Sibiraea complex's evolutionary history on the Qinghai-Tibetan Plateau (QTP) was confusing and could not be distinguishable on the molecular level. In this study, the genetic structure and gene flow of Sibiraea laevigata and Sibiraea angustata on the QTP was examined across 45 populations using 8 microsatellite loci. Microsatellites revealed high genetic diversity in Sibiraea populations. Most of the variance was detected within populations (87.45%) rather than between species (4.39%). We found no significant correlations between genetic and geographical distances among populations. Bayesian cluster analysis grouped all individuals in the sympatric area of Sibiraea into one cluster and other individuals of S. angustata into another. Divergence history analysis based on the approximate Bayesian computation method indicated that the populations of S. angustata at the sympatric area derived from the admixture of the 2 species. The assignment test assigned all individuals to populations of their own species rather than its congeneric species. Consistently, intraspecies were detected rather than interspecies first-generation migrants. The bidirectional gene flow in long-term patterns between the 2 species was asymmetric, with more from S. angustata to S. laevigata. In conclusion, the Sibiraea complex was distinguishable on the molecular level using microsatellite loci. We found that the high genetic similarity of the complex resulted from huge bidirectional gene flow, especially on the sympatric area where population admixtures occurred. This study sheds light on speciation with gene flow in the QTP.Studying closely related species and divergent populations provides insight into the process of speciation. Previous studies showed that the  Understanding the effect of gene flow in genetic evolution could shed light on speciation Slatkin, . During Gene flow among populations can be estimated using population genetic analyses  Hand.-Mazz. and Sibiraea laevigata (L.) Maxim. (Rosaceae) are the 2 common Sibiraea species in the QTP  Whether the complex could be genetically distinguished using microsatellite loci and (ii) What the reason was for the high genetic similarity and whether gene flow contributed to it. This study addresses these questions through genetic variation analysis based on 8 unlinked microsatellite loci.S. angustata and S. laevigata, respectively, with a total of 681 individuals from 33 sites throughout the QTP . The reaction profile included an initial denaturation at 95°C for 5 min, followed by 36 cycles of initial denaturation at 95°C for 50 s, an annealing temperature of 53°C for 50 s, extension at 72°C for 30 s, and a final extension step at 72°C for 7 min. We analyzed amplified fragments using the QIAxcel DNA High Resolution cartridge and method OM800 on the QIAxcel Advanced System . Allele sizing was performed using QIAxcel ScreenGel software version 1.0.2.0  by comparing alleles with a molecular alignment marker  and size marker .For this study, we used the 8 microsatellite loci  described by Arranz et al. . PCR reaHO), expected heterozygosity (HE), and pairwise FST using ARLEQUIN version 3.5  was regressed against log-geographical distance and tested for significance using the Mantel test with 10,000 permutations.The genetic structure among populations and individuals was investigated using the Bayesian clustering algorithm implemented in STRUCTURE version 2.3.1  analysis  contained S. laevigata populations, the second (named ANS) contained S. angustata populations that were sympatric with S. laevigata, and the third (named ANR) contained the remaining S. angustata populations. A total of 3 evolutionary scenarios were compared  were uniformly high, and ranged from 0.564 to 0.895 (Table P < 0.05). In contrast to S. laevigata, more S. angustata locations showed significant departure from Hardy-Weinberg equilibrium with a significant multilocus heterozygosity deficiency . Populations in the sympatric area of the complex were more inbred than those from the non-sympatric area. Pairwise FST values . AMOVA analysis showed that 4.39% of the variance was found between species (Va = 0.166), 8.16% among populations within species (Vb = 0.309), and 87.45% within populations (Vc = 3.315)  Table .K) increased with each K-value and did not plateau when the complex was analyzed together  and favored this as the most probable scenario Figure . The medS. laevigata, r = 0.457, P = 0.997; for S. angustata, r = 0.236, P = 1.000). The assignment test assigned all individuals to populations of their own species rather than their congeneric species. GENEGLASS detected no first-generation migrant between the 2 species. Ten first-generation migrants were detected within S. laevigata populations and 41 first-generation migrants were detected within S. angustata populations  was much lower than the recent immigration rate from S. angustata to S. laevigata (m21 = 0.0234) (Table S. laevigata to S. angustata was only slightly lower (m12 = 0.0106) than that from S. angustata to S. laevigata (m21 = 0.0187).There were no significant correlations between genetic and geographical distances among the populations analyzed in this study . Then, STRUCTURE provided a comprehensive understanding of the Sibiraea populations under study. The K = 2 model seems to support the interpretation that each cluster represents a single geographical region; one is the sympatric region, while the other is the left region. The K = 7 model in STRUCTURE clearly separated S. laevigata populations from S. angustata populations. Furthermore, STRUCTURE grouped all S. laevigata populations into a unique cluster (the blue one), and S. angustata populations under another clusters . However, population S13 was not geographically adjacent to the 5 populations. Since a significant bottleneck effect was detected in population S13, the geographical disjunction between population S13 and other populations of the red cluster might be due to a genetic remnant following the genetic bottleneck.Even though the complex was genetically distinguished, they shared some genetic variability. There was evident structuring regarding 5 populations (P8–P12), where approximately 60% of genetic variability was attributed to the blue cluster, and 40% to the red cluster Figure , which aFST < 0.05 as indicator of little genetic differentiation. The FST between the Sibiraea complex is low (0.049), meaning that there is not much genetic differentiation between them. At the same time, shared genetic variation was observed and most genetic variance was detected within populations rather than between species. However, the complex has been divergent in stable morphological characteristics, such as pubescent or peduncles and pedicels  between the Sibiraea complex. Coincidentally, AMOVA analysis also indicated that the majority of diversity was within populations (87.45%) rather than among populations (12.55%). Moreover, MCMC searches indicated high gene flow between S. laevigata and S. angustata, especially on the sympatric area  contributed to the admixture more than the southern S. angustata (39%). Significant bidirectional gene flow between them, especially on the sympatric area, contributed to their high genetic similarity. When generation time of Sibiraea was set to 5 years, according to our field observations, the admixture occurred about 23,000 years ago, i.e., when the QTP was in the Last Glacial period. Phylogeographic study indicated that the Sibiraea complex had 3 independent refugia in the QTP, 2 of which were high altitude , the area covered by glaciers was larger than that of former glaciations in the southern part of Himalaya and Southeastern Tibet (Zheng et al., Sibiraea divergence and decreased population size. The population size decrease was observed in our previous study using the Bayesian skyline plot, which showed that Sibiraea populations in the QTP decreased 23-fold during the last 0.12 Ma (Fu et al., Studying closely related species and divergent populations gives insight into how genetic differences in speciation proceed (Surget-Groba and Kay, Sibiraea from the QTP. The Sibiraea complex, S. angustata and S. laevigata, which could not be distinguished by DNA sequence data in previous research, were genetically distinguished based on the microsatellite markers applied in this study. We found that the high genetic similarity of these 2 species is the result of huge bidirectional gene flow, especially on the sympatric area where population admixtures occurred. The Sibiraea complex may be on an incomplete process of speciation with gene flow. Our study suggests that microsatellite loci are helpful for species population genetics, especially for closely related species. This study greatly enhanced our knowledge on the genetic diversity and evolutionary history of Sibiraea in the QTP.In conclusion, this study examined the population genetic structure of 45 populations of PF conceived and designed the experiments, performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables. QG reviewed drafts of the paper, revise the manuscript. FZ analyzed the data, prepared figures and/or tables, reviewed drafts of the paper, revise the manuscript. RX, JW, and HL performed the experiments, analyzed the data. SC conceived and designed the experiments, reviewed drafts of the paper. PF, FZ have contributed equally to this work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Monttea aphylla, an endemic species of the Monte Desert, to understand the evolutionary history of vegetation communities inhabiting the South American Arid Diagonal. We obtained sequences of three chloroplast  and one nuclear (ITS) intergenic spacers from 272 individuals of 34 localities throughout the range of the species. Population genetic and Bayesian coalescent analyses were performed to infer genealogical relationships among haplotypes, population genetic structure, and demographic history of the study species. Timing of demographic events was inferred using Bayesian Skyline Plot and the spatio-temporal patterns of lineage diversification was reconstructed using Bayesian relaxed diffusion models. Palaeo-distribution models (PDM) were performed through three different timescales to validate phylogeographical patterns. Twenty-five and 22 haplotypes were identified in the cpDNA and nDNA data, respectively. that clustered