeulations –86. Howeulations . As a re C. spinosa was profoundly affected not only by temperature variation but also by aridification in Eastern Central Asia; thus xeric species may well prefer relatively arid areas during the warm and moist interglacial stages. Considering the network diagram, genotypes H1 and H8 were found with high rates of incidence on the south side of Tianshan group, indicating that these two were predominant haplotypes in Group V , extensive intraspecific differentiation is probably connected with range fragmentation or long-distance dispersal [ C. spinosa is a xeromorphic species that prefers to distribute itself in eroding rocky mountains and arid piedmont proluvial or alluvial gravelly fans  in twenty-four haplotypes of Capparis spinosa.Supplementary Table S2: Variable sites of three chloroplast DNA sequences (


Plasmodium vivax has the widest worldwide distribution of the human malaria species and imposes a serious impact on global public health, the investigation of genetic diversity in this species has been limited in comparison to Plasmodium falciparum. Markers of genetic diversity are vital to the evaluation of drug and vaccine efficacy, tracking of P. vivax outbreaks, and assessing geographical differentiation between parasite populations.Even though P. vivax populations (n = 543) was investigated by using two microsatellites (MS), m1501 and m3502, chosen because of their seven and eight base-pair (bp) repeat lengths, respectively. These were compared with published data of the same loci from six other P. vivax populations.The genetic diversity of eight P. vivax samples from 14 countries on three continents were compared. There was highest heterozygosity within Asian populations, where expected heterozygosity (He) was 0.92-0.98, and alleles with a high repeat number were more common. Pairwise FST revealed significant differentiation between most P. vivax populations, with the highest divergence found between Asian and South American populations, yet the majority of the diversity (~89%) was found to exist within rather than between populations.In total, 1,440 P. vivax population comparisons and their seven and eight bp repeat length facilitated population comparison using data from independent studies. A complex spatial pattern of MS polymorphisms among global P. vivax populations was observed which has potential utility in future epidemiological studies of the P. vivax parasite.The MS markers used were informative in both global and local The online version of this article (doi:10.1186/1475-2875-13-392) contains supplementary material, which is available to authorized users. Plasmodium vivax has the widest global distribution of the five human malaria species and imposes a serious impact on global public health, descriptions of global genetic diversity of this parasite are more limited than for Plasmodium falciparum. A recent study comparing the total genomic diversity among six P. vivax isolates drawn from Asia, Africa and South America with a comparable set of six P. falciparum isolates reported twice as much single nucleotide polymorphism (SNP) diversity and a far deeper divergence among the P. vivax geographic strains . ComparP. vivax genome [1-∑pi2], where n is the number of samples and pi is the frequency of the ith allele. The estimation of He for each of the two loci in P. vivax in PNG was taken from the original publication [FST pairwise population genetic distances; 3) analysis of molecular variance (AMOVA) attributes the proportion of the total genetic variance which is found within populations (countries), between populations within groups (continents), and among groups; and, 4) isolation by distance (IBD) was used to test for any correlation between FST estimates and geographic distances using Mantel’s test [FST/(1-FST) against the natural logarithm of the geographical distance (in km). Evaluation of IBD was performed by showing R squared (R2), indicating the percentage of the variance explained by the model, the correlation coefficient r as a measure of the degree of linear relationship between two variables and the P-value. The calculations in all tests were performed by including both mono- and polyclonal samples.The analysis of allelic diversity measured in the lication ; 2) compl’s test . The cale was Excel add-in MS Toolkit® software, whereas the program FSTAT was used to perform the pairwise FST estimates and IBD calculations with 10,000 permutations [The software used for the calculations of Hutations . Lastly,utations , and sigTo enable calculations of IBD, geographical distances in km were determined by use of Google Earth . The same at each locus in every site is compared. A high He was observed at both loci in all the sites, with variations among sites being broadly consistent across the two loci. The South American sites had lower diversity  than Asian  and African sites .In total, 543 samples from Sri Lanka, Nepal, Pakistan and Afghanistan, Venezuela, Ecuador, Sudan and Sao Tome were analysed. The number and percentage of samples successfully amplified at each locus are shown in Table P. vivax in Thailand, Laos, India and Colombia published by Imwong et al. [et al. [For the analysis of genetic differentiation between populations, independently derived data from published studies of g et al.  and from [et al.  were inc [et al.  in the mA few m1501 alleles from Sri Lanka and Pakistan samples and m3502 alleles in samples from Laos and Sri Lanka showed irregular spacing caused by a fragment size shift of a few nucleotides, presumably caused by insertion/deletion within the PCR amplified fragment. These irregular alleles were classified under the same repeat number as those of similar size elsewhere but are distinguished in the Tables by appending ‘a’ and ‘b’ to the repeat number. In the allele frequency and Fst calculations subgroups a and b were combined [see Additional files P. vivax populations were highly polymorphic at both loci, with the exception of the Korean P. vivax population. This contrasted with the South American P. vivax populations, which were less diverse. Diversity at m1501 was highest in Pakistan where 27 alleles were detected among 315 samples, while only two were seen in Ecuador (n = 17) and three in Korea (n = 58) [see Additional file The m1501 marker discriminated 29 alleles in the global dataset while m3502 discriminated 18 alleles. The number of alleles differed remarkably between populations. Asian Alleles with long fragment lengths, above 12 repeats for m1501 and 10 repeats for m3502, were detected more commonly in Asian than elsewhere  showed evidence of population structure. FST using the m1501 marker revealed a significant difference between Colombia and the two