Dracaena cambodiana, a plant species distributed in paleotropical forests.The origin of extraordinarily rich biodiversity in tropical forests is often attributed to evolution under stable climatic conditions over a long period or to climatic fluctuations during the recent Quaternary period. Here, we test these two hypotheses using atpB-rbcL and trnD-trnT) regions and genotype data of six nuclear microsatellites from 15 populations  distributed in Indochina Peninsular and Hainan Island to infer the patterns of genetic diversity and phylogeographic structure. The population bottleneck and genetic drift were estimated based upon nuclear microsatellites data using the software programs BOTTLENECK and 2MOD. The lineage divergence times and past population dynamics based on cpDNA data were estimated using coalescent-based isolation-with-migration (IMa) and BEAST software programs.We analyzed nucleotide sequence data of two chloroplast DNA  and genetic differentiation among populations were detected. Bottleneck analyses and Bayesian skyline plot suggested recent population reduction. The cpDNA haplotype network revealed the ancestral populations from the southern Indochina region expanded to northward. The most recent ancestor divergence time of D. cambodiana dated back to the Tertiary era and rapid diversification of terminal lineages corresponded to the Quaternary period.A significant phylogeographic structure (D. cambodiana was an outcome of Tertiary dispersal and rapid divergence during the Quaternary period under limited gene flow influenced by the uplift of Himalayan-Tibetan Plateau and Quaternary climatic fluctuations respectively. Evolutionary processes, such as extinction-recolonization during the Pleistocene may have contributed to the fast diversification in D. cambodiana.The results indicated that the present distribution of genetic diversity in  Understanding the origin of extraordinary biological diversity in tropical forests remains as one of the greatest challenges in evolutionary biology. In general, two contrasting hypotheses have been put forward to explain the origin of high biological diversity in tropical ecosystems. The persistence hypothesis stipulates that the gradual evolution during the long-term survival of organisms and low extinction rates under stable climatic conditions may have lead to the present day high biological diversity in the tropics. Alternatively, the refugia hypothesis suggests that the current diversity in the tropics could be attributable to successive isolation and subsequent expansion of populations in response to frequent oscillations of the climate during the Quaternary period The limited understanding of the evolution of tropical plants during the Quaternary period could be attributable to paucity of reliable data from the tropical regions. The fossil records of vascular plants in the tropics representing the Quaternary period are rare Dracaena cambodiana Pierre ex Gagnepain (Asperagaceae), commonly known as the dragon blood tree is a vulnerable species and generally distributed in the escarpments of island-like limestone mountains in paleotropical Southeastern Asia. Current dragon trees mainly distributed in Asia and Africa D. cambodiana was the representative of dragon trees in Asia. Dispersal of tropical elements between Asia and Africa has two possible ways D. cambodiana, this species should exist in Asian Paleotropics during Tertiary. Thus, D. cambodiana serves as an ideal model species to test the biodiversity-original hypotheses of plant species in Paleotropics. The objectives of this study are (i) to illustrate the genetic diversity, gene flow and past population demographics using cpDNA and nSSR; (ii) to determine the ancestral populations and past migration patterns through phylogeographic analysis of cpDNA; (iii) to date the genetic divergence under different molecular clock methods.Dracaena cambodiana is national second class protected plants in China. This species is listed in the Inventory of Rare and Endangered Plants of China and the Key Protected Inventory of Wild Plants of China (http://db.kib.ac.cn/eflora/View/plant/ZXBWSpecies.aspx), but it is not evaluated by the International Union for Conservation of Nature (IUCN) and it is also not recognized as the endangered or protected species in Thailand, Cambodia, Laos and Vietnam. Furthermore, the locations in Thailand, Cambodia, Laos and Vietnam are not privately-owned or protected in any way except the Pumat National Park of Vietnam. We got the permission of the Wildlife Protection and Administration Office under the Forestry Department of Yunnan, Guangxi and Hainan Province in China and the permission of the Administration of Pumat National Park of Vietnam. We also got the permission of local forestry department in other locations of Thailand, Cambodia, Laos and Vietnam. The sampling process was under the guidance of local rangers. This plant was solely used for scientific research and our sampling will not affect the regular growth of D. cambodiana.D. cambodiana plants from 15 localities representing four geographical regions covering most of the natural distribution range of the species in Asia . Based upon sequence variability, atpB-rbcL trnD-trnT D. cambodiana and outgroups were deposited in the GenBank (accessions: JF784389–JF784419).Ten individuals representing different geographic regions were surveyed for the nucleotide sequence variation at four cpDNA regions  and nucleotide diversity (π) were calculated for each group and overall level using the program DNASP V5.10 HT) and within population genetic diversity (HS) were calculated using PERMUTCPSSR V2.0 FST-cp) among all fifteen populations and among four groups was calculated using ARLEQUIN V3.1 For cpDNA data, the haplotype diversity (HO and expected HE) and test for linkage-disequilibrium were calculated using GENEPOP V3.3 FIS values for each population and each locus. The statistical significance of deviations was assessed by randomization for 1000 times. The proportion of genetic variation partitioned among populations and among groups of populations were quantified using analyses of molecular variance (AMOVA) as implemented in ARLEQUIN and the statistical significance was tested with 10000 permutations.For microsatellite data, the presence of null alleles, large allele dropout and scoring errors due to stuttering were tested using MICRO-CHECKER V2.2.3 GST) with an estimate of population subdivision based on phylogenetically ordered alleles (NST) using the program PERMUTCPSSR. Because GST may be problematic for the interpretation of genetic differentiation, we employ the standardized genetic differentiation ('GST) to measure genetic differentiation among populations according to the formula 'GST = GST(1+ HS)/(1 - HS) The presence of phylogeographic structure of cpDNA was tested by comparing the level of population differentiation based upon the infinite allele model  and the natural logarithm of geographic distance for all pairs of populations et al.FST-SSR and RST which are expected to be equal under the null hypothesis of no phylogeographic signal. 'GST of nSSR was estimated by SMOGD Using nuclear microsatellite data, we assessed the genetic structure based on two differentiation indices namely k) ranging from 1 to 20 et al. k by plotting the log probability of L(k) and the Δk of the data over multiple runs using the program STRUCTURE HARVESTER k, we calculated the symmetric similarity coefficients (SSC) using the Greedy algorithm as implemented in the CLUMPP software We used nuclear microsatellite data to test for the existence of cryptic population structure employing the Bayesian model based software program STRUCTURE V2.3.3 www.fluxus-engineering.com) following the median-Joining method Agava sp., Yucca gloriosa and Asparagus setacea as outgroups for the evolutionary network analysis. Phylogenetically Asparagus is basal to Agava and Yucca, which are closely related to DracaenaAsparagus as the basal outgroup to root the evolutionary network.The evolutionary network reconstruction and ancestral haplotype inference based on cpDNA data were carried out using the software program NETWORK V4.5.1.6 (ti (i as the node of diverging groups) and the time since most recent common ancestor (TMRCA) of the species and each group (tTMRCA). The resulting values were converted to absolute time scale, T in years using the formula T = t/µk, where μ is the number of substitution per site per year (s/s/y) and k is the length of the sequence μ as 0.146476±0.063280×10−9 s/s/y based on uncorrelated exponential clock model  coalescent model as implemented in the program IMa2 r groups  was consDSFTTMRCA were further analyzed through Bayesian skyline plot The departure from population demographic equilibrium was assessed using Tajima's He) as compared to the expected heterozygosity under mutation drift equilibrium (Heq) for a given allelic diversity is an indication of recent reduction in the effective population size He>Heq) through Wilcoxon sign-rank test and sign test with 5000 replications. We performed the analysis under Infinite allele model (IAM) and two-phase model of microsatellite evolution with the proportion of single step stepwise mutation (SMM) set to 70% and the variance to 30%. The population bottleneck is also expected to change the allele frequency distribution The excess in heterozygosity , one 17 bp, one 5 bp and two 1bp indels. After excluding gaps, the length of combined cpDNA consensus sequence length was 1814 bp, which included 15 polymorphic sites and 18 haplotypes  estimated from nSSR was from 0.474 to 0.706 (average was 0.637). Expected heterozygosity (HE) estimated from nSSR was from 0.747 to 0.919 (average was 0.948).Overall, only 4  of the 18 haplotypes were shared among populations . Only onectively . The totFST-cp = 0.850, P<0.05; FST-SSR = 0.329, P<0.01) and within–group population divergence . Although cpDNA data revealed a significant genetic differentiation among four groups , microsatellite data showed no significant genetic differentiation among groups . The gene flow (Nm) among populations based on cpDNA and microsatellite data was 0.0882 and 0.5099 respectively. The range-wide isolation by distance (IBD) in D. cambodiana was statistically non-significant . A significant phylogeographic structure based on both cpDNA and microsatellites data was detected . The occurrence of nine genetic clusters (K = 9), a value higher than the geographically defined four sampling regions suggests a high genetic differentiation among populations than among regions. The bar-plot depicting ancestry of samples ) gradually increased reaching the highest value corresponding to K = 9 . Similar samples  further The rooted phylogenetic network analysis of 18 cpDNA haplotypes  showed tTTMRCA) based on the results of IMa2 and BEAST program were 11.48 (5.46–22.39) million years ago (Ma) and 9.77 (3.69–18.64) Ma respectively (TTMRCA of the 18 cpDNA haplotyes was 15.80 (5.29–29.88) Ma. The posterior probability distributions of all parameters showed a high degree of convergence  also supports the demographic equilibrium. Moreover, both Tajima’s D value  and Fu’s SF  value also in agreement with the demographic equilibrium.The mismatch