into two main genealogical lineages following a latitudinal pattern, the northern and the southern Monte (south of 35° S). The northern Monte showed two lineages of high genetic structure, and more relative stable demography than the southern Monte that retrieved three groups with little phylogenetic structure and a strong signal of demographic expansion that would have started during the Last Interglacial period (ca. 120 Ka). The PDM and diffusion models analyses agreed in the southeast direction of the range expansion. Differential effect of climatic oscillations across the Monte phytogeographic province was observed in Monttea aphylla lineages. In northern Monte, greater genetic structure and more relative stable demography resulted from a more stable climate than in the southern Monte. Pleistocene glaciations drastically decreased the species area in the southern Monte, which expanded in a southeastern direction to the new available areas during the interglacial periods.Effects of Pleistocene climatic oscillations on plant phylogeographic patterns are relatively well studied in forest, savanna and grassland biomes, but such impacts remain less explored on desert regions of the world, especially in South America. Here, we performed a phylogeographical study of  Quaternary climatic oscillations profoundly impacted the abundance and distribution of organisms due to changes in landscape configuration, habitats and resource availability . PaleoflMunroa argentina Fig 1A). , with other intense glaciations occurring 0.5 Ma, 70–60 thousands of years (Ka), and finally the Last Glacial Maximum (LGM) around 26–21 Ka. During this last glaciation period, palynological records suggest that the climate was colder, drier and windier than today in the Monte Desert  ), trnH–pnd psbA; ) and trnrpL16x1; ), and thence see . The proloops) in networks were resolved using the frequency and topological criteria  while diversification of haplotypes within the northern Monte began at 2.97 Ma (95% HPD = 1.65–6.27 Ma). The individual with H13 haplotype of cpNDA, located at the southwestern corner of northern Monte, appears as the sister lineage of a clade distributed exclusively in the southern Monte  all demographic analyses showed evidence of recent demographic expansion based on chloroplast and nuclear markers, as indicated by the negative and significant values for Fu‘s and Tajima’s neutrality tests, and by the SSD index, which indicates that the observed mismatch distribution did not differ from an expected sudden expansion model Tables .The BSP was run independently for the two genetic groups of the northern Monte (N-1 and N-2), while BSP was evaluated jointly for all localities of the southern Monte because the group was retrieved as monophyletic and and the three genetic groups retrieved by Geneland shared haplotypes among them. Results showed congruent patterns between nuclear and chloroplast signals; outcomes evidenced a constant population size over the last 250–800 Ka for N-1 group, and 100–900 Ka for N-2 group . The BSPM. aphylla distribution, while for the N-1 genetic group in the northern Monte, neutrality tests and mismatch distribution evidenced signals of relatively recent demographic expansion. Therefore, the phylogeographic history of spatial diffusion was inferred for the lineage located in the southern Monte and N-1 in the northern Monte.Considering the results of demographic analyses, the four approaches used retrieved clear signals of recent demographic expansion in the southern edge of M. aphylla experienced four main colonization phases: (1) around 120 Ka a short distance colonization to the southeast established the ancestors of the current locations of La Pampa Province . The RRW diffusion model inferred that the expansion of the southern group would have begun at around 150–180 Ka and inferred its, geographic origin in southwestern La Pampa Province bordering with Río Negro river, in the north of southern Monte  froProvince ; (3) cpDProvince ; (4) befProvince , being m aphylla .For the N-1, the spatial diffusion rate was 457.776 km/Ma (95% HPD = 330.002–599.370) based on the cpDNA matrix. The origin of the expansion revealed by the cpDNA would have began 50 Ka, around the northwestern area of La Rioja Province . The origin of the expansion showed great uncertainty and would have begun 2 Ma between western La Pampa Province and eastern of Río Negro Province,  and the origin of the diffusion revealed by the nDNA began around 1.6 Ma in the center of La Rioja Province  suggests a slight disjunction in relation to its current geographical range, with areas of high probability of occurrence in the northern and southern edges of the distribution connected by areas of moderate to low probability along mid-latitudinal areas of the Monte , while diversification within each genetic group occurred mainly in the limit Pliocene-Pleistocene (ca 2.7 Ma). These results are highly congruent with previous estimations performed in south american desert plants . Ver Desert: .Although the calibration of a molecular clock allows associations between demographic processes and paleoenvironmental events, limitations and biases of this kind of analyses are known due to the use of mutation rates estimated for taxa different from the focus group (reviewed in ). HoweveLiolaemus darwinii complex ), but also other key reproductive processes such as flowering and germination, limiting the southernmost range of the species.The great differences observed in paleoclimatic and demographic patterns for the northern and southern Monte may be due to changes in air currents that regulated rainfall between these two Monte ecoregions. To the north, moisture primarily comes from air masses from the Atlantic anticyclone center, while to the south the humidity is determined by the entry of air masses from the Pacific and Atlantic Ocean ,31. DuriAlthough this study is the first one considering the whole extension of Monte Desert, our results are congruent with previous phylogeographical studies, including co-distributed animals and a plant species of the Monte Desert and will be useful for elucidating common patterns shared among these taxa. Such common patterns will enable regional and landscape-level patterns of biodiversity to be detected, which are important for understanding macroecology and evolution in a geographical mosaic against a backdrop broadly impacted by geological and paleoclimatic events.S1 DocPermission to publishmap shapefiles.(DOC)Click here for additional data file.S1 Fig(DOC)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S5 Table(DOC)Click here for additional data file.S6 Table(DOC)Click here for additional data file.


Pantholops hodgsonii), or chiru, is an endangered antelope, distributed in China , and India (Aksai Chin and Ladakh). There is a global demand for the species prized wool, which is used in weaving shahtoosh shawls. Over the years, the population of the Tibetan antelope has drastically declined from more than a million to a few thousand individuals, mainly due to poaching. Field studies undertaken in Ladakh, India also indicated winter migration of the population to Tibet. Migration between winter and calving habitats is well established to be female-biased across the Qinghai Tibetan Plateau (QTP). For effective conservation planning, genetic characterization is considered the best way to understand the likely impact of threats for ensuring the long-term viability of the population. In this regard, genetic characteristics of all Chinese populations are well-studied using mitochondrial and microsatellite markers, but information is lacking for the Indian population. Therefore, using the control region marker, we document for the first time the genetic variation of the Indian population of the Tibetan antelope, the extent of migration and its relationships with other populations of China.The Tibetan antelope  diversity were observed in the Indian population when compared to Chinese populations (π = 0.01357–0.02048 and hd = 0.889–0.986). Commonly used indices (Tajima's D and Fu's Fs) were analyzed for inferring the demographic history of the Indian populations, and all values were negative indicating population expansion or demographic equilibrium, though nonsignificant. We observed five haplotypes in the Indian population, and these were not reported in previously studied populations of QTP. Bayesian-based phylogenetic analysis indicates the presence of four clades, however, the posterior probability support for three of these clades is weak (<0.5). Of these, the Indian population formed a distinct clade, whereas the Chinese populations exhibited shared haplotypes, and no geographic structure was observed. Median-joining network analysis was conducted for 46 haplotypes in the overall population, except the samples from India which showed a star-like topology. The Indian population is separated by one median vector from the Chinese population.The partial fragment of control region (259 bp) marker was successfully amplified in 30 Tibetan antelope samples that were collected from the Chang Chenmo Valley in eastern Ladakh, India. We also retrieved control region sequences (n = 88) available in the public domain from GenBank of different Chinese populations. Low levels of nucleotide  and haplotype  under the proposed transboundary agenda between India and China and assess relationships with other populations. Such understanding would enable the planning of conservation strategies for ensuring long-term survival of westernmost populations in its range, and if required, it would establish connectivity with the other populations. Pantholops hodgsonii), also known as chiru has very recently been reclassified on the International Union for Conservation of Nature (IUCN) Red list as near threatened due to the recovery of some populations , 1413, 1Tibetan antelopes are poached for their wool, known as ‘shahtoosh,’ a layer of dense, fine wool on their skin that traps warm air, from which fine yarn is produced. This commands a high price , 28. ShaA limited number of studies have been conducted so far on the genetic aspects of Tibetan antelope populations, and these studies have revealed that Tibetan antelopes have female-biased dispersal with a high mutation rate of mitochondrial DNA (mtDNA) , 19, whiFour hair samples and 26 fecal samples