other South American countries (FST = 0.05-0.21), while FST using the m3502 marker, found significant differences between Venezuela and the two other South American countries (FST = 0.26-0.27) [see Additional file P. vivax populations from these three countries were significantly different from all the Asian countries, with few exceptions. Low, but significant pairwise FST estimates were found among the Asian countries; 0.02-0.31 (m1501) and 0.02-0.14 (m3502). The analysis of IBD was done by using pairwise genetic and geographic distances. Evidence for IBD was found for both loci by Mantel’s test; m1501: , and m3502: .FST estimates using the two loci combined were slightly higher and pairwise estimates between P. vivax populations from Korea, Nepal, Pakistan, Sri Lanka, Venezuela, Ecuador, Sudan, and São Tomé, revealed FST between 0.01-0.40 [see Additional file P. vivax populations in Ecuador and Sudan and the lowest between P. vivax populations from Nepal and Sri Lanka. The two-locus haplotypes were often not specific to a single sample set from one country, but present in more than one P. vivax population as for instance the ‘107-142’ haplotype seen in Nepal, Pakistan and Sri Lanka [see Additional file P. vivax populations was estimated by He. The average He per locus was 0.90 (m1501) and 0.84 (m3502). The He varied considerably from site to site, but was generally lowest in the three South American countries (He = 0.22-0.76) and highest in the Asian populations (He = 0.80-0.94) with the exception of Korea, which possessed very low He values of 0.42 (m1501) and 0.73 (m3502)  , thereafter Africa  whereas Asia had the most diverse P. vivax populations in this study . The same ranking was seen using the m3502 marker: South America , Africa  and Asia .Comparison of the three continents  showed that the South American P. vivax populations by examining the two MS, m1501 and m3502 in P. vivax samples collected from Ecuador, Venezuela, Sri Lanka, Afghanistan, Pakistan, Nepal, São Tomé, and Sudan (n = 543). Although some of these counties were represented by a large P. vivax sample set, the limited available sample set from Ecuador (n = 17), São Tomé (n = 4) and Sudan (n = 8) means caution should be applied to the interpretation of the results regarding these sites. Furthermore, there is a significant time interval between the collection of the Venezuelan samples in 1996–1997 and the other South American samples some years later. Where possible, the resulting data were compared with published data from studies of the same markers in Colombia, India, Laos, Thailand, PNG, and Korea (n = 897).The main objective of the current study was to evaluate the global genetic diversity of P. vivax populations was shown to be highly diverse, with the majority of the diversity found to be present within the populations (~89%). In total, 29 m1501 and 18 m3502 alleles were detected with average He estimates per allele of 0.90 (m1501) and 0.84 (m3502), lowest in the South American samples and highest in Asia. The He values found in this study by use of only two MS (He = 0.78-0.98) were high compared to He estimates reported by other studies (He = 0.48-0.87), which have analysed samples from the same three continents including nine to 14 MS [e estimates of 0.26 to 0.91 reviewed by others [The geographical genetic diversity of the to 14 MS , 20, 32,FST, whereas the African population generally could not be distinguished statistically from most other populations, most probably this was due to the limited sample size. The FST estimates ranged from 0.02-0.63 (m1501) and 0.03-0.28 (m3502) per site, and a Mantel test correlating pairwise genetic and geographic distances between populations showed evidence for IBD.South American and Asian populations were found to be significantly different from each other by pairwise P. vivax populations, and the rarer long-repeat fragments were mainly restricted to Asia. Only a limited number of samples from Africa were analysed, but these suggest Africa is intermediate between South American and Asian populations. The long repeat-alleles appearing in the Asian P. vivax populations might relate to factors, such as higher P. vivax endemicity in Asian countries.The high frequency of alleles with low repeat number (one to six repeats) was common for all the e estimates and MOI values were measured among the individual populations sampled. The maximum MOI for an individual sample was six clones and this was found in Pakistan. The mean MOI in Pakistan was 1.55 and rates of heterozygosity were also high. The complexity of the Pakistan/Afghanistan population is striking, since these areas are not generally considered highly endemic. Similar rates of heterozygosity were reported in PNG [P. vivax genome is unknown, but the high rates of complexity observed here are fully consistent with that reported from previous studies of P. vivax[Malaria endemicity is expected to have a significant influence on genetic diversity and levels of inbreeding/outbreeding. Among the populations sampled in the present study, Asian populations were the most diverse, although a range of Hd in PNG  and the d in PNG . This maP. vivax, 32, 35.P. falciparum research, and is not the complete explanation of the mechanisms that contribute to P. vivax diversity. Genome wide analyses of MS diversity have shown P. vivax diversity is significantly greater than P. falciparum[P. vivax than P. falciparum[P. vivax isolates suggesting P. vivax diversity is more ancient. Although just six P. vivax isolates were compared, similar geographic trends were observed. South American isolates Brazil 1, Salvador 1 and Peru shared a more recent common ancestor with each other than with isolates from Asia  and Africa (Mauritania), which had deep branch lengths and did not cluster. The data presented in this study also showed high level of diversity and differentiation between the South American and Asian populations and the findings indicate that further investigation of genome-wide diversity among P. vivax populations from Central and Southeast Asia may reveal even greater levels of genetic diversity.The above model of polyclonality and diversity is developed based on extensive and cumulative lciparum, but alslciparum. The samP. falciparum-model’ may not entirely predict and explain genetic diversity of P. vivax populations. Important biological differences between P. vivax and P. falciparum may also be at play. In P. vivax, gametocytogenesis occurs earlier in clinical episodes, and reticulocytes are essential to both invasion and relapse [P. vivax epidemics.A ‘ relapse . Signifi relapse , the Col relapse , the Kor relapse , and by  relapse  and Braz relapse  and thesP. vivax population structure, irrespective of geographical origin, although more markers might be needed in hypo-endemic areas if distinguishing between individual parasites is a priority. Ultimately, whole- genome analyses will provide detailed estimation of the total genetic diversity –the results presented in this study suggests that the greatest diversity will be in Southeast Asia.The data presented in this study show the utility of the MS m1501 and m3502 in studies of Plasmodium genes were approved by London School of Hygiene Tropical Medicine and Hygiene Ethics Board, locally by Bioethics Committee, Pakistan Medical Research Council and Directorate of Public Health, Jalalabad, Nangahar, Comitee de Bioetica Universidad San Francisco de Quito and the Nepal Health Research Council.Clearance for analysis of Allelic diversity at the m1501 locus ofP. vivaxsamples obtained from various malaria endemic countries.