Papaver rhoeas, an annual plant species in the Papaveraceae family, is part of the biodiversity of agricultural ecosystems and also a noxious agronomic weed. We developed microsatellite markers to study the genetic diversity of P. rhoeas, using an enriched microsatellite library coupled with 454 next-generation sequencing. A total of 13,825 sequences were obtained that yielded 1795 microsatellite loci. After discarding loci with less than six repeats of the microsatellite motif, automated primer design was successful for 598 loci. We tested 74 of these loci for amplification with a total of 97 primer pairs. Thirty loci passed our tests and were subsequently tested for polymorphism using 384 P. rhoeas plants originating from 12 populations from France. Of the 30 loci, 11 showed reliable polymorphism not affected by the presence of null alleles. The number of alleles and the expected heterozygosity ranged from 3 to 7.4 and from 0.27 to 0.73, respectively. A low but significant genetic differentiation among populations was observed . The 11 validated polymorphic microsatellite markers developed in this work will be useful in studies of genetic diversity and population structure of P. rhoeas, assisting in designing management strategies for the control or the conservation of this species. Papaver genus in the Papaveraceae family comprises 100 species distributed in various countries around the world, from central and south Europe to temperate Asia, America, Oceania and South Africa [Papaver rhoeas L. (corn poppy) is one of the most well-known members of this genus, easily identified by its scarlet flowers. This species has been the symbol for Remembrance Day (or Poppy Day) since the end of WWI, which makes it a heritage species. P. rhoeas is a semelparous species that thrives in disturbed land. It has a high reproductive potential through its seeds, which are dormant and have long viability, thus facilitating its persistence and spread [P. rhoeas can dominate agricultural fields, where it is listed as a serious annual weed of winter cereal crops that can cause significant yield losses because of its high competitive ability [P. rhoeas also includes indirect harmful effects on crops through the harbouring of phytopathogenic viruses [P. rhoeas [The d spread –4. Its ad spread  as well d spread ,7. On th ability . The agr viruses .Various P. rhoeas is an insect-pollinated, diploid hermaphroditic species (2n = 14) with high self-incompatibility [P. rhoeas has been reported to exhibit high phenotypic plasticity and high genetic variation, which for example results in variable petal pigmentation and leaf shape [tibility . Outcrostibility . P. rhoeaf shape .P. rhoeas is important for the study of the ecology and population structure of this species. Such studies can assist in implementing management strategies in the context of either conservation ecology or weed control. Simple sequence repeat (SSR) markers, also called microsatellite markers, are popular tools used to assess the genetic diversity of species [P. rhoeas populations. The aim of this work was to develop and validate polymorphic microsatellite markers for P. rhoeas, and to assess the genetic variation in 12 P. rhoeas populations from France.Quantification of genetic diversity within and among populations of  species . Their aP. rhoeas enriched library. Average sequence length was 240 nucleotides, with a maximum length of 570 nucleotides. After discarding short sequences (less than 80 nucleotides) and sequences without microsatellite motifs, the remaining sequences were filtered for redundancy and multiple copies in the sequence data set. In total, 1795 validated microsatellite loci were identified. Automated primer design was successful for 829 loci. Loci with less than six repeats of the microsatellite motif were discarded, yielding 598 loci for which the QDD pipe-line generated 4453 primer pairs.A total of 13,825 sequences were obtained from the P. rhoeas plants used for screening. Among the 30 loci, 20 carried a dinucleotide motif and 10 a trinucleotide motif. The remaining 44 loci were not further considered because no amplicon could be obtained, or amplicons were not obtained for all seven P. rhoeas plants used for screening, or no polymorphism was detected after sequencing the amplicons.We selected 74 of the 598 microsatellite loci based on the lowest value of the “penalty” index computed by QDD. A total of 97 primer pairs were tested to amplify the 74 loci. An amplicon was consistently obtained from 30 of the 74 selected loci. The 30 amplicons were sequenced in both directions with the corresponding primer pair. They were all confirmed to contain the expected microsatellite motif, and polymorphism was observed for all loci among the seven P. rhoeas plants . The software MicroChecker [i.e., short allele dominance due to PCR artefacts). There was no evidence for large allele drop-out in any loci. Null alleles can cause deviations from Hardy-Weinberg equilibrium, and can introduce substantial bias in population genetic analyses. Loci for which null alleles were detected in a majority of populations (more than seven populations among the 12 studied) were, therefore, not further considered. This screening resulted in a final set of 11 microsatellite loci, the characteristics of which are summarised in The 30 microsatellite loci were further tested on 384 oChecker  was usedP. rhoeas collected in cultivated fields in France (32 plants per population) were genotyped at the 11 microsatellite loci. The average number of alleles per population (NA) was between 3.0 (locus PMS073) and 7.4 (locus PMS002). The mean expected heterozygosity per population (HE) also varied widely among loci, ranging from 0.271 (locus PMS073) to 0.731 (locus PMS037). Significant pairwise linkage disequilibrium was detected for only three pairs of loci: PMS005 and PMS073 , PMS015 and PMS073  and PMS039 and PMS054 .A total of 384 plants from 12 natural populations of FIS values (HE) observed over all loci in each population. Four other loci  also showed some deviation from Hardy-Weinberg equilibrium. However, for each locus, this was only observed in one or two populations among the 12 studied , varied between 0% and 24% . For several samples, the estimated rates of autogamy seemed to conflict with the self-incompatible mating system described in P. rhoeas [i.e., mating among relatives due to limited seed dispersal and restricted pollinator flight distances), or from population subdivision at the level of each sampled field . This remains to be investigated.Considering all loci except PMS002, the mean FST, was 0.04. This indicated large population sizes and/or high gene flow, as expected in an allogamous plant species [p-value < 0.001). Pairwise FST values computed for all possible pairs of populations ranged from 0.009 to 0.087. A significant linear relationship was observed between pairwise FST/(1 − FST) values and the natural logarithm of geographical distances between populations  . The genetic differentiation among all populations, as measured by  species . Althoug< 0.001) . This inP. rhoeas populations were sampled in agricultural fields in the Burgundy region (France) during the summer of 2011 (Twelve  of 2011 . SamplesThe approach used has been described elsewhere . BrieflyMicrosatellite loci of interest were selected among those identified by running the QDD pipe-line using the following criteria: (1) a target microsatellite sequence containing at least six repeats of the microsatellite motif, and (2) an expected amplicon of 90 to 350 bp in length. Editing of the primer sequences generated by QDD was performed when necessary to ensure an annealing temperature around 60 °C, and the presence of a C or G nucleotide at each primer 3′ end to increase the stability of primer binding to its target. The edited primers were then checked for self-complementarity and complementarity between primers in a same pair to avoid the formation of primer dimers. Primers sequences were further edited as needed.P. rhoeas plants originating from five populations: FE (3 plants), BE (1 plant), ST (1 plant), CS (1 plant) and PY (1 plant). Geographic origins of the populations are shown in 2, 17 mM (NH4)2SO4, 10 mM beta-mercaptoethanol, 0.05% (wt/vol) polyoxyethylene-ether W1, 0.2 mg/mL bovine serum albumin, 200 mM of each dNTP, 10 ng genomic DNA, 0.5 units of Taq DNA polymerase, and 0.2 μM each of reverse and forward primers. The PCR program used consisted of 5 min at 95 °C, followed by 37 cycles of 5 s at 95 °C, 10 s at 60 °C and 30 s at 72 °C [P. rhoeas populations of 32 individuals each  and assayed on an ABI 3730XL sequencer  using 500 liz  as a size standard. Amplicon sizes were analyzed with GeneMapper 4.0 .NA), observed (HO) and expected (HE) levels of heterozygosity as well as the inbreeding coefficient FIS were estimated for each population. Genetic differentiation among populations was estimated using Weir and Cockerham’s FST [FST was tested, based on 5000 random permutations of genotypes among populations.Micro-Checker v2.2.3  was usedam’s FST . DeviatiP. rhoeas proved reliable for population genetic studies. Based on a sample of populations clustered within roughly 150 km, we observed a level of genetic differentiation that was low but that increased significantly with geographical distances. This suggests that addressing population structure in P. rhoeas at a geographical scale larger than the scale considered here should reveal more genetic polymorphism and higher levels of genetic differentiation. On the other hand, fine-scale studies are necessary to elucidate the existence of some genetic structure within cultivated fields. Future studies, based on the 11 polymorphic microsatellites we developed, will enable a better understanding of the patterns of genetic diversity of P. rhoeas, providing information on population structure and dynamics that can be useful for guiding management strategies.The 11 polymorphic microsatellites developed for


