Bactrocera dorsalis (Diptera: Tephritidae) is mainly distributed in tropical and subtropical Asia and in the Pacific region. Despite its economic importance, very few studies have addressed the question of the wide genetic structure and potential source area of this species. This pilot study attempts to infer the native region of this pest and its colonization pathways in Asia. Combining mitochondrial and microsatellite markers, we evaluated the level of genetic diversity, genetic structure, and the gene flow among fly populations collected across Southeast Asia and China. A complex and significant genetic structure corresponding to the geographic pattern was found with both types of molecular markers. However, the genetic structure found was rather weak in both cases, and no pattern of isolation by distance was identified. Multiple long-distance dispersal events and miscellaneous host selection by this species may explain the results. These complex patterns may have been influenced by human-mediated transportation of the pest from one area to another and the complex topography of the study region. For both mitochondrial and microsatellite data, no signs of bottleneck or founder events could be identified. Nonetheless, maximal genetic diversity was observed in Myanmar, Vietnam and Guangdong (China) and asymmetric migration patterns were found. These results provide indirect evidence that the tropical regions of Southeast Asia and southern coast of China may be considered as the native range of the species and the population expansion is northward. Yunnan (China) is a contact zone that has been colonized from different sources. Regions along the southern coast of Vietnam and China probably served to colonize mainly the southern region of China. Southern coastal regions of China may also have colonized central parts of China and of central Yunnan. Determining the source area and understanding the colonization routes of invasive pests are key issues when developing management strategies Bactrocera dorsalis (Hendel), the oriental fruit fly, is one of the most notorious species in the family Tephritidae. This species belongs to the B. dorsalis species complex K-selected demographic strategy B. dorsalis would be adapted for growth and establishment in near optimal environmental conditions, taking advantage of the most productive niches B. dorsalis has been regarded as a typical invasive species causing high economic losses, and many research programs have been already carried out in the fields of ecology and biology B. dorsalis in these regions is complex, and its interpretation was not straightforward. Some populations at the western limit of the range  or in which the fly was recently introduced  were somewhat differentiated, while high gene flow was suggested among Southeast Asian countries, namely Laos, Thailand and Cambodia. Aketarawong et al. B. dorsalis, and that genetic structure is weak, even at this geographical scale. The authors The question of identifying the source of origin and understanding the colonization pathway for this species has received less attention. Recently, two studies focused on this issue. One paper was based on microsatellites only B. dorsalis over a large geographical scale in China and Southeast Asia. We also aimed to identify the native region of this pest species and its colonization pathways in Asia.The present study is based on an extensive geographical sampling of the oriental fruit fly, as it includes most regions studied by Akeratawong et al. Bactrocera oleae (Gmelin), Bactrocera cucurbitae (Coquillett), or Ceratitis capitata (Wiedemann) represent cases of successful invasive species, which have caused tremendous economic losses in many countries Bactrocera invadens, a recently described species of Tephritidae, has expanded throughout Africa within only two years after its first discovery and is currently causing extensive agricultural losses Phytophagous flies of the Tephritidae family, also called “true fruit flies”, are among the most important pests of fruits and vegetables. One-third of the species in this large family (more than 4000 known species) attack soft fruits, including many commercially and economically important species B. dorsalis typically occurs in tropical areas of South, East, and Southeast Asia  as well as in some Pacific islands  B. dorsalis was first reported in 1934 in Hainan province B. dorsalis infestations were reported in southwestern and southeastern parts of China B. dorsalis is now distributed mainly in provinces located south of the Yangtze River in mainland China No specific permits were required in our studies of this widespread agriculture pest. We confirm that the study locations were not privately owned or protected. This work did not involve endangered or protected species.B. dorsalis adults were collected from 21 localities in China and Southeast Asian countries. In China, we sampled 9 provinces, namely Yunnan, Guizhou, Guangxi, Fujian, Guangdong, Hainan, Jiangxi, Hubei and Hunan. From Southeast Asia we sampled in Myanmar, Vietnam, Laos, Thailand and Cambodia. Details about sampling sites and sampling sizes are given in A 601 bp fragment of the mitochondrial cytochrome oxidase gene (COI) was amplified and sequenced using the protocols described in Shi et al. Seven microsatellite loci were used in this study. Technical details are given in Dai et al. Numbers and distribution of haplotypes, numbers of unique haplotypes, polymorphic sites, within-population mean number of pairwise differences between pairs of haplotypes (π) and nucleotide diversity na); number of private alleles (np); frequency of private alleles (AP); observed heterozygosity (HO) and expected heterozygosity (HE). Gene diversity (HS) and allelic richness (RS) were calculated using FSTAT 2.9.3.2 For each population, the following genetic diversity estimates were calculated as averages over all microsatellite loci with GENEPOP 4.0 ST estimates for both types of markers. They were calculated using ARLEQUIN for mitochondrial sequences, and estimated using the ENA (Excluding Null Alleles) correction described in Chapuis & Estoup The degree of genetic differentiation between pairs of populations was measured by pairwise FST values) were compared with the matrix of geographic distances by means of a simple Mantel test To detect isolation by distance, matrices of pairwise estimates of genetic differentiation (FDs) calculated with microsatellite data Genealogical relationships between mitochondrial haplotypes were reconstructed using TCS 1.21 CT values should reach a maximum or a plateau. Second, the configurations with one or more single-population groups should be excluded because this indicates that the group structure is disappearing Spatial analysis of molecular variance (SAMOVA) was performed with both types of markers separately using SAMOVA 1.0 For microsatellite markers, the Bayesian approach implemented in STRUCTURE 2.3.3 AMOVAs were run on the mitochondrial data set as well as on the microsatellite data to test the effect of the host plants on the genetic structure of the fly. Flies were thus grouped depending on the host they were sampled from . Such analyses were performed using ARLEQUIN.D and Fu's SF for the 8 identified SAMOVA groups. We then tested whether these indices significantly deviated from 0. All demographic analyses were performed using ARLEQUIN.To study the demographic history of the groups of populations identified previously, we studied mismatch distributions The program BOTTLENECK 1.2.02 The coalescent-based strategy implemented in MIGRATE 3.2.17 The GENECLASS 2.0 software B. dorsalis from 21 populations. We merged these sequences with previously published sequences  from five populations in Yunnan province  and three Southeast Asian populations  B. dorsalis flies from 29 localities. The total alignment was 519 base pairs (bp) long. There were 85 polymorphic sites and 73 haplotypes were observed , number of haplotypes and number of unique haplotypes were calculated to measure genetic diversity within population see . Among tB. dorsalis flies sampled across 21 populations and genotyped at 7 microsatellite loci. Number of alleles per locus ranged from 12 to 27. After the sequential Bonferroni correction Our data were based on 420 ST estimates were used to measure the genetic structure of the 29 B. dorsalis populations using mitochondrial sequences, and 21 populations genotyped with microsatellites. ST values that ranged from 0.01 (YK/YY) to 0.4 (YL/YQ). Concerning microsatellite data, ST values after ENA correction, that ranged from 0.01 (TB/TC and FX/VP) to 0.22 (HC/YQ). Most values were significant.Pairwise FST were not significant , indicating the absence of IbD.The Mantel tests performed with both kinds of markers showed that the correlations between geographic distances and pairwise FAn unrooted NJ tree  was consThe NJ tree of populations constructed with the microsatellite data  also showed five monophyletic clades . Three pCT value was highest for K = 8 , as shown by the likelihood curve  and subjected to AMOVA. The results based either on mitochondrial or on microsatellite data showed that no significant differences were found among groups (see To test the effect of host plant species on the genetic structure of oups see . Most moD and Fu's Fs) were performed on mitochondrial data for the whole data set and for each of the 8 groups identified by SAMOVA. Significant negative values were found in the whole dataset  and three of the SAMOVA groups, namely Mt-C, Mt-G and Mt-F  was found for TC (P = 0.023) and GJ (P = 0.012). On the contrary, a significant heterozygote deficit  was detected in YY (P = 0.039), YK (P = 0.023), HA (P = 0.027), HC (P = 0.034) and JA (P = 0.019), suggesting a recent population expansion for these populations. However, under the more relaxed hypothesis of a certain proportion of mutations encompassing more than a single repeat unit (TPM), the results suggested that no populations had faced a bottleneck. The population expansion signal was confirmed in JA (P = 0.039) and HC (P = 0.043).We tested the hypothesis of a recent bottleneck or a recent population expansion for each of the 21 genotyped We also tested the hypothesis of a recent bottleneck or expansion for each of the 7 microsatellite SAMOVA groups. Under the SMM model, population groups Msat-A (P = 0.016) and Msat-E (P = 0.016) showed signs of recent significant bottlenecks, while groups Msat-C (P = 0.0195), Msat-F (P = 0.039) and Msat-G (P = 0.008) experienced a recent population expansion. When using the TPM model, no bottleneck was found for any of the 7 groups, but groups Msat-C (P = 0.039), Msat-E (P = 0.039), Msat-F (P = 0.039) still showed signs of recent expansion.GENECLASS 2.0 was used to estimate the proportion of immigrants (m) into each population see . The diaB. dorsalis in Asia to unravel the patterns of genetic differentiation of an important insect pest over a large area of its geographical distribution, including its potential invasive range. Our results go well beyond previous studies In this study, we obtained data using both mitochondrial and nuclear DNA markers of an extensive sampling of Mitochondrial and microsatellite data both revealed a weak but significant genetic structure that corresponds to a geographic pattern see , 3. The B. dorsalis was already discussed in previous studies Our data allowed for the identification of some patterns that did not appear previously, probably because of the limited sampling studied in previous papers. Some of the identified groups  correspond either to geographically distant sites, sometimes at the limit of the fly's range  and range expansion (see below). Even though we did not find any significant genetic structure due to host groups .(TIF)Click here for additional data file.Figure S2CT and LnP(D) for SAMOVA groups and STRUCTURE clusters.Values of F A: FCT values from K = 2 to 10 based on mitochondrial SAMOVA results. B: FCT values from K = 2 to 10 based on microsatellite SAMOVA results. C: Log-likelihood probability LnP(D) of the number of inferred clusters (K) as a function of K using STRUCTURE, for K = 2 to 13, with 10 independent runs for each K.(TIFF)Click here for additional data file.Table S1ST values based on mitochondrial sequences  and microsatellite loci .Pairwise F Some of the values were not significantly different from zero (ns)(XLS)Click here for additional data file.Table S2Effective immigration rate between populations pairs estimated from mitochondrial data using MIGRATE. Instances of asymmetrical gene flow are indicated in bold. The source population is indicated in columns, the target population in row.(XLS)Click here for additional data file.