(PDF 11 KB)Additional file 1:Allelic diversity at the m3502 locus ofP. vivaxsamples obtained from various endemic countries.(PDF 19 KB)Additional file 2:Population differentiation at m1501 and m3502 allelic estimated by pairwise fixation index, FST.(PDF 13 KB)Additional file 3:Population differentiation estimated by pairwise fixation index, FSTusing the combined MS data from m1501 and m3502.(DOCX 16 KB)Additional file 4:Allelic diversity for the combined MS genotype “m1501-3502” haplotypes per study site and in total.(DOCX 18 KB)Additional file 5:Expected Heterozygosity (He) twoP. vivaxmicrosatellites, m1501 and 3502 in each site.(DOCX 15 KB)Additional file 6:Global analysis of molecular variance (AMOVA) amongP. vivaxpopulations and continents (groups).(DOCX 16 KB)Additional file 7:


AbstractAnastrephafraterculus across Brazil. To this aim, we used Simple Sequence Repeat (SSR) markers, which may capture the presence of differentiative processes across the genome in distinct populations. Spatial analyses of molecular variance were used to identify groups of populations that are both genetically and geographically homogeneous while also being maximally differentiated from each other. The spatial analysis of genetic diversity indicates that the levels of diversity among the six populations vary significantly on an eco-geographical basis. Particularly, altitude seems to represent a differentiating adaptation, as the main genetic differentiation is detected between the two populations present at higher altitudes and the other four populations at sea level. The data, together with the outcomes from different cluster analyses, identify a genetic diversity pattern that overlaps with the distribution of the known morphotypes in the Brazilian area.We used a population genetic approach to detect the presence of genetic diversity among six populations of PageBreak Anastrephafraterculus Wiedemann (Diptera: Tephritidae) belongs to the fraterculus group, which comprises a total of 34 formally described species   . Althougfraterculus group and the Anastrephafraterculus complex have a recent evolutionary history, and thus molecular markers with a higher power of resolution were required to help understand the specific/subspecific differentiation within and between populations of this group  (Anastrephaobliqua (Macquart)  , and foracquart) . Microsaterculus  and haveSterile Insect Technique (SIT) against this fruit fly complex . For this purpose we used highly informative SSR markers, which may capture the presence of eventual differentiative processes across the genome in different populations.This paper is centred on the assessment of genetic diversity among Anastrephafraterculus by Dr. Elton L. Araujo (UFERSA), Dr. Keiko Uramoto (USP), Dr. Miguel Francisco Souza Filho (Instituto Biológico) and Dr. Roberto A. Zucchi (USP) using the aculeus shape following Zucchi (2000)  following the standard DNeasy protocol. DNA samples were screened using the following ten microsatellite loci: AfD4, AfD105, AfA7, AfA112, AfA115, AfA120, AfA122, AfA117, AfA10, and AfC103  , expected and observed heterozygosity were estimated using GENEPOP version 4.0.7 (Polymorphic Information Content (PIC) was derived using CERVUS  and mean null allele frequency   of populations that exist and generates F-statistics  using an AMOVA approach. Different numbers of groups (K) were tested, and a simulated annealing procedure permitted the identification of the composition of each of the K groups that maximizes the FCT index . The program was run for two to five groups (K = 2 to K = 5) each time with the simulated annealing process repeated 100 times, starting each time with a different partition of the population samples into the K groups. The analysis of molecular variance (AMOVA) was carried out using ARLEQUIN software version 3.11 . Principal Coordinate Analysis (PCoA) in the program GenAIEx 6.5 (er (MSA)  was applulations . The staMOVA 2.0 . This apAIEx 6.5  was applPageBreakAnastrephafraterculus populations are shown in Table PIC) estimate for each locus ranges from 0.44 (A10a) to 0.88 (A120a), and the across loci average is 0.74, suggesting that this set of loci is informative for population analyses.The variability estimates describing the suitability of the ten SSR loci  for detecting the presence of differentiation among the Hardy-Weinberg equilibrium (HWE) using Fisher’s exact test with the sequential Bonferroni correction (HWE) at most loci. The very few observed locus/populations combinations that were not in HWE were not concentrated at any locus or in any population. Significant linkage disequilibrium was not detected between genotypes at the ten loci. As no evidence of linkage disequilibrium between loci was assessed, these 10 loci can be considered to be independent.Tests for rrection  revealedPageBreaktotal variation is detected among populations. Indeed as shown in Table FCT values were observed when we increased the group number (K): four of the five simulated groupings  produced non-significant FCT values. This implies that the molecular variance due to differences between populations within each group is weak, confirming the AMOVA data. Only in one case did we observe an FCT estimate which statistically maximized the differences between groups. This is the grouping configuration which splits the six populations into three groups: i) Una, Porto Seguro and São Mateus, ii) Monte Alegre, iii) Campos do Jordão and Vacaria, . These data indicate that the mountain populations of Campos do Jordão (state of São Paulo) and Vacaria (state of Rio Grande do Sul) are genetically homogeneous and differentiated from the group of the three coastal populations , and from the other more distant coastal population of Monte Alegre. The pairwise FST estimates (Table PCoA) was performed to better clarify the genetic relations among the