Echinococcus granulosus by analyzing the complete mitochondrial NADH dehydrogenase subunit 2 (ND2) gene in 51 isolates of E. granulosus sensu stricto metacestodes collected at three locations in this region. We detected 19 haplotypes, which formed a distinct clade with the standard sheep strain (G1). Hence, all 51 isolates were identified as E. granulosus sensu stricto (G1–G3). Genetic relationships among haplotypes were not associated with geographical divisions, and fixation indices (Fst) among sampling localities were low. Hence, regional populations of E. granulosus in the southwest China are not differentiated, as gene flow among them remains high. This information is important for formulating unified region-wide prevention and control measures. We found large negative Fu's Fs and Tajima's D values and a unimodal mismatch distribution, indicating that the population has undergone a demographic expansion. We observed high genetic diversity among the E. granulosus s. s. isolates, indicating that the parasite population in this important bioregion is genetically robust and likely to survive and spread. The data from this study will prove valuable for future studies focusing on improving diagnosis and prevention methods and developing robust control strategies.We evaluated genetic diversity and structure of  Echinococcus granulosus complex  was higher than the frequency of G + C [32.07%]) [All 51 isolates were successfully amplified for the complete 32.07%]) . OverallND2 formed one clade and was not divided into regional clades according to the allopatric distributions of the isolates (E. granulosus sensu stricto (genotypes G1–G3). Results from the Bayesian tree analysis depicted a similar topology to the maximum-likelihood phylogram  within the 51 isolates , although nucleotide diversity was low (Pi = 0.005 < 5%). The genetic distance between haplotypes (Kimura 2-parameters) ranged from 0.001 to 0.028 , one (H2) was shared between the Qinghai and Sichuan populations and two (H5 and H7) were shared between the Qinghai and Tibet populations, while no haplotypes were shared between the Sichuan and Tibet populations. The haplotype phylogenetic tree showed that E. multilocularis was genetically distinct from E. granulosus s. s.  and that all 19 haplotypes  grouped with the standard sheep strain (G1) and were thus classified as E. granulosus s. s. (genotypes G1–G3). This finding is similar to previous studies that found that E. granulosus s. s. (G1–G3) is the major strains of E. granulosus throughout China [Of the 19 haplotypes we defined  and high haplotype diversities (Hd > 0.5) consistent with the hypothesis of sudden demographic expansion . This hyribution . This prND2 gene similarity of the 51 E. granulosus isolates was found to be high (96.71% to 100%), though no sequence displayed 100% homology with the G1 genotype. The genetic diversity observed among the isolates in this study was significantly higher than that reported in previous studies, which used the CO1 gene to investigate genetic polymorphisms in E. granulosus in China [ND2 gene than the CO1 gene of E. granulosus. Nevertheless, the high degree of genetic diversity in E. granulosus in this region revealed by all the studies indicates that the populations are genetically robust and likely to survive and spread.The in China . HoweverE. granulosus populations and transmission dynamics of echinococcosis in these regions. Having the useful information, focus should now be directed to strengthen disease surveillance in these regions and improve diagnostic and prevention methods, in addition to developing a robust control strategy.In summary, our study provides the basic information about genetic diversity analysis of a wider range of isolates from different regions of Southwest China in order to understand in detail the genetic structure of


Paeonia rockii and the role of the Qinling Mountains as a barrier that has driven intraspecific fragmentation were evaluated using 14 microsatellite markers.Tree peonies are great ornamental plants associated with a rich ethnobotanical history in Chinese culture and have recently been used as an evolutionary model. The Qinling Mountains represent a significant geographic barrier in Asia, dividing mainland China into northern (temperate) and southern  regions; however, their flora has not been well analyzed. In this study, the genetic differentiation and genetic structure of P. rockii. Significant population differentiation was suggested . Moderate genetic diversity at the population level (SH of 0.516) and high population diversity at the species level (TH of 0.749) were detected. Significant excess homozygosity (ISF of 0.076) and recent population bottlenecks were detected in three populations. Bayesian clusters, population genetic trees and principal coordinate analysis all classified the P. rockii populations into three genetic groups and one admixed Wenxian population. An isolation-by-distance model for P. rockii was suggested by Mantel tests  and supported by AMOVA (P<0.001), revealing a significant molecular variance among the groups (11.32%) and their populations (21.22%). These data support the five geographic boundaries surrounding the Qinling Mountains and adjacent areas that were detected with Monmonier's maximum-difference algorithm.Twenty wild populations were sampled from the distributional range of P. rockii has resulted from the fragmentation of a formerly continuously distributed large population following the restriction of gene flow between populations of this species by the Qinling Mountains. This study provides a fundamental genetic profile for the conservation and responsible exploitation of the extant germplasm of this species and for improving the genetic basis for breeding its cultivars.Our data suggest that the current genetic structure of  Ailuropoda melanoleuca) revealed that limited gene flow occurs between the Qinling population and other populations of this species The patterns of genetic structure in plants are the result of many interacting factors, including climatic fluctuations, complicated landforms, soil types and human activities; however, a more important factor is the evolutionary history of a species. The Qinling Mountains represent an important geographic barrier in Eastern Asia that divides the current mainland of China into southern and northern and temperate and semi–tropical regions. Additionally, these mountains are a major watershed of the Yellow and Yangtze Rivers. These regional divisions have a huge impact on numerous geographic, climactic and agricultural factors Moudan of the genus Paeonia in the family Paeoniaceae, and all 9 species of these plants are endemic to China Paeonia jishanensis, P. suffruticosa, P. cathayana, P. ostii, P. qiui, P. decomposita, P. rockii, P. delavayi and P. ludlowii, are distributed in the Yunnan, Sichuan, Gansu, Shaanxi, Shanxi, Henan, Hubei, Anhui and Xizang (Tibet) provinces Paeonia rockii is endemic to the Qinling Mountains and the adjacent area; additionally, this species is associated with the widest distribution range and the greatest numbers of extant plants among all nine species P. rockii is one of the most important ancestral species that has contributed to establishing the cultivated tree peonies. It includes two allopatric infraspecific taxa, P. rockii ssp. rockii (subspecies rockii) and P. rockii ssp. atava (subspecies atava), based on their morphological characteristics rockii is found in the western and eastern parts of the Qinling Mountains and has been observed in the Bashan Mountains in Hubei province, whereas subspecies atava is limited to only the northern slopes of the Qinling Mountains and further northward in this vicinity atava presents ovate (or rounded) and totally (or mostly) lobed leaflets, whereas subspecies rockii exhibits lanceolate to ovate–lanceolate and totally (or mostly) unlobed leaflets Tree peonies are an important group of plants that are referred to as ‘the king of flowers’ due to their rich palette of horticultural varieties and deep ethnobotanical history in Chinese culture; in addition, this group of plants has recently been used as an evolutionary model P. rockii closely coincide with the geographic distribution of this species, as revealed by three chloroplast genes, suggesting that the high Qinling Mountains have acted as a geographic barrier contributing to this phylogeographic structure atava, which is located on a northern slope of the Qinling Mountains, is more closely related genetically to the clades of the populations of the western group of subspecies rockii than to the clades of other populations  of the northern group of subspecies atava. In addition, the clade of the Ganquan population (GQ) of subspecies atava that is located to the north of the Qinling Mountains is much closer to the clades of the Luanchuan population (LC) of the eastern group, which is located in the Xun'er Mountains of the eastern Qinling Mountains PaeoniaP. rockii in greater detail.Molecular phylogeographic approaches examining both nuclear and organellar genomes have become popular for understanding the patterns of population genetic structure P. rockii has recently been decreasing, similar to that of all other tree peony species, and it has been listed as an endangered species in the Chinese Red Data Book P. rockii in breeding new cultivated tree peonies and because it is currently facing endangered status, continuing study of the genetic diversity and genetic structure of this species is worthwhile. The original development of landforms of the Qinling Mountains was promoted by Yanshanian tectonic events in the latest Cretaceous approximately 