were collected from different locations in Chang Chenmo Valley (Ladakh District), Jammu and Kashmir, India IND 1–30) , and approximate 20 ng of genomic DNA. To check for DNA contamination, a negative control was set up with a PCR master mix. The PCR cycling conditions were the following: initial denaturation (94 °C) for 5 min, followed by 40 cycles of denaturation (40 s at 94 °C), annealing (50 s at 56 °C), extension (40 s at 72 °C), and a final extension at 72 °C (10 min). Amplification of PCR was checked on gel electrophoresis using 2 % agarose gel.Genomic DNA was extracted from hair samples using a hair extraction kit , and pellet genomic DNA was isolated using a commercially available kit  according to the manufacturer’s instructions. To avoid contamination, each sample was processed separately using new sterile blade during DNA extraction, and negatives control were included to monitor contamination. Amplification of the hypervariable fragment of control region (259 bp) marker was amplified using an ABI 2720 thermocycler with a pair of primers . The polCapra hircus) was used as an outgroup in our study. The alignments of the control region sequences of Tibetan antelopes with the complete control region of goat (AF533441) were aligned using Clustal W as implemented in BioEdit version 7.0.9.0 [hd) and nucleotide diversity (π) using DnaSP version 3.5.1 [http://www.fluxusengineering.com).The PCR products were purified using ExoSAP  and finally sequenced on an ABI 3130 genetic analyzer using a Big Dye Terminator v 3.1 Kit . Sequences of the control region (n = 88) of different Chinese populations of Tibetan antelope were retrieved from the work of Ruan et al.  and Zhan 7.0.9.0 . The seq 7.0.9.0 . We calcat AF5334 were alion 3.5.1  and DnaSat AF5334 were alion 3.5.1 . A media7 generations and were sampled every 1000 generations with the first 2500 samples discarded as burn-in. Other parameters were set as default values, and results were visualized in TRACER version 1.6 [Patterns of historical demography were inferred from the estimates of effective population size over time using the Bayesian skyline plot (BSP) implemented in BEAST version. 2.1.3. We used the substitution rate according to Zhang et al.  which wasion 1.6 .The partial fragment of the control region (259 bp) was sequenced successfully for all 30 samples of the Indian population. Nine polymorphic and two singleton variable sites were found Table  which dehd and π within the Indian population were 0.543 and 0.00458. The combined analysis of the Indian and Chinese samples revealed that the overall haplotype and nucleotide diversity were 0.927 and 0.01810, respectively (Table hd = 0.889–0.956). Additionally, these diversity indices were also lower when compared with the data set of Ruan et al. [hd 0.997) based on more than 1 kb pair  [Saiga tatarica; π 0.00478; hd 0.918) [Cervus elaphus hanglu; π 0.008; hd 0.589) [A comparison of diversity indices to other endangered populations of antelope and deer species indicate similar ranges of diversity viz. Roan antelope (00–1.00) , Saiga ad 0.918) , and Kasd 0.589) . HoweverD and Fu’s Fs tests for understanding demographic history in Indian and other Tibetan antelope populations  between the Indian and the Xinjiang populations in comparison to the other three Chinese populations  network Fig.  analysisDemographic history analysis and star-like topology in the median-joining network analysis undertaken in the present study and other studies , 25 havePresence of H in different clades as well as topology in MJ network analysis clearly suggest the absence of any geographic structure in the population of QTP , 19, 25,Schaller , 21 has Analysis of the mtDNA control region (259 bp) indicates the presence of relatively low genetic diversity in the surviving Tibetan antelope population in India in comparison to what has been reported for Tibetan antelope populations of QTP. Five H were observed in the Indian population, and so far, these have not been reported in previously studied populations on QTP. Bayesian phylogenetic tree analysis reveals the presence of four clades and indicates lack of population divergence in majority of populations. The absence of Indian H in other populations and a separate major clade in topology probably indicates the absence of female biased migration from India to QTP but requires studies to document the extent of migration present in these populations. The Chinese populations studied so far have a limited geographic structure, and correspondingly, the estimated gene flow among the four meta-populations  indicates strong historical connections between the populations and making QTP as panmictic population. For effective conservation of the western-most population in India, our priority should be to plan genetic studies to include patterns of genetic diversity, relatedness, and population connectivity using mtDNA and nuclear markers of the reported populations around Ladakh and Aksai Chin to determine their relationship with other populations of QTP and identify if any barriers exist. This would enable us to understand and plan for effective conservation of Tibetan antelopes in the meta-population framework and plan adaptive management for restoring the genetic diversity of the eastern most populations. Besides, Tibetan antelopes will also require strong legislation because continued poaching for high wool demand may influence genetic diversity by reducing effective population size as well as population demography.


Cnesterodon decemmaculatus, a freshwater fish species that occurs throughout most of the Pampa biome. We characterized mitochondrial and autosomal genetic lineages in populations sampled from Southern Brazil and Uruguay to investigate (1) the correspondence between current drainage systems and evolutionary lineages, (2) the demographic history for each genetic lineage, and (3) the temporal depth of these lineages. Overall, we found that the major evolutionary lineages in this species are strongly related to the main Pampean drainage systems, even though stream capture events may have affected the distribution of genetic lineages among drainages. There was evidence for recent population growth in the lineages occupying drainages closest to the shore, which may indicate the effect of quaternary sea-level changes. In general, divergence time estimates among evolutionary lineages were shallow, ranging from 20,000 to 800,000 years before present, indicating a geologically recent history for this group, as previously reported in other Pampean species. A Bayesian phylogeographical reconstruction suggested that an ancestral lineage probably colonized the Uruguay River Basin, and then expanded throughout the Pampas. This evolutionary scenario may represent useful starting models for other freshwater species having a similar distribution.The Pampas is a Neotropical biome formed primarily by low altitude grasslands and encompasses the southernmost portion of Brazil, Uruguay, and part of Argentina. Despite the high level of endemism, and its significant environmental heterogeneity, Pampean species are underrepresented in phylogeographic studies, especially aquatic organisms. The Pampean hydrological system resulted from a long history of tectonism, climate, and sea level changes since the Neogene. In this study, we examined the population genetic structure of  The South American Pampa, or “Pampas,” is a Neotropical biome dominated by natural grasslands spreading over plains of Uruguay, Northern Argentina, Southern Brazil, and part of Paraguay . HoweverAustraloheros, Cnesterodon, Jenynsia, and Corydoras  is endemic to the Pampa biome and is one of the most widespread freshwater fish species in this region. It is found in the freshwater Ecoregions 332 (lower Uruguay) and 334 (laguna dos Patos basin) described by C. decemmaculatus populations from the southern Pampean region showed low genetic divergence and evidences of recent colonization of this area  How strong is the correspondence between current drainage systems and evolutionary lineages? (2) Is there evidence of ancient population growth or reduction for each genetic lineage in this species? (3) What is the temporal depth for the evolutionary divergence among lineages and populations? (4) What is the colonization history for this taxon in the Pampas?In this study, we used mitochondrial and nuclear genetic markers to evaluate the genetic structure of Figure 1), covering a significant part of the species’ range. Our sampling ranged from one to eight individuals per locality  (ND2) (SH3PX3) and myosin heavy chain 6 (Myh6)  and sequenced using the Sanger method at Macrogen Inc. . Both DNA strands were sequenced, checked, and aligned with GeneiousPro v.4.8 .Total genomic DNA was isolated from muscle tissue with cetyltrimethyl ammonium bromide (CTAB) as described by COI)  and NADHI) (ND2)  and two 6 (Myh6) . Howeverro v.4.8 . HaplotyHASE 2.1  using 10HASE 2.1 . All seqFigure 1 and Supplementary Table Dispersal of obligatory freshwater fish requires connections of aquatic habitats between adjacent basins and, most of taxa have their distribution range narrowly coincident, if not equal, with the hydrographic basins boundaries in which they live . Based o1. The resulting genetic structure was quantified using an analysis of molecular variance (AMOVA)  to 37, the number of sampled local populations. Because stream capture events may affect gene genealogies and molecular diversity patterns within drainages, we decided to run several analyses excluding localities for which we may have evidence for stream capture. We inferred a possible stream capture event whenever we found shared haplotypes between neighbor drainages, especially when populations closely related to watersheds were involved (see the section “Discussion” for further details for each case).To determine the correspondence between current drainage systems and evolutionary lineages, we inferred the evolutionary relationship among haplotypes using a median-joining network  estimate (AMOVA)  and pairquin 3.5 . In addiquin 3.5 . We usedH), followed by Tajima’s D (FS (NE) and the population growth parameter (G) for each population without the localities with evidence for headwater capture , using both markers in the program LAMARC v. 2.1.6  we first estimated descriptive statistics of genetic