distribution was multimodal , indicatP<0.05; sign test, P<0.05) from mutation drift equilibrium .The population bottleneck analysis showed a mode-shift in allele frequency distribution in three populations  from Northern Indochina . These tilibrium . Among tD. cambodiana were higher than most high-altitude tropical, subtropical and temperate tree or shrub species in Southeast Asia [mean cpDNA HT = 0.79 in 76]. The diversity parameters (HE) and genetic differentiation (FST) estimated using nuclear microsatellites were also high as compared to many tree species Lithocarpus sp. in tropical Asia D. cambodiana is in agreement with the postulation that high genetic diversity could be attributable to long-term evolution of plant populations isolation in refugia under fluctuating climatic conditions The genetic diversity estimates of D. cambodiana indicate that the gene flow through seeds between populations is limited despite possessing berry type fruits adapted for bird-mediated long-distance dispersal. The flower of D. cambodiana is entomophilous and therefore pollen flow among distant populations is unlikely. The significant population genetic differentiation and pronounced larger NST than GST is an indication of limited gene flow among populations. High G'ST of cpDNA and nSSR support the strong population structure with limited gene flow among populations.The limited seed dispersal and low levels of gene flow may act as a driving force of divergence among populations FST-SSR and RST-SSR values indicated that population subdivision between Hainan Island and Indochina Peninsular reflect a phylogeographic structuring. Similarly, F-statistics and Bayesian model based clustering also reveled strong differentiation between populations in Hainan Island and Indochina Peninsular  corroborating the cpDNA-based results and providing evidence for limited gene flow between populations. The low Nm value (<1) also suggests that the limited migration among populations may not offset the divergence due to genetic drift FIS for some of the microsatellite loci is an indication that inbreeding occurs in several populations D. cambodiana populations appears to be the result of the combined effect of distinctly limited gene flow and significant inbreeding within populations.Although our cpDNA haplotype data showed a high genetic divergence between populations in Hainan Island and Indochina Peninsular, the microsatellite data provided insight into contemporary gene flow. The significant differences between corresponding D. cambodiana is closely linked to the topographical features of the Indochina Peninsula  is considered as one of the most important periods for genetic diversification and speciation e period   indicatFigure S1The posterior probability convergence of t0, t1, t2 and tTMRCA. A: convergence of time parameters for each node in (PDF)Click here for additional data file.Figure S2The phylogenetic network of haplotypes with outgroups. O-1 = Agava sp., O-2 = Yucca gloriosa, O-3 = Asparagus plumosus. Red dots are missing haplotypes or missing samples.(TIF)Click here for additional data file.Figure S3The bayesian tree for evolutionary rate estimation based on uncorrelated lognormal relaxed clock. Haps are the cpDNA haplotypes. The numbers above branches are the median rates of substitutions per site per million years. The numbers below the branches denotes the Bayesian posteriors. The height-median divergence time (boldfaces) and the 95%HPD time ranges (blue bars) marked at the nodes for the posteriors are more than 0.6. The coalescent times of the most recent common ancestor (TTMRCA) of 18 cpDNA haplotypes is 15.80 (5.29–29.88) Ma.(TIF)Click here for additional data file.Figure S4The bayesian tree of D. cambodiana individuals based on Bayesian Skyline model. The height-median divergence time (boldfaces) and the 95%HPD time ranges (blue bars) marked at the nodes. The coalescent times of the most recent common ancestor (TTMRCA) of all individuals is 9.77 (3.69–18.64) Ma.(TIF)Click here for additional data file.Table S1log10 Bayes factors of three different relaxed clock models.(DOCX)Click here for additional data file.Table S2Pairwise genetic differentiation (FST) among four groups. The permutations for significance tests were 1023. P-value for all FST is less than 0.001.(DOCX)Click here for additional data file.Table S3The diversity and genetic structure parameters  corresponding to each of the six microsatellite loci.(DOCX)Click here for additional data file.Table S4The genetic parameters of populations of D. cambodiana in Hainan Island and Indochina Peninsular: expected and observed heterozygosity (HE and HO), and mean observed number of alleles per polymorphic locus (A), mean allelic richness per polymorphic locus (AR), number of private alleles (AP), FIS and FST-SSR per population. HE in * indicate significant deviations from HWE (at 0.05 significance level). The negative value of FIS indicates heterozygosity excess.(DOCX)Click here for additional data file.Table S5Analysis of population bottleneck based on IAM and TPM model of microsatellite evolution and Mode shift test for allele frequency distribution. Significance of gene diversity excess (He>Heq) was tested using Sign test and Wilcoxon signed ranks test  based on 5000 replications. *P<0.05. NL = normal L-shaped distribution and MS = mode-shift in allele frequency distribution.(DOCX)Click here for additional data file.File S1Supplementary (PDF)Click here for additional data file.


Puccinia striiformis f.sp. tritici (PST), capable of rapid spread to new regions and crop varieties. Although a range of recent PST invasions at continental scales are well documented, the worldwide population structure and the center of origin of the pathogen were still unknown. In this study, we used multilocus microsatellite genotyping to infer worldwide population structure of PST and the origin of new invasions based on 409 isolates representative of distribution of the fungus on six continents. Bayesian and multivariate clustering methods partitioned the set of multilocus genotypes into six distinct genetic groups associated with their geographical origin. Analyses of linkage disequilibrium and genotypic diversity indicated a strong regional heterogeneity in levels of recombination, with clear signatures of recombination in the Himalayan  and near-Himalayan regions (China) and a predominant clonal population structure in other regions. The higher genotypic diversity, recombinant population structure and high sexual reproduction ability in the Himalayan and neighboring regions suggests this area as the putative center of origin of PST. We used clustering methods and approximate Bayesian computation (ABC) to compare different competing scenarios describing ancestral relationship among ancestral populations and more recently founded populations. Our analyses confirmed the Middle East-East Africa as the most likely source of newly spreading, high-temperature-adapted strains; Europe as the source of South American, North American and Australian populations; and Mediterranean-Central Asian populations as the origin of South African populations. Although most geographic populations are not markedly affected by recent dispersal events, this study emphasizes the influence of human activities on recent long-distance spread of the pathogen.Analyses of large-scale population structure of pathogens enable the identification of migration patterns, diversity reservoirs or longevity of populations, the understanding of current evolutionary trajectories and the anticipation of future ones. This is particularly important for long-distance migrating fungal pathogens such as  Domestication of ecosystems, climate change and expanding global trade have accelerated the pace of disease emergence, caused by their introduction into new areas with susceptible hosts or the spread of new damaging pathogen genotypes. The wheat yellow rust pathogen (PST) is a pathogen with recent reports of invasions, significantly affecting worldwide wheat production. However, its origin and ancient migration routes remained unclear and the source areas of newly spreading strains were largely unknown. This information is important to understand the trajectories of current invasions and forecast the future spread, and more generally develop risk-assessment models of pathogen emergence. We analyzed a set of worldwide representative isolates of PST, which enabled to identify six different area-specific populations. Using population genetics tools, we identified its centre of diversity in the Himalayan and near Himalayan region. We also identified the source of the recently emerged populations; Middle East-East Africa as the source of high-temperature-adapted strains spreading worldwide; Europe as the source of American and Australian populations; and Mediterranean-Central Asian populations as the origin of South African populations. We demonstrate the influence of human activities on the recent long-distance spread of the disease, though most geographic populations are not markedly affected by recent dispersal events. Domestication of ecosystems, climate change and the expanding global trade have accelerated the pace of pathogen emergence and spread Puccinia striiformis f.sp. tritici (PST), is present in most wheat-growing regions of the world Yellow (stripe) rust on wheat, which is caused by th century, with an unknown origin Yr9, with a first report in 1986 in Eastern Africa (Ethiopia) and subsequent invasions of the Middle East, Pakistan and India, reaching Bangladesh in only 12 years Despite the capacity for long-distance migration, the worldwide spread of PST is relatively recent, with most emergences reported only within the last decades. The pathogen reached South Africa in 1996 from an unknown source, but the first pathotypes detected were similar to those present in the Middle East and Mediterranean regions Several recent studies, using different genetic markers, investigated the population structure of PST over relatively large geographic scales. Analyses revealed an overall clonal population structure with low genetic diversity worldwide In the present study, we assembled a representative set of isolates from a larger collection of PST populations from the worldwide geographical range of the fungus and analyzed their genetic variability using a single set of highly variable genetic markers. Our objectives were: (i) to identify the main genetic groups in modern PST populations; (ii) to test the Himalayan region as putative centre of diversity; and (iii) to identify the geographical origin of recently emerged populations and investigate the ancestral relationships among geographically spaced populations.tructure analyses, and were therefore not included in population-based analyses. Details regarding the number of isolates are shown in A set of 409 isolates was selected to represent 11 geographical regions on six continents  from a collection of more than 4,000 isolates available at Institut National de la Recherche Agronomique (INRA), France and the Global Rust Reference Centre, Aarhus University, Denmark. The selection was made to maximize the representation of each population  under panmixia, using GWe used