70 Ma (million years ago) and the subsequent lateral extrusion of the Tibet plateau in the early Cenozoic (approximately 40 Ma). Additionally, its relatively stable landforms (with a gentle hillside and flat lands) formed in the early Quaternary period (2.4–1.2 Ma). Then, the present high topographic gradients with a height drop of 1,000–2,000 m in the Qinling Mountains and the highest peak, Taibai Mountain, which is 3767 m, resulted from the latest rapid uplift activated by the Himalaya orogenic belt during the Pleistocene (approximately 0.7 Ma) P. rockii in this region could provide a good model for examining the intraspecific evolutionary processes and dynamics associated with this history of complicated landform development in the Qinling Mountains. Furthermore, these studies could provide fundamental genetic information for developing effective and sustainable conservation plans and material for the breeders of this important ornamental plant. Therefore, the major objectives of the present study were 1) to reveal the genetic diversity and population genetic structure of P. rockii using nuclear markers (microsatellite markers) and compare the similarities and differences between the results obtained from chloroplast DNA (cpDNA) and nuclear microsatellite markers and 2) to determine whether and how the Qinling Mountains as a geographic barrier has deeply impacted the population evolution of the endemic species P. rockii.The distribution of Paeonia rockii is a deciduous shrub that grows up to 1.8 m tall, flowers from late April to May, and fruits from late July to August P. rockii (including both subspecies rockii and subspecies atava) and immediately placed in silica bags. The samplings included 335 wild individuals of 20 populations that contained four populations  of subspecies atava and the other 16 populations of subspecies rockii, as shown in P. rockii collection was carried out using 14 microsatellite markers, as shown in PaeoniaFor each sampling site, the altitude, longitude and latitude were recorded using a GPS  . The gen4), 2.5 mM MgCl2, 0.2 mM each dNTP, 0.25 µM reverse primer and 0.25 µM forward primer tagged with JOE, NED, FAM or PET fluorescent dye to facilitate post-PCR multiplexing. The PCR amplifications were performed using an initial denaturation at 94°C for 3 minutes, followed by 25 cycles of 30 seconds of denaturation at 94°C, 30 seconds of annealing at 54°C and 1 minute of polymerization at 72°C, with a final extension step of 10 minutes at 72°C. The PCR products were detected using an ABI 3730xl DNA Analyzer with GeneScanTM-600 LIZ  as an internal size standard, and each locus was genotyped with GeneMapper ver. 4.0 . Samples in which the alleles were uncertain were double-checked by repeating the amplification and proofreading the raw data.PCR amplifications were performed in 10-µL reaction volumes containing 1× PCR buffer , observed heterozygosity (oH), average genetic diversity (SH) both within populations and among populations and total gene diversity (TH) OA), observed heterozygosity (OH), unbiased expected heterozygosity (EH), private alleles (AP) and allelic richness (AR) AR[10]) was standardized for 5 individuals  using FSTAT version 2.9.3.2 For each microsatellite locus, the genetic diversity was analyzed to calculate the total number of alleles (ISF) P-values ( = 0.05) were adjusted using a sequential Bonferroni correction The significance of any deviations from Hardy-Weinberg equilibrium, as indicated by deviations of the fixation index (AR and EH) and their geographic locations (latitude and longitude) were tested by linear regression. Reduction in the effective population size was estimated using Wilcoxon's signed-rank tests (one-tailed) with the null hypothesis, EH>EQH, and the alternative hypothesis, EH<EQH, under the assumption of mutation-drift equilibrium in the infinite-alleles model (IAM) and the stepwise mutation model (SMM) using BOTTLENECK version 1.2.02 To examine the geographic gradients in the genetic diversity within populations, the relationships between the genetic diversity within populations (STF using GenAlEx version 6.2 STF/(1−STF) according to Rousset The genetic differentiation among populations was estimated at each locus and across all of the loci by calculating K) without using prior information on the individual sampling locations. This program distributes the individuals among K clusters based on their allelic frequencies and estimates the posterior probability of the data given each particular K. STRUCTURE was run for the K = 1 to K = 30 clusters. Each run was pursued for 106 MCMC iterations, with an initial burn-in of 5×105, and an ancestry model that allowed for admixture, with the same alpha for all of the populations. To assess stability, 10 independent simulations were performed for each K. The final posterior probability of K, lnP(x|K), was computed, as suggested by Pritchard et al. K. lnP(x|K) usually plateaus or increases slightly after the ‘right’ K is reached. Thus, following Evanno et al. K was calculated. The sensitivity of the final result to the specific previous assumptions of alpha and with regard to the independence of the allelic frequencies was also computed. The second method was a distance-based clustering analysis using a neighbor-joining tree based on genetic distance (AD) The pattern of genetic variation among populations was assessed using three different methods. First, the software package, STRUCTURE 2.1 The genetic barriers associated with each geographical location and population were investigated using Monmonier's maximum-difference algorithm SH) among the loci was 0.516 and ranged from 0.245 to 0.648. Similarly, the average overall genetic diversity (TH) estimate among the populations was 0.749, ranging from 0.288 to 0.899. The mean number of alleles per locus was 15.9 and ranged from 1.71 (TC) to 24.71 (JL). The mean values for the observed heterozygosity (OH) and unbiased expected heterozygosity (EH) were 0.459 and 0.492, respectively. The value of the private alleles per population (AP) was 2.3, and the mean within the population allelic richness (AR[10]) was 2.65, as shown in ISF over all the loci was found to be significant in eight populations , whereas the ISF within populations was significant for eight loci , as shown in Estimates of the diversity among the microsatellite loci are shown in AR within populations (or EH) and their geographic locations (latitude and longitude). According to Wilcoxon's signed-rank test performed using BOTTLENECK, three populations  showed a significant deviation (excess heterozygosity) from the mutation-drift equilibrium under the IAM , as shown in No significant relationships existed between the genetic diversity in terms of the atava  and subspecies rockii  were observed after 1000 permutation tests (P>0.05).The genetic diversity was found to be relatively high, but no significant differences between subspecies STF value of 0.302 detected, although the STF ranged from 0.095 (pdel07) to 0.584 (jx05-2). The measure of the relative genetic differentiation for the pairwise STF values between populations was found to be significant  and ssp. rockii (STF = 0.284) was detected at fourteen microsatellite loci .As shown in  P<0.05, , except 2R = 0.369, P<0.001; P<0.001, The Mantel test revealed a significant and strong correlation between the geographic and genetic distances of the populations (K) was not straightforward because log-likelihood values for the data conditional on K, and lnP(x|K), increased progressively as K increased, as shown in K. However, the ▵K values K classes indicated a strong signal at K = 3, as shown in P. rockii.When using the Bayesian-based clustering approach of Pitchard et al. AD are presented in The results of the distance-based clustering analysis using a neighbor-joining tree based on the The PCA clustered these populations into three groups, as shown in Analysis of the genetic boundaries between the populations supported the five boundaries shown in STF values (averaging 0.302); additionally, we detected moderate genetic diversity at the population level  and high population diversity (TH = 0.749) at the species level. The finding of high genetic diversity at the species level was similar to the results obtained from three cpDNA sequences in a previous study P. rockii. The cold climatic fluctuation during the glacial cycles of the Quaternary Period might have been responsible for the whole population bottlenecks, leading to genetic drift and further increases in population differentiation. This prediction was also supported by the cpDNA data reported by Yuan et al. P. rockii would have subsequently diverged rapidly due to founder events, habitat fragmentation, or human disturbance. The fact that clear signals of recent bottlenecks were found in only three of the twenty populations might be attributed to the biparental inheritance characteristics of the microsatellite loci that prevented the detection of additional bottleneck events because of homoplasy characteristics or to most populations reaching a new mutation-drift equilibrium at a new effective population size following the last bottleneck events.Significant population differentiation was indicated by the high P. rockii populations, as indicated by both the ISF value over all the loci  equilibrium was also observed in the the loci  and the er locus . The maiP. rockii, comprising three major genetic distinct groups, P1 (the western clade), P2 (the eastern clade) and P3 (the northern clade), and only one mixed population, was detected by Bayesian analysis, Neighbor-joining tree and PCA on the basis of microsatellite data. Additionally, the genetic relationships among the populations of P. rockii reflecting the natural geographic locations of the populations were supported by an