diversity, such as nucleotide (π) and haplotype diversity , which has been suggested in the literature for Cyprinodontiformes based on a dataset which included the ND2 gene . We excluded localities showing evidence for headwater capture, as the species-tree method does not allow gene flow among terminals. We used a rate-reference prior to calibrate the molecular clock for Myh6 (ND2) is taken as a reference to estimate the relative molecular clock associated with alternative gene trees . This is a useful approach when different partitions evolve at different rates, but there is no prior information on the molecular clock for all independent partitions in the matrix  yielding 31 different haplotypes defined by 42 variable sites (28 parsimoniously informative). For the autosomal Myh6 gene we had 188 sequences (681 bp) resulting in 25 different haplotypes defined by 21 variable sites (16 parsimoniously informative). In general, the mtDNA haplotype network showed little haplotype sharing among populations, and a close relationship among haplotypes from the same population . Populations of MIR and SOU exhibited related haplotypes, except for two SOU localities that had haplotypes H21 and H30, which are more closely related to haplotypes found in URU. Concerning Myh6 haplotypes , URU, MIR, and SOU shared the most frequent haplotypes (H5 and H1), and H9 was the only haplotype present in populations of all drainage systems. The close relationship among haplotypes for both markers is suggestive of a shallow genealogical history for this species (see below).Ninety-nine sequences were obtained for the mtDNA K = 6 genetic populations . There was a marked difference in the number of different genetic population occurring in each major drainage. Five genetic populations occurred in URU, three in NEG, and two in both SOU and MIR. Not considering samples possibly affected by stream capture events, we found four genetic populations in URU, two in SOU, and one in both NEG and MIR, suggesting significant genetic structure within URU mainly due to three genetic groups with a restricted geographical distribution in this drainage .Considering all samples, the Bayesian clustering analysis found C. decemmaculatus  suggests the most recent common ancestor (MRCA) of all mitochondrial lineages dates from 0.8 mya [95% credible interval (CI) 0.5–1.2 mya], and were most probably located in URU (PP = 0.58). A well-supported clade (posterior probability PP = 1.00), sister to all other lineages, occurred in isolated populations inhabiting elevated plains from three basins: URU, MIR, and NEG . These haplotypes represented one of the Bayesian genetic populations. However, the location of the MRCA for this clade was uncertain . Its sister clade, which had moderate support (PP = 0.82) had URU as the most likely location (PP = 0.75), and showed H25 (another Bayesian genetic population) sister to a well-supported clade (PP = 0.99) that also has URU as its most likely location (PP = 0.80). In turn, this clade harbored four Bayesian genetic populations that corresponded to three well-supported clades (PP > 0.90) plus a “mixed” genetic population that included H1, H7, H8, H30, and H21. Regarding the most likely ancestral location, all remaining genetic groups had URU as the most likely location (PP > 0.85), with one exception. The clade, which was almost exclusive to MIR and SOU , had SOU or MIR as its most likely ancestral location .The mtDNA genealogy for H = 0.94 for ND2 and H = 0.79 for Myh6). Both haplotype and nucleotide diversity were higher for URU and SOU compared with NEG and MIR (Table 1). There was significant genetic population structure . Excluding localities that may represent stream capture events increased ΦST estimates . For ND2, this was still lower than the ΦST estimate among the genetic populations delimited with BAPS , which is expected, given that Bayesian populations were defined based on ND2 data. However, this trend was not followed by Myh6 . Pairwise ΦST values indicated that geographic groups were significantly different from each other except MIR vs. SOU (Table 2). Taking into account possible events of stream capture resulted in increased ΦST values between NEG and the remaining populations for both genetic markers, and between the other pairs only for ND2 (Table 2). Because localities from URU might represent different genetic populations, we also quantified the genetic structure between them. For both ND2 and Myh6, we found significant genetic structure in URU , which was robust to the exclusion of some localities due to stream capture events .Considering the full dataset, both markers showed high haplotype diversities (Table 1). On the other hand, maximum likelihood-based demographic estimates revealed population growth for SOU and MIR (Table 3). Effective population size was also larger for SOU and MIR , while NEG had the lowest value . The combined analysis of mtDNA and nDNA markers suggested that the split between NEG and the clade URU+MIR+SOU, which was also the root of the tree, dates back from ∼0.6 mya (95% CI 0.27–0.95 mya). This was followed by the divergence between URU and MIR+SOU around 0.12 mya (95% CI 0.04–0.19 mya), and the subsequent divergence between MIR and SOU <30,000 years ago  .Neutrality tests provided mixed evidence for population growth in some populations. However, they were dependent on which statistic or genetic marker were used . However, there is no simple relationship between clades and basins, such that a single basin may contain more than one mtDNA clade, and different basins may share the same mtDNA haplotype . Thus, understanding population structure imposed by the drainage system is necessary, but not sufficient, to account for the genetic patterns observed in our study. This is reinforced by the Bayesian clustering of populations, which suggested K = 6 as the bet number of genetic populations. Three alternative (but not exclusive) scenarios that may explain the discrepancy between drainage and genetic structure are: (1) migration among neighbor populations , (2) headwater capture , or (3) shared ancestral polymorphism. The first scenario seems unlikely because C. decemmaculatus has a poor swimming capacity in high-speed currents . Even though URU has received higher support as the most probable location of the MRCA, mtDNA network analysis indicates that this clade evolved in NEG with subsequent stream capture events toward URU and MIR. First, there are seven exclusive substitutions in this clade , suggesting it may have evolved in relative isolation. Second, the sampling points in URU and MIR containing these lineages are adjacent to NEG. Third, H14 and H19 are probably descendent from H11, which occurs in NEG . Under this interpretation, this clade would reflect three independent stream capture events: NEG would have been colonized from URU between 0.2 and 0.8 mya (see below), while MIR and URU would have received migrants from NEG more recently (<0.2 mya). Even in the alternative scenario in which URU is the true location for the MRCA of this clade, two headwater capture events would have occurred, since this lineage would have reached NEG from URU by ∼0.2 mya, dispersing to MIR later on. Two other examples of haplotype sharing that may reflect stream capture toward NEG include H8 (from URU) and H10 (from SOU). In both cases, these haplotypes are more closely related to other lineages from specific drainages, and their sequence identity suggests a recent arrival in NEG. However, while H10 occurs in a site close to SOU, H8 occurs relatively distant from the watershed with URU. An alternative hypothesis for the occurrence of H8 in NEG would involve upstream active dispersal from lower URU populations despite the low dispersal ability of Cnesterodon . The mtDNA genealogy suggests that from URU, C. decemmaculatus would have colonized NEG and SOU basins independently . The high genetic diversity in URU is consistent with its role as the ancestral location. Even though we have found a case of headwater capture from NEG to MIR, the most likely colonization route leading to MIR is through SOU, given that most haplotypes found in SOU and MIR belong to the same mtDNA haplogroup. The Bayesian delimitation of genetic populations also suggested that most localities in MIR and SOU belong to the same genetic population, which is also in agreement with pairwise indices of genetic structure (Table 2). These finding also highlight that, for this species, the genetic diversity in the lower Uruguay ecoregion would be much higher than in Laguna dos Patos . We also found evidence for population growth in MIR. However, differences in genetic diversity and NE estimates between MIR and SOU may reflect different population histories after marine transgressions. Alternatively, some founder effect may have reduced genetic diversity in MIR following its colonization from SOU. Coastal populations from SOU may have also benefited from an extended coastal plain during marine retractions, facilitating population growth during these periods, as have been suggested for other species occurring along the coastal rivers of southern and southeastern Brazil (While the Pampean region has been always dominated by grasslands during the whole Pleistocene , changesxpansion . On the xpansion . In thisxpansion  that coun Brazil .AR-F, LM, and NF designed the study, interpreted the results, and contributed to the final version of the manuscript. AR-F did the laboratory work. AR-F and NF analyzed the data.