six populations. The first two axes explain a relatively high amount of the genetic variation (88%). The first axis (77.91%) separates Campos do Jordão and Vacaria from the other PageBreakpopulations. The second axis (10.39%) mainly differentiates São Mateus from the group of Una, Porto Seguro, and Monte Alegre, but also Campos do Jordão from Vacaria. It is interesting that there is a certain correspondence of the genetic grouping with the known morphotype classification  on genetic differentiation of mountain Vacaria and Campos do Jordão, than on the remaining samples belonging to the coastal plain populations.An estimate of variability distribution in and among the six tested populations (AMOVA) indicates that 90% of the variation occurs within populations while only about 10% of fication . The mol axis 10.9% mainlye Figure  and the e Figure , respectAnastrephafraterculus complex, an analysis of the underpinning genetic architecture and its interaction with correlated ecological, biological and morphological traits. In this context, we have used microsatellite markers to perform a genetic analysis of populations from a complex ecological area such as Brazil.With this paper we initiated, in the polymorphic Although the chromosomal location of the considered SSR loci remains unknown, the assessed linkage equilibrium between them suggests they are statistically independent and that their variability patterns might reflect genome-wide patterns across populations. The six considered ecogeographic populations are here represented by highly polymorphic samples, which reflect a high degree of intrapopulation genetic diversity. Indeed only 10% of the total variability (AMOVA) is represented by the differences between the six geographic populations, while greater variability was found within populations.PCoA data. More than by geographical distance, the genetic differentiation is influenced by altitude. The multivariate analysis of ten microsatellites depicts a structural pattern, which clearly separates populations on climatic distribution both on latitudinal and altitudinal basis. Particularly, altitude seems to represent a differentiating adaptation, as the main genetic differentiation is that detected between the populations present at higher altitudes (Campos de Jordão and Vacaria) and those populations from sea level. Genetic divergence between populations from low and high altitude areas has already been observed for populations within the Anastrephafraterculus complex using isozymes  is the observed population differentiation contributing to the underpinning genetic architecture of the morphotypes associated to these populations? and 2) do morphotypes track environmental variability? In retrospect, a further aim is to clarify the evolutionary relationships between populations, ecotypes, and morphotypes.One interesting observation, which arises from our data, is that the observed structure of Brazilian populations is entangled with the presence of morphotypes. The actual number of these entities and their respective geographic range are questions that remain to be further elucidated. At the moment three different morphotypes are identified in Brazil . As it aAnastrephafraterculus complex, is also important from an applied perspective. The overall level of genetic variability and the presence of differentiation that we detected among the Brazilian populations of Anastrephafraterculus constitute an important contribution for any potential future application of SIT for the control of populations of this fruit fly pest in Brazil.The population genetic approach, in addition to improving our knowledge of the underpinning genetic architecture of the


Fusarium graminearum (Fg) is a ubiquitous pathogen of wheat, barley and maize causing Fusarium head blight. Large annual yield losses and contamination of foodstuffs with harmful mycotoxins make Fg one of the most-studied plant pathogens. Analyses of natural field populations can lead to a better understanding of the evolutionary processes affecting this pathogen. Restriction site associated DNA sequencing (RADseq) was used to conduct population genomics analyses including 213 pathogen isolates from 13 German field populations of Fg.Fg field populations and low differentiation (FST = 0.003) was found among populations. Linkage disequilibrium (LD) decayed rapidly over a distance of 1000 bp. The low multilocus LD indicates that significant sexual recombination occurs in all populations. Several recombination hotspots were detected on each chromosome, but different chromosomes showed different levels of recombination. There was some evidence for selection hotspots.High genetic diversity was found within Fg is consistent with a high degree of sexual recombination that is not equally distributed across the chromosomes. The high gene flow found among these field populations should enable this pathogen to adapt rapidly to changes in its environment, including deployment of resistant cultivars, applications of fungicides and a warming climate.The population genomic structure of The online version of this article (doi:10.1186/s12864-015-2166-0) contains supplementary material, which is available to authorized users. Population genomics analyses can provide insight into the evolutionary history of populations by providing rich, genome-wide measures of genetic variation and its related parameters including gene flow, population size and reproductive system , 2. PopuDrosophila melanogaster reported an extremely rapid decay of LD within the surprisingly small distance of 10 bp [Recombination has an important effect on both genotypic diversity and evolutionary potential. The evolutionary potential reflects a pathogen’s response to control measures such as fungicide applications and deployment of resistant cultivars. Both homothallic inbreeding and asexual reproduction can reduce the effective recombination rate relative to random outcrossing , 4, geneof 10 bp , 12.Population genomic studies that search for associations between markers and quantitative traits require a large number of genetic markers , 2, ofteFusarium, including F. graminearum (anamorph: Gibberella zeae (Schwein.) petch) and F. culmorum. F. graminearum sensu stricto (Fg ss) is one of 14 cryptic species within the F. graminearum species complex [Fg ss is a homothallic fungus with a mixed reproductive system including inbreeding, outcrossing and asexual reproduction. Sexual reproduction in this fungus is highly dependent on temperature, with an optimum of 25–28 °C [Fusarium head blight (FHB) disease causes significant annual losses in cereal production around the world, in addition to frequent contamination of foodstuffs with harmful mycotoxins such as