isolation-by-distance model for P. rockii using the Mantel test P<0.001), which revealed significant molecular variance both among groups (11.32%) and within groups (21.22%)  . Only poP. rockii populations. The first boundary, between the northern clade and the eastern clade, was located between the TC population of the Weihe Basin and population LC of the Xun'er Mountains in the eastern-to-central Qinling Mountains. This boundary was obvious in nature and was supported by the existence of the tectonic boundary of the Luonan-Luanchuan Fault P. rockii in the Eastern Qinling Mountains and the Bashan Mountains from the other populations in the Central Qinling Mountains. The second boundary, between the northern clade and the western clade, was located between the TM population in the Taibai Mountains at the northern boundary of the Qinling Mountains and the LD population in the mountain area of Southern Gansu in the western part of the Qinling Mountains. As a result of the normal fault growth and footwall flexure of the mountains, especially after a large-magnitude rapid uplift in 0.7 Ma, the Taibai Mountain became the highest peak of the Qinling Mountains Five boundaries, consisting of two boundaries between three groups of populations  and three additional boundaries within the three groups, were detected using BARRIER software P. rockii would be so perfectly in line with the existing geographical barriers. The current landform and topographical features of the Qinling Mountains mainly formed by beginning with a small-magnitude uplift of 70–40 Ma and a rapid large-magnitude uplift beginning at 1.2–2.4 Ma (or 0.7 Ma), especially with respect to the higher mountain features of the Taibai Mountains P. rockii populations using cpDNA sequences ranges from 0.4 to 1.6 Ma according to Yuan et al. P. rockii populations is greatly related to the rapid, large-magnitude uplift of the Qinling Mountains. Although the estimation of divergence rates or times using the molecular clock hypothesis has been based on uncertain assumptions and approximate values with respect to the lack of Paeonia fossils, these estimations are still useful for attempts to understand the rate of evolution and the historical biogeography of Paeonia. In addition, geographic isolation has been proposed by Sang et al. Paeonia species, and the timing of the intercontinental disjunction of peonies had been roughly estimated to 16.6 Ma in the Miocene based on cpDNA sequences P. rockii populations that were detected based on both microsatellite and cpDNA data were merely random coincidence; however, it is clear that the geographic isolation that has occurred in the Qinling Mountains has contributed to limiting the gene flow between the P. rockii populations It was highly unexpected that the genetic subpopulations of P. rockii? We hypothesize that a formerly continuously distributed panmictic large population of P. rockii has fragmented into small, isolated populations. Such changes would lead to an erosion of the genetic variation and an increase of genetic differentiation among populations due to enhanced genetic drift and reduced gene flow P. rockii, based on the Mantel test, indicated a significant correlation between the genetic distance and the geographic distance. The recent bottleneck events detected, the high degree of population differentiation among three gene pools and the existence of five natural geographic barriers suggest that the genetic differentiation among the studied populations has been impacted by impediments to gene flow and that geographic distance has played an important role in the current genetic pattern .Phenotypic variation followed the geographical distribution of this species. The populations that are geographically distributed in the north of Qinling Mountains and further north in its vicinity  exhibit ovate or rounded and totally or mostly lobed leaflets, classified as P. rockii because the isolated gene pools of this species might have accumulated a number of mutations to adapt to the different ecologic niches within the different climates after independently evolving for almost 0.7–2.4 Ma.Cultivated tree peonies, respected as ‘the king of flowers’ in China, are very popular ornamental plants, and breeding programs will greatly benefit from this study. More new cold-resistant, wet-resistant, or hot-resistant varieties with more ornamental value could be bred in the future using the different genetic germplasms from the different gene pools of P. rockii in this study, suggesting the occurrence of significant subpopulation and population bottlenecks among the populations of this species. More interestingly, five geographic barriers were identified between and within three genetic groups geographically located in the Western, Eastern and Northern Qinling Mountains. It was also proposed in this report that the current genetic structure of P. rockii could be the result of the fragmentation of a formerly continuously distributed panmictic large population. Furthermore, our results provide a fundamental genetic profile for the conservation and responsible exploitation of the extant germplasm of this species to improve the genetic basis for breeding its cultivars. Finally, this study offers new insight into the abundant biodiversity of plant species that are endemic to the Qinling Mountains of China and provides a model case for studying their complex evolutionary history.In conclusion, significant population differentiation and clear genetic structure were revealed in Figure S1Mantel test for matrix correlation between the genetic distance and log geographical distance.(TIF)Click here for additional data file.Figure S2Clustering analysis of the 20 Paeonia rockii populations based on genetic distance. The phylogenetic tree was constructed with the POWERMARKER software package  based on the populations' pair-wise genetic distances (Nei 1983). The shapes near site names on the tree indicate STRUCTURE grouping.(TIF)Click here for additional data file.Figure S3Principal coordinate analysis (PCA) of Paeonia rockii populations based on microsatellite data.(TIF)Click here for additional data file.Table S1Population locations and sample sizes for Paeonia rockii.(DOC)Click here for additional data file.Table S2Repeat motifs, annealing temperatures (Ta), primer sequences and the range of alleles detected per locus for the microsatellite loci.(DOC)Click here for additional data file.Table S3A pair-wise matrix of geographic distance (upper right) and STF (below left)_between the 20 populations of Paeonia rockii.(DOC)Click here for additional data file.


Eucalyptus, and well exemplified by the Eucalyptus globulus complex. This ecologically and economically significant complex comprises four taxa  that are geographically and morphologically distinct, but linked by extensive “intergrade” populations. To resolve their genetic affinities, nine microsatellites were used to genotype 1200 trees from throughout the natural range of the complex in Australia, representing 33 morphological core and intergrade populations. There was significant spatial genetic structure (FST = 0.10), but variation was continuous. High genetic diversity in southern ssp. maidenii indicates that this region is the center of origin. Genetic diversity decreases and population differentiation increases with distance from this area, suggesting that drift is a major evolutionary process. Many of the intergrade populations, along with other populations morphologically classified as ssp. pseudoglobulus or ssp. globulus, belong to a “cryptic genetic entity” that is genetically and geographically intermediate between core ssp. bicostata, ssp. maidenii, and ssp. globulus. Geography, rather than morphology, therefore, is the best predictor of overall genetic affinities within the complex and should be used to classify germplasm into management units for conservation and breeding purposes.Forest trees frequently form species complexes, complicating taxonomic classification and gene pool management. This is certainly the case in  The most widely accepted species concept defines species as reproductively isolated units , feed on the nectar of its flowers , and open-pollinated seed (where available) were sampled from 1200 adult trees representing 103 natural populations, 33 regions, and the four taxa of the ergrades . Trees sergrades . LocalitHindIII molecular weight standard.Total genomic DNA was extracted from fresh or frozen leaf tissue using a CTAB method  designed by Steane et al. , two EM, and one complex .All statistics calculated at the locality or regional level were based on the 103 localities or the 33 regions shown in A), observed and expected heterozygosity , and Wright's Fixation Index (F). Null allele frequencies in each region were estimated using the Population Inbreeding method in INEst  in the E. globulus complex, and the affinities of individuals to these groups (Q) independent of a priori morphological assumptions, were determined using STRUCTURE 2.2.3 , using 100 000 MCMC repetitions following a burnin of 120 000 repetitions (at which point stationarity had been reached). Twenty independent runs for each value of K were performed, and results were interpreted using the five runs with the highest likelihoods. The Greedy algorithm in CLUMPP (Jakobsson and Rosenberg #bK from K = 2 to K = 6. DISTRUCT (Rosenberg Q) of each individual into each of the inferred clusters. The average proportion of membership of each of the 33 regions into the K = 6 clusters, from the analysis of all sampled individuals of the E. globulus complex, was plotted on a map.The number of groups of genetically similar individuals  for populations of “core” globulus, bicostata, pseudoglobulus, and maidenii . These core individuals were therefore duplicated in the analysis. Ten independent runs at K = 