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The structure of haplogroup H reveals significant differences between the western and eastern edges of the Mediterranean, as well as between the northern and southern regions. Human populations along the westernmost Mediterranean coasts, which were settled by individuals from two continents separated by a relatively narrow body of water, show the highest frequencies of mitochondrial haplogroup H. These characteristics permit the analysis of ancient migrations between both shores, which may have occurred via primitive sea crafts and early seafaring. We collected a sample of 750 autochthonous people from the southern Iberian Peninsula . We performed a high-resolution analysis of haplogroup H by control region sequencing and coding SNP screening of the 337 individuals harboring this maternal marker. Our results were compared with those of a wide panel of populations, including individuals from Iberia, the Maghreb, and other regions around the Mediterranean, collected from the literature.Both Andalusian subpopulations showed a typical western European profile for the internal composition of clade H, but eastern Andalusians from Granada also revealed interesting traces from the eastern Mediterranean. The basal nodes of the most frequent H sub-haplogroups, H1 and H3, harbored many individuals of Iberian and Maghrebian origins. Derived haplotypes were found in both regions; haplotypes were shared far more frequently between Andalusia and Morocco than between Andalusia and the rest of the Maghreb. These and previous results indicate intense, ancient and sustained contact among populations on both sides of the Mediterranean.Our genetic data on mtDNA diversity, combined with corresponding archaeological similarities, provide support for arguments favoring prehistoric bonds with a genetic legacy traceable in extant populations. Furthermore, the results presented here indicate that the Strait of Gibraltar and the adjacent Alboran Sea, which have often been assumed to be an insurmountable geographic barrier in prehistory, served as a frequently traveled route between continents.The online version of this article (doi:10.1186/s12863-017-0514-6) contains supplementary material, which is available to authorized users. The particular spatial distribution patterns of specific mitochondrial lineages in contemporary human populations offer insights into human origins, past migration events and gene flow with defined directions and demographic consequences –5. MitocEuroasiatic marker par excellence) clearly dominates the mitochondrial DNA (mtDNA) gene pool of Europeans (~40-45% on average) . These proportions were similar to those observed in other southwestern European populations . Both H1Clade H1 was more diverse in western (0.940 ± 0.013) than eastern (0.865 ± 0.442) Andalusians. Curiously, most of the identified H1 derived branches were not shared between these two southern Iberian populations; for example, H1a1, H1c3, H1k and H1ah1 were only found in Huelva, whereas H1b1, H1e1a3, H1t1a1 and H1ba were only detected in Granada. H1 sub-lineages were generally observed at frequencies <5% of the whole Hg H group   : 4%, present study; Catalonia: 5%], and higher proportions (~10%) have been recorded in mainland Italy and Sardinia. H20  had few representatives in Iberia , 17. H36FST = 0.0065; P-value = 0.0180), and genetic differentiation was significant according to a χ2 test . The analysis of the corrected typified residuals further suggested that sub-Hgs H18, H10, H1 and H13 (in descending order of significance) were the main contributors  to the observed differences. The above values closely matched those obtained with sequence data . When combining control region sequences and coding region polymorphisms, we found 158 different haplotypes among the 337 H individuals (46.9%), with only a small percentage of them  shared between western and eastern Andalusians.Hg H profiles in Andalusia exhibited substructuring (D) and Fu’s (Fs) neutrality tests. The non-significant sum of squared deviations (SSD) between observed and expected distributions of pairwise differences, along with the Harpending’s index (r) value also support the above statement. The entire distribution of observed Hg H control region haplotypes (n = 337) had a high goodness of fit (R2 = 0.879), which serves as a quality indicator reflecting the lack of sampling bias. Haplotype diversities [0.923 ± 0.021 (Huelva) and 0.969 ± 0.006 (Granada)] were higher than those found in the northern extremes of the Peninsula . In Nor± 0.042, . In consFigure FCT = 0.065, P-value <0.001), with H1 contributing more prominently to that structure. Roostalu et al. , together with others from the rest of Europe and North Africa . C1 was characterized by sub-Hgs H1  and H3 . C2 (n = 10) could be described as the “Basque” cluster, with H1 showing strong statistical significance  in defining the Basque mtDNA gene pool. C3 was primarily characterized by Hg H5  and comprised a high number of populations (n = 20) with a major Italian component along with samples from Tunisia  was significantly influenced by H*  and more weakly by H13 and H20. The mosaic of populations that made up this grouping originated primarily in the Middle East, the Caucasus and southwestern Asia (n = 12), with a few others (n = 5) from Italy and central Europe. The H10, H3 and H1 lineages were particularly underrepresented in C4.Cluster 1 (C1) was the most numerous n = 2 and was , where Morocco (#85) and Algeria (#87)  and those from the southern end of the Peninsula   and source (donor of genes) population in relationship to Africa. The role of Iberia in the spread of genes from Africa to the rest of Europe would also be paralleled by the transmission of other genetic traces southward along the Mediterranean coast and across the Strait of Gibraltar. The lower demographic size in northwestern Africa in relation to Iberia during past millennia would have favored processes increasing the frequency of Hg H in the former region.Today, there is empirical evidence that the contribution of European and African mtDNA lineages is not equivalent on the opposite coasts of the western Mediterranean. The traces left by African mtDNA clades U6, M1 and L in the European gene pool are greatest in the Iberian Peninsula, with the highest frequencies of occurrence (~15%) in western Andalusia . In contAs the climate warmed after the end of the LGM, wide ranging movements of populations through Europe became possible . Those pPhylogeographic and phylogenetic analyses performed in the present study suggest that the main H sub-Hg indicators of post-glacial population expansions from the Iberian refuge were H1 and H3, the most frequent maternal lineages in Iberia. The time depths calculated for these sub-clades in Iberia and the Maghreb, together with the presence of specific H variants in some North African populations –revealed at a deep phylogenetic resolution – it wouInterestingly, the patterns of variation displayed by Hgs H1, H3 and H5  have been found [The first maritime contact would have been associated with fishing in waters more distant from the coast and with increasingly larger watercrafts used for the capture of large fish. In Nerja cave , the remains of large sea mammals  and Y-SNPs –55, CaldHere we present arguments that the western Mediterranean has not been a barrier to human gene flow and, more specifically, that the Strait of Gibraltar and adjacent areas acted as an active bridge between Africa and Europe. A pertinent example is found in Andalusia and its autochthonous contemporaneous populations, whose genetic composition is notably influenced both by the close proximity to North Africa and by intense involvement in the history of the Mediterranean.Additional file 1:TaqMan design for genotyping H and H1 lineages. (XLSX 10 kb)Additional file 2:Amplification primers and probes for the three SNaPshot multiplex reactions. (XLSX 62 kb)Additional file 3:Mitochondrial molecular characterization of 337 Andalusians belonging to haplogroup H. Control region information is showed jointly with coding SNPs. (XLSX 65 kb)Additional file 4:H) for each population. (XLSX 16 kb)Literature compilation for haplogroup H comparative analyses. Geographic affiliations are as follows: WME (Western Mediterranean Europe), WEU (Western Europe), CEU , NEU (North Eastern Europe), EEU (Eastern Europe), CME , CAU (Caucasus), ANA (Anatolia), NES (Near East), ARA (Arabian Peninsula), NAF (North Africa), WAF (Western Africa). Those studies that reached a deep level of resolution inside clade H (towards the definition of specific sub-clades by coding SNP testing) are underlined. The table shows the total sample size (N) and the number of individuals classified as belonging to haplogroup H . (PDF 722 kb)Geographic location of the 93 populations used for comparative purposes and haplogroup frequency maps. A. Europe and the Mediterranean space. B. Detailed view of the Iberian Peninsula. See codes and references in Additional file Additional file 6:http://www.naturalearthdata.com/). (PDF 2058 kb)Interpolation frequency maps (% of the population) of broadly distributed H sub-clades . Map templates were taken from Natural Earth free map repository . (PDF 791 kb)Interpolation frequency maps (% of the population) of sub-clades with a western European/Mediterranean preferential distribution (H17 and H18). Map templates were taken from Natural Earth free map repository . (PDF 1261 kb)Interpolation frequency maps (% of the population) of sub-clades with a central European preferential distribution . Map templates were taken from Natural Earth free map repository . (PDF 889 kb)Interpolation frequency maps (%) of sub-clades with an eastern European/Mediterranean preferential distribution (H13 and H20). Map templates were taken from Natural Earth free map repository  and non-significant as white circles for 10 distance classes. Distances are shown in kilometres. The analysis was performed for the whole database (see Additional file Spatial Autocorrelation Analyses Correlograms of clade H and sub-clades with significant global values of Moran’s I after Bonferroni correction. Significant points are indicated as red circles (