approximate Bayesian computation (ABC) to compare different competing scenarios describing ancestral relationship among populations. The approach bypasses the calculation of exact likelihoods rendering it efficient for complex population genetic models such as those underlying biological invasions iyabc5 simulated data sets was generated for each scenario under the generalized stepwise mutation model, with two parameters: the mean mutation rate (I) and the mean parameter (P) of the geometric distribution used to model the length of mutation events (in number of repeats). The mean mutation rate was drawn from a uniform distribution of 10−4 to 10−3, while the mutation rate of each locus was drawn from a gamma distribution . The parameter P was kept in the range of 0.1 to 0.3. A range of 40 contiguous allelic states was kept for each locus, characterized by individual value of mutation rate (Ll) and the parameter of the geometric distribution (LP), which were obtained from Gamma distribution . Mean number of alleles per locus, mean genetic diversity STF2δμSimulations were performed using D5 simulated datasets used for model selection and treated them as observed datasets . Posterior probabilities of competing models were evaluated for each pseudo-observed dataset, using all remaining simulated datasets and the same methodology as described for the observed dataset. Confidence in model choice was then estimated using the number of pseudo-observed dataset that gave higher posterior probability to the model they had been simulated with. In tests of goodness-of-fit , we simulated 100 datasets of similar number of markers as observed datasets and calculated for each dataset the average across loci of several test quantities. The set of test quantities included the summary statistics used in analyses of the observed dataset. However, because using the same statistics in parameter inference and model checking can overestimate the quality of the fit Relative posterior probabilities of different scenarios were estimated by fitting a multinomial logistic regression between the summary statistics and a polychotomous variable corresponding to the different model indexes We performed multilocus genotyping of 409 PST isolates, representatives of a worldwide collection, using a set of 20 microsatellite markers. Plotting the multilocus genotypes detected against the number of loci re-sampled showed that the full set of SSRs was sufficient to discriminate clonal lineages  Europe and Central Asia-Mediterranean region  analysis. The Bayesian information criteria (BIC) curve in the DAPC analyses supported n region .tructure also identified an optimal number of clusters (K) equal to 6, based on the rate of change in the log probability of data across successive K values K = 2, Middle Eastern, Mediterranean and Central Asian populations were assigned to one group; the Chinese population was assigned to the other group; and Nepalese, Pakistani and NW European populations had a mixed assignment of the two groups  and near Himalayan (Chinese) populations not departing from HWE, suggesting the occurrence of recombination within the populations , and shoWe detected only a few recent migrants, admixed and unassigned isolates in each geographical region in clustering analyses . The exiIsolates of the recently emerged aggressive strain PstS1, associated with the post-2000 epidemics in the USA and Australia, consisted of only a single multilocus genotype (MLG-99) resampled in other geographical regions as well. PstS1 was closely related to the other recently emerged aggressive strain reported in Asia, Africa and Europe, the PstS2, which consisted of different multilocus genotypes, including this MLG-99. Both PstS1 and PstS2 were assigned to the Middle Eastern-East Africa group, suggesting a source in Middle East-East Africa for these strains . An oldeWe used approximate Bayesian computation (ABC) analyses to infer on the ancestral relationship among populations. To limit the number and complexity of the scenarios to be compared, we used a sequential approach: we defined nested subsets of competing scenarios based on our understanding of the population structure in different regions, and analyzed these subsets sequentially. The origin of recently emerged populations was investigated using clustering and differentiation analyses and it has been presented in the previous section.We started by analyzing the historical relationships among the three populations displaying recombining population structures and located in the region of highest diversity . The four competing scenarios assumed three different population-trees, and admixture in the ancestry of the Nepalese population  as this The origin of Central Asian, Middle Eastern and Mediterranean population was investigated jointly, based on the relatedness of these populations in population structure analyses , and theThe most likely scenario identified in analyses of Central Asian, Middle Eastern and Mediterranean populations was then used as a backbone to investigate the origin of the NW European population. Six scenarios were compared, assuming three different populations of origin  and admixture . The scePuccinia striiformis f.sp. tritici (PST) using a comprehensive set of isolates from six continents and 20 highly informative SSR markers. We showed that despite the long-distance dispersal and the recent global spread of aggressive strains, the worldwide PST population has a clear genetic structure and is separated into six groups corresponding to main epidemic areas. The maintenance of a clear genetic structure despite substantial gene flow results from both strong clonality in many PST populations, and strong founder effects in recently emerged populations.We investigated the origin, migration routes and population structure of wheat yellow rust pathogen Yr (resistance) genes, the population structure of PST was considered to be shaped by a continual replacement of pre-existing populations by the newly emerged and spread pathotypes, or aggressive strains. This process is well known as the boom and bust cycle Yr9 resistance gene , and it also provides an integrative scenario for the worldwide spread of PST including new findings regarding the origins of South American and European populations . More exThe existence of a high genotypic diversity, a high ability for sexual reproduction as well as the independent maintenance of strongly differentiated populations in the Himalayan region suggests this region as the putative centre of origin of PST. Differences in the levels of diversity and mode of reproduction among geographically distant populations are particularly relevant in the context of risk-assessment for disease emergence: Asian populations  with a high level of recombination, diversity and ability for sexual reproduction could serve as possible sources for the emergence of new, virulent and aggressive strains. The maintenance of populations specific to geographical regions in Asia suggests a survival of local populations in these regions, potentially through sexual reproduction. For integrated disease management, it would be important to quantify the relative contribution of sexual vs. asexual reproduction to the diversity in different world populations and identify sexual host(s) or clonal over-summering/-wintering pathways. Finally, this study emphasizes the potential role of human travel and commerce as a major driver in the emergence of PST. The intensification of business and tourism activities in regions known as major sources of pathogen diversity should be considered in the context of risks associated with the emergence and worldwide spread of plant disease.Figure S1Box-plot of number of PST genotypes detected as a function of the number of loci re-sampled 1000 times within the 20 microsatellite markers (from 0–20) using GENCLONE software. The box represents the average, minimum and maximum numbers of MLGs detected when re-sampling on loci.(DOC)Click here for additional data file.Figure S2Assignment of PST isolates from worldwide geographical regions to genetic groups, using the STRUCTURE software and different K-values (genetic groups). The chart represents the consensus assignment obtained by analysis of result from 20 runs of the STRUCTURE analysis with the CLUMPP software. Each color represents a different genetic group.(DOC)Click here for additional data file.Figure S3Microsatellite distance-based Neighbor-Joining tree for PST isolates from worldwide geographically spaced populations. Agr = Aggressive strain, CA = Central Asia, Ch = China, Erit = East Africa, MdlEst = Middle East, Med = Mediterranean region, Nep = Nepal, NWEu = NW European group, Pk = Pakistan and SA = South Africa.(DOC)Click here for additional data file.Figure S4Puccinia striiformis f.sp. tritici.Scenarios regarding the evolutionary relationship among the three recombinant populations from the centre of diversity of (DOC)Click here for additional data file.Figure S5Scenarios regarding the evolutionary relationship between the two ancestral populations of Pakistan and China with the Middle Eastern and Central Asian-Mediterranean populations.(DOC)Click here for additional data file.Figure S6Scenarios regarding the evolutionary relationship between the two ancestral populations of Pakistan and China with Middle Eastern, Central Asian-Mediterranean and NW European populations.(DOC)Click here for additional data file.Figure S7Scenarios regarding the evolutionary relationship between the two ancestral populations of Pakistan and China with Middle Eastern, Central Asian-Mediterranean and NW European populations along with a ‘ghost’ population.(DOC)Click here for additional data file.Table S1Gene diversity for 20 SSR loci in PST populations of diverse geographical origin.(XLS)Click here for additional data file.Table S2Prior distributions of model parameters.(XLS)Click here for additional data file.Table S3Demographic and mutation parameter estimated using Approximate Bayesian Computation. Only parameter esimtates for the scenarios having highest posterior probabilities are shown. Demographic parameters are introduced in corresponding Figures. Composite parameters scaled by the mutation rate are also shown. The mutation parameters are μ (mean mutation rate) and p .(XLS)Click here for additional data file.Table S4Model checking for demographic scenarios analysed using Approximate Bayesian Computation.(XLS)Click here for additional data file.Table S5Confidence in model choice for the demographic scenarios compared using approximate Bayesian computations. Confidence in model choice was assessed using a leave-one-out method. For each scenario 100 of the 500,000 simulated datasets used for model selection were randomly drawn and treated as pseudo-observed datasets. Posterior probabilities of competing scenarios were evaluated for each pseudo-observed dataset, using all remaining simulated datasets. Confidence in model choice was then estimated as the proportions of simulated data assigned to each scenario.(XLS)Click here for additional data file.Table S6Posterior probabilities (PP) of the different sets of scenarios compared using Approximate Bayesian Computation.(XLS)Click here for additional data file.