4 were performed using 50 000 MCMC repetitions and a burnin of 50 000 repetitions, and the individual Q-values from the run with the highest likelihood were used to test for correlations with morphological affinities (see below) and were not presented graphically. Only individuals for whom both morphological and molecular data were available were included in this analysis.A second STRUCTURE analysis was undertaken to directly compare molecular and morphological affinities of individuals. This analysis used a model with For 446 of the trees sampled in this study, 12 capsule, inflorescence, and leaf variables (defined in x0.75). Multivariate analyses were undertaken at the region level using Canonical Discriminant Analysis, an ordination technique that maximizes differences between groups relative to within-group variation, using the PROC DISCRIM procedure in the statistical package SAS . The locality level mean canonical variate scores were placed within the multivariate discriminant space defined by the region level analysis. The vectors indicating the direction of variation in each variable were constructed from the standardized canonical coefficients. The lengths of the vectors were proportional to the analysis of variance (ANOVA) F-values  were calculated using PROC DISCRIM.Analyses were undertaken using the means of the five capsule replicates from each tree. The variables were transformed to optimize population normality and remove the correlation between locality or region means and variances. The transformations were the same as in Jordan et al. , with foTo test for the effects of drift and selection, a Mantel test was conducted using XLSTAT  to test the relationships between Mahalanobis distance , Nei's  genetic bicostata, globulus, pseudoglobulus, and maidenii individuals (see M) of these individuals to each of the core taxa  were then calculated using PROC DISCRIM  based on the classification functions derived from PROC DISCRIM. PROC CORR was then used to test whether the morphological affinities of individuals were significantly correlated with their molecular affinities, in each region in Victoria and NSW with more than 10 individuals, using Pearson's correlation coefficient. The molecular affinities had been calculated in the STRUCTURE analysis (above) that used a priori population information for the same core individuals, and calculated the affinities of individuals (Q) to each of the four core groups .Using morphological data, the core uals see  were useHe) or allelic richness (Rt), was highest in the 11-South East Forests region, in the southern part of the maidenii distribution . This was driven partly by the large geographic disjunction and low genetic diversity of the Mt. Bryan locality as well as the lower diversity in Tasmania, but when the Mt. Bryan population and Tasmanian samples were excluded, the correlations were still significant at both the regional and locality levels (data not shown). As the distribution of E. globulus is interrupted by geographic barriers  and southern  ends of the highway (data not shown).Recent hybrid origin would be expected to impart intergrade populations with increased genetic diversity. Genetic diversity in these intergrade populations was not higher than in their putative parental populations. For example, 15-Buchan, 16-Mitchell River, and 17-Lerderderg, which were intermediate in morphology between FST  was 0.10  and pseudoglobulus  and highest among regions of bicostata . Differentiation was moderate among regions of globulus , increasing slightly when globulus intergrade regions were included . Pairwise FST values among the four taxa revealed that pseudoglobulus was the least differentiated taxon .Differentiation was lowest among regions of globulus and its intergrades clustered tightly together in the Neighbor-Joining radial tree, distinct from the maidenii cluster and the bicostata cluster  were on long branches, as was the population at 24-Wilson's Promontory . At each increase in K, the group of regions that split into a new cluster were geographically contiguous, but at each given K, there were intermediate individuals that linked the K clusters .The Δo et al.  detectedat K = 2 , correspat K = 2 . There w2  indicating that isolation-by-distance and genetic drift is an important process in the E. globulus species complex. The major outliers on this plot (not shown) were the pairwise comparisons of regions that were separated by a strong geographic barrier over a short geographic distance . There was no association between Mahalanobis distance  and geographic distance . Nei's . Outliers on this plot (not shown) were mainly pure Victorian globulus populations in South Gippsland compared with geographically proximal, but morphologically differentiated, intergrade populations, indicating that selection is acting over a steep gradient in this area.There was a highly significant association between Nei's  genetic ). Nei's  genetic globulus . Fourteen individuals had strong and similar morphological and molecular affinities to Victorian globulus in this region. No significant correlations involving the other taxa were observed and individuals with morphological and molecular affinities to Victorian globulus were not geographically clustered (data not shown).As a recent hybrid zone would be expected to have correlated morphological and molecular affinities, the morphological and molecular affinities of individuals in regions of NSW and Victoria were tested for significant correlations in each region in which there was clear variation in morphological affinities. The only region that had a significant correlation between morphological and molecular affinities was 20-Strzelecki Ranges. In this region, there was a strong correlation between morphological and molecular affinity to Victorian E. globulus species complex. Across the broad geographic range, continuous molecular and morphological variation link the four recognized taxa. These continua appear to be driven by the interaction of the key evolutionary processes of genetic drift, selection, and, to a lesser extent, hybridization. These processes, occurring at multiple geographic locations and scales, result in a poor relationship between molecular and morphological affinities in many intergrade regions, which has implications for gene pool management and exploitation.This combined analysis of molecular and morphological data, sampled over a large scale and focusing on morphologically intermediate populations as well as “pure” populations, revealed that multiple evolutionary processes have driven the patterns of genetic differentiation in the E. globulus. Across the broad geographic range, there is a continuum in molecular genetic variation and a significant and positive association between genetic and geographic distance. While weak, the observed relationship with distance (r2 = 0.37) is comparable to that reported in other eucalypts , but most of the intergrade populations appear to be a result of primary differentiation as suggested by Kirkpatrick , a major pollinator of globulus  to winter  rainfall zones  to local population level, indicating that natural selection has also played a significant role in shaping variation patterns in the E. globulus complex. At the subspecies level, core bicostata and maidenii are poorly adapted to the cool temperate climate of Tasmania  and quantitative (QST) traits, Steane et al. (QST > FST). Subsequent studies have revealed other traits where QST > FST  are relatively well-defined genetic entities, consistent with their geographic separation. The key problem is the lack of morphological definition of a large fraction of the gene pool that includes pseudoglobulus and the populations south of the Great Dividing Range in Victoria that comprise morphological intergrades between the cores . Thus, geographic location would be the most pragmatic approach to classify germplasm in this region into taxonomic units for breeding and conservation purposes.The molecular perspective on the E. globulus complex from several perspectives. First, the genetic entities defined at various levels with the STRUCTURE analysis can guide translocation of E. globulus germplasm within its native range for forestry, agro-forestry, and restoration purposes. The level of molecular differentiation between a local and a non-local seed source can provide an early predictor of the genetic impact of introgression between translocated and native populations (Potts et al. globulus, together with some Victorian intergrade populations, which are here classified with pseudoglobulus, forms the base populations of E. globulus breeding programs in many countries (Potts et al. E. globulus breeding program, the subrace classification of Dutkowski and Potts (The study has provided a molecular framework for the management of the genetic resources of the nd Potts  is used nd Potts . Howevernd Potts , some su