ST distances among the Central-western Persian speaking populations of Iran testify to the common origin of these populations from one proto-population. Mismatch distribution suggests that this proto-Iranian population started to colonize Iran about 30000 years ago which is almost consistent with the timing of arrival and colonization of western Asia by the anatomically modern human. Star-like haplotype network structures, significant and negative Tajima’s D  and unimodal mismatch distributions support the genetic effects of this expansion. Iranian populations presented mtDNA lineages that clearly belong to the European gene pool (i.e. H and U), while the Mashhad population was characterized by the presence of eastern and central Asian mtDNA lineages . Furthermore, the low diversity (h=0.428) observed in Mashhad may indicated the presence of inbreeding, drift or bottleneck events. The application of Monmonier’s maximum differences algorithm revealed a geographic zone of genetic discontinuity between the Arab people of Khuzestan and rest of Iranian populations. Geographical factors, in cooperation with cultural/linguistic differences, are the main reasons for this differentiation. The lack of a sharp geographical or ethno-linguistic structure for mtDNA HV2 sequence diversity was statistically supported by AMOVA and Mantel  tests.In order to survey the evolutionary history and impact of historical events on the genetic structure of Iranian people, the HV2 region of 141 mtDNA sequences related to six Iranian populations were analyzed. Slight and non-significant F Present-day Iran has played a key role in the distribution of the modern human, and has acted as a corridor and natural inter-continental passageway for the expansion of genes . The NeoThe analysis of DNA samples provides a powerful tool for the reconstruction of evolutionary history in extinct and extant species , 15. RecDespite the fact that the Iranian corridor still hosts and is surrounded by populations with very different backgrounds in terms of origins, languages, religions and modes of subsistence, the genetic diversity of very few Iranian ethnic groups has been investigated so far -31. ThesStudy populations: HV2 nucleotide sequences of 141 individuals from six Iranian populations including five Persian speaking populations from Tehran [m Tehran , Esfahanm Tehran  includinm Tehran , Iraqi Km Tehran  and AdygSequence alignment: Sequence alignment was first performed using the ClustalW procedure implemented in Mega, version 5.2, and then by hand [ by hand .Statistical analysis: Basic parameters of molecular diversity such as the number of haplotypes (H), the number of polymorphic sites (s), the mean number of nucleotide differences (k) [h) diversity [nces (k) , and nuciversity  were caliversity . Mega veiversity  and deteiversity . Evolutiiversity  using thiversity . Thus, Siversity -45. ST genetic distance values were calculated based on the number of pairwise differences between sequences and the K2P model of nucleotide substitutions. The statistical significance of pairwise FST genetic distances was estimated by permutation analysis using 10000 random permutations. These values were used to evaluate the genetic differentiation of different populations. A neighbor-joining tree [ST distance matrix. The distance matrix was also represented by non-linear multidimensional scaling (NM-MDS) using the STATISTICA 10 package (StatSoft Inc.) [The best probabilistic model of sequence evolution was determined using the software JModeltest version 2.1.3  and the ing tree  was builft Inc.) .rg) [SSD<0.05) and raggedness index (Prg<0.05) between the observed and simulated mismatch distributions indicated that the population was at a mutation-drift equilibrium (i.e. in a non-expansion phase) [SSD≥0.05) using Arlequin version 3.5, the time since the beginning of the expansion (t) was estimated from the peak of the distribution (i.e. τ) as t = τ/2μ [ Changes of effective population size through time were examined following two different approaches; 1) a neutrality test against population growth and 2) the distribution of pairwise differences (mismatch distribution or MMD). First, potential departures from a null hypothesis of the mutation-drift equilibrium and constant population size were estimated by computing the Tajima’s D test for selective neutrality . Thus, nrg) , 54. Sign phase) , 52. Whet = τ/2μ , where μ, algorithmic analysis of molecular variance (AMOVA) was applied to the groups classified according to their geographic and linguistic affiliation. This test calculates fixation indices  [ST genetic distance matrices was evaluated by the Mantel test [Based on a Delaunay triangulation connectivity network, Monmonier’s maximum-difference algorithm -58 was uand ΦCT) , analogoand ΦCT) , allowintel test  with 100tel test . The Geotel test .Genetic diversity: Using 294 bp long sequences comprising nucleotide positions 48 to 342 of the mtDNA control region, we recognized 125 haplotypes in 1694 individuals, which 34 of them observed in the Iranians (h), nucleotide diversity (π), and mean number of pairwise differences (k) are listed in Iranians . In addiHaplogroup definition: Each mtDNA haplotype was assigned to a particular mitochondrial haplogroup on the basis of mtDNA TreeBuild, version 15, of the PhyloTree. The frequency distribution of the mtDNA haplogroups was inferred from data on HV2 nucleotide sequences. Data on the distribution of mtDNA haplogroups in all Iranian populations under study are summarized in Demographic analysis: Mismatch distribution analyses (MMD) were carried out using goodness-of-fit tests based on sum of squared deviations and raggedness index (SSD<0.05). Tajima's D was strongly negative and significantly different from zero for the pooled data set  and almost all single Iranian ethnic groups with the exception of Esfahan  and Arabs from Khuzestan province  (ss index . When apss index . Poolingss index . Mismatc P>0.05) . Thus, t0=2N0µ and θ1=2N1µ, where N0 and N1 indicate the effective population size in the past and present, respectively, and μ denotes the mutation rate of the human mtDNA D-Loop region [t = τ/2µ, where t is the time since expansion and µ is the per nucleotide mutation rate multiplied by the sequence length. Estimates of the time elapsed since the beginning of expansion for the pooled data set and each Iranian population (with the exception of Mashhad) are given in From the observed distribution of pairwise differences (MMD), it is possible to estimate the parameters of the theoretical model (i.e. τ) proposed by Rogers and Harpending (1992) or its simplified version  Table 3Table 3. p region  . The pairwise FST estimates between the Persian speaking populations including Tehran, Esfahan, Shiraz and Yazd were insignificantly low, suggesting little genetic differentiation between population pairs, which could be possibly attributable to high gene flow or the recent common origin of Persian speaking populations. In addition, Iranians, with the exception of those from Mashhad, showed high levels of differentiation with the Central Asian groups. The people from Mashhad showed lower levels of differentiation with the Azeris (-0.011) from Azerbaijan, Central Asian Kazakhs (-0.0069), Kirgizes (-0.0066) and Pathans (-0.006) from northern Pakistan. Based on the pairwise FST genetic distances, previous study shows that the Central Asian mtDNA sequences presented features between those of the Europeans and eastern Asians [n Asians . SeveralST genetic distance matrix for HV2 sequence data using the Neighbor-Joining (NJ) algorithm  or geography (pattern 1: the western (the Caucasians), the Central (the Iranians), and the eastern  groups; pattern 2: the western (the Caucasians), the Central (the Iranians), the southeastern (the Pakistanians), and the northeastern  groups), the among-groups component of variance (i.e. ΦCT) was always low , indicating that the levels of genetic resemblance between populations is weakly dependent on geographic distances.ways low . The majST genetic distances. Localities were connected according to adjacency criteria, thus defining a so-called Delaunay triangulation  [ST genetic distances for mtDNA data indicate close genetic relationships among the four Iranian populations , while Mashhad and Khuzestan differ from these and from each other. The high genetic similarity of these populations suggests the existence of a single proto-Iranian population in the distant past from which greatly diverse modern Iranian ethnic groups (i.e. the Persian speaking populations) originated. The major mtDNA lineages in Iranians all exhibit a star-like network (The Iranian populations of this study come from different regions and speak different languagesPersian)  despite  network  structurThe lack of a sharp geographical or ethno-linguistic structure for mtDNA HV2 sequence diversity was statistically supported by the AMOVA test. Additionally, the Mantel test revealed a small but significant correlation  indicating that the level of genetic resemblance between populations was slightly dependent on geographic distance . These rST genetic distances, the Mashhad population showed a close genetic relationship with the Central Asian populations; suggesting the presence of a gene flow from these populations. Central Asian mtDNA sequences presented features between European and eastern Asian sequences [ST genetic distances. Several hypotheses could explain the intermediate position of Central Asia between Europe and the eastern Asia, but the most plausible would involve extensive levels of admixture between the Europeans and eastern Asians in Central Asia, possibly enhanced during the Silk Road trade and clearly after eastern and the western Eurasian human groups had diverged [h= 0.428), which indicates the effect of evolutionary forces such as inbreeding, drift or bottleneck events during the evolution of this population.Furthermore, five populations from Iran including Tehran, Yazd, Esfahan, Shiraz and Mashhad have the same linguistic origin; nevertheless, the Mashhad population is genetically different from the others, being characterized by an mtDNA pool composition similar to that of eastern and Central Asia  while the western-central Iranian populations were characterized by an mtDNA pool composition similar to that of Europe and western Asia, a finding consistent with previous studies . Accordiequences  in severdiverged . In addiST genetic distances, we showed differentiation among the Arab people of Khuzestan province and other Iranians. Geography [Furthermore, Yazd, Shiraz, Esfahan and Khuzestan province populations reside in geographical proximity, but the Arab population of Khuzestan is genetically distant from the others, reflecting their different origin. The Persian language belongs to the Iranian branch of the Indo-European family, whereas the Arabic language spoken in parts of Khuzestan province belongs to the Semitic family . In the eography  and culteography , 75 can