nivalenol, deoxynivalenol, and zearalenone. FHB is caused by different species related to the genus  complex , 7. Fg s25–28 °C . The pat25–28 °C , 12 depeFg included a limited number of isolates or a limited number of markers that were not equally distributed over the four Fg chromosomes [Fg ss to assess the reproductive system and the degree of population subdivision occurring over regional spatial scales; (ii) Determine the extent of linkage disequilibrium in the genome and conduct a genome-scale search for recombination hotspots; (iii) Search for evidence of selection hotspots that may contain candidate genes under strong selection.Several studies of population genetic structure have been conducted in different countries , 13–17 tomosomes , 18–20. Fg ss coming from different geographical areas and environments across Germany (Table ST) was 0.003 while pairwise differentiation between populations ranged from 0 to 0.036 . Though the first 8 coordinates were consistent with a continuous distribution, the second and third coordinates explained the highest overall amount of genetic variation , whereas it ranged from −0.41 to 0.11 when analyzing each field population separately (Table r2 < 0.10) across the four chromosomes. Linked markers showing r2 values > 0.5 dropped rapidly to r2 < 0.2 within a physical distance ranging from 600 to 1000 bp on different chromosomes  using a sliding window of 50 kbp revealed different patterns of LD on different chromosomes Fig. . The cormes Fig. . The sloA) and its standardized form (rd) for each population and each chromosome we found that all populations showed very low values of disequilibrium, on average, across all chromosomes. However, some field populations showed varying values of IA, including some with significant P-values, on different chromosomes within the same population. These differences may reflect variation in the degree of sexual recombination experienced by different populations and/or variation in frequencies of recombination among chromosomes  ranged from 0.3 × 10−5 to 7.3 × 10−5 crossovers per bp per generation , but in the combined population it was 0.4 × 10−5 crossovers/bp/generation over the four chromosomes (genome-wide recombination rate). By analyzing SNP markers on a finer scale, hotspots of recombination were revealed across the genome for the combined population  was highest on Chr1 followed by Chr3, Chr4 and was lowest on Chr2  to −0.05 (KEL: Chr4). The selection hotspots in the combined population had D values ranging from −1.4 to 1.2. High negative values are consistent with selective sweeps while high positive values are consistent with balancing selection.We used Tajima’s D test to determine the type and strength of selection operating in genomic regions that were in recombination hotspots. Measures of selection were based on departures of SNP allele frequencies from neutral expectations , 21. A sFg ss. Though geographical distances among the 13 field populations ranged from ~10 to 540 km, the overall population differentiation in Germany was very low (FST = 0.003), consistent with high gene flow across Germany and indicating that the individual field populations are part of a single metapopulation. Zeller et al. [Fg ss sampled over a spatial scale of ~330–1670 km, that found significant divergence among populations in the Upper Midwest of the USA for 10 polymorphic RFLP probes [ST = ~0.02–0.44) was also found for chemotypes in the North American populations based on nine variable number of tandem repeat (VNTR) markers using 130 isolates [We used RADseq to conduct the first population genomics study in P probes . Signifiisolates .ST values  [ST values (0.09–0.76) were also reported by Gale et al. [In an earlier study using the same German isolates, we reported much higher F = 0.20) . We beli = 0.20) , 24 and  0.09–0.7 were alse et al.  in a popFg ss populations in Europe identified three different chemotypes without any related clustering [F. graminearum isolates analyzed in the USA, suggesting the presence of a distinct subpopulation [Principle coordinate analysis (PCoA) was shown to be a suitable tool to monitor structure in human populations , 6, 25. ustering , 26, 27.pulation , 7, 28. pulation , 28) on r2 = 0.2 was found within a distance of ~600–1000 bp for different chromosomes. A similar rapid decay of LD was reported across a 10 bp scale on autosomes and across a 30 bp scale on the X chromosome of Drosophila melanogaster [F. graminearum is consistent with a high degree of recombination in these populations. Similar findings of very low LD and frequent recombination were reported [F. culmorum using multilocus sequence typing of 111 isolates from Australia, West Africa/North Asia, and Europe (14 countries). FST was 0.002 among these populations, similar to our results. In our dataset, some SNPs on chromosome 3 maintained moderate LD (r2 = 0.50–0.30) despite being separated by long physical distances . This may result from epistatic selection acting on the sites in disequilibrium [The pattern of LD across a genome reflects a population’s history, including the effects of genetic drift, selection, recent population admixture and frequency of recombination , 8, 29. nogaster , 11, 12 reported –17, 31 filibrium –20, 32 oilibrium , 33.Fg populations from Southern Louisiana and the Gulf Coast based on analyzing 10 PCR-RFLP loci in 534 isolates [A value of 0.01, but this value was significant at P = 0.001. A similar value of multilocus linkage disequilibrium (IA = 0.02) was reported in Chinese populations (169 isolates from 15 provinces) of Fg ss using 12 VNTR markers [d was low (0.03), HOH1 exhibited significant disequilibrium across all chromosomes even after applying a Bonferroni correction. We postulate that this reflects recent population admixture. HOH1 was sampled from naturally infected wheat within an experimental research station operated by University of Hohenheim. The naturally infected field included in our analyses was located near an artificially infected field that was inoculated with strains from across Germany, hence we expect that some ascospore movement among fields would introduce inoculated strains into our sampled field, generating a significant degree of population admixture. Overall, we believe that most of the observed disequilibrium in the combined population as well as the individual field populations likely reflects admixture rather than a significant degree of asexual reproduction within the German field populations.The importance of sexual recombination is cryptic for many homothallic fungi , 34, yetisolates , 23. Our markers . The inder) were measured among chromosomes in different field populations  also identified recombination hotspots [−11 to 22.9 × 10−2 