Lunella granulata from mainland China, Penghu Archipelago, Taiwan Island, and Japan using mitochondrial COI and 16S markers. A total of 45 haplotypes were obtained in 112 specimens, and relatively high levels of haplotype diversity (h = 0.903) and low levels of nucleotide diversity (π = 0.0046) were detected. Four major phylogenetic lineage clusters were revealed and were concordant with their geographic distribution, agreeing with the haplotype network. These results suggested that geographic barrier isolating effects were occurring among the populations. This hypothesis was also supported by a significant genetic differentiation index (FST = 0.709) and by a spatial analysis of molecular variance (SAMOVA) analysis. A mismatch distribution analysis, neutrality tests and Bayesian skyline plots found a single significant population expansion. This expansion occurred on the coast of mainland China before 20–17 ka. Consequently, although the dispersal ability of the planktonic stage and the circulation of ocean currents generally promote genetic exchanges among populations, L. granulata has tended to maintain distinct genetic groups that reflect the respective geographic origins of the constituent lineages. Although the circulation of ocean currents, in principle, may still play a role in determining the genetic composition of populations, long-distance migration between regions is difficult even at the planktonic stage.We examined the genetic variation and phylogeographic relationships among 10 populations of  Information on population structure is required for management and conservation strategy design in marine species facing overexploitation and extinction risk . Based oLunella granulata Gmelin, 1791), distributed primarily in the Indo-West Pacific area and ranging from Japan and Korea to China, preferentially inhabits rocky shores in the intertidal zone [L. granulata is one of the most important species for the residents of the Penghu Archipelago and Taiwan due to its abundance and widespread distribution. The life history of the moon turban snail begins with the planktonic larval stage, which begins one day after hatching. The congener L. coreensis maintains its planktonic larval stage for 2.5 days [L. granulata populations.The moon turban snail  cornutus, have revealed a clear genetic structure that corresponds to the pattern of warm currents along the Japanese coastline [The inherent dispersal ability of species and the lack of significant oceanic geographic barriers result in high levels of gene flow among marine populations, reducing the potential for detectable genetic structure over a large geographic scale . Previouoastline ,9. Severoastline ,11.Littorina spp. [Haliotis cracherodii) [It has been proposed that the occurrence of a pelagic larval stage in benthic marine species and the dispersal capacity of the larvae act jointly to shape genetic structure. This hypothesis has been examined in marine gastropod species such as ina spp.  and abalherodii)  and in oherodii) –16. Moreherodii) . Howeverherodii) .L. granulata in southeast Asia, the aim of this study is to examine the population genetic structure and phylogeographic patterns of L. granulata throughout mainland China and adjacent islands, including the Penghu Archipelago and Taiwan, based on concatenated mtDNA nucleotide sequences from COI and 16S genes. This region is also characterized by the complex circulation patterns of ocean currents as well as the occurrence of the last glacial maximum (LGM) approximately 10,000 years ago. A hierarchical scale-based analysis of molecular variation and descriptive genetic information would be helpful to define the evolutionary history of L. granulata and to provide evidence about the effects of environmental dynamics on intra-species genetic structure.The development of molecular techniques and analytical tools has provided high-resolution genetic information and the potential to determine the genetic structure of populations. These tools include the polymerase chain reaction (PCR), sequencing of DNA fragments, and phylogenetic analysis of allelic variants. Given the broad distribution of L. granulata were selected from 10 populations including mainland China, Taiwan, and Japan (h = 0.899), with values ranging from 0.154 in Ishigaki (IS) to 0.933 in Wangan (WA). The samples from Japan (IS) presented the lowest nucleotide diversity (π = 0.0001) (Specimens of nd Japan . The comnd Japan . The seq 0.0001) , similar 0.0001) .We conducted a phylogenetic analysis based on the combined nucleotide sequences of the COI and 16S genes. A neighbor-joining (NJ) tree reconstructed from the concatenated nucleotide sequences (COI-16S tree) is shown in Based on a network analysis, the observed mtDNA haplotypes were separated into one 1-step clade (1-1) and one 2-step clade (2-1); the last group included all haplotypes in three 3-step clades  . The majD and Fu’s Fs tests supported demographic expansion, with the latter test showing particularly clear statistical significance [p > 0.05). In terms of the beginning of population expansion, the τ value of 1.156 derived by Arlequin suggested a time approximately 19,000–66,000 years before the present, occurring during the last glacial maximum (LGM) in the Pleistocene. For the populations in Taiwan and Penghu Archipelago, the non-significant value of both Tajima’s D and Fu’s Fs values obtained from intra-populational and intra-regional data implied a scenario based on stable population demographics. Interestingly, except for the populations from the coast of China, the analysis of the mismatch distribution for the total populations did not identify any significant deviations from the expected distribution of differences under a stepwise expansion model .The observed moderate to high haplotype diversity coupled with a low nucleotide diversity in all lineages  indicateificance . In addiL. granulata lineages. The results revealed stable population sizes through time for lineages A and B, but a pattern of population growth occurring during 20–17 ka was detected in lineage C (A Bayesian skyline plot (BSP) analysis was used to date the shifts in population size of the ineage C .NST and GST. A comparison of NST and GST revealed a strong correlation between phylogeny and geography, with NST substantially greater than GST . The overall differentiation among populations was very high (FST = 0.70). An AMOVA revealed a high degree of structuring, with the largest proportion of the variation  among geographic groups and significantly lower proportions of variation among populations within geographic district  and within populations  (CT was four (K = 4) based on a spatial analysis of molecular variance (SAMOVA) . The θ values from MIGRATE for each lineage showed that Penghu Archipelago maintained the largest effective population size, whereas the smallest effective population size was found for Taiwan Island  . The num(SAMOVA) . This co(SAMOVA) . These fn Island .The surface plots produced from the genetic landscape interpolation with different distance weighting parameters were qualitatively similar to one another. Overall, there was a strong pattern of high genetic differentiation (indicated by peaks) among the Penghu Archipelago samples .L. granulata populations. Grant and Bowen [L. granulata is much lower than that of other marine gastropod species . Althouoitation  and a shoitation . The higcurrents . The netL. granulata identified genetic groups that partially corresponded to geographic origins. This result suggested the dominant effects of geographic isolation due to the lower sea level in Taiwan Strait during the Pleistocene era, preventing gene flow among Taiwan Island, Penghu Archipelago and mainland China. The phylogeographic signal based on the presence/absence of haplotypes in the MSN  to the mainland China coast (clade 3-1), and the other from the Penghu Archipelago through Taiwan (clade 3-2) to Japan. The MIGRATE analysis also supported that gene flow occurred only from Penghu Archipelago (clade 3-3) to Taiwan (clade 3-2) and Japan (clade 1-1).In this study, the phylogenetic reconstruction performed for  the MSN  revealedThe distribution of marine fauna along the coast of southern China is primarily driven by the circulatory pattern of complex ocean currents ,31. DuriL. granulata. The dominant geographic structure detected in L. granulata indicates limited gene flow among lineages, most likely reflecting limited dispersal ability during the early life history. Otherwise, most lineages also harbored “unique” haplotypes, indicating a very low connectivity among geographic groups. A Mantel test detected a weak but significant correlation between genetic dissimilarity and geographic distance, indicating that the isolation by distance pattern might not be the driving force for the differentiation of the lineages. For L. granulata, the results suggested a good agreement between hydrographic barriers and the genetic differentiation of geographically distinct populations. In addition, a clear pattern of both demographic and range expansion was observed along the coast of mainland China. The beginning of rapid population growth at 17,000–20,000 bp indicated that the most recent expansion occurred during the interglacial period prior to the LGM, 23,000–18,000 bp [2 was exposed on the East China Sea Shelf during the Pleistocene ice ages, and the contraction and expansion of the available land masses are thought to have greatly influenced the distribution of species, including that of L. granulata . Theref species .L. granulata . A small piece of foot tissue was cut off from each specimen. The tissue samples were then preserved in 95% ethanol and stored at 4 °C.A total of 112 snails were collected from four regions including mainland China, Taiwan and Japan. These samples included 45 individuals from four sites  in Taiwan; 33 samples from three sites  in Panghu Archipelago; 22 individuals from two sites (Kinmen and Matsu) on the coast of mainland China; and 12 samples from Ishigaki, Ryukyu, Japan . For twoWe extracted genomic DNA with a High Pure PCR Template Preparation Kit (Roche) from approximately 10 mg of tissue sample. The mitochondrial COI and 16S genes were used as molecular markers in this study. A fragment of COI (658 bp) was amplified by primers LCO 1490: 5′-GGTCAACAAATCATAAAGATATTGG-3′ and HCO 2198: 5′-TAAACTTAGGGTGACC AAAAATCA-3′ , and a 1h) [GST and NST was performed with DnaSP 5.0 [All sequences were edited with the program DNAStar software  and checked manually. After the sequences were aligned with MEGA 5 , genetich)  and nuclh) . Prelimih) . The relh) , using 1h)  to link naSP 5.0 .D [Fs test [t, time in generations) was calculated from the relationship τ = 