G. p. palpalis, is known to be present in the Ivory Coast. G. p. palpalis has recently been reported to occur in several places within the town of Abidjan, including: (i) the Banco forest, (ii) the Abobo Adjamé University campus and (iii) the zoological park. Could these three places be treated sequentially, as separate tsetse populations, or should they be taken as one area comprising a single, panmictic population?Sleeping sickness, transmitted by The amount of gene flow between these places provides strategic information for vector control. It was estimated by the use of both microsatellite DNA and morphometric markers. The idea was to assess the interest of the faster and much less expensive morphometric approach in providing relevant information about population structure. Thus, to detect possible lack of insect exchange between these neighbouring areas of Abidjan, we used both genetic (microsatellite DNA) and phenetic (geometric morphometrics) markers on the same specimens.Using these same markers, we also compared these samples with specimens from a more distant area of south Ivory Coast, the region of Aniassué (186 km north from Abidjan).G. p. palpalis samples. Thus, the null hypothesis of a single panmictic population within the city of Abidjan could not be rejected, suggesting the control strategy should not consider them separately. The markers were also in agreement when comparing G. p. palpalis from Abidjan with those of Aniassué, showing significant divergence between the two sites.Neither genetic nor phenetic markers detected significant differentiation between the three Abidjan Both markers suggested that a successful control of tsetse in Abidjan would require the three Abidjan sites to be considered together, either by deploying control measures simultaneously in all three sites, or by a continuous progression of interventions following for instance the "rolling carpet" principle. To compare the geometry of wing venation of tsetse flies is a cheap and fast technique. Agreement with the microsatellite approach highlights its potential for rapid assessment of population structure. Tsetse flies (Diptera: Glossinidae) are the main vectors of trypanosomes (Kinetoplastida: Trypanosomatidae), which cause human and animal trypanosomiases in subsaharan Africa. These diseases have a considerable impact on public health and economic development, althougTsetse populations may be reduced using a variety of techniques, including insecticide impregnated traps and targets, live-baits, sequential aerial spraying, and sterile male release-13. HoweGlossina austeni Newstead on Unguja Island, Zanzibar[G. p. palpalis are known to be present in Ivory Coast[G. p. palpalis has been reported to occur within the city of Abidjan[This has fueled debate as to whether in some instances "eradication" (defined by FAO as the creation of a tsetse free zone) may be more cost-effective than "suppression" where tsetse densities are reduced to a level minimizing the risk of disease transmission. Decisions on eradication or suppression strategies will be facilitated when the population structure within the target region, in particular the degree of genetic isolation of the target population from adjacent populations is clearly understood. For isoZanzibar. But forZanzibar-17. Humary Coast,18 and G Abidjan,20. Due  Abidjan.G. p. palpalis populations in the three affected areas within Abidjan, we used both genetic (microsatellite DNA) and phenetic (geometric morphometrics) markers on the same specimens, and compared these populations to G. p. palpalis populations from another area of southern Ivory Coast in the region of Aniassué. The idea was to assess the interest of the faster and much less expensive morphometric approach in providing relevant information about population structure.To detect possible evidence of isolation between The expected outcome of this study was to help the national control program to decide which is the best strategy of vector control in the town of Abidjan: can these three localities be treated sequentially (i.e. are the tsetse populations isolated between the three sites), or should they be taken as one area comprising a single, panmictic population?For the total sample (n = 141) of genotyped tsetse, the seven microsatellite loci displayed 17 (Pgp1), 17 (PgP13), 14 (PgP24), 25 (B104), 19 (B110), 7 (C102), and 9 (GPCAG) alleles, respectively. The mean number of alleles was 9.71 (Banco), 11 (University) and 10.85 (Zoo) in Abidjan, and 10.00 in Aniassué. Mean observed heterozygosities were 0.68, 0.76 and 0.77 for Banco, University and Zoo, respectively, and 0.70 in Aniassué (no significant difference).Fis values were 0.12, 0.09 and 0.05 for Banco, University, and Zoo, significant at p<0.0001, p<0.001, and p<0.05 respectively. In Aniassué, Fis was 0.15, p<0.0001. The heterozygote deficit was mainly due to two loci (PgP1 and B110) for the three populations of Abidjan . Hence the null hypothesis of panmixia in Abidjan could not be rejected. In Aniassué, Fis on these 5 loci was 0.18 (p<0.0001), indicating consistant heterozygote deficiency. The heterozygote deficiency found in Aniassué confirmed earlier observations on G. p. palpalis in the forested areas of Ivory Coast, which attributed such deficiency to a combination of null alleles and genetic structuring at local scale due to Wahlund effects[This suggested locus had specific technical problems , because when these loci were removed from the analysis,  effects.Fst value for the 5 loci among the four populations was estimated at θ = 0.017 (CI95: 0.011 <θ< 0.023), p<0.0001. For the Abidjan samples it was θ = 0.007 (CI95: 0.00150 <θ< 0.01184) and was not significant, meaning that most of the differentiation was due to differences between Aniassué and Abidjan. Looking at paired Fst values between sites  from those of University and Zoo, whereas the latter two were genetically similar.The mean The specimens from Aniassué were significantly smaller compared to those from Abidjan, whereas within Abidjan there was no significant size difference between flies from the three sites Figure.The first two discriminant factors derived from the shape variables showed that the polygon representing the Aniassué population tended to separate from the Abidjan sites Figure. The recThe Mahalanobis distances between the Abidjan samples Table were notThe validated reclassification scores confirmed this pattern, since Aniassué had the highest score (86%). However, in spite of the lack of significant differentiation within Abidjan, the reclassification score obtained for Banco (77%) was much higher than for the University (37%) and Zoo (33%), suggesting a relatively higher level of shape divergence in the Banco forest.Correlation between metric and genetic distances was high. Regression of the Mahalanobis distances on the genetic distances indicated that 79% of the morphometric variation could be explained by the genetic variation Figure.From an epidemiological point of view, our study aimed at knowing whether tsetse populations from three sites in Abidjan could be considered to be isolated from each other. Such information is relevant for designing an adequate tsetse control strategy. For example, an insecticide application could be sequential in case of separation between sites, working on each site separately without risk of reinvasion to the next, or it should simultaneously cover all three sites if no evidence for separation is found.We used a population genetics approach to analyG. p. palpalis from sites within Abidjan, but both markers agreed in showing strong differentiation between individuals from Aniassué and those from Abidjan.Both the phenetic (geometric morphometrics) and genetic (microsatellite loci) markers showed no evidence for differentiation between At the scale of Abidjan, our data showed that males from the three sites showed no genetic differentiation, and accordingly had similar metric properties (size and shape).The microsatellite markers did not show any significant departure from the null hypothesis of panmixia, i.e. we did not observe any genetic differentiation between the 3 populations within Abidjan. There was however a slight, non significant trend for the population of Banco to diverge from the two others. A possible explanation is then a slow, on-going process for this population of Banco to have less genetic exchanges with the two others, due to urbanization which restricts tsetse movements. It may be possible, as observed in other studies in Burkina Faso, that the molecular markers used are not sensitive enough to detect it, since this is a recent, on-going phenomenon whereas what the molecular markers show is the result of a genetic history over several generations. This lack of sensitivity of molecular markers for recent genetic changes has already been observed in tsetse studies, and mayThis idea is reflected by the much higher shape-based reclassification score obtained for Banco (77%), compared to the two other sites (37% and 33%). This indirect evidence for some morphometric specificity in the forest might be due to an environmental effect ("forest" versus "city"), although in tsetse most of the pre-imago development is relatively protected from external influences as tsetse larvae grow in the uterus of their mother during the three first stages, buffering morphometric variations against external influences.G. p. palpalis in forested areas of Ivory Coast is governed by seasonal climatic effects[Temperature and humidity do become influential factors at the time when pupae are in the soil. The effect has been studied for size (not shape), indicating that higher temperatures tend to result in smaller individuals, whereas effects. In AbidBy contrast, both molecular (microsatellite loci) and morphometric (centroid size and shape variables) data showed significant differences between tsetse from Abidjan and Aniassué. This was expected due to the geographic distance between the two sites (186 km), to the differences of biotopes, and to the fact that the tsetse belt in South Ivory Coast is discontinuous as a consequence of anthropic pressure on habitats.The tsetse from Aniassué were smaller than those from Abidjan. This was in agreement with both the slightly higher temperature and dryeG. p. palpalis from Abidjan and Aniassué also involved shape, which may reflect genetic variations[G. p. gambiensis, a similar parallel was observed in natural populations of different biotopes from West Africa[The differences between riations,27, esperiations-30. Thist Africa, Guineat Africa, Burkinat Africa and Senet Africa. Here, 7t Africa) study).t Africa.G. p. palpalis in the forested areas of Ivory Coast, which attributed such deficits to a combination of null alleles and genetic structuring at local scale due to Wahlund effects[The heterozygote deficits found in Aniassué confirmed earlier observations on  effects.G. palpalis gambiensis) were isolated from the mainland and structured according to the island[How can the knowledge of population structure help to choose a control strategy? Since microsatellite and morphometric markers did not show significant differentiation between tsetse from the three sites in Abidjan, there would appear to be no significant