crossovers/bp/generation. Tsai et al. [Saccharomyces cerevisiae and S. paradoxus, finding shared recombination hotspots with 9–45 × 10−4 crossovers/bp. We detected several shared recombination hotspots among the individual field populations when they were analyzed individually (data not shown).Different degrees of recombination , Tajima’s D values ranged between −0.58 and 0.38 and a t-test was used to show that the region with the highest negative value had been affected by a selective sweep [Analyses of nucleotide diversity can be used to infer processes affecting population evolution, based on deviations from the null hypothesis of neutral variation in an isolated population of constant size. We tested for departures from neutrality using Tajima’s est Fig. . Generalve sweep . Genome ve sweep . Recombive sweep . The recFg ss to adapt to a broad array of environmental stresses, including climate warming, deployment of resistant cultivars and fungicide applications. This study provides a baseline measure of recombination in natural field populations and a first indication of genomic regions that may be under selection. The low LD found over short physical distance coupled with a lack of population structure among these field populations of Fg ss optimizes all the parameters needed to perform a genome wide association study using these isolates.We found that field populations of Fusarium graminearum in Germany belong to a single, highly recombined meta-population. We identified ~240 recombination hotspots in this German meta-population. The high recombination rate observed across the genome coupled with the high gene flow observed among populations may enable Fg ss based on morphology [Thirteen field populations composed of an average of 16 isolates were sampled from naturally infected wheat fields in major wheat growing regions of Germany. All 213 single spore isolates were identified as rphology .PstI. We adapted the RADseq protocol developed by Baird et al. [DNA was extracted using DNeasy Plant Mini Kits (QIAGEN) and quantified using a Qubit 2.0 fluorimeter (Invitrogen). DNA concentrations were adjusted to ~50 ng/μl and digested with d et al.  with minF. graminearum PH-1 . SNPs were called using the Genome Analysis Tool Kit (GATK)-3.1-1 [The raw data were quality checked using the FASTX toolkit 0.13. Sequences were quality trimmed from the 3’ end using Trimmomatic 0.32 , omittinK)-3.1-1  from bamK)-3.1-1  was usedAll isolates were grouped into field populations according to their source. GenAlEx 6.5  was usedK = the proposed number of populations, L = average value of LnP (D) for the 100 permutations of Kth, and s = average of corresponding permutations.where Saccharomyces that showed a pairwise LD decay over 25–50 kbp [After taking into account the physical position of each SNP, the pairwise linkage disequilibrium was calculated using VCFtools v.3  using a 5–50 kbp . Multilo5–50 kbp  based onFg isolates as a single large population; (ii) by considering each field population separately. These analyses took into account the physical position of each SNP. The correlation between LD and physical distance was calculated using 1000 permutations. Recombination hotspots were detected using Fearnhead’s method [P < 0.001. Estimates of recombination rate were based on a Bayesian reversible jump MCMC model [S) [P < 0.01 to generate the results shown in Table The recombination rate in the German population was analyzed using LDhat 2.2  in two ws method , which dMC model . Tajima’odel [S) . Visualiodel [S) . EnrichmNo human or animal subjects or genetically modified organisms were included in this study.A VCF file containing the nucleotide variation among the used isolates and basic information about the isolates is available at the European Nucleotide Archive under the accession number PRJEB11357. Using the data require citation.


Liriodendron chinense, an important temperate Asian tree species, as a model system to evaluate the roles of biogeographic history and marginal population genetics in determining range limits. We examined the demographic history and genetic diversity of 29 L. chinense populations using both chloroplast and nuclear microsatellite loci. Significant phylogeographic structure was recovered with haplotype clusters coinciding with major mountain regions. Long-term demographical stability was suggested by mismatch distribution analyses, neutrality tests, and ecological niche models (ENM) and suggested the existence of LGM refuges within mountain regions. Differences in genetic diversity between central and marginal populations were not significant for either genomic region. However, asymmetrical gene flow was inferred from central populations to marginal populations, which could potentially limit range adaptation and expansion of L. chinense.Species ranges are influenced by past climate oscillations, geographical constraints, and adaptive potential to colonize novel habitats at range limits. This study used  Species range limits reflect demographic and evolutionary responses to spatially and ecologically varying habitats even in the absence of geographic barriers12453et al.10The central–marginal hypothesis, which is related to the ‘abundant-center’ hypothesis11From the perspective of gene flow, the related asymmetrical gene flow hypothesis posits disproportionate levels of gene flow from central to peripheral populations81618816610In addition to gene flow, biogeographic history has profoundly influenced species’ geographic distributions and population genetic structure23242527Phylogeographic patterns used in identifying refugia and colonization histories have been extensively reported in Europe322234Liriodendron chinense (Hemsl.) Sarg. (Magnoliaceae), also known as the Chinese tulip tree. The most recent common ancestor of L. chinense and its North American sister species, Lirodendron tulipifera, had a broad Northern Hemisphere distribution during the globally warmer Mid-Tertiary period, and contracted into its current range disjunction, leading to allopatric divergence of the two Liriodendron species in the mid Miocene (ca. 14 mya)Liriodendron is comprised of tall deciduous tree species and are shade intolerant. The two species can hybridize under experimental conditions. Liriodendron tulipifera is a dominant species in early succession and is widespread in eastern Northern American broad-leaf forests, with its greatest abundance in the southern Appalachian uplandsLiriodendron chinense, in contrast, although with similarly wide geographical range, is scattered in subtropical China extending to Northern Vietnam mostly at elevations around 450–1800 mL. chinense makes it an ideal case study of range limit dynamics.Here, we focus on a Tertiary