2 ut [u was calculated from u = μk, where μ was the mutation rate per nucleotide and k was the number of nucleotides of the analyzed fragment.To check for deviations from neutrality, the sequence data were tested using Tajima’s D  and Fu’s[Fs test  in DnaSP[Fs test . A signi[Fs test . To comp[Fs test . The timτ = 2 ut , where τL. granulata female effective population size (Nfe) since the time to the most recent common ancestor (TMRCA) of the sampled mitochondrial haplotypes. Because the mtDNA exhibited clock-like properties and we had no well-defined calibration points, we adopted a conservative approach and used a range of sequence divergence rates (0.7%–2.4% per Myr) based on data from the related family Trochidae [We used a model-free coalescent-based method  implemerochidae –58.K = 2 to 7. Given an a priori number of groups (K), SAMOVA uses a simulated annealing procedure to define the group composition in which the populations within a group are as genetically homogeneous as possible (FSC minimized) and the groups are maximally differentiated from each other (FCT maximized). The best-fit grouping pattern was subsequently applied in an AMOVA  using Arlequin  to obtaion 3.5; ). Pairwiion 3.5; ).M were estimated jointly from the data), which was compared to a restricted model . A migration matrix model with unequal population sizes and different migration rates was assumed [To determine the direction of historical gene flow, we used MIGRATE software version 3.2.6 , applyin assumed .To test for a correlation between genetic differentiation and geographic distance, a Mantel test and a spatial autocorrelation analysis were performed using Alleles in Space . This meL. granulata that we analyzed exhibit a strong genetic structure corresponding to their geographic origins. L. granulata was divided into four population groups by SAMOVA. This result was consistent with the lineage clusters  depicted in the NJ tree. As a consequence, the dominant geographic structure in L. granulata indicated a limited level of gene flow among geographic lineages, suggesting that geographic distance, rather than a mobile planktonic stage and circulating ocean currents, was the factor that determined the genetic distribution. Interestingly, the demographic pattern based on BSP and mismatch analysis revealed a population expansion only on the coast of mainland China during the glacial retreat, in contrast with the results of other studies conducted in the same region. As a result, L. granulata tends to maintain a stable population structure without suffering from the dynamic effects of population size and glacial change. The distinct geographic groups identified by the analysis were a result of the inefficiency of dispersal at the planktonic stage and the limited ability of circulatory ocean currents to drive genetic exchanges among populations of L. granulata.Our study shows that the populations of


Shorea parvifolia is one of the most common tropical lowland rainforest tree species in Southeastern Asia. To infer population structure and demographic history of S. parvifolia, as indicators of temporal changes in the distribution and extent of tropical rainforest in this region, we studied levels and patterns of nucleotide polymorphism in the following five nuclear gene regions: GapC, GBSSI, PgiC, SBE2, and SODH. Seven populations from peninsular Malaysia, Sumatra, and eastern Borneo were included in the analyses. STRUCTURE analysis revealed that the investigated populations are divided into two groups: Sumatra-Malay and Borneo. Furthermore, each group contained one admixed population. Under isolation with migration model, divergence of the two groups was estimated to occur between late Pliocene (2.6 MYA) and middle Pleistocene (0.7 MYA). The log-likelihood ratio tests of several demographic models strongly supported model with population expansion and low level of migration after divergence of the Sumatra-Malay and Borneo groups. The inferred demographic history of S. parvifolia suggested the presence of a scarcely forested land bridge on the Sunda Shelf during glacial periods in the Pleistocene and predominance of tropical lowland rainforest at least in Sumatra and eastern Borneo.Distribution of tropical rainforests in Southeastern Asia has changed over geo-logical time scale, due to movement of tectonic plates and/or global climatic changes.  Distribution of tropical rainforests in Southeastern Asia has changed over geological time scale, due to movement of tectonic plates and/or global climatic changes . These cRecent studies revealed considerable fluctuations in the distribution and extent of tropical rainforests in this area coinciding with glacial cycles in the Pleistocene . At the Lithocarpus: Trigonobalanus: Macaranga: Shorea leprosula: S. leprosula and S. parvifolia: S. parvifolia and some Macaranga species suggesting gene flow and/or maintenance of the ancestral polymorphism. These two events have completely different historical causes. The former attributes to migration or admixture between formerly separated populations, while the latter attributes to recent divergence of populations.Phylogeographic analysis, which deals with the geographic distribution of genetic variation, is a powerful tool to infer past demographic events that include population expansion or bottlenecks as well as population structure and migration. Few studies of tropical tree species have been performed on a regional scale in Southeastern Asia. Two montane rainforest tree genera  in peninsular Malaysia was investigated by D values in S. curtisii, S. leprosula, and S. parvifolia and relatively low levels of population differentiations in S. acuminata and S. curtisii suggested recent expansion of the investigated species in peninsular Malaysia. This result posed new questions about the regional scale of population structure and demographic histories of these species.Evaluation of the levels and patterns of nucleotide polymorphism in multiple nuclear loci is useful in inferences of population structure and estimations of demographic parameters, as well as in detecting natural selection e.g., . StudiesShorea parvifolia Dyer collected from peninsular Malaysia, Sumatra, and eastern Borneo. Shorea parvifolia is one of the most abundant canopy tree species in lowland evergreen rainforest in Southeastern Asia and distributed throughout peninsular Malaysia, Sumatra and Borneo , two populations from Sumatra , and three populations from eastern Borneo, Kalimantan . Twelve individuals were analyzed for each population, except for the population ITCI Karya Utama, where only six individuals could be sampled. In total, 78 individuals of S. parvifolia were analyzed in this study. Most individuals from the population Seremban were the same as those studied by Information about the locations and sample sizes of the investigated populations is summarized in S. parvifolia and S. acuminata and one putative hybrid between S. parvifolia and S. leprosula were found among S. parvifolia individuals from peninsular Malaysia based on genotypes of the GapC and the PgiC gene regions  as S. parvifolia . Sequences of the PCR products amplified using these primers were determined. Gene regions that contained single-nucleotide repeats (>10) or many indels were excluded. Specific primers for the remaining gene regions were designed based on the sequences of S. parvifolia obtained during trial PCR amplifications. Finally, the following five nuclear genes were used: GapC , GBSSI , PgiC , SBE2 , and SODH . Names of all loci were assigned according to the corresponding homologues of Arabidopsis thaliana.Several enzymes that commonly exist in plants and whose functions are well known were chosen as candidate loci for this study. Trial PCR primers were designed based on the known sequences of PgiC gene region from population Seremban of S. parvifolia obtained by PgiC sequence for one individual of S. maxwelliana obtained by Genomic DNA was isolated from ∼300 mg of leaves using a modified cetyl trimethyl ammonium bromide method  or DNeasHd; Nec, where eN is the effective population size and c is the recombination rate per generation per site) was estimated for each locus using the composite-likelihood method (http://www.stats.ox.ac.uk/~mcvean/LDhat/index.html). To test for deviation from selective neutrality and other assumptions , Tajima's D . In the multilocus Tajima's D test, P-values for average Tajima's D statistic over five loci were obtained. All calculations and coalescent simulations (except for estimation of ρ and multi-locus Tajima's D and HKA tests) were performed using the DnaSP program ver. 4.10.9 . Multiple sequence alignment for individual loci was performed using the Clustal W program ver. 1.4  and corrd method  implemenjima's D  test wasal model  with no al model  and amonal model . The rat. 4.10.9 .FST; Da; http://pritch.bsd.uchicago.edu/structure.html) to detect population structure and assign individuals to populations. Related haplotypes were grouped and treated as single alleles. Haplotype grouping was performed using the TCS program ver. 1.18 (K) was selected based on the ΔK statistic of To investigate the degree of population differentiation, fixation indices FST;  between er. 1.18 . All modNeu of Sumatra-Malay group (θsm), Borneo group (θb), and their ancestral population (θA), migration rate from Borneo group to Sumatra-Malay group (msm), migration rate from Sumatra-Malay group to Borneo group (mb), and divergence time (t). The program implements Markov chain Monte Carlo simulations for generating genealogy fitting the ‘‘isolation with migration’’ (IM) model  model  to data M) model  was usedM) model . First, Ne, effective population size; T, divergence time in years; 2Nem, population migration rate). For the estimate of T, t must be divided by the geometric