Mnemiopsis leidyi entered the Black Sea in the early 1980s. The invasion was followed by the Azov, Caspian, Baltic and North Seas, and, most recently, the Mediterranean Sea. Previous studies identified two distinct invasion pathways of M. leidyi from its native range in the western Atlantic Ocean to Eurasia. However, the source of newly established populations in the Mediterranean Sea remains unclear. Here we build upon our previous study and investigate sequence variation in both mitochondrial (Cytochrome c Oxidase subunit I) and nuclear  markers in M. leidyi, encompassing five native and 11 introduced populations, including four from the Mediterranean Sea. Extant genetic diversity in Mediterranean populations  preclude the occurrence of a severe genetic bottleneck or founder effects in the initial colonizing population. Our mitochondrial and nuclear marker surveys revealed two possible pathways of introduction into Mediterranean Sea. In total, 17 haplotypes and 18 alleles were recovered from all surveyed populations. Haplotype and allelic diversity of Mediterranean populations were comparable to populations from which they were likely drawn. The distribution of genetic diversity and pattern of genetic differentiation suggest initial colonization of the Mediterranean from the Black-Azov Seas (pairwise FST = 0.001–0.028). However, some haplotypes and alleles from the Mediterranean Sea were not detected from the well-sampled Black Sea, although they were found in Gulf of Mexico populations that were also genetically similar to those in the Mediterranean Sea (pairwise FST = 0.010–0.032), raising the possibility of multiple invasion sources. Multiple introductions from a combination of Black Sea and native region sources could be facilitated by intense local and transcontinental shipping activity, respectively.Gelatinous zooplankton outbreaks have increased globally owing to a number of human-mediated factors, including food web alterations and species introductions. The invasive ctenophore  Introduction of non-indigenous species (NIS) beyond their native range is considered a principal threat to marine ecosystems worldwide In recent years, gelatinous zooplankton outbreaks have raised concerns regarding the health of aquatic ecosystems The Mediterranean Sea has an enormously rich native biodiversity, though it is also the world's most invaded marine ecosystem Mnemiopsis leidyi A. Agassiz 1865 to the Mediterranean Sea. Mnemiopsis leidyi is native to the western Atlantic Ocean from Massachusetts, USA to Argentina. The species is a simultaneous hermaphrodite capable of self-fertilization, may reach maturity at two weeks of age, and can release up to 10,000 eggs per day Knowledge of the source and pathways of NIS introductions is essential for developing management strategies to prevent invasions. A focus on areas at high risk of biological invasions is crucial and should be considered a management priority M. leidyi were reported throughout the Mediterranean Sea in 2009, from eastern to western coastal areas M. leidyi from its native region to Eurasia excepting the Mediterranean Sea M. leidyi was introduced to Eurasia via at least two pathways. The first invasion occurred from the Gulf of Mexico to the Black Sea, followed by secondary spread to the Caspian Sea M. leidyi population in the Mediterranean Sea remains unclear. Several possibilities can be envisaged. It is possible the species has spread exclusively from the Black Sea c Oxidase subunit I; COI) and nuclear ribosomal  genes.Blooms of No specific permits were required for the described field studies in Eurasia, North America or South America. The species collected is an invasive pest in Eurasia and is not protected throughout its range. Sampling points did not include any protected or private lands.M. leidyi individuals were sampled from five native  and 11 introduced populations . Individuals were preserved separately in 95% ethanol prior to genetic analysis.A total of 286 5′- TGTCGCCCAAATTACTGTTTC-3′ and Ml-COIR: 5′- TGACGGGGTAAACCTCATAAA-3′). Primers were designed in this study according to the available sequenced M. leidyi mitochondrial genome (GenBank accession no: NC016117). The universal primer pair, (ITS5F and ITS4R) Taq DNA Polymerase (QIAGEN), 1 x PCR buffer, 2.5 mM of MgCl2, 0.2 mM of dNTPs, and 0.4 µM of each primer. PCR was performed with an initial denaturing step at 95°C for 1 min, followed by 35 amplification cycles , and a final elongation step at 72°C for 7 min.Genomic DNA was isolated from gelatinous lobe tissue of the ctenophores using the automatic extraction protocol described by Elphinstone et al. We purified PCR products, which were then sequenced for both COI and ITS markers with forward (Ml-COIF) and reverse primers (ITS4R), respectively, using Big Dye terminator sequencing chemistry with an ABI 3130XL genetic analyzer (Applied Biosystems). Sequences were inspected, manually edited, and aligned using Codon Code Aligner 2.0 . Sequence of alleles containing double nucleotide calls (overlapping peaks) were cloned using Cloning and Amplification Kit  according to Ghabooli et al. n), haplotype diversity (h) and nucleotide diversity (π) Pleurobrachia pileus (GenBank accession no JF760211) as an outgroup. We generated a parsimony network of haplotypes using TCS 1.0 We assessed diversity indices within populations, such as the number of haplotypes (aN), observed (oH), and calculated expected heterozygosity (eH) using GENEPOP (online version http://genepop.curtin.edu.au) and Arlequin version 3.1 FST using Arlequin.Using the protocol described above, we processed four new populations from Mediterranean Sea  as well as one more from the native range (MH from North Carolina) in addition to our previously published dataset which consisted of 190 individuals analyzed for ITS marker To estimate the sufficiency of our sampling, we generated rarefaction curves using ECOSIM and 5000 random iterations Analysis of a 656-bp fragment of COI obtained from 241 individuals resulted in 17 different haplotypes in surveyed populations (GenBank accession nos KF435105–KF435121). In total, we detected 29 variable sites (4.42%), 16 of which were specific to the divergent haplotype Ml01 from Peninsula Valdes, Argentina (2.43%). Ml03 and Ml09 were the most common haplotypes. We found haplotype Ml03 in all populations except in Peninsula Valdes, while Ml09 was not recovered from Peninsula Valdes, Limfjorden, or the Baltic Sea.We found twelve different haplotypes in native populations, all of which were present in introduced populations except for Ml01 from Peninsula Valdes, the single private haplotype at this site. We detected a total of 16 haplotypes among the introduced populations. Black-Azov Sea populations contained 11 haplotypes, which was higher than in all other introduced regions. Mediterranean Sea populations contained eight haplotypes, while those from the Caspian and Baltic seas had four haplotypes each. Out of eight haplotypes observed in the Mediterranean Sea, only Ml11 was not recovered from native populations in North America. Six haplotypes including Ml11 were detected in Black-Azov Seas. Two haplotypes from Mediterranean Sea populations were not found in either the Black or Azov Sea, though they were present in the native region, mainly in Florida and Morehead , Table 1The Black-Azov Seas shared six haplotypes with native populations, while the other five haplotypes from this region were either private for one population  or shared with France in Mediterranean Sea (Ml11). All four haplotypes found in Caspian Sea populations were present in both the Black Sea and North America. The Baltic Sea and Limfjorden (Denmark) shared all of their haplotypes with the native region, mainly Narragansett Bay, and only one haplotype with other introduced populations , Table 1n = 7) . Native populations from Morehead and Peninsula Valdes exhibited the highest (n = 7) and lowest (n = 1) number of haplotypes, respectively (The introduced population (BL) from Black Sea contained the highest number of haplotypes (n = 7) . Among iectively .h) and nucleotide diversity (π) in all introduced populations were 0.704±0.059 and 0.0020±0.0003, respectively. Comparable values in Mediterranean Sea populations were nearly identical, 0.702±0.008 and 0.0019±0.0001, respectively. Native populations exhibited higher values for each of these indices . We excluded the non-diverse individuals of Peninsula Valdes of South America from this calculation.Mean COI haplotype diversity . The rest of the haplotypes formed the third group . The comChao-1 COI haplotype richness estimates were moderately higher than obtained values in Black-Azov Sea populations , indicating undersampling of these regions, although the lower 95% confidence interval limit (11.7) was marginally higher than observed diversity in the Black-Azov Seas . Chao-1 M. leidyi - including the 190 individuals analyzed in our previous study Analysis of the 619 bp DNA fragment comprising the complete ITS1, 5.8 S rRNA and ITS2 regions obtained from 286 individuals of We detected thirteen different alleles in native populations, all of which were recovered from introduced populations, except for the private allele G from Peninsula Valdes . MediterThe Chao-1 allele richness estimate for the Black-Azov Seas  indicates reasonably comprehensive sampling of this region . For theoH) was lower in introduced populations (0.56±0.011) than in native ones (0.62±0.12)  . Pairwispulation .FST values with those from the Black and Azov Seas (FST = 0.001–0.028). However, Mediterranean Sea populations were also very similar to those from the Gulf of Mexico (FL) and North Carolina (MH) in the native region , while those in Italy and Israel were most divergent (FST = 0.033). Individuals from Limfjorden, Denmark had the lowest FST with Baltic Sea (FST = 0.015), and with Narragansett Bay (FST = 0.021) in the native region  and nuclear (ITS) markers. Our results support a multiple source model, composed by at least two different introduction pathways. One source of M. leidyi in the Mediterranean appears to have originated from Black Sea, consistent with the view of Bolte et al. In this study, we build upon our previous study to explore genetic diversity, and determine the source(s) of, Introduced populations in the Mediterranean Sea exhibited lower values of haplotype diversity (0.702±0.008) and observed heterozygosity (0.58±0.05) relative to native ones . However, none of the Mediterranean populations exhibited erosion of genetic diversity for either of the analyzed markers relative to their putative source populations. This pattern could be driven by repeated introductions from the native range