barrier to gene flow at this scale. From a control perspective, this means that intervention against tsetse in any one site is likely to face reinvasion from the other two. This is different from a similar study conducted on the Loos archipelago, Guinea, which showed that tsetse populations  and old cocoa plantations.In Abidjan, the three study sites were the Banco forest (Banco), the University of Abobo Adjamé (University) and the zoological park (Zoo). The Banco forest is in the north-western part of the city of Abidjan, at 5°N latitude and 4°W longitude. East of Banco are two small relicts of the forest which have now been substantially degraded by urbanisation: the Abobo Adjamé University and the zoo of Abidjan. These three sites, although geographically close (less than 500 meters between sites), are separated by roads and urbanisation Figure. For comThere is little temperature difference between Abidjan (from 24.2°C to 27.7°C) and Aniassué (from 24.3°C to 27.9°C), but relatively more variation in relative humidity (RH), which decreases from south (RH on average 90%) to north (RH between 60% and 70%). In both areas, there are two rainy and two dry seasons during a year.Tsetse flies were caught using Vavoua traps in AprilWright's F-statistics, the parFis is a measure of the inbreeding of individuals (hence I) resulting from non-random union of gametes within each sub-population (hence S).Fst quantifies the differentiation between subpopulations in the total population (hence S and T) as a measure of the relatedness between individuals resulting from non-random distribution of individuals between sub-populations, relative to the total population.Fit is a measure of the inbreeding of individuals resulting both from non-random union of gametes within sub-populations, and from population structuring .f  (for Fis), θ (for Fst) and F (for Fit)[Fis was tested by randomizing alleles between individuals within sub-samples. The significance of Fst was tested by randomizing individuals among sub-samples.These F-statistics were estimated by Weir and Cockerham's unbiased estimators for Fit). The sigG. p. palpalis males (M) were used, i.e. 9 from Banco, 16 from University, 15 from Zoo and 15 from Aniassué.The tsetse specimens used for geometric morphometrics constituted a subsample of those on which the molecular analyses were done. Out of the 141 flies used for microsatellites, 55 had non-damaged wings allowing morphometric analyses. The analyses were conducted only on males, and focused on the right wing, which was generally the wing in best conditions. A total of 55 right wings of Wings were dry-mounted between two microscope slides and scanned at 1800 ppp at dimensions of 0.90 x 0.50 cm, using a multifunction scanner HP Deskjet F 2180. From this picture, the coordinates of 10 landmarks (LM) defined by vein intersections were recorded for each wing, by the same person in the same order ,45, prodThe size variable was the isometric estimator known as centroid size (CS) derived from coordinate data and defined as the square root of the sum of the squared distances between the center of the configuration of landmarks, and each individual landmark. StatistThe 16 shape variables were the "partial warps" (PW). To circumvent the problem of small sample sizes relative to the large number of shape variables (16 PW), we used the first 6 principal components of the PW  as input for discriminant analyses, as these represented 84% of the total variation and had the highest discriminatory power.Mahalanobis distances computedMahalanobis distances based re-classification scores were computed according to a validation procedure whereby each individual was assigned to its closest group without using that individual to help determine a group centre, althoughttp://www.mpl.ird.fr/morphometrics/clic/index.html. PHYLIP software with "neighbor" module[Fst with Fstat[Collections of anatomical landmarks of the wings, general Procrustes analysis (GPA), multivariate and discriminant analyses, were performed using the CLIC package, freely " module and NJPL" module were use" module and Fsta" module). The ovth Fstat.The authors declare that they have no competing interests.Genetic techniques: SR, KA, LG, EKN'G, PS. Morphometric techniques: DK, HB-V, J-PD. Data analyses: DK, SR, PS, J-PD. Field collections: DK, MK, GA-Y. Text: DK, PS, J-PD, CJS. All authors read and approuved the final version of the manuscript.


Abies chensiensis and Abies fargesii, in China, at sites where they are parapatric relative to where they are allopatric. We examined genetic divergence across 126 amplified fragment length polymorphism (AFLP) markers in a set of 172 individuals sampled from both allopatric and parapatric populations of the two species. Our analyses demonstrated that AFLP divergence was much greater between the species when comparisons were made between parapatric populations than between allopatric populations. We suggest that selection in parapatry may have largely contributed to this increased divergence.Because of introgressive hybridization, closely related species can be more similar to each other in areas of range overlap (parapatry or sympatry) than in areas where they are geographically isolated from each other . Here, we report the reverse situation based on nuclear genetic divergence between two fir species,  Introgression commonly occurs between closely related species in areas where their distributions overlap Abies cheions Liu . Neverthions Liu .Based on the distribution of cpDNA and mtDNA diversity within and between both species, it has been suggested that parapatric populations likely represent secondary contact zones between the species, which formed after the penultimate glacial period , with an internal size standard. All loci between 100 and 500 bp were then visually inspected in all individuals. Only unambiguously scorable loci and individuals were included in the analysis, and peaks found in <3% of individuals were excluded. In total, 172 individuals were scored for 126 AFLP markers, with all scoring being performed blind to population of origin. To ensure high repeatability of analyzed AFLP loci, we ran a subset of 30 individuals twice from the preselective amplification step. The average per-locus genotyping error rate for the AFLP data, measured as recommended by Bonin et al.  procedure  were determined. We also estimated total diversity (HT), within-population diversity (HS), between-population diversity (HB), and population differentiation (FST) for allopatric and parapatric populations of each species, taken separately and in combination, using the method of Lynch and Milligan (f) cannot be calculated directly due to the dominant nature of AFLPs, we employed an alternative Bayesian approach developed by Holsinger et al.  for dominant markers, which accounts for uncertainty in f and simultaneously provides accurate and reliable estimates of genetic differentiation ; (2) f = 0 model (θB is unknown and no inbreeding occurs); (3) f-free model (f was not estimated but was chosen at random from the prior distribution). The posterior distribution of θB was estimated through Markov Chain Monte Carlo (MCMC) methods by HICKORY v1.1, with a burn-in of 5000 iterations and a sampling run of 25,000 iterations from which every fifth sample was retained for posterior calculations. The deviance information criterion (DIC) was subsequently used to determine the model showing the best fit to the data  and population type  using AFLP-SURV 1.0 and HICKORY 1.1. The significance of FST values was calculated based on comparison with values obtained from 10,000 randomly permuted individuals among the populations and/or between groups. We then constructed a neighbor-joining (NJ) tree with the program NEIGHBOR incorporated in the software package PHYLIP version 3.6  on a genetic distance matrix generated from the binary presence–absence matrix as implemented in GENALEX 6.2   as implemented for dominant markers in GENALEX 6.2 . Pairwise genetic distance between populations was estimated using the Slatkin's linearized FST values (FST/1−FST) in ARLEQUIN 3.5  and locus-specific effects (αi) on genetic differentiation and has been shown to be robust to complex demographic models.We checked whether genetic structure between parapatric and allopatric populations of  Rousset . Then, aHe) per population ranged between 0.199 and 0.284 for A. chensiensis, and between 0.169 and 0.303 for A. fargesii (Table HS) was much higher than average between-population diversity (HB) for both allopatric and parapatric populations whether considered separately or together , and thus, we only present the θB values from the full model. Population differentiation (FST and θB) between parapatric or allopatric populations treated separately in both species was much lower than values of FST and θB obtained when all populations within species were considered together  than when comparisons were made only between parapatric populations  , than were allopatric populations of A. chensiensis to parapatric populations of A. fargesii . There was greater divergence between allopatric and parapatric populations in A. fargesii  than within A. chensiensis   were polymorphic. Values of average gene diversity  was very low in all comparisons . The lack of evidence for IBD in both allopatric and parapatric populations of each species . Thus, rather than being restricted solely to outlier loci, it appears that interspecific divergence in parapatry is characterized by widespread genetic differentiation across the genome.Despite the strong population structure observed in both Abies, A. chensiensis and A. fargesii might be reduced due to introgression, we found that divergence based on amplified fragment length polymorphism was much greater between parapatric populations than between allopatric populations. This difference could result from demographic processes associated with range expansions of the two species (Freedman et al. In contrast to an initial expectation that genetic divergence between parapatric populations of two species of A. chensiensis and A. fargesii in parapatry may reflect the effects of divergent selection (Barton and Bengtsson A. chensiensis occurs mainly at low altitudes while A. fargesii is found above 2200 m (Guan A. chensiensis and A. fargesii, especially in areas of parapatry, indicating that hybridization occurs between these two species. If hybrids were formed frequently in the past and had low fitness relative to parental species, selection for reinforcement might have occurred at parapatric sites causing barriers to gene flow to strengthen (Hopkins A. chensiensis and A. fargesii.Based on our results, it seems more likely that increased genetic divergence between 0 m Guan . However0 m Guan . We have Hopkins . Third,  Hopkins . AlthougAbies species in parapatry and allopatry are now required to establish the exact level and pattern of genomic divergence, which exists between them.The very low level of AFLP divergence recorded between species based on comparisons between allopatric populations was somewhat surprising given their morphological divergence in allopatry Liu . This su