relict species in this region, L. chinense had previously been constrained by low numbers of sampled wild populationsL. chinense. Specifically, we aimed to: (i) infer genetic signatures of refugia and possible colonization during the past climate oscillations; (ii) investigate genetic structure and genetic diversity across the whole geographic range; (iii) evaluate the central-marginal and asymmetry gene flow hypotheses. In addition, we sampled haplotypes from the North American tulip tree L. tulipera in order to compare the demographic histories within this iconic disjunction between forests of eastern North America and eastern Asia.The measure of geographic genetic diversity of L. chinense , whereas the average haplotype diversity within population was relatively low (hS = 0.259). The highest population haplotype richness was in population YY located in Wuling Mountains (HR = 3.194), followed by population HGS in the west Wuyi Mountains (HR = 3.000). Population haplotype diversity (H) ranged from 0 to 0.667 , indicating phylogeographic structure across the range of L. chinense. The NJ tree  harbored haplotypes  from a separate branch in cluster C. Populations in the northwest (Daba Mountains) contained the remaining haplotypes in cluster C closest to L. tulipifera (cluster F). Haplotypes from populations in the east of Wuyi Mountains comprised cluster B, while haplotypes from the west of Wuyi Mountains (HGS) formed one branch in cluster D. The other branch of cluster D was distributed in Wuling  and Nanling (ZY) mountain regions, located on the east edge of Yungui Plateau, and also shared haplotypes from cluster A with populations in Yungui Plateau.Cluster ountains  and 2. Cr statistics could reject the sudden expansion model (p > 0.05), the mismatch distribution pattern over the whole range was multimodal  exhibited this deviation in two or three loci. Ten population-locus pairs showed significant LD, but no locus pair showed significant LD after Bonferroni correction. Two genetic diversity hotspots were identified: one was along the Wu Mountains and Wuling Mountains toward the western edge of Nanling Mountains; the other was located in the east, near the Tianmu Mountains and northeast of the Wuyi Mountains. Allelic richness (AR) was highest in population JS  in the western Nanling Mountains. The extracted genetic diversity (H’nSSR) accounted for 90.1% of the five genetic diversity  variances. Populations with high genetic diversity typically occurred at mid-latitudes closer to the current northern range edge, and it was obviously low in the southwest ranges  of L. chinense was 0.694 and 0.970 for nSSR and cpSSR markers, respectively, while the genetic differentiation measured as FST was 0.308 and 0.905, respectively. Pairwise FST values were all significant (p < 0.05), and ranged from 0.708 to 0.056 for nSSRs, and from 1.000 to 0.258 for cpSSRs. The AMOVA analyses of L. chinense revealed most nuclear genetic variation resided within populations . The predicted present habitat niches nearly fit the actual geographic distribution of chinense . The prehe south . The conL. chinense were classified by their geographic locations  in marginal populations was not significantly higher than in central populations in either genome .Over the whole range of Liriodendron species despite similarities in ecological niche. In North America, L. tulipifera, as a dominant part of the deciduous broadleaf forest, is distributed in higher latitudes and lower altitudes than L. chinense, and it experienced a major southward range shift during the Pleistocene glaciations. Genetic and morphological data suggested the existence of isolated refugia in the Apalachicola River basin and in north-central Florida for L. tulipifera, while the southeastern coastal plains were implicated as a zone of genetic admixturein situ scattered refugia in subtropical China during the LGM32L. chinense also indicated no sign of large scale habitat shifts to the south and no obvious colonization to the north after the LGM, but instead supported a model of geographic stability, partially leading to lower latitude distribution compared with its sister species in North America.The eastern Asian and eastern North American intercontinental floristic disjunction is of longstanding scientific interestL. chinense suggested at least six scattered subtropical refugia in the Yungui Plateau and the mountains of Daba, Dabie and Northern Luoxiao, Nanling, Tianmu and Wuyi. These putative refugia have been recognized as centers of plant diversity and possible glacial refugia for other ancient relict tree species such as Taxus wallichianaGinkgo bilobaTapiscia sinensisGeographical haplotype distributions of L. chinense in the eastern region remained fragmented and without admixture during the cold period as predicted by ENM and genetic data. Populations in the Dabie and adjacent Luoxiao Mountains harbored a unique haplotype branch with the most mutation steps to other branches, suggesting isolation from other regions. Central populations (JGS) within this region harbored the most diverse and potentially ancestral haplotypes that may have preceded colonization of adjacent regions. The Tianmu and Nanling Mountains have acted as refugia for many temperate tree species including Ginkgo bilobaFagus englerianaL. chinense. The habitat of the western region was continuously ecologically suitable for L. chinenseTapiscia sinensis, Zhang et al.L. chinense were found. The Yungui Plateau, which extends to highlands in warm Northern Vietnam, was also an important LGM refuge for L. chinense. Populations there comprised a cluster without much haplotype loss (cluster A), also suggesting that Yungui Plateau was environmentally stable. Populations in the south edge contained putative ancient haplotypes; their expansion to north Yungui Plateau and adjacent mountains was supported by demographic tests, and contributed to the genetic diversity hotspots in the Wuling and Nanling Mountains  and genetic heterozygosity  than north marginal populations. It has been shown that the south margin of temperate species, such as L. chinense, functioned as refugia during glacial intervals leading to population differentiationL. chinense and maintenance of within population diversity was favored over population differentiation. Although neutral genetic diversity may have no direct connection with adaptive traits20L. chinense to adapt to novel environments beyond these range limits12L. chinense in the south edge of its range.However, populations of L. chinense, the asymmetrical gene flow hypothesis was supported and asymmetrical gene flow from central to both south and north marginal edges over the whole range was inferred. Hence, the large influx