as well as from the adjacent Black Sea area, given intense vector activity between these regions and the high diversity of source populations Two Mediterranean Sea populations (Spain and France) exhibited deviation from Hardy-Weinberg equilibrium . Both poFST with populations from the Black-Azov Seas (FST = 0.001–0.028). However, Mediterranean populations also exhibited low genetic differentiation with those from Florida and Morehead (FST = 0.010–0.032) in the native range. The highest genetic differentiation occurred among introduced populations in Mediterranean or Black-Azov-Caspian seas and those in the Baltic Sea and Limfjorden (Denmark), ranging from 0.177 to 0.417 , suggesting some structuring and limited gene flow in the native region. The private haplotype Ml01 was separated from other haplotypes by at least 15 mutation steps. All other haplotypes had only one or a few mutation steps between them. Pairwise genetic differentiation, parsimony network analysis, and phylogenetic reconstruction of haplotypes demonstrate high genetic divergence between South America and all other locations, notwithstanding the paucity of samples available from the former region. Long-term isolation of populations could explain the observed divergence. Pleistocene glacial periods in the northern hemisphere could drive high genetic divergence between South America and North America, resulting in population fragmentation M. leidyi between South America and Eurasia.Genetic differentiation among native populations was relatively high (M. leidyi introduction into major Eurasia Seas M. leidyi entered the Black Sea via ships' ballast water from the Gulf of Mexico region. Spread of M. leidyi into the Sea of Azov occurred via the natural connection between these basins M. leidyi into the Baltic Sea, with subsequent spread into the North Sea M. leidyi in the Mediterranean Sea. However, in this study, genetic differentiation of North American and Mediterranean Sea populations was only slightly greater than that with populations from the Black Sea (M. leidyi in the Mediterranean Sea.Genetic analyses have revealed pathways of lack Sea . In addilack Sea . Finally


Alnus glutinosa Gaertn., a European tree species that plays a significant ecological role as a keystone of riparian ecosystems. We genotyped 551 adults from 19 populations located across North Africa at 12 microsatellite loci and applied a coalescent-based simulation approach to reconstruct the demographic and evolutionary history of these populations. Surprisingly, Moroccan trees were tetraploids demonstrating a strong distinctiveness of these populations within a species otherwise known as diploid. Best-fitting models of demographic reconstruction revealed the relict nature of Moroccan populations that were found to have withstood past climate change events and to be much older than Algerian and Tunisian populations. This study highlights the complex demographic history that can be encountered in rear-edge distribution margins that here consist of both old stable climate relict and more recent populations, distinctively diverse genetically both quantitatively and qualitatively. We emphasize the high evolutionary and conservation value of marginal rear-edge populations of a keystone riparian species in the context of on-going climate change in the Mediterranean region.Populations located at the rear-edge of a species’ distribution may have disproportionate ecological and evolutionary importance for biodiversity conservation in a changing global environment. Yet genetic studies of such populations remain rare. This study investigates the evolutionary history of North-African low latitude marginal populations of  Alnus glutinosa Gaertn., a monoecious self-incompatible wind-dispersed European tree species with fruits dispersed by wind at small distance and by water at longer distance and which acts as a keystone of riparian ecosystems A poleward shift in the geographical distribution of many species is expected as one of the major consequences of on-going global climate change A. glutinosa over most of Europe was achieved in the early Holocene, as shown by pollen and macrofossil records which indicate its presence in Southern Switzerland by 14,000 cal. BP A. glutinosa in central and eastern Europe A. glutinosa, Although different responses to past climatic events have been reported for species with contrasted ecological characteristics Stable rear-edge populations A. glutinosa are good candidate climate relicts. Firstly, pollen records indicate a long-term presence of A. glutinosa in North Africa North-African rear-edge populations of A. glutinosa, however their evolutionary history remains unclear. In this paper, we consider three alternative hypothetical scenarios. The first one hypothesises that North-African populations constituted LGM refugia that contributed to postglacial recolonisation of Europe. A second scenario considers that North-African populations are pre-LGM stable rear-edge climate relicts that did not contribute to postglacial recolonisation. In these two scenarios, populations would be expected to display a relatively low genetic diversity within populations but high regional genetic diversity and genetic differentiation. However expected differential characteristics of climate relicts are high genetic distinctiveness resulting from a longer term isolation A. glutinosa population dynamics would follow a metapopulation process where local populations are transient. This third scenario would consider the current North-African distribution to represent a subset of the genetic diversity of the core post-LGM distribution.Contemporary North-African populations clearly lay at the rear-edge of the current distribution of A. glutinosa in North Africa, we use recently developed species-specific nuclear microsatellite markers A. glutinosa in the region from best-fitted models. Our results demonstrate the complex demographic history that can be encountered in rear-edge populations and contribute to emphasize the high evolutionary and conservation value of rear-edge populations in the context of future climate change.In order to discriminate between those alternative hypotheses and clarify the conservation status of No permits were required for the field study as only a few leaves of tree were collected with no effect significant to individual and ecosystem health. The sampled species is not protected nor is the accessed land.Alnus glutinosa populations throughout the current known species distribution in North African and persisting in locally wet habitats in pockets of riparian forests, small floodplains and alder carrs, located from sea level upwards to altitudes nearing 1000 m  with one 3 mm-diameter tungsten bead (Qiagen) following two disruption cycles of 60 s each, using Isolate Plant DNA minikit (Bioline) according to manufacturer instructions and stored at −20°C until further use. The 551 individuals were subsequently genotyped at twelve independent microsatellite loci, assumed to evolve neutrally, described in full in ype-it Microsatellite PCR Master Mix (Qiagen), with unequal concentrations of each of the 12 fluorescent forward primers labelled with one of 6-FAM, VIC, PET or NED dyes and reverse unlabelled primers  and consisted of an initial denaturation step of 5 min at 95°C, followed by 30 cycles of 95°C for 30 s, 58°C for 180 s and 72°C for 30 s and a final elongation step of 30 min at 60°C. PCR products were sent for fragment analysis on an ABI3730xl capillary sequencer to DNA Sequencing and Services  with a 1∶50 dilution before run and using GeneScan 500 LIZ internal size standard (Applied Biosystems).Total genomic DNA was isolated from 20 mg of silica dried mers see  and 5 ngandA. glutinosa individuals  in R.Unexpectedly, individuals from the four Moroccan populations exhibited up to four alleles at each of the 12 loci genotyped suggesting a difference in ploidy level between populations sampled in Morocco and those sampled elsewhere (tructure v.2.3.3 K) formed by the 587 individuals sampled in North Africa and in PGM with no a priori partitioning of individuals into groups defined according to their geographic location because such partitioning may not reflect true panmictic populations and may affect both estimates of genetic structure and their interpretation Regional genetic structure was first described using the individual-based Bayesian genetic assignment method implemented in Structure user guide Recessive Alleles model tructure that these genotypes are diploids.We followed the instructions provided in the SAdmixture model of ancestry  which allows for individual to have mixed ancestry and a Correlated Allele Frequency model (parameter λ set to 1 and not assuming same Fst among populations) which assumes that populations have each undergone independent drift away from a hypothetical ancestral population tructure Harvestertructure was used to build a neighbour joining tree using the ape package in R The most-likely number of genetic clusters (K) was determined inspecting the variation of the mean logarithm of the probability of the data (Ln P(D)) across replicated runs. We ran 20 independent runs for K = 1 to K = 10 using a burn-in period of 50,000 steps followed by 100,000 Markov Chain Monte Carlo iterations, with an tructure. We used a discriminant analysis of principal components  while keeping all principal components to maximize the variation. A k-means clustering was then applied to identify the best number of clusters K that minimize the variation within clusters using Bayesian Information Criterion (BIC). A discriminant analysis (DA) was then applied using the dapc function using 30 PCs explaining more than 90% of the total variance of the data and retaining all discriminant functions  and membership probabilities of each individuals to each clusters was then computed. The relationship between clusters was analysed building a neighbour joining tree based on the dapc distance between centroids of the different clusters with the ape package In order to validate those results, regional genetic structure was then described using a multivariate statistics based clustering method that requires fewer assumptions than population genetics model based methods such as Sts dapc,  implemendiyabc v.1.0.4.46beta structure and dapc but showing limited admixture (diyabc.The Approximate Bayesian Computation (ABC) method implemented in dmixture  thus resSeven demographic scenarios characterised by divergence times in generations , effective population size of standing  and putative ancestral (Ne and Ns) populations were compared . In the −3 and 10−4 mutation per haploid genome and generation (SSRµ∼U[10−4–10−3]) while