Pinus nigra Arn. populations from Southern Spain and Northern Morocco were examined using inter-simple sequence repeat markers to characterize the genetic variability amongst populations. Pair-wise population genetic distance ranged from 0.031 to 0.283, with a mean of 0.150 between populations. The highest inter-population average distance was between PaCU from Cuenca and YeCA from Cazorla, while the lowest distance was between TaMO from Morocco and MA Sierra Mágina populations. Analysis of molecular variance (AMOVA) and Nei’s genetic diversity analyses revealed higher genetic variation within the same population than among different populations. Genetic differentiation (Gst) was 0.233. Cuenca showed the highest Nei’s genetic diversity followed by the Moroccan region, Sierra Mágina, and Cazorla region. However, clustering of populations was not in accordance with their geographical locations. Principal component analysis showed the presence of two major groups—Group 1 contained all populations from Cuenca while Group 2 contained populations from Cazorla, Sierra Mágina and Morocco—while Bayesian analysis revealed the presence of three clusters. The low genetic diversity observed in PaCU and YeCA is probably a consequence of inappropriate management since no estimation of genetic variability was performed before the silvicultural treatments. Data indicates that the inter-simple sequence repeat (ISSR) method is sufficiently informative and powerful to assess genetic variability among populations of P. nigra.Eight  Pinus nigra Arn.) is a tertiary relictual species belonging to the group of Mediterranean pines [P. nigra is one of the oldest European pine species, descending from a group that already existed in the Cretaceous (100 million years ago) [P. nigra is a widespread species, with a discontinuous range that extends from North Africa through the northern Mediterranean and eastwards to the Black Sea. It is also found on the islands of Corsica and Sicily. The fragmented distribution has led to morphological variations, which are difficult to interpret and have resulted in several diverse classifications. Relatively little is known about its long history, although in the Tertiary it was more widespread than today and it seems to have shifted over time from coastal areas to its current mountain locations, since the dry cold climate of these areas resembles that of bygone glacial periods [European black pine (an pines . Their fan pines –4. P. niars ago) . P. nigr periods . Migrato periods .P. nigra is divided into five subspecies: P. nigra ssp. nigra from the Alps; P. nigra ssp. laricio from Corsica and Sicily; P. nigra ssp. pallasiana from Turkey and Crimea; and both P. nigra ssp. salzmannii and P. nigra ssp. mauretanica from North Africa. In Spain, the predominant subspecies is ssp. salzmannii, with two varieties that are also identified within the subspecies P. nigra ssp. Salzmannii: the hispanica variety located in the Iberian Central System and Betic Cordilleras and the pyrenaica variety, which extends throughout Castellón, Aragón and Catalonia [The species atalonia .in situ conservation, exploitation of genetic resources and forest management [Trees are more vulnerable than annual plants to rapid climate changes since they are not able to respond by migration or genetic selection within a short period of time. High genetic variability of forest species is therefore important for the processes of adaptation to biotic and abiotic stresses in order to ensure the viability of the species. Since the pressure on natural resources has brought about significant losses in diversity, especially in tree species, it is essential to gain a comprehensive idea of genetic variability amongst populations in order to provide a basis for nagement –11. In rnagement –16. FurtP. nigra using DNA markers [P. nigra from seven populations in Southern Spain and one population in Northern Morocco, in order to develop the genetic background for their conservation and restoration.Numerous studies have researched the genetic variation in natural populations of  markers ,7,17. ThPinus nigra samples were selected for preliminary experiments to determine optimal amplification reaction conditions and primer screening for ISSR. 12 primers did not result in any amplification, 15 gave unclear profiles in some samples and only eight out of the 35 primers tested resulted in well separated bands. On the basis of this data, the 160 samples included in the study were analyzed with eight primers (Two  primers . Most lo primers .P. nigra in this study (HE = 0.175) was lower than other Pinus species similarly researched with ISSR markers, such as Pinus tabuleaformis , Pinus , Pinus , Pinus , and hi . The diThe populations from the Cuenca region showed the highest Nei’s genetic diversity except the population PaCU. TrCU and ArCU are natural populations and have been neither exploited nor managed.i.e., where and when to apply silvicultural treatments, were defined. According to the shelterwood method context, some seedling trees (from 1 to 4 per ha) were left standing. Thus, for each regeneration period, the new trees were seeded naturally with seeds from the seedling trees. This silvicultural system has continued in Cuenca and Cazorla for forest stand regeneration, leading to a decrease in genetic diversity in these populations since before the silvicultural treatments no estimation of genetic variation of PaCU and YeCA has been projected to provide an appropriate strategy for sampling and propagation.The low diversity of PaCU and YeCA can be explained by the forest management practices applied to this population. Since the end of the 19th century, the Spanish black pine forest stand at PaCU and YeCA has been managed under the shelterwood system with a 100–120-year rotation and 20–30-year regeneration period. This Spanish black pine stand was divided into compartments up to 111 hectares in surface, delineated by roads, streams, rocky outcrops and other spatial features. Individual compartments or a number of aggregated ones were established as management units, and for each management unit tactical planning considerations, P. nigra from Cuenca’s region (TrCU and ArCU). Tiscar-oliver et al. [P. nigra populations. The authors concluded that the number of large trees and the diameter classes decrease in both populations over time; however the uneven-aged silvicultural method showed higher values of both variables. No management data were recorded for TaMO, MA and PaCA.In 1893, NaCA was first managed under the shelterwood method with a shelter-phase of 20 years and a rotation of 120 years. In 1920 an uneven-aged system was applied and in 1944, an ideal reverse-J diameter distribution was achieved to resolve the unsuccessful natural regeneration. This silvicultural treatment may explain the lower Nei’s genetic diversity in comparison with the two natural populations of r et al.  studied Pair-wise Nei’s distances  were calThe low distance between TaMO and MA is logical since Southern Spain and Northern Morocco have the same geological origin. Several authors have highlighted the floristic continuity of both sides of the Strait of Gibraltar and have thus proposed the creation of a Tingitanian-Aljibean floristic sector . ExchangThe average distance between populations from the Cuenca region (0.064) was lower than the populations from Cazorla (0.120) suggesting different patterns of genetic exchange and genetic divergence between the two regions. Based on these distances, principal component analysis (PCA) analysis was computed showing the presence of two major groups . Group 1The genetic differences between the Cazorla and Cuenca regions may be due to several factors such as natural barriers, agricultural and forest harvesting, local adaptation and management methods .P. nigra inferred using the method of Pritchard et al. [The population structure of d et al. , was carP. nigra population, while 52% is attributed to the differences among individuals within populations and 39% among regions. The contributions from each of the three sources were significantly greater than 0, indicating statistically significant genetic differentiations among and within populations (P < 0.01 in the AMOVA tests). A relatively high level of differentiation among populations in the studied regions was found. Our data showed that 9% of the total genetic variation was attributed among populations which were much higher than the one reported by Naydenov et al. [P. nigra from Bulgaria. The most genetic variation in P. nigra is within populations rather than between them, indicating relatively restricted population differentiation. Such a pattern of population genetic structure has been found in all P. nigra populations in different regions [Based on the AMOVA analysis, only 9% of the total variation resides among the v et al.  analyzin regions ,7,17. Th regions  who sugg regions ,34.r = 0.3) and a medium negative correlation with a mean temperature (r = −0.4) were found. Geographic distances between population-pairs were not correlated with the corresponding genetic distance  with the population from Sierra Mágina (MA). The climate changes during the late Pliocene and the Pleistocene in the Mediterranean region are highly complex and palaeoenvironmental data have demonstrated century-to-millennial climate variability, with rapid changes [i.e., 50%) occurring within the hotspots. Hotspot 2, which encompasses the Baetic–Rifan complex, supports the relatedness of the Morocco population with those of southern Spain.Both a small positive correlation of genetic diversity with a mean annual precipitation , 2-Talassemtane (Morocco), 3-Sierra Mágina (Spain) and 4-Cazorla (Spain) (One hundred sixty samples of  (Spain) . All theDNA was extracted from 150 to 300 mg of needle/bud material using a modified method . Leaf ma2, 200 μM each of dATP, dCTP, dGTP, and dTTP, 0.4 μM primer, and 1 unit of Taq DNA polymerase using a thermal cycler . The cycling program began with an initial 2 min at 94 °C followed by 40 cycles at 94 °C for 45 s, 52–62 °C for 45 s and 72 °C for 2 min plus a final 10 min at 72 °C and storage at 4 °C. Water was the negative control while the positive control showed an amplicon of expected size = 320 bp. The sequences of primers are shown in 15 and 30 ng of genomic DNA were amplified in a volume of 25 μL containing 10 mM Tris-HCl, pH 9.0, 1.5 mM MgClPPL), number of alleles per locus (na), effective number of alleles per locus (ne), genetic diversity (HE = expected heterozygocity), Shannon’s index diversity (I) and genetic differentiation among populations (GST).We analyzed ISSR data based on both allele and phonotypic frequencies. Polymorphic bands were selected at the 95% level (two-tailed test) for use in further analyses. Data matrices were analyzed using POPGENE version 1.32  with ther) was calculated and assessed by permuting the arrangement of distance matrices. The P value associated to the observed correlation is the proportion of such permutations that lead to a higher correlation coefficient than the one observed. The Pearson correlation coefficients between pairs of genetic diversity and climate factor (temperature and precipitation) for these populations were analyzed with Excel . We tested the null hypothesis that genetic distance and geographical distance matrices are not correlated against the alternative hypothesis that the two matrices are correlated using GenAlEx 6.41. Genetic relationships among populations were studied via principal component analysis (PCA) using GenAlEx 6.41 [We examined the hierarchical genetic variation across the geographic range of the populations studied using an analysis of molecular variance (AMOVA) determined with GenAlEx 6.41 . To testlEx 6.41 .K ranging from 2 to 10. The most likely value for K was calculated with Structure Harvester [ΔK, which represents the greatest rate of change between each subsequent K value [Bayesian assignment tests were applied to estimate the number of genetic clusters and to evaluate the degree of admixture among them using Structure v2.3.3 . Structuarvester  using th K value .Pinus nigra in the studied regions is essential for conservation of genetic resources. If genetic diversity is to be preserved an estimation of genetic variability of populations should be achieved before initiating a planting strategy. The low genetic diversity observed in PaCU and YeCA is probably a direct consequence of inappropriate management since no estimation of genetic variability was carried out before the silvicultural treatments. We strongly recommended testing the genetic variability of populations before any management. Our results indicate that ISSR are sufficiently informative for assessing genetic variability. Because of the dominant nature of the ISSR markers, the application of codominant molecular markers should be undertaken to complement the data reported here.Genetic differentiation among populations of