mean of mutation rate per year per locus. Unfortunately, it is difficult to estimate mutation rate of Shorea species due to the absence of precisely dated fossil records. Thus, the minimum and maximum mutation rates per site per year for synonymous nucleotide substitutions in nuclear genes studied in other tree species: usyn = 0.7 × 10−9 in Pinus  for S. parvifolia were computed as 1.55 × 10−5 and 5.78 × 10−5 for five loci, and 1.72 × 10−5 and 6.42 × 10−5 for four loci excluding the GBSSI gene. Population migration rate 2Nem per generation was computed by multiplying θ by m/2.The six parameters estimated by the IMa program were converted to the actual demographic parameters  in the nested models. For one of the nested models, msm = mb = 0, namely no migration after divergence (isolation model), the LLR is expected to follow a mixed χ2 distribution. However, this is not a good approximation (IM discussion group: http://groups.google.com/group/isolation-with-migration). Therefore, deviations of the LLR of the full and nested models (msm = mb) from a χ2 distribution (df = 1) were tested first. Subsequently, the deviation of the LLR of the nested models (msm = mb) and (msm = mb = 0) was tested from a mixed χ2 distribution: one half of the values is zero and the other half follows a χ2 distribution (df = 1). If neither test was rejected the isolation model was accepted as a null model.To test several different demographic models, log-likelihood ratio (LLR) tests between the full model and nested models were performed using the results of simulations performed using IMa program. These tests are also implemented in the IMa program . The fulGapC, GBSSI, PgiC, SBE2, and SODH, were obtained for 78 individuals of S. parvifolia from seven populations, and a single individual of each S. acuminata, S. leprosula, S. curtisii, and S. maxwelliana. As sequences of both haplotypes for each individual were determined, in total, we obtained 156 sequences of S. parvifolia and two sequences of each individual of other species for each gene region. The alignment length was 1111, 1229, 1248, 1075, and 1277 bp, respectively .Sequences of the partial regions of the five nuclear genes, SODH gene region, three haplotypes were observed in the individual of S. maxwelliana, suggesting the existence of more than one copy of the SODH gene in this species. To determine whether the SODH gene is duplicated in S. maxwelliana, we constructed NJ tree of this gene using all sequences obtained in this study. The three haplotypes from S. maxwelliana formed a separate group on the NJ tree, which was distinct from haplotypes of the other Shorea species (data not shown), indicating this gene duplication was specific to this species. Shorea maxwelliana was used as outgroup in the MK and HKA tests, which require that haplotypes from a single copy of a gene are used. Because it is difficult to identify which haplotype combination constitutes a genotype of a particular copy of the SODH gene, we considered all six possible allele combinations as genotypes of a single copy of the SODH gene. Namely, three heterozygous combinations, and three homozygous combinations, and tested each genotype in the neutrality tests. Results for the acceptance or rejection of null hypothesis were the same for each genotype. Therefore, one of these genotypes was used in further analyses. In S. parvifolia, only two haplotypes were observed at any locus indicating that only a single gene copy was amplified for all loci.For the S. parvifolia individuals investigated, one putative F1 hybrid between S. parvifolia and S. acuminata was found in population Nanjak Makmur from Sumatra. This individual was a heterozygote harboring one S. parvifolia haplotype and one S. acuminata haplotype at all five investigated loci. Haplotype sharing with different species was not observed at any locus in other individuals. Because inclusion of a putative hybrid will bias estimation of the summary statistics, the aforementioned putative hybrid was excluded and consequently 154 sequences of S. parvifolia were used for all further analyses.Of the 78 Hd), nucleotide diversity (π), and population recombination rate per site (ρ) over five loci are summarized in sil) varied about twofold between the five investigated loci, from 0.0053 (GapC) to 0.0113 (GBSSI), with an average of 0.0075 for a total sample of S. parvifolia. For the nonsynonymous sites (πrep), it ranged from 0.0001 (SBE2) to 0.0014 (GBSSI), with an average of 0.0006, which is more than 10-fold smaller than silent polymorphism. The levels of polymorphism also differed among populations. Especially, populations Seremban and Mersing from peninsular Malaysia had slightly lower levels of nucleotide diversity (π) and haplotype diversity (Hd) compared to the other populations included in our present study.Average haplotype diversity  to 0.0262 (SODH). In comparisons among populations, ρ was low in populations Seremban and Mersing from peninsular Malaysia and population Nanjak Makmur from Sumatra. In population Mersing, ρ was 0 except for the SODH gene region. The lower levels of nucleotide diversity and recombination rate in populations from peninsular Malaysia and Sumatra suggested smaller effective population size of those populations.Similar to nucleotide diversity, population recombination rate (ρ) per site differed among the five loci as well as among populations. For the total sample of D statistics are summarized in S. parvifolia, significant negative Tajima’D values were observed in all five loci. At population level, the values of Tajima's D were negative in almost all loci and populations and some were significantly negative. Such genome-wide patterns indicated the influences of demographic factors rather than locus specific factors such as natural selection. The MK test detected significantly higher nonsynonymous to synonymous ratio for polymorphism (29/23) than for divergence (1/12) in the GBSSI gene region  and within Borneo populations (FST < 0.169) were observed (FST > 0.250) were observed at some loci between populations Nanjak Makmur and Asialog from Sumatra, between peninsular Malaysia and Borneo populations, and between Sumatra and Borneo populations. The NJ tree based on the Da values revealed two major groups of populations  was larger than the migration rate from the Sumatra-Malay group to the Borneo group (2Nmb = 0.25). The marginal distribution of t had clear peak (t) of the two groups were very similar between the two datasets: 0.685 and 0.635 for five loci and four loci, respectively. For the dataset with five loci, the estimated divergence time of the Sumatra-Malay and Borneo groups was 0.71 MYA and 2.65 MYA, when the maximum and minimum mutation rates were used, respectively. Estimates of the six parameters are only slightly different between the datasets with five and four loci excluding the GBSSI gene region  was rejected for the dataset with five loci (P = 10−17) but not for the dataset with four loci (P = 0.13730). The models in which a pair of or all of θsm, θb, and θA are the same were rejected in both datasets (P < 0.01), which also supported the relationship of θA < θsm < θb. In addition, a clear peak in marginal distribution of the divergence time (t) of these groups  is in the lower range of those reported for herbaceous species and quite similar to values reported for other woody species  were observed between peninsular Malaysia or Sumatra and Borneo populations. STRUCTURE analysis revealed that the investigated S. parvifolia individuals could be divided into two genetically distinct groups: Sumatra-Malay and Borneo with one admixed population in Sumatra  and one admixed population in Borneo (ITCI Karya Utama). Clear separation of the two groups except for one individual from ITCI Karya Utama  in Southern and Southeastern Asia.How big fraction of GBSSI gene region , conditions of Southeastern Asia were warm and humid with widespread rainforest , and seaPopulation expansion of the Sumatra-Malay and Borneo groups after their divergence indicated an abundance of favorable habitats during Pleistocene such as moist and warm forests in these areas. In fact, recent study revealed that southwest Sumatra and substantial region of Sunda Shelf and Borneo were covered by rainforests during much of the last million years . In the Shorea parvifolia has been reported to be pollinated by thrips (Nem per generation <2) revealed in our present study, imply that contacts of the two population groups were limited. Therefore, when Sunda Shelf was exposed, the expanding savanna must have been strong but imperfect barrier to gene flow between the two groups. In summary, our results suggest the existence of a temporary savanna corridor across Sunda Shelf during the glacial periods of the Pleistocene, and a predominance of tropical lowland rainforest at least in Sumatra and eastern Borneo.In the LLR tests, the isolation model was rejected when all five loci were included, suggesting that there were migrations after divergence of the Sumatra-Malay and Borneo groups. y thrips  or beetly thrips , which ay thrips . These fy thrips , the conTrigonobalanus verticillata were suggested to have diverged around 17 MYA in Pliocene  during the glacial periods in the Pleistocene. Contrasting pattern of natural selection acting on nonsynonumous mutations between populations from peninsular Malaysia and other regions was also suggested, which is likely due to differences in the effective population size. Analysis of additional gene regions and populations will enable to test more complicated demographic models such as a model with different timings of population expansion between two groups. Application of similar methods to other tropical tree species in Southeastern Asia would reveal more detailed picture of biogeographic history of this region.