using a Uniform prior distribution bounded between 0.1 and 0.3 for P (P∼U[0.1–0.3]), the parameter of the Geometric distribution in which the number of repeats of the mutation is drawn. Within this general parameterisation of the mutation model, mutation parameters were allowed to vary independently for individual loci using a hierarchical scheme to account for heterogeneity in mutation rate and model across loci .For each simulated scenario, priors were set as a wide Uniform distribution bounded between 10 and 20,000 individuals for N1, N2, N3, Ne and Ns and between 1 and 20,000 generations for divergence times with the additional constraint of t1<t2<t3a,b. Default values were used for genetic parameters, assuming a Generalized Stepwise Mutation model diyabc to compute observed summary statistics. In order to include information content from the 30 tetraploid individuals sampled in BOU in analyses, allele frequencies at each locus for BOU were first estimated using the function simpleFreq in the R package polysatsample in R to generate 30 diploid multilocus genotypes assuming random mating as input data for diyabc to generate equivalent summary statistics.Input data consisted of diploid multilocus genotypes scored at 11 microsatellite markers for 30 (TIT) to 36 (PGM) individuals per population which are used by The observed and simulated genetic datasets were summarised using mean number of alleles, mean size variance and M index 6 simulations were run providing 10.106 simulations for each scenario. The 7,000 simulations closest to the real genetic dataset were subjected to a weighted polychotomous logistic regression 70.10locfitDemographic parameters were inferred from the most-likely scenario using the normalized Euclidian distance between the observed and simulated summary statistics to select the 10,000 simulated datasets closest to observed data (0.1% of the simulations). Parameters’ posterior distributions were estimated using a local linear regression method Alnus glutinosa individuals were genotyped at 12 microsatellite loci across 20 populations  with Morocco showing the highest mean gene diversity (rHs = 0.72) followed by Tunisia (rHs = 0.55) and Algeria (rHs = 0.50).Overall mean gene diversity in North African populations of alder was relatively low for a tree species with Hs ranging for 0.48 to 0.78 ; scenario 4: P = 0.32 (CI95% = 0.27–0.37)). Yet, in simulations made to access the power of our dataset to discriminate between alternative scenarios, one third of pseudo-observed datasets assigned to scenario 5 were simulated under scenario 4  to closely related A. incana or A. cordata species (results not shown) and the genetic distance between the tetraploid Moroccan populations and the diploid populations is within the same magnitude that the genetic distance found between Scottish and Tunisian/Algerian diploid populations. Given the high genetic diversity within tetraploid populations, a scenario of autopolyploidy by hybridization between differentiated A. glutinosa populations seems more likely than a chromosome doubling within individual. Indeed the high genetic diversity standing in tetraploid population is not due only to the numeric increased in genome number alone as genetic indices computed on a rarefied sampled size still show a statistically significantly higher genetic diversity within Moroccan populations , the high distinctiveness of these relict populations may be accompanied with distinctiveness of the whole dependent ecosystem. Indeed, series of studies in community genetics initiated in the riparian ecosystem shaped by cottonwoods (Populus spp.) have demonstrated that foundation species genetics have impact not only for the species phenotype but also for the community and/or ecosystem phenotype A. glutinosa. The high ecological and evolutionary values of these A. glutinosa populations may thus extend beyond the tree species to the whole community standing there probably as relict ecosystem that persisted throughout the ages.The relict nature and the striking distinctiveness of Moroccan Overall our study illustrates the complexity of rear-edge population history Figure S1Examples of microsatellite electropherograms leading to categorisation of individuals as diploids or tetraploids.(PDF)Click here for additional data file.Figure S2Comparison of observed and expected distribution of the number of alleles per individual by ploidy level.(PDF)Click here for additional data file.Table S1Power to discriminate between alternative demographic scenarios tested by coalescence.(PDF)Click here for additional data file.Table S2Confidence and precision of parameter estimates under the most-likely of seven tested scenarios of population demographic history.(PDF)Click here for additional data file.


HvABCG31/Eibi1 is an ABCG transporter gene, involved in cuticle formation that was recently identified in wild barley (Hordeum vulgare ssp. spontaneum). To study the genetic variation of HvABCG31 in different habitats, its 2 kb promoter region was sequenced from 112 wild barley accessions collected from five natural populations from southern and northern Israel. The sites included three mesic and two xeric habitats, and differed in annual rainfall, soil type, and soil water capacity.The cuticle is an important adaptive structure whose origin played a crucial role in the transition of plants from aqueous to terrestrial conditions. HvABCG31 promoter sequences clustered the majority of accessions (69 out of 71) from the three northern mesic populations into one cluster, while all 21 accessions from the Dead Sea area, a xeric southern population, and two isolated accessions (one from a xeric population at Mitzpe Ramon and one from the xeric ‘African Slope’ of “Evolution Canyon”) formed the second cluster. The southern arid populations included six haplotypes, but they differed from the consensus sequence at a large number of positions, while the northern mesic populations included 15 haplotypes that were, on average, more similar to the consensus sequence. Most of the haplotypes (20 of 22) were unique to a population. Interestingly, higher genetic variation occurred within populations (54.2%) than among populations (45.8%). Analysis of the promoter region detected a large number of transcription factor binding sites: 121–128 and 121–134 sites in the two southern arid populations, and 123–128,125–128, and 123–125 sites in the three northern mesic populations. Three types of TFBSs were significantly enriched: those related to GA (gibberellin), Dof (DNA binding with one finger), and light.Phylogenetic analysis of the aligned HvABCG31 promoter. Abiotic stresses may be involved in the HvABCG31 gene transcription regulations, generating more protective cuticles in plants under stresses.Drought stress and adaptive natural selection may have been important determinants in the observed sequence variation of  Eibi1 is the only full ABC transporter gene for releasing cuticle compounds reported in cereals. It was identified in wild barley, (Hordeum spontaneum), as “HvABCG31” by analogy with the rice gene [HvABCG31 gene was cloned from a spontaneous mutant of wild barley from Israel. This mutant had a drought-hypersensitive phenotype, with a 50% reduction in the major cutin monomers and an incomplete cuticle surface. HvABCG31 gene was mapped and sequenced and its major function was related to cuticle secretion [Over their long evolutionary history, plants have evolved morphological, physiological, biochemical, and genetic traits that enable them to respond to abiotic and biotic stresses . Reportsce gene ,10. The cretion .Hordeum vulgare ssp. spontaneum, the progenitor of cultivated barley, lies in the “Fertile Crescent”, starting from Israel and Jordan to southern Turkey, Iraq, Kurdistan, and south-western Iran [The distribution centre of rn Iran ,14, and rn Iran -19. Extrrn Iran ,19-21. Wrn Iran ,22,23. THvABCG31 are conserved in monocot and dicot plants [The coding regions of  plants . Increas plants -26. Gene plants . Transcr plants .HvABCG31 promoter region were analyzed using 112 wild barley accessions from five natural populations from different habitats in Israel, ranging from the southern xeric Negev desert to the northern mesic Golan Heights. The phylogeny of the HvABCG31 promoter sequence suggested that adaptation to ecogeographic environmental stresses, including aridity, was an important explanatory factor for the observed variation and history of HvABCG31. The analysis of TFBSs in the HvABCG31 promoter region indicated that gibberellin (GA), light, and abiotic stresses may be involved in the HvABCG31 transcriptional regulation.In this study, the genetic diversity, phylogenetic relationships, and distribution of TFBSs in the HvABCG31 promoter were compared among 112 wild barley accessions from five natural populations: Mitzpe Ramon , Dead Sea , "Evolution Canyon" , Arbel , and Yehudiyya   with the accession numbers AB709909–AB709930. Over 90% of the haplotypes were unique to a population: 20 haplotypes were population-specific, while only two were shared between populations in this study , or low frequencies of harmful mutations [The genetic diversity analysis and the neutrality test results in different populations are summarized in Table tations ,29,30. HHvABCG31 homologue from the wheat (Triticum aestivum var. Chinese Spring) 3B chromosome. Most accessions (69/71 = 97.2%) from the three northern mesic populations  formed one cluster (Cluster 1). This cluster was further divided into two major sub-clusters with high support values: Cluster 1-a from P3 and P4 and Cluster 1-b from P5. Interestingly, the structure of the sub-clusters was in accordance with the soil types of their collection sites . The second cluster (Cluster 2) included all 21 accessions from P2, one accession from the xeric population of P1, and one accession from the xeric ‘African Slope’ of P3. One shared haplotype (Hap 1) from both P1 and P3 proved very distant from both clusters and represented most of the accessions from P1 (19/20 = 95%) and one accession from the ‘European Slope’ of P3.To investigate the relationships among the different populations, a phylogenetic tree based on SNP haplotypes was constructed using maximum likelihood as implemented in MEGA5 software Figure . The treThe distances among the haplotypes in Cluster 1 (including Clusters 1-a and 1-b) were closer than those in Cluster 2  and 121–134 (P2) TFBSs in the two southern arid populations, and 123–128 (P3), 125–128 (P4), and 123–125 (P5) sites in the three northern mesic populations. The TFBSs could be classified into 8 categories according to their basic functions   were reported in a study of wild