Afghanistan has held a strategic position throughout history. It has been inhabited since the Paleolithic and later became a crossroad for expanding civilizations and empires. Afghanistan's location, history, and diverse ethnic groups present a unique opportunity to explore how nations and ethnic groups emerged, and how major cultural evolutions and technological developments in human history have influenced modern population structures. In this study we have analyzed, for the first time, the four major ethnic groups in present-day Afghanistan: Hazara, Pashtun, Tajik, and Uzbek, using 52 binary markers and 19 short tandem repeats on the non-recombinant segment of the Y-chromosome. A total of 204 Afghan samples were investigated along with more than 8,500 samples from surrounding populations important to Afghanistan's history through migrations and conquests, including Iranians, Greeks, Indians, Middle Easterners, East Europeans, and East Asians. Our results suggest that all current Afghans largely share a heritage derived from a common unstructured ancestral population that could have emerged during the Neolithic revolution and the formation of the first farming communities. Our results also indicate that inter-Afghan differentiation started during the Bronze Age, probably driven by the formation of the first civilizations in the region. Later migrations and invasions into the region have been assimilated differentially among the ethnic groups, increasing inter-population genetic differences, and giving the Afghans a unique genetic diversity in Central Asia. Afghanistan is a landlocked country at the intersection of Central Asia, South Asia, and the Middle East that has held a strategic position throughout history. It was a crossroad of ancient trade routes and human migrations. The main east-west trade routes passed through its northern and southern plains, and through its mountain passes before the ascendancy of waterborne trade between Europe and the Far East.Paleolithic humans probably inhabited the caves of Afghanistan as long as 50,000 years ago (ya). In northern Afghanistan, flake tools found in Dara Dadil, Darra Chakhmakh, and elsewhere indicate the probable existence of Middle Paleolithic industries The second and first millennia BCE were also marked by the influx of Iranian tribes, later ruling Afghanistan as part of the Achaemenid Empire established by Cyrus the Great (550 BCE) The present population of Afghanistan contains many diverse elements, the result of large-scale migrations and conquests that influenced its culture and demography. Pashtuns are the largest ethnic group in Afghanistan, accounting for about 42 percent of the population, with Tajiks (27%), Hazaras (9%), Uzbeks (9%), Aimaqs (4%), Turkmen people (3%), Baluch (2%), and other groups (4%) making up the remainder While previous theories about the origin of the Afghans are usually based on oral traditions or scanty historical information , few stuAll participants recruited and genotyped in the present study had at least three generations of paternal ancestry in their country of birth, and provided details of their geographical origin and written consent for this study, which was approved by the IRB of the Lebanese American University.th and 14th century. In addition, we have also included populations from the Pontic-Caspian steppe region, from West Russia and East Europe, which were possibly involved in the Indo-European migrations that reached the Iranian plateau and Northern India.The modern populations selected for this study were those from regions with ancient historical importance to Afghanistan through conquest or migration, including Iranians, Greeks and Indians, in addition to populations with more recent impacts, such as the Arab expansion in the 7th century and the East Asian invasions in the 13A total of 8,706 samples were used in the analyses including 204 newly genotyped samples from Afghanistan. The genotyping results and the subjects' paternal province and their city or village of origin when available are listed in DNA was extracted from blood or buccal swabs using a standard phenol-chloroform protocol. Samples were genotyped using the Applied Biosystems 7900HT Fast Real-Time PCR System with a set of 52, highly informative, custom Y-chromosomal binary marker assays  from the non-recombining portion of the Y chromosome which define 32 different haplogroups. A total of 19 Y-chromosome STR loci were analyzed for each sample in two multiplexes on an Applied Biosystems 3130xl Genetic Analyzer. The first multiplex contained the standard 17 loci of the Y-filer™ PCR Amplification kit . The remaining two loci, DYS388 and DYS426, were genotyped in a custom multiplex. STR alleles were named according to previous recommendations Fisher's exact tests were performed on haplogroups vs populations to identify which haplogroups were significantly over- or under- represented in Afghanistan's ethnic groups. A principal component analysis (PCA) ST distances between populations computed by ARLEQUIN Non-metric multidimensional scaling (MDS) Monmonier's maximum difference algorithm Significance of population structures created by Barrier was tested using AMOVA Second, populations were grouped according to the identified barriers; 1- Pashtun, Tajik, North India, West India. 2- Hazara, Uzbek 3- Caucasus: Avar, Darginian, Lezgi. 4- Caucasus: Circassian, Abkhazian, 5- Iran: East Azerbaijan, Markazi, Mazandaran, Qazvin, Sistan and Baluchistan. 6- Belarus, West Russia. 7- Middle East and Europe: Greece, Turkey, Lebanon, Syria.Reduced Median (RM) Networks We applied BATWING The mutation rate priors applied to these calculations were those proposed in Xue et al. The data were partitioned into multiple runs . The indThe Indian populations structures resulted in slower equilibration than was seen among the other populations. After equilibration, the Indian populations showed older splits among them than is shown between India as a whole and the other populations when India is pooled. This older split may have resulted partly from differences in weights among candidate trees that the Metropolis-Hastings algorithm samples based on the likelihood ratios derived from the population configurations that will lead to different modal trees with different split times. Alternatively, the older split may have also resulted from violations of the assumption of isolation after population splitting. These complications led to the separate treatments of India BATWING runs from the western populations runs.Genotyping revealed 32 halpogroups present in Afghanistan's ethnic groups among our samples. Haplogroups R1a1a-M17, C3-M217, J2-M172, and L-M20 were the most frequent when Afghan ethnic groups were pooled, together comprising >66% of the chromosomes. Absolute and relative haplogroup frequencies are tabulated in −9) in Uzbeks (41.18%) and Hazaras (33.33%) than it was in Tajiks (3.57%) and Pashtuns (2.04%). On the other hand, R1a1a-M17 was significantly more frequent (p = 3.00×10−6) in Pashtuns (51.02%) and Tajiks (30.36%) than in Uzbeks (17.65%) and Hazaras (6.67%). RM networks of C3-M217 , has the highest observed diversity among populations of the Indus Valley MDS and Barrier analysis have identified a significant affinity between Pashtun, Tajik, North Indian, and West Indian populations, creating an Afghan-Indian population structure that excludes the Hazaras, Uzbeks, and the South Indian Dravidian speakers. In addition, gene flow to Afghanistan from India marked by Indian lineages, L-M20, H-M69, and R2a-M124, also seems to mostly involve Pashtuns and Tajiks. This genetic affinity and gene flow suggests interactions that could have existed since at least the establishment of the region's first civilizations at the Indus Valley and the Bactria-Margiana Archaeological Complex.Furthermore, BATWING results indicate that the Afghan populations split from Iranians, Indians and East Europeans at about 10.6 kya , which marks the start of the Neolithic revolution and the establishment of the farming communities. In addition, Pashtun split first from the rest of the Afghans around 4.7 kya , which is a date marked by the rise of the Bronze Age civilizations of the region. These dates suggest that the differentiation of the social systems in Afghanistan could have been driven by the emergence of the first urban civilizations. However, the dates suggested by BATWING should be treated with care, since BATWING does not model gene flow and differential assimilation of incoming migrations. These events could alter the time of split. However, it was previously shown that topologies and times of splits in the modal trees generated by BATWING are insensitive to in-migration BATWING's hypotheses model mutations and coalescent events, reflecting ancestral structures from which the current populations have emerged. Later expansions into the region would have assimilated the ancestral population, granting the Afghans distinctive genetics from the expanding source populations even though they shared general genetic features. This is evident in the Afghan Hazara and Afghan Uzbek who have always been associated with expanding Mongols and Turco-Mongols. Although we have found that at least third to half of their chromosomes are of East Asian origin, PCA places them between East Asia and Caucasus/Middle East/Europe clusters.Historical expansions and invasions appear to have had differential contribution in shaping Afghanistan population structures. We have found limited genetic evidence of expansions previously thought to have left specific imprints in current populations.The