Sogatella furcifera(Horváth) is a serious pest causing grievous damage to rice plants. In the present study, inter-simple sequence repeats were employed to investigate the genetic diversity of 108 samples from 27 WBPH geographic populations in China. Ten primers were screened out with 147 amplified bands, average percentage of polymorphic bands, polymorphic information content, and marker index were 78.9, 0.456, and 6.753% respectively. The results indicated that genetic diversity was different among populations, but genetic variation was as low as 0.2% among the populations and as high as 99.8% within the same geographic population. Among the examined WBPH populations, genetic distances were weakly correlated to geographic distance, and there was no correlation between genetic identity and elevation. Cluster analysis showed that the 27 WBPH populations studied could be lumped into four clusters, with which the results of principal coordinate analysis , Sogatella furcifera(Horváth) (Hemiptera: Delphacidae), is a serious pest of rice worldwide in distribution (The white-backed planthopper (WBPH),ibution . It cauibution , even cibution . In recibution . This hibution , but thHigh-altitude aerial netting, ship-catching, recapture of marked insects, dissection of female ovaries, radar monitoring, and trajectory analysis studies have found that the original source of WBPH in China is the Indo-China Peninsula aided by the southwest monsoon . HoweveInter-simple sequence repeats (ISSRs) is a technique of molecular markers, which was developed byDeepening our knowledge on WBPH, including that on genetic structure, genetic diversity, and migration, is important for the development of integrated control strategies and to reduce economic losses. In this study, ISSRs were employed to investigate the genetic diversity of 108 samples from 27 WBPH geographic populations. This study was undertaken to enrich the genetic and migratory knowledge on WBPH, which could provide information to develop regional strategies of integrated control.One hundred and eight samples  were collected from 27 locations of rice-planting areas where WBPH causes serious damage in 14 provinces of China . The coTotal genomic DNA was extracted from WBPH using the modified sodium-dodecyl sulphate (SDS) method . DNA coOne hundred primers were selected from the ISSR primer set developed by Michael Smith Laboratories at the University of British Columbia (UBC)and synthesized by Sangon . Ten primers that produced bright, clear, and repeatable bands were screened for further studies .2+, 250 μmol/L dNTPs, 0.9 μmol/L primer, and 1 U Taq DNA Polymerase , and addition of ddH2O to make a total volume of 20 μl.Optimized reaction system of ISSR-PCR: each PCR amplification reaction mixture comprised 50 ng of DNA, 2 μl of reaction buffer , 1.5 mmol/L MgAmplification was performed in an iCycler Thermal Cycler (Bio-Rad) under the following conditions: an initial denaturation of 94°C for 4 min, followed by 40 cycles of denaturing at 94°C for 1 min, annealing for 1 min (temperature depending on primers used), extension at 72°C for 2 min, and a final extension at 72°C for 10 min. The PCR products were separated on 2% agarose gels in 0.5× TBE buffer and detected by staining with GoldView I . Bands sizes were compared with 100 bp DNA ladder marker (TaKaRa), and the intensities of bands were determined by spectrophotometry using ImageQuantity One (Bio-Rad).Band patterns were recorded with a Universal Hood II Imaging System (Bio-Rad). Amplified products were scored from the gel images according to presence or absence of bands . Thus a matrix  was obtained.MI = Allele × PIC, Allele is the number of allele) of each primer were calculated according to the information in the matrix , and Shannon’s index (I) within a population and genetic distance and genetic identity among populations were calculated using POPGENE Version 1.31 software , Nei’s gene diversity ] and principal coordinate analysis (PCOA) were performed according to genetic diversity. Mantel tests were performed between genetic diversity and cluster analysis. All analyses mentioned earlier were performed with NTSYS-pc 2.10e software .Mantel tests were performed with TFPGA Version 1.3  to examAccording to the brightness, clarity, and repeatability of bands, 10 primers  were seH) values from 0.1718 ± 0.2049 (ahhs) to 0.2849 ± 0.2052 (scnj), and Shannon’s index (I) values from 0.2514 ± 0.2977 (ahhs) to 0.4125 ± 0.2930 (scnj). Results ofH,Ivalues indicated that genetic diversities were different among populations:Based on the matrix of amplified fragments, the genetic diversity indexes calculated using POPGENE Version 1.31 are shown inP = 0.152, > 0.05), which was confirmed byFSTvalues (0.00199).AMOVA indicated that 99.8% of genetic variability was within the same geographic population, and only 0.2% among different geographic populations . A randResults of a Mantel test on genetic diversity and a cluster analysis yielded a correlation index of 0.75, which indicated that the cluster was representative of the genetic relationship among populations .Results demonstrated that there is genetic differentiation among the 27 geographic populations of WBPH. The tree of cluster analysis and3 gResults of PCOA on 27 geographic populations showed that the contributions of the anterior three principal coordinates were 32.13, 29.25, 17.58, respectively, and the accumulation was 78.96 . Accordr = 0.3216,P = 0.001] . Correlation analysis of 27 WBPH geographic populations in China showed that genetic and geographic distances were weakly correlated [Mantel test :r = 0 0.001] . The diPrevious research has shown that WBPH can survive winter at only a few places in the south part of the Yunnan, Guangxi, and Hainan provinces . The anIn this study, experimental procedures were standardized to control the effect of confounding factors that were observed in previous studies . A numbFSTvalues revealed no obvious differentiation. Mantel test results showed that the correlation between geographic and genetic distance, and genetic diversity and elevation were both weak. All results above showed that broad gene exchange is occurring among sampled populations. The possible reason is likely the long-distance migratory capability of WBPH  strengthen international cooperation and communication for management of insect populations in the source region and for production of accurate yearly premigration forecasts; 2) strengthen regional cooperation between western and eastern China, to detect seasonal initial landing of WBPH, and then issue timely forecasts to secondary seasonal landing sites; 3) strengthen application of control measures of WBPH in Guangxi, Guangdong, Yunnan, Guizhou, to decrease its quantity in fields, and therefore reduce the size of remigrant populations at its Chinese initial seasonal source to secondary seasonal migrating sites. Given the migrant nature of this important pest, best management results can only be obtained through cooperative area-wide coordinated control programs.


Corythucha ciliata (Say), an invasive pest originating from North America, causes severe damage on sycamore trees. However, little is known about the population genetics and evolutionary forces underlying the invasiveness of this important pest. In the present study, we use three mitochondrial genes  and nine microsatellite markers to investigate the population genetics of C. ciliata and retrace its spread through China. The results suggest a low level of genetic diversity in Chinese and European populations of C. ciliata. Our results indicate that populations of C. ciliata have obvious genetic structure, and genetic differentiation is not caused by geographic isolation. In median-joining networks, we observed a higher frequency of shared haplotypes in groups 1 and 3. Based on gene flow and approximate Bayesian computation analyses, we discovered that C. ciliata first invaded the east coast of China and subsequently moved inland. Demographic analysis suggested that populations of C. ciliata in China may have undergone a recent bottleneck effect. Finally, our results suggest that population structure, high gene flow and environmental conditions have favored the broad invasiveness of this important pest. Corythucha ciliata (Say) (Hemiptera: Tingidae), is an invasive insect species that originated from the eastern and central regions of North America1. It spread to Italy in 19602 and then became widely-distributed throughout Europe3. Shortly after 1990, it successively invaded South America4, Asia5 and Australia6. C. ciliata causes damage to sycamore trees by feeding on the foliage, thus reducing photosynthesis and promoting disease. Once established, it can form fairly stable, high density populations10. C. ciliata has become a primary pest of trees in the genus Platanus13, and effective controls are lacking during widespread outbreaks14. For example, C. ciliata caused serious damage throughout Europe in the 1980 s, and this resulted in the formation of a special working group to study its biology, ecology, nature enemies and chemical control.The sycamore lace bug, C. ciliata was first discovered in Changsha in 200215 and then in Wuhan17. In a relatively short time, it dispersed to at least 23 cities in China and to the Yangtze River basin20. Why is C. ciliata so successful in invading new places? It is helpful to note that this insect has a strong reproductive capacity, and adult females can produce 250–350 eggs20. Secondly, C. ciliata can withstand both high and low temperature extremes. In laboratory experiments, the Ltemp50 and Ltemp90 of C. ciliata adults and nymphs were 41.4 °C and 43.8 °C, respectively, after one hour of high temperature stress21. C. ciliata adults can also survive after exposure to very low temperatures; e.g. −23.3 °C. Additionally, C. ciliata is easily dispersed; although its wings are relatively weak, it can migrate thousands of meters via wind currents22 and has been extensively disseminated by human activity1. For example, it may have spread from North America to Italy as a result of the shipping industry10. In Turkey, C. ciliata has dispersed to large cities located along highways23, which indicates that human transportation is a major factor in dissemination. Finally, the host trees, Platanus spp., are widely planted in urban areas. C. ciliata has presumably co-evolved with Platanus spp.24, which contains seven tree species originating from North America, Europe, and Asia.In China, 27, and the combined use of these two genetic markers may result in more accurate information than that obtained from a single marker28. In the present study, three