barley populations from Israel [HvABCG31 promoter sequences grouped most accessions (69/71 = 97.2%) of the northern populations  into one cluster, whereas all accessions from the Dead Sea (P2), one accession from Mitzpe Ramon (P1), and one accession from the ‘African Slope’ of “Evolution Canyon” (P3) formed a second cluster. Interestingly, there were many indel polymorphisms in our five natural populations but the basic phylogenetic structure was similar whether or not indels were considered (results not shown). The major ecological factors differentiating the northern  and southern (P1 and P2) populations, excluding geographic distance, were average annual rainfall and soil type. Water availability is the most important environmental factor for plant growth [High genetic diversity was found in the ) Table  in the p Israel -36. Drou Israel ,38. The  growth ,39. Drou growth -42; howehttp://evolution.haifa.ac.il/index.php/component/content/article/29).Different selection pressures in different regions are important in adaptation to local habitats [HvABCG31 promoter are maintained by variable drought and salinity.In the current study significant evidence against neutrality (based on Tajima's D test and Fu & Li's F* test) was found in the two southern populations P1 and P2) and in one northern populations (P3), while no significant deviations from neutrality were found in the other two northern populations (P4 and P5). Significant positive values of the test statistics indicate balancing selection, while significant negative values may suggest purifying selection and demographic effects (population expansion)  and in o,30,43-45abitats ,49. Our abitats . HoweverHvABCG31 is involved in cuticle formation [HvABCG31 promoter. Previous studies showed that cuticle formation is affected by drought and light [HvABCG31 promoter, most base changes (44%) added or deleted GA-binding sites. Of note, a recent microarray analysis of drought-stressed wild emmer wheat roots indicated that GA may be involved in drought resistance [HvABCG31 transcription, which may indirectly indicate that HvABCG31 affects abiotic stress tolerance, although other processes may influence HvABCG31.Although rmation , only thrmation . The TFBd light -53. The d light . GA is ad light . In the istance . In addiistance . GA, ligHvABCG31 promoters of minor haplotypes, mainly in the southern (xeric) populations. An early paper reported that TFBSs can appear or disappear among closely related species and even within populations [HvABCG31; more expression experiments are required to further understand its regulation.Base variation can dramatically change TFBSs and thus affect gene regulation . We founlations . Neverthlations . Loss oflations . It remaHvABCG31 gene in five natural populations of H. vulgare ssp. spontaneum in Israel suggests that aridity may be an important factor affecting the observed sequence variation. The analysis of TFBSs in the HvABCG31 promoter region indicated that GA, light, and abiotic stresses may be involved in the HvABCG31 transcription regulation and that HvABCG31 may be involved in abiotic stress responses, both locally and regionally, thereby generating adaptive structures in response to environmental stresses.Our phylogenetic analysis of eco-geographical variation in the 2kb promoter region of the H. vulgare ssp. spontaneum were collected from five natural populations in Israel during April 2007. The seeds were collected randomly from individuals situated at least 1–2 m apart [http://www.biogis.huji.ac.il/Map.aspx). All the data were checked using a relevant reference [http://ims.gov.il/IMS/CLIMATE).Seeds of 112 accessions of m apart . We analm apart ,46,47,60ference  and the HvABCG31[http://frodo.wi.mit.edu/primer3/).Seeds were germinated in Petri dishes on filter paper with purified water, treated with cold (4°C) for 7 d then held at 25°C for 3 d. Seedlings were then transplanted into pots  with a cycle of 16 h, 22°C day and 8 h, 18°C night. Genomic DNA was extracted from young leaves using a small-scale isolation method . Based oHvABCG31,12, one 2 (25 mM), 4 μL dNTP (2.5 uM), 1.5 μL each primer (10 uM), 3 μL DMSO  and water. The PCR procedure consisted of an initial denaturing step ; followed by 30 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 2 min, and completed by an incubation at 72°C for 15 min. Amplified DNA products were electrophoresed on 1–2% w/v agarose gels and sequenced on an ABI 3100 DNA sequencer .The PCR amplifications were carried out in a C1000TM thermal cycler . Each 50 μL PCR reaction contained 20 ng template DNA, 5 μL 10× PCR buffer, 1.25 U ExTaq DNA polymerase , 5 μL MgClhttp://en.bio-soft.net/format/DNAMAN.html), and ambiguous sites were checked manually in accordance with the DNA peak files in Chromas2.01 (http://www.technelysium.com.au/chromas_lite.html). Sequences were aligned with the MUSCLE feature in MEGA5 (http://www.megasoftware.net/) [http://www.ub.edu/dnasp/). Gaps were considered a fifth state [http://cmpg.unibe.ch/software/arlequin3/) based on the haplotype frequencies [Overlapping DNA sequences were assembled using DNAMAN6.0 (e.net/) ,64. Geneh state . Haplotyuencies . Geneticuencies ,68. Polyuencies ,70. An auencies .H. vulgare ssp. spontaneum HvABCG31 promoter were scanned for TFBSs using the PlantPan database (http://plantpan.mbc.nctu.edu.tw/index.php), which combines several plant TFBS databases, such as PLACE, TRANSFAC, JASPER, and AGRIS [A phylogenetic tree of haplotypes sequences was constructed using the maximum likelihood (ML) algorithm in MEGA5, with 1000 bootstrap replicates to gauge support, and a 60% cut off was used in the analysis. The phylogenetic tree was rooted using 2 kb of the putative homologue from wheat which was obtained from a BLAST search of the 3B chromosome sequences . All haplotypes of the d AGRIS . The sead AGRIS . A speciThe authors declare that they have no competing interests.XM, GJ, and GC had the main responsibility for the study and participated in all aspects, including developing the main idea, hypotheses, setting up the study, collecting some of the data, analyzing the data, and preparing and revising the manuscript. CL, AW, MP, SS, and TK carried out the molecular genetic studies, including DNA extraction, sequencing and data analysis. HS, DW, TK, XM, EN, and AK interpreted data and helped to revise the manuscript. All authors read and approved the final manuscript.Table S1. Detailed information for the polymorphic sites sample.Click here for fileTable S2. Detailed information for 22 haplotype in wild barley (Hordeum vulgare ssp. spontaneum).Click here for fileTable S3. Detailed information for TFBSs classification.Click here for file


Opisthorchis viverrini. Here, we used microsatellite DNA analyses to examine the genetic diversity and population structure of O. viverrini from four geographically close localities in Khon Kaen Province. Genotyping based on 12 microsatellite loci yielded a mean number of alleles per locus that ranged from 2.83 to 3.7 with an expected heterozygosity in Hardy–Weinberg equilibrium of 0.44–0.56. Assessment of population structure by pairwise STF analysis showed inter-population differentiation (P<0.05) which indicates population substructuring between these localities. Unique alleles were found in three of four localities with the highest number observed per locality being three. Our results highlight the existence of genetic diversity and population substructuring in O. viverrini over a small spatial scale which is similar to that found at a larger scale. This provides the basis for the investigation of the role of parasite genetic diversity and differentiation in transmission dynamics and control of O. viverrini.Khon Kaen Province in northeast Thailand is known as a hot spot for opisthorchiasis in Southeast Asia. Preliminary allozyme and mitochondrial DNA haplotype data from within one endemic district in this Province (Ban Phai), indicated substantial genetic variability within  (Opisthorchis viverrini) is a risk factor for cholangiocarcinoma (CCA), which is highly prevalent in Khon Kaen Province, Thailand. Within this Province, there is considerable variation in the epidemiology of opisthorchiasis among districts. Preliminary allozyme and mitochondrial DNA data indicate that genetic variation in O. viverrini occurs even over a small endemic area within the province. Here, we used microsatellite DNA analyses to examine the population genetic structure of O. viverrini from four geographically close localities. Analyses of adult worms based on 12 microsatellite loci revealed varying levels of genetic diversity and population substructuring between the four localities. Worms originating from one locality (Phu Wiang) had significantly higher genetic diversity than the other three localities. Data on the population genetic structure observed in these localities are similar to those found at a larger geographic scale. This provides background data to further investigate the biological and epidemiological significance of these genetic variants.Infection with the liver fluke  Opisthorchis viverrini is a food-borne trematode endemic in Southeast Asia, including Thailand, Lao PDR, Vietnam and Cambodia with more than 10 million people infected O. viverrini infection is a significant medical problem because of its involvement as a major risk factor causing bile duct cancer  The liver fluke, O. viverrini does not represent a single species but consists of at least two morphologically similar but genetically distinct (i.e. cryptic) species from Thailand and Lao PDR O. viverrini from different wetlands in Thailand and Lao PDR has been detected. For instance, worm recovery as well as the fecundity of O. viverrini from the Songkram River in Thailand was significantly different from other wetland systems  in Thailand and Lao PDR (Nam Ngum River) O. viverrini has to date only been studied from a single locality , but the results indicated considerable genetic diversity and heterozygote deficiency occurring within a small geographical area Recently, we reported that O. viverrini is, however, needed to fully determine whether population substructuring and/or differential genetic diversity are associated with geographical differences in distinct wetlands, river systems and flooding patterns O. viverrini and provided evidence of population subdivision over a large spatial scale with the maximum distant apart of up to 770 km More detailed information on the population genetic structure of O. viverrini populations occurring within and between four geographically close localities  less than 60 km apart in Khon Kaen Province, northeast Thailand. Comparisons were also made with data