E1b1b1-M35 lineages in some Pakistani Pashtun were previously traced to a Greek origin brought by Alexander's invasions th century CE left an immense cultural impact on the region, with reports of Arabs settling in Afghanistan and mixing with the local population The Islamic invasion in the 7We also note that three Hazara subjects belonged to haplogroup B-M60, which is very rare outside Africa. RM network shows that the subjects had a recent founding ancestor from East Africa, which could have been brought to Afghanistan through slave trade. This shows that the genetic ethnic boundaries have been selectively permeable, however the history of the rules of assimilation in this region over time are not yet clearly understood.th century CE Language adoption and spread in Afghanistan also seem to have been a complex process. The Afghan genetic structure tends to correlate Hazara and Uzbek which belong to two different language families. Hazara, like Pashtun and Tajik, belong to the Indo-Iranian group of the Indo-European family, while the Uzbek language is in the Turkic family. The form of Turkic spoken by the Uzbek appears to be a direct descendent of an extinct Turkic language that was developed in the 15In conclusion, Y-chromosome diversity in Afghanistan reveals major differences among its ethnic groups. However, we have found that all Afghans largely share a heritage of a common ancestral population that emerged during the Neolithic revolution and remained unstructured until 4.7 kya . The first genetic structures between the different social systems started during the Bronze Age accompanied, or driven, by the formation of the first civilizations in the region. Later migrations and invasions to the region have been differentially assimilated by the ethnic groups, increasing inter-population genetic differences, and giving the Afghan a unique genetic diversity in Central Asia.Figure S1Reduced median networks. (A) C-M130, (B) R1a1a-M17, (C) E1b1b1-M35, and (D) B-M60 showing STR haplotype distributions among populations; area is proportional to haplotype frequency, and color indicates populations. Connecting lines represent putative phylogenetic relationships between haplotypes.(TIF)Click here for additional data file.Table S1Suggested origins of the main ethnic groups in Afghanistan.(DOC)Click here for additional data file.Table S2Y-chromosome haplogroups and haplotypes in 204 unrelated individuals from Afghanistan.(XLS)Click here for additional data file.Table S3Populations selected for this study.(XLS)Click here for additional data file.Table S4Y-chromosome haplogroups frequencies in Afghanistan's ethnic groups.(XLS)Click here for additional data file.Table S5AMOVA results. Comparing populations grouped according to their country or region of origin with populations grouped according to Barrier structures.(DOC)Click here for additional data file.Table S6BATWING topologies and dates with 95% confidence intervals of population splits derived from multiple combinations of population subsets.(XLS)Click here for additional data file.


Fraxinus angustifolia Vahl, within a recognised glacial refugium in Croatia. We sampled 11 populations from environmentally divergent habitats within the Continental and Mediterranean biogeographical regions. We combined genetic data analyses based on nuclear microsatellite loci, multivariate statistics on environmental data and ecological niche modelling (ENM). We identified a geographic structure with a high genetic diversity and low differentiation in the Continental region, which contrasted with the significantly lower genetic diversity and higher population divergence in the Mediterranean region. The positive and significant correlation between environmental and genetic distances after controlling for geographic distance suggests an important influence of ecological divergence of the sites in shaping genetic variation. The ENM provided support for niche differentiation between the populations from the Continental and Mediterranean regions, suggesting that contemporary populations may represent two divergent ecotypes. Ecotype differentiation was also supported by multivariate environmental and genetic distance analyses. Our results suggest that despite extensive gene flow in continental areas, long-term stability of heterogeneous environments have likely promoted genetic divergence of ashes in this region and can explain the present-day genetic variation patterns of these ancient populations.Tree species with wide distributions often exhibit different levels of genetic structuring correlated to their environment. However, understanding how environmental heterogeneity influences genetic variation is difficult because the effects of gene flow, drift and selection are confounded. We investigated the genetic variation and its ecological correlates in a wind-pollinated Mediterranean tree species,  Understanding how environmental heterogeneity influences the distribution of genetic variation among natural populations along different spatial scales remains a central question in evolutionary biology and population genetics Yet, greater genetic divergence than expected among populations inhabiting different environments has been used to suggest that contrasting ecological conditions may have a strong influence on the genetic differentiation of local populations Ecological niche modelling (ENM) allows the generation of biogeographical hypotheses and, when coupled with genetic data, provides new insights into the evolutionary history of animal and plant species Fraxinus angustifolia Vahl, Oleaceae), a wind-pollinated tree species with a predominantly Mediterranean distribution, but found frequently in habitats of both Continental and Mediterranean biogeographical regions of Europe. It occurs naturally throughout Southern and Eastern Europe, from Portugal in the west to the Black Sea in the east. Although the phylogeography and genetic structure of the closely related species, the common ash (Fraxinus excelsior L.), and the hybridisation process between the two species have been well studied F. angustifolia populations. Unlike F. excelsior, which is widely distributed in mixed deciduous forests all over Europe, F. angustifolia is a habitat specialist associated with surface and ground waters, and thus, its dispersal ability is more restricted. In Central Europe, the Pannonian Basin and the Balkans, it occurs mainly in lowlands, riparian and floodplain forests along large rivers and their tributaries , where it forms large and continuous populations. The distribution of this species in the Mediterranean region is patchy and reduced to smaller and more isolated populations on drier sites at higher altitudes or on wetland sites For this study, we selected the narrow-leaved ash , ssp. oxycarpa (M. Bieb. ex Willd.) Franco and Rocha Afonso , and ssp. syriaca (Boiss.) Yalt. (in Turkey and eastwards to Iran). Fukarek F. angustifolia in Croatia and the surrounding area of the Western Balkans. Continental populations along the rivers and floodplains of the Danube River Basin were named Fraxinus angustifolia ssp. pannonica (Fuk.) Soó and Simon. This putative new taxon is morphologically closer to ssp. oxycarpa, whereas Mediterranean populations along rivers and wetlands of the Adriatic River Basin probably belong to the typical ssp. angustifolia. Such a division differs from the more accepted general geographical classification, suggesting a possible increased within-region differentiation in the Western Balkan area.There are several different points of view regarding the taxonomic status of this species, but in general, the prevailing opinion is that Although located at a relatively high latitude and with a relatively small size, Croatia has been highlighted as having one of the most genetically divergent forests among 25 European forests investigated based on chloroplast genetic diversity F. angustifolia populations. We focused on answering these specific questions: 1) What are the levels of genetic diversity and divergence within and among natural populations of Fraxinus angustifolia distributed across the Croatian Continental and Mediterranean areas? 2) Does the neutral spatial genetic variation correlate with the environmental variation? and 3) Are areas predicted as suitable by ENM concordant with patterns of population genetic divergence?In this study, we combined population genetic analyses of neutral loci, landscape genetic analysis using multivariate environmental data and ENM to examine genetic variation and its ecological correlates in Collections of samples from protected areas were permitted by the authority of The Krka National Park and Lonjsko Polje Nature Park. For other locations no specific permits were required for the described field studies because sample collection did not involve endangered or protected plant species or privately-owned locations.2 land surface). Sampling was carried out in 11 natural populations of F. angustifolia . In this region, stands are influenced by hot summers and cold winters with medium precipitation levels. In contrast, coastal stands are small and fragmented, influenced by the Mediterranean climate which is characterised by hot, dry summers and mild, rainy winters. These forests grow along the rivers of the Adriatic catchment area (21.405 km2), the Dinaric karst fields and wetland sites .About 9% of Croatian land area is frequently flooded Fraxinus spp. studies: Femsatl4, Femsatl11, Femsatl12, Femsatl16, Femsatl19 and M2–30 Total DNA was extracted using DNeasy 96 Plant Kit (Qiagen) from up to ten mg of dried leaf tissue following the manufacturer's protocols. We used six polymorphic microsatellite markers that had been extensively used in Na), observed heterozygosity (HO), and expected heterozygosity (HE). The program MICRO-CHECKER HEnull). GENEPOP was also used to test for Hardy-Weinberg equilibrium (HWE) for each locus in each population and to test the loci for linkage disequilibrium. The probability tests were based on the Markov chain method Nar), which yields allele counts standardised to the minimum sample size, and to test the significance of the differences in average values of Nar, HO and HE between the Continental and Mediterranean populations. The number of private alleles (Npr) per population was assessed by MICROSAT GENEPOP 4.0 FST) and their significance were calculated using FSTAT. Pairwise FST values were also estimated after correcting for the presence of null alleles using a method implemented in FREENA F-statistics using Weir and Cockerham's method Pairwise genetic distances between populations , the National Forest Inventory , whilst keeping the variables useful in predicting the distribution limits of trees, such as climatic averages and extremes Climate data for current conditions were obtained from the WorldClim database with a spatial resolution close to a square km http://www.cro-nen.hr/map/index_en). All topographic variables were based on a 90-m spatial resolution digital elevation model (DEM)  and prepared in ArcGIS® 9.3 (ESRI).Ten environmental variables were selected to describe the ecological characteristics of the sampled stands, for the Principal Component Analysis (PCA) and for the calculation of environmental