of foreign individuals may have swamped local adaptive alleles of the edge populations, and impact the range limit of L. chinense. However, special attention should be paid to the genetic effects of gene flow to marginal populations apart from the symmetry pattern. The relative contribution of swamping local adaptive alleles and providing potential adaptive alleles for marginal populations was crucial in determining the asymmetry gene flow hypothesis for range limits8There are few empirical tests of the assumptions that asymmetrical gene flow may swamp local adaptation in peripheral populations with potentially maladapted genes, thereby precluding range expansionL. chinense over its geographical range, attempting to uncover the potential impacts of historical climate oscillation on its current spatial genetic structure and evaluate expectations of two longstanding evolutionary hypotheses involving range limits by molecular population genetic investigation. Inferred from the phylogeographic history of L. chinense, Quaternary glacial cycles have played a prominent role in shaping the geographic distribution and genetic structure of L. chinense. In accordance with most tree species in subtropical forests of China, no massive range shifts but multiple local refugia were suggestedL. chinense after the LGM, resulting a relatively even genetic diversity distribution pattern over the central and marginal populations. The expected asymmetrical gene flow from central to marginal populations was observed in L. chinense, which may partly explain the range limits of L. chinense. However, whether lower genetic diversity and asymmetrical gene flow is associated with lower population fitness performance beyond the range limits remains uncertain. As a complement to molecular studies, field studies investigating population fitness performance should be used in future studies to provide deeper insight into evolutionary mechanism involved in the maintenance of species’ range limits.In summary, in the present study, we investigated the phylogeographic pattern and genetic structure of L. chinense in 2013 (N = 24) for each population were sampled, except for population XF and HGS, from which only nine and six wild individuals were obtained respectively. Twenty-seven L. tulipifera samples collected from a mixed population introduced from the U.S. were used for comparative purposes. The genomic DNA was extracted from young leaves using the CTAB method.We collected young leaves of 693 individuals from 29 wild populations across the entire range of  in 2013 . FifteenL. chinensede novo characterized nuclear microsatellite primers in L. chinenseL. tulipifera52Ten mononucleotide chloroplast SSR primers developed for NA), effective number of haplotypes (NE), the haplotype richness (HR), and the haplotype diversityH) were calculated using the program GenAlEx 6.5et al.Unique combinations of cpSSR alleles within a chloroplast genome were defined as haplotypes. The number of haplotypes  under 10 000 permutations in Arlequin version 3.1RST  was significantly higher than the GST (depending on frequencies of haplotypes) under 1000 random permutations in PermutCpSSR version 2.0The presence of phylogeographic structure was determined by whether the value of L. tulipifera included as the outgroup using PowerMarker v3.25L. chinense was assessed by neutrality tests with Tajima’s D and Fu’s Fs and mismatch distribution analysis  tree was constructed based on 1 000 bootstraps of Nei’s distanceanalysis . ChloropMicro-checker (available at http://www.norwichresearchpark.com/ourresearch/researchgroups/elsa/software/microchecker.aspx). Linkage disequilibrium (LD) and Hardy-Weinberg equilibrium (HWE) for each population and microsatellite locus pair was assessed in Genepop version 1.2NE), allelic richness (AR), expected genetic diversity (HE) and observed genetic diversity (HO) were calculated using the software GeneAlEx 6.5Genotyping errors were identified by the software L. chinense was determined using STRUCTURE v2.3.3K (1–33) were carried out by 50 000 burn-in and 100 000 iterations of the MCMC. The suitable number of clusters (K) was chosen at the largest rate of change in the log probability of data between successive K valuesThe extraction of genetic diversity and estimation of population differentiation were performed as described in cpSSR markers  curve switches from a sharp decrease to a plateauAn additional cluster analysis was performed with TESS v2.0et al.−4 per locus per generation8 and a logging frequency of 1 × 106.Extended Bayesian Skyline Plots (EBSPs) were used to test the hypothesis of demographic expansion at Yungui Plateau. The EBSPs were constructed in BEAST v1.80L. chinense was carried out to evaluate the habitat suitability at present and during the Last Glacial Maximum . One hundred and forty-eight occurrence localities including three from Northern Vietnam  and local flora. Nineteen bioclim variables were extracted from the WorldClim databasebioclim variables  with lower level of correlation (r2 < 0.75) were used in the climatic niche model building. The ENM was executed using Maxent version 3.3.3kMaxent was run using the default settings with ten repeats, and the final maps of prediction results were visualized in arcgis 9.3 . The projected present habitat suitability scores for each sampled population were extracted.The ENM of  Vietnam  were obtn r2 < 0. were useL. chinense by their geographic locations. Geographical locations of L. chinense based on extensive field surveys and herbarium records were digitized into the range maps in arcgis 10.2. We then divided species’ range into peripheral and central regions by equal areavs. marginal populations in both genomes were compared using t-tests or Mann-Whitney U tests depended on the distributed mode of each parameter , and ANOVA was performed to evaluate the difference of central, south marginal and north marginal followed by Duncan post hoc test in SPSS 13.0 program. The genetic differentiation between the two groups was compared using a 1 000 permutation test in the FSTAT version 2.9.3We defined the central and marginal populations of Neμ) and directional mutation-scaled effective migration rate (M = m/μ) were calculated in Migrate v3.5.1 (available at http://popgen.dcd.fsu.edu/Migrate-n.html) using nuclear microsatellite data under the suggested Brownian microsatellite mutation model. Considering different situations along species’ edgesM were generated from FST values, and we conducted searches using ten short chains of 200 000 sampled trees and three long chains each with 70 000 record trees out of 3.5 million sampled trees, and discarded the initial 20 000 trees as burn in.Mutation-scaled effective population size .