previously reported for populations separated by much greater distances . Finally, potential population mechanism(s) related to the host and environmental factors that may contribute to the current population structure were discussed.In this study, we examined the genetic diversity and population structure of O. viverrini for this study were obtained from four reservoirs located within three adjacent districts namely Khon Kaen, Ban Phai and Phu Wiang in Khon Kaen Province, Thailand as shown in Khon Kaen Province is geographically centrally located in northeast Thailand and by political division it currently consists of 26 districts. Samples of O. viverrini were recovered from hamsters experimentally infected with metacercariae obtained from pools of naturally infected cyprinid fish (Cyclocheilichthys armatus). In each sampling locality, approximately 500 fish weighing at least five kilograms were processed by a pepsin digestion method to isolate metacercariae Samples of adult The worms were identified based on standard morphological methods and washed several times with 0.85% NaCl. A minimum number of hamsters was used to provide a sufficient number of worms for different experiments that we are undertaking. For microsatellite analyses a random sample of 30 individual worms from a mean of 26–34 worms per hamster per locality were selected.O. viverrini microsatellite loci (12) were used in this study 2+, 0.2 mM of each nucleotide, 0.2 pmol of each primer, and 0.05 units Taq polymerase . PCR amplifications were carried out in a BIORAD thermocycler  in a total volume of 25 µl. The cycling conditions included 30 cycles of 1 min at 94°C, 1 min at the optimized annealing temperature, and 3 min at 72°C. PCR products were diluted in HIDI formamide with internal GeneScan size standard, ROX-400HD (PE Applied Biosystems) then loaded on the ABI 3100 DNA sequencer (PE Applied Biosystems). Before analysis, the PCR products were denatured in the thermocycler at 95°C and rapidly cooled on ice. Allele sizes were determined using ABI Prism GeneScan Analysis 3.1 and Genotyper 2.5 (PE Applied Biosystems). PCR reactions were redone in all cases where samples could not be amplified and in each case non amplification was confirmed. To avoid scoring artificial bands resulting in scoring errors, all PCR of samples with electropherograms with many peaks or non-specific products were repeated until unambiguous results were obtained. Furthermore, only clear electropherograms with one or two peaks of the expected size were considered in the analysis.Individual worms were homogenized on ice in a microcentrifuge tube using a handmade glass pestle. Genomic DNA was extracted by GenomicPrep Cells and a Tissue DNA Isolation kit following manufacturer recommendations . DNA concentration and purity were determined by spectrophotometry . Previously isolated and characterized ISF) and genetic differentiation between populations (STF) based on Weir and Cockerham F-statistics STF values was evaluated STF and geographical distances by a Mantel test. All the calculations described above were conducted using GENEPOP Version 3.4 software ISF of the parasite populations were calculated by using FSTAT For each locus, the number of alleles, allelic frequencies, and linkage disequilibrium among polymorphic loci using the Markov chain approach This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council of Thailand. The protocol of animal experimentation was approved by the Institutional Animal Ethics Committee, Khon Kaen University (AEKKU20/2551). All surgery and necropsy was performed under sodium pentobarbital anesthesia, and every effort was made to minimize pain and suffering to the animals.O. viverrini (P>0.05) (data not shown). However, linkage disequilibrium was found for Ovms14 and 15, but this was not significant after sequential Bonferroni's adjustment (P>0.01) O. viverrini populations unique alleles are of low frequency (<6%). Three of the four Khon Kaen populations  exhibited unique alleles  across five loci (Allele distribution patterns at 12 microsatellite loci varied greatly among samples of iverrini . For theive loci . Widely ive loci . HoweverO. viverrini populations as shown in P>0.05). Expected heterozygosity per locus (EH) for the four localities ranged from 0.44–0.56. The mean allelic richness which is a measure of allelic diversity based on the number of alleles per locus for KPv was 3.56, significantly higher than that for KBp and KLp (1.88) and KBs (1.83) .With respect to genetic diversity among Khon Kaen IFS values were calculated  genetic differentiation between all pairs of localities at all geographic scales  0.002–0.105], and that of the more widely spaced populations in the previous study was 0.043 (CI = 0.016–0.075) STF  = 0.165) occurred in only a single comparison, between LP and NP. Comparisons between Khon Kaen and widely spaced populations yielded 60% and 40% of populations with little and moderate differentiation, respectively. A Mantel regression test was done to determine the correlation between the STF and geographical distance between populations. No correlation between genetic and geographic distance was found among populations from Khon Kaen as well as between Khon Kaen and widely spaced populations, whether distances were calculated in straight lines or along river courses . Of these hosts, snails in particular show high levels of genetic diversity Bithynia snail intermediate host, which in turn may play a role in the observed genetic, biological and/or morphologically variation in this parasite.The population genetic data on Schistosoma mansoni, that parasite genetic diversity is reduced after praziquantel treatment An alternate explanation of relatively low genetic diversity in three localities , as opposed to high diversity in KPv, may be due to the history of the parasite control program by chemotherapy. All of these areas are endemic for opisthorchiasis and there are records of praziquantel treatments O. viverrini sensu lato contains at least three species each genetically distinct with two morphologically similar (hence cryptic species) in river wetlands in Thailand and Lao PDR and one morphologically and biologically distinct isolate in Sakon Nakhon and Nakhon Phanom in Songkram River wetland, Thailand O. viverrini. In the case of other parasites, such as S. mansoni, a similar range of frequency of unique (private) alleles (1.1–4.1%) was observed Interestingly, even with the sample size of 30 adult worms per Khon Kaen locality analyzed in this study, a private or unique allele was observed in five of the 12 microsatellite loci we examined, albeit at low frequency (<6%). It is possible that with a larger sample size more unique alleles can be detected and could be used as markers to differentiate between populations of ISF values for all samples. This could be the result of possible extensive migration rates of the second intermediate host causing cross fertilization between distinct populations of O. viverrini.Only a single locus (Ovms10) showed significant negative STF suggest mostly low to moderate differentiation between populations. Gene flow between the closely spaced populations analysed in Khon Kaen Province and the populations of O. viverrini from other wetlands in Thailand and Lao PDR is unlikely via river flow and flooding patterns of snails and fish hosts as each wetland is distinct. Additionally, the Nam Ngum River, which contains a genetically very distinct O. viverrini cryptic species, enters the Mekong River 578 km upstream from where the Mun River meets the Mekong River. Whether, human and/or food (in this case fish) cross border movement may provide an avenue for parasite gene flow in Thailand and Lao PDR requires further investigations.Most pairs of populations presented in O. viverrini (10 to 60 km separation) within the Chi River wetland in Khon Kaen and more distant populations (up to 770 km separation) including Thailand and Lao PDR river wetland indicates the existence of intra-specific population structuring O. viverrini is not a panmictic population but rather is differentiated genetically into different gene pools, indicating the existence of intraspecific population structures that may be associated with different cryptic species within defined wetlands that make up the O. viverrini species complex.The observed levels of genetic differentiation between the four spatially close populations of STF within a single species for the Khon Kaen populations in this study was not different from that between cryptic species in the widely spaced populations STF estimated from the overall loci of the cryptic species populations (0.043) was higher than that for the closely spaced Khon Kaen populations (0.038). Substantial genetic differentiation (STF  = 0.165) was observed herein only between the cryptic species at a large geographical scale. This provides further independent evidence to support the hypothesis of the existence of cryptic species of O. viverrini within Thailand and Lao PDR S. japonicumAlthough the average pairwise O. viverrini infection, the life style and behavior, as well as genetic polymorphism in human genes and the frequency of praziquantel treatment may influence the transmission dynamics of O. viverrini and thus the incidence of CCA O. viverrini have been found between localities in close proximity and even within the Lawa Lake, Ban Bhai district O. viverrini from different reservoirs and streams, and hence different ecological and geographical environments, have different levels of gene/allele frequency and/or number of genotypes, as has been shown for schistosomes Several factors such as the intensity of O. viverriniO. viverrini using eggs from feces, cercariae from snails and metacercariae from fish should be compared to the adult worms to determine whether there is any affect of host selection or not as has been shown for schistosomes Although expulsion chemotherapy to collect adult worms from humans is possible for In conclusion we have shown that substantial genetic diversity and population genetic differentiation exists between four geographically close localities in the northeast of Thailand. Significantly higher allelic richness was found in worms from the KPv locality compared to worms from the other three localities. The overall genetic diversity and population structure observed at a relatively small spatial scale with a maximum population separation of 60 km was largely similar to that found at a much larger scale where the populations analyzed were separated by a distance of up to 770 km. The level of genetic differentiation was, however, significantly correlated with the distance between populations.