distances. The eight bioclimatic variables included averages, extremes and seasonal variation in precipitation and temperature, and the two topographic variables altitude and terrain slope see . Three a2) between the populations were computed to obtain a matrix of environmental distances among the populations. Mahalanobis distances are analogous to Euclidian distances but also account for covariance among variables. Mantel tests FST/(1-FST) ratios and (2) the matrix of environmental distances (D2) and the matrix of pairwise FST/(1-FST) ratios. Only individuals that had information about all three parameters  were used for the correlation tests (n = 335). In addition, a three-way Mantel test was applied between the matrix of environmental distances and the matrix of pairwise FST/(1-FST) ratios while accounting for geographical distances among populations. The significance level was assessed after 10,000 permutations as implemented in NTSYS-pc Ver. 2.02 To generate the environmental distance matrix, we performed a canonical discriminant analysis (CDA) based on the ten environmental variables see  using PR2) were visualised by constructing two neighbour-joining trees. Pairwise Nei's standard genetic distances Finally, the relationships between the populations based on both genetic distances and environmental distances . There was no evidence of allele dropout in the data according to MICRO-CHECKER. Null alleles were suggested in seven out of 66 locus×population combinations. Estimated null allele frequencies using FREENA ranged from 0.052 (Femsatl12 in population Trnjani) to 0.146 (Femsatl16 in population Lonjsko Polje). The HE values increased slightly when recalculated using adjusted allele frequencies . Therefore, all subsequent analyses were conducted using the original data set. Significant differences (P<0.05) in genetic diversity  were found between the Mediterranean and Continental populations, with lower values observed in the Mediterranean stands. Moreover, 57 private alleles were identified in the Continental region, whereas there were only nine in the Mediterranean region (P<0.01) from the HWE were observed at any loci in any population after applying sequential Bonferroni corrections. Finally, among a total of 165 tests for linkage disequilibrium between pairs of loci, no test was found significant after applying sequential Bonferroni corrections (P<0.01).A total of 176 alleles were observed across the six markers, with the number of alleles per locus ranging from nine (Femsatl16) to 48 (M2–30) and a mean value of 29.33. to 17.15 . High lequencies , but no n region . No signFIS = 0.024, P = 0.0025). Moreover, mean multilocus values of FIS in the Mediterranean region were almost four times higher and significant than that for the Continental region (FST) was 0.022. The within-region FST, however, was higher in the Mediterranean populations (FST = 0.027) than that among the Continental populations (FST = 0.012), showing that the coastal populations were more structured.Although testing for HWE within each population showed no significant departures, the average multilocus inbreeding coefficient of the overall sample was slightly positive but significant , suggesting that null alleles did not affect this analysis. Most population pairs from the Continental region had non-significant pairwise FST values, with the exception of the Čakovec population. In contrast, most population pairs within the Mediterranean were significantly differentiated. Pairwise differentiation was also detected between the Mediterranean and Continental populations, with the exception of the Istrian population Mirna, which was not significantly differentiated from several Continental populations. The AMOVA analysis    even after accounting for the effect of geographical distance in a three-way Mantel test . Therefore, our populations show a clear “isolation by environmental distance” pattern, rather than IBD as such.The analysed populations showed both significant levels of IBD (= 0.026)  and even= 0.004) . The cortel test . On the The first principal component (PC1) explained 60.32% of the total variation and clearly separated the Continental and Mediterranean localities along temperature and precipitation gradients . ElevatiF. angustifolia in Croatia  found in many European F. excelsior populations  was not observed in the present study. This characteristic of common ash was often attributed to the presence of null alleles, biparental inbreeding or the Wahlund effect. Unlike common ash populations studied in Europe FIS, which provides information on the cumulative effect of inbreeding, was low but significant at the level of the overall sample (average FIS of 0.024), but at the regional level only for the Mediterranean stands (average FIS of 0.046). Although positive FIS values may reflect the presence of null alleles, which are commonly suspected for microsatellite loci, controlled crosses carried out by Morand HE and FST values calculated with and without corrections for null allele frequencies. A plausible explanation for this positive inbreeding would be a Wahlund effect at the level of the overall sample population, mainly due to the differentiation between subpopulations, even though subpopulations themselves are in HWE.The genetic analysis results agree with the expectations of high polymorphism within populations and low genetic differentiation between populations, as observed in ashes and wind-pollinated trees in general FST, even between most distant populations. On the other hand, Mediterranean populations are reduced to a few smaller suitable sites associated with Dinaric karst fields, short karst rivers and rare natural wetlands with no apparent above-ground connections due to the limestone base. In consequence, populations are more isolated from each other and also from the Continental part of the distribution, limiting dispersion and favouring the maintenance of an intra-regional genetic structure.Local population sizes determined by suitable habitat availability may explain the contrasting genetic structures found within and among biogeographical regions. The observed higher genetic diversity in the Continental populations can possibly be maintained due to larger effective population sizes compared to their Mediterranean counterparts. Longitudinal distribution along rivers and floodplains typical for the Pannonian lowlands with no barriers to gene flow allows free pollen and seed dispersion between the populations, which explains the lack of significant genetic structure in this region based on pairwise FST values and multilocus estimates of F-statistics suggest that each region has different evolutionary constraints. For example, some very close Mediterranean populations (such as Zrmanja and Krka) have significant pairwise FST values although they are not geographically distant (only 40 km apart), suggesting more restricted gene flow in this region probably caused by isolation of suitable habitat and/or habitat differentiation. Environmental distinctiveness and habitat discontinuities among Mediterranean populations are apparent from the PCA and niche models, revealing a similar differentiation pattern compared to pairwise FST. The Mediterranean is known for pronounced environmental heterogeneity over very short distances because of factors such as slope, exposure, distance from sea, and rock type. In contrast, most of the Continental populations that occur in a rather homogenous environment exhibit non-significant pairwise FST values (except for the Čakovec population), even in distant populations (>240 km). Similar trends were observed in Taxus baccataLarge amounts of neutral population genetic variation were explained by environmental variation rather than by simple geographic distances, suggesting a strong role of environmental heterogeneity in the genetic divergence of populations Fraxinus spp. Second, we found a significant correlation between genetic and environmental variation. IBED patterns may be arising from a neutral process of temporally disrupted gene flow among individuals living in environmentally distinct habitats, leading to phenological differences F. angustifolia has a wide distribution, both latitudinally and altitudinally, this is a confounding factor that needs to be kept in mind.Using an ENM approach, we tested whether the predicted distributions of the species corresponded with the patterns of population genetic structure. In particular, we searched for areas where the Continental and Mediterranean ENMs overlap, as they could highlight populations from different biogeographical regions with lower levels of pairwise genetic differentiation. The overall ENM detected a separation of the two putative ecotypes by the mountainous region, representing an unsuitable habitat for the survival of the species and a potential barrier to gene flow. The Continental and Mediterranean ENM barely overlapped, indicating a clear divergence in the ecological niche space occupied by the populations in each region. An overlap of independent ENMs suggests only one point of contact in the Istrian peninsula at the Mirna population site . This stFrom our observations, we cannot exclude the fact that historical migration processes in this refugial area could have raised the observed pattern of genetic variation in studied populations. Mediterranean tree populations have persisted in the southern refugia without significant geographical movements due to long-term stable environmental conditions in the Mediterranean F. angustifolia is a thermophilic tree species with distinct moisture requirements that could have survived in situ during the last glacial period in several river valley sites along the Dalmatian coast at lower to mid-altitudes until today. These humid but not too cold sites provided continuous moisture availability and shelter for Mediterranean tree species during the Adriatic Sea level drop in the LGM, leaving the Northern half of the Adriatic Sea basin exposed and unsuitable for the survival of moisture-dependent species F. angustifolia haplotype lineages meet at the vicinity of the investigated populations (H01, H02 and H03, sensuQuercus robur in Europe during the LGM in situ. Assuming that F. angustifolia followed a similar distribution during the LGM, this species likely survived for long time in this area, allowing enough time for the differentiation of distinct populations to occur through a processes of ecological isolation.Overall, our results suggest that long-term stability of heterogeneous environments at regional spatial scales may explain current levels of genetic diversity and population genetic divergence in narrow-leaved ash in these ancient refugia. Environmental differences between the regions may have led to the general subdivision into two ecotypes, with the pronounced environmental heterogeneity in the Mediterranean further promoting the genetic differentiation of the coastal populations. Thus, the local genetic structure in the narrow-leaved ash is more complex than a simple allopatry divergence model as the populations are not clear-cut differentiated but rather in a complex genetic cline, probably resulting from the environmental heterogeneity over the studied geographical area.


The role of European wild horses in horse domestication is poorly understood. While the fossil record for wild horses in Europe prior to horse domestication is scarce, there have been suggestions that wild populations from various European regions might have contributed to the gene pool of domestic horses. To distinguish between regions where domestic populations are mainly descended from local wild stock and those where horses were largely imported, we investigated patterns of genetic diversity in 24 European horse breeds typed at 12 microsatellite loci. The distribution of high levels of genetic diversity in Europe coincides with the distribution of predominantly open landscapes prior to domestication, as suggested by simulation-based vegetation reconstructions, with breeds from Iberia and the Caspian Sea region having significantly higher genetic diversity than breeds from central Europe and the UK, which were largely forested at the time the first domestic horses appear there. Our results suggest that not only the Eastern steppes, but also the Iberian Peninsula provided refugia for wild horses in the Holocene, and that the genetic contribution of these wild populations to local domestic stock may have been considerable. In contrast, the consistently low levels of diversity in central Europe and the UK suggest that domestic horses in these regions largely derive from horses that were imported from the Eastern refugium, the Iberian refugium, or both. The domestication of horses was a fundamental step in the history of humankind, providing horse-centred societies with enormous advantages over agricultural societies with regard to long-distance travel, warfare and trade. Consistent with the preference of horses for predominantly open landscapes, the earliest evidence for horse domestication  appears in the Eurasian steppes around 3500 BCE Around the time when the first domesticated horses appeared in the Eurasian steppes, large parts of Europe were still covered by vast expanses of dense forest To identify primary areas of horse domestication in Europe, we investigate spatial patterns of genetic diversity in horse breeds for which empirical evidence demonstrates a historic origin in a distinct region of mainland Europe or the UK . For the purpose of this paper, we define primary areas of horse domestication as regions where local domestic populations largely descend from local wild stock, be it through their initial recruitment to found domestic populations (“independent” domestication), their extensive introgression into local domestic populations, or both.If there were only a few, geographically restricted regions in Europe where the genetic contribution of local wild horses to domestic stock was substantial, and if domestic populations from such areas were imported into regions where local wild stock was scarce, we would expect the former areas to have retained high levels of genetic diversity, and the latter areas to be characterised by low levels of diversity. The rationale behind this reasoning is that, as populations expand out of origins, genetic diversity will be lost as a consequence of the  small population sizes involved in such expansions (“founder effect”), see To investigate spatial patterns of autosomal genetic diversity in European horses, we assembled a unique dataset of more than 1100 horses typed at 12 autosomal microsatellite loci , using bH) reveals two hotspots of diversity, one in the Caspian region of western Asia, our easternmost sampling location, and one in the Iberian Peninsula  than the former  Iberia: 0.035 (0.007–0.052); cE/UK: −0.009 (−0.037–0.028)), implying that breeding practices are unlikely to explain the observed pattern in diversity. Furthermore, cE/UK breeds, but not Iberian ones, would have had to undergo extreme recent contraction, to average effective population sizes (Ne) of between ten and 20 individuals, to generate the observed pattern (equation (3)). While a few individual breeds are known to have undergone such severe bottlenecks in the recent past, these include breeds from the proposed refugia The observed genetic pattern could be a consequence of recent demographic processes: high diversity in Iberia might reflect disproportionally high levels of admixture from high-diversity non-Iberian breeds. Similarly, low diversity in central Europe and the UK (cE/UK) might reflect disproportionally severe recent bottlenecks or higher levels of inbreeding in breeds from these areas. Since domestication, horses from the Middle East have been among the most widely used to “improve” horse breeds across Europe Y –C and exY . This is consistent with the fossil record for horses, which, in turn, reflects the ecology of this large, group-living animal. While our results do not imply that wild horses were entirely absent from forested parts of Holocene Europe, we suggest that their presence in these regions was spatially and temporally discontinuous, with local extinctions and re-colonisations occurring in response to natural forest gap dynamics.E. ferus persisted in refugial steppe habitat in the East In this study, we have confirmed previous claims whereby populations of In this paper, we present new genotyping data supplemented by microsatellite data from four published studies For our final dataset, we excluded all non-European breeds as well as breeds that are known to have been introduced to various European islands in recent times. In order to maximise our chances to detect signals of domestication, we furthermore excluded modern “warmblood” breeds which, by definition, are composite breeds with varying contributions of “heavy” draft horses and “light” riding horses DNA was extracted from blood or hair. For DNA extraction from blood see the For DNA extraction and PCR amplification protocols of the previously published datasets, please refer to the original publications HFISN = 17. Private alleles were determined using GDA Nei's gene diversity i to each sample location j. United Kingdom, Ireland, and Shetland were connected to the rest of the graph by creating suitable “landbridges”. For each grid point i we then estimated the value of genetic diversity :a was chosen to a = 23, such that the kernel The observed genetic pattern could be a consequence of recent demographic processes rather than a signal of domestication. We considered three major confounding factors that would invalidate the interpretation of genetic hotspots as centres of origin: admixture, recent population declines, and population substructure.mYFS. mY coefficients and standard deviations were computed as averages of 1,000 random bootstrap samples using the programme ADMIX mY is based on the assumption that allele frequencies in the admixed populations are linear combinations of those in the parental populations; contrary to other admixture coefficients, mY takes into account allele frequency differences as well as the degree of molecular divergence between alleles and has been shown to be appropriate for use with microsatellite data mY values merely describe the relative contribution of the surrogate parental populations to the admixed populations, not their absolute contributions.In the recent past, breeds from the Middle East have been widely used to “improve” horse breeds throughout Europe FISFIS values in the former.If mating is non-random, substructure within breeds may arise, causing a reduction in overall heterozygosity (Wahlund effect). This reduction can be measured using Recent bottlenecks might have contributed to the low diversity observed in central Europe and Great Britain (cE/UK), as compared to Iberia and western Asia. We explored the magnitude of the bottlenecks that would have been necessary to produce the lower median diversity found in cE/UK using the recursiont+H1 = tH*(1-1/2tN), (3)where tH is the within-population heterozygosity and Nt the effective population size in generation t. We set the initial diversity Ht equal to the median diversity observed in the putative refugial populations. This is a very conservative estimate, since it (incorrectly) assumes that the latter did not experience recent declines in population sizes.N = 10, 20, 30, 40, or 50 either six or three generations ago, and then recovered at an annual growth rate r equal to 1.1. Using a generation time of 12 years, the bottlenecks coincide with the 1940s and the 1970s, two periods in which many native horse breeds in Europe experienced dramatic declines in population sizes We considered scenarios in which central European and British populations were reduced to minimum effective population sizes of Table S1Admixture coefficients mY for all breeds using (A) Arab and Hucul, (B) Akhal Teke and Hucul, and (C) Caspian and Hucul as parental populations.(PDF)Click here for additional data file.Table S2Expected homozygosity (SF) of different European horse breeds with the Arab, Akhal Teke, and Caspian, respectively.(PDF)Click here for additional data file.