mtDNA genes , NADH dehydrogenase subunits 1 and 5 (ND1 and ND5) and nine microsatellite loci were used to study the genetic structure of 21 Chinese and two European populations of C. ciliata. The primary goals of this study were to: (1) investigate the genetic structure and diversity of C. ciliata populations in China and Europe; (2) identify the potential routes of dispersal in China; (3) define the demographic profile associated with the expansion of C. ciliata; and (4) analyze the genetic and environmental factors contributing to the invasiveness of C. ciliata.Mitochondrial DNA sequences and microsatellites are frequently used to investigate the genetic structure of populationsCOI, ND1 and ND5  ranged from 0.000–0.889, 0.000–0.711, 0.000–0.533, and 0.000–0.644 in concatenated sequences, COI, ND1 and ND5, respectively. Based on concatenated sequences, the H values of the ZJ and SV populations were the highest (H = 0.889) and the GY, WH, YZ and YC populations were the lowest (H = 0.000)  and lowest for the GY, WH, YZ and YC populations (π = 0.00000) based on the concatenated sequences  were identified for –H5 were es Table .Figure 1C. ciliata from the two European populations  was relatively higher than most of the other populations .A total of 158 alleles were detected at the nine microsatellite loci, and genetic variability is shown in Supplementary Table O) ranged from 0.47–0.77, mean expected heterozygosity (HE) was 0.50–0.75, and the average inbreeding index (Fis) ranged from −0.17 to 0.28. We also analyzed two groups , and the genetic diversity of GI was lower than GII (P < 0.0001)  when K was four; consequently, the 23 populations were divided into four groups designated G1, G2, G3 and G4. G1 included the following populations: HF, YCC, GY, WH, HY, ZZ, CS, TC, YC, JA, CD, HZ, XA, YW, SV and IT. G2 included NJ and YZ, G3 contained LY, SQ, XZ and TA, and G4 consisted of a single population, ZJ. Although G3 was an exception, the occurrence of G1, G2, and G4 was not consistent with geographic distribution.With respect to concatenated mtDNA sequences, SAMOVA analysis indicated that the FCOI, ND1, and ND5  (P < 0.05)  Table .Table 2PFor microsatellite data, we implemented Bayesian cluster analysis with the Structure program. This analysis revealed that the most likely K value was 2 (chosen with Evanno’s ΔK method) Fig. , which sC. ciliata at various hierarchical levels  (Table FST = 0.19958**) was found within populations based on microsatellites, which was different from mtDNA analysis where a high level of differentiation was found between groups.According to the two clusters assigned by Structure, AMOVA suggested that significant genetic structure was present for s) Table . A high C. ciliata populations was estimated by pairwise FST values between populations based on concatenated mtDNA sequences and microsatellite data  .Additionally, we constructed a NJ tree for We also constructed a NJ tree from microsatellite data Fig. . AnalysiThe effective mutation scaled population size (Θ) was estimated for each population and group based on concatenated mtDNA sequences and microsatellite data. The bidirectional mutation-scaled immigration rate (M) was estimated for 253 population pairs  to 742.2 (G4 → G2) .Additionally, we also observed higher migration rates (M) and asymmetric migration between population pairs based on microsatellites. The M ranged from 131.93 (GY → CS) to 888.44 (HF → YW), and 48 population pairs (18.9%) had asymmetric migration rates (Supplementary Table P = 0.6943), COI , ND1  and ND5  and microsatellites .The Mantel test indicated that there was no correlation between genetic and geographic distance (Km) for concatenated mtDNA sequences  analysis31. The first step of ABC analysis was undertaken to define the most likely area for the initial invasion by C. ciliata. These results unambiguously pointed to a scenario where invasion initiated in the eastern coastal region  (logistic regression: 0.5192 [95% CI: 0.4556–0.5827])  for the test data was 0.994, indicating a high fit of the modelled and observed distributions33. As evident from Fig. C. ciliata. The Jackknife evaluation indicated that precipitation during the warmest quarter (Bioclim 18) and temperature seasonality (Bioclim 4) were the main factors influencing the distribution of C. ciliata .To evaluate the abiotic environmental factors that have influenced the distribution of COI, ND1, and ND5 .MJ networks were reconstructed from haplotypes of ND5 Fig. . Some haD and Fu’s Fs values of concatenated sequences, COI, ND1 and ND5 were positive and not significant. However, exceptions included the Fs value for G1 in the concatenated sequences, and Tajima’s D values for G1 and G3 in the concatenated mtDNA sequences, COI, ND1 and ND5, respectively  and one population (XZ) had a statistically significant excess of heterozygotes when analyzed using the infinite allele model (IAM) and two-phased model of mutation (TPM), respectively  to analyze bottleneck effects in ly Table . Howeverly Table , suggestC. ciliata populations in China is relatively low  and generally less than the diversity exhibited in European populations   exhibited a high level of genetic diversity (H = 0.889 and π = 0.00376) and a high level of gene flow with the other three groups (9.135 to 21.450). The increased genetic variability due to the admixing between G2 and G3  Table . InvasivC. ciliata likes to congregate at feeding sites and exhibits generational overlap after the second generation38. Besides, we could not rule out possibility that homozygote excess was related to HWE because most of the loci had a tendency to slight homozygote excess , and sampling bias and overlapping generations could result in the inbreeding phenomenon. In support of this, we observed that 7–0.28, aC. ciliata exhibits obvious genetic structure in China based on Structure analysis. SAMOVA indicated that the 23 populations were defined as four groups based on mtDNA sequences, and AMOVA indicated that significant genetic differentiation existed among groups, among populations within groups, and within populations. Additionally, the NJ tree revealed a pattern of genetic differentiation similar to the SAMOVA analysis. Conversely, Structure analysis indicated that the 23 populations formed two clusters based on microsatellites; AMOVA results showed significant genetic differentiation among groups, among populations within groups, and within populations. The NJ tree based on microsatellites revealed genetic differentiation similar to Structure analysis. The pairwise FST values also indicated that the Chinese populations of C. ciliata have obvious genetic differentiation. Moreover, genetic differentiation was not caused by geographic isolation because genetic and geographic distances were not correlated.We discovered that C. ciliata migrated from the eastern regions  to the western regions of China (G1)  was an admixture derived from G2 and G3, which was then introduced to inland China (G3). This route of invasion is supported by the fact that sycamore trees were transplanted to China. Thus, we hypothesize that C. ciliata invaded China from seaports located along the east coast, possibly due to the importation of sycamore trees. However, it is important to note that our hypotheses are somewhat inconsistent with the initial reports of C. ciliata in Changsha15 and Wuhan17, which are inland cities.The direction of asymmetric gene flow can potentially reveal migration patterns and routes of invasion. Our analysis of gene flow indicated that P = 0.2411). This is likely to be another example of the Bridgehead effect39, which was predicted to be a common occurrence40 and formed the bridgehead populations that act as a source of western populations in China. The Bridgehead effect which refers to widespread secondary invasions stemming from a particular primary invasive population has been observed for the dispersal patterns of several invasive insects, including Frankliniella occidentalis41 the fire ant Solenopsis invicta42 and the western corn rootworm, Diabrotica virgifera virgifera43.Some populations located in eastern China (e.g. ZJ) exhibited a high level of genetic diversity. These populations exhibit an asymmetric migration relative to other populations and have a slighter higher genetic diversity in mtDNA than other populations  were collected from leaves of Platanus spp. in 23 urban locations, including Italy and Slovenia. The 21 Chinese populations  and stored at −20 °C until needed for PCR.In this study, ons Fig.  encompasCOI, ND1 and ND5, respectively) were amplified using DNA from 10 individuals per population. Primer pairs included COI-F (5′GGTCAACAAATCATAAAGATATTGG) and COI-R (5′-TAAACTTCAGGGTGACCAAAAAATCA)44, ND1-F (5′-ATTCAGACTCTCCTTCAGCA) and ND1-R (5′-GGATAACAGCCTAATCTTCT), and ND5-F (5′-AAAATCACCTCAACTATCAT) and ND5-R (5′-GCTCCTACACCAGTTTCTTC). Primers for ND1 and ND5 were designed based on the complete mitochondrial genome sequence of C. ciliata45. Conditions for PCR amplification were as follows: initial denaturation for 5 min at 95 °C, followed by 35 cycles at 94 °C (1 min each), annealing for 1 min at 45–50 °C, elongation for 1–3 min (depending on predicted fragment length) at 68 °C, and a final extension step at 72 °C for 10 min. Amplification products were purified and then sequenced by Invitrogen Biotechnology Co. .Three fragments of the mitochondrial genome . Amplified PCR products were analyzed by Sangon Biotechnology Co.  using the ABI 3730 automated DNA sequencer (Applied Biosystems). GENEMAPPER v. 3.5 (Applied Biosystems) was used to obtain allele designations.The nine microsatellite loci, primer sequences, and PCR conditions used in the current study were previously described by Yang 47. The number of variable sites and haplotypes, haplotype diversity, and nucleotide diversity of the three mtDNA genes  were calculated by DnaSP v. 5.048. The expected and observed heterozygosity, polymorphic information content, and estimated null frequency were calculated for each locus in individual populations using CERVUS v. 3.0.349.Fragments comprising the three mitochondrial genes were initially aligned using ClustalX v. 2.0E) were calculated by FSTAT v. 2.9.3.250. The deviation from Hardy-Weinberg equilibrium for each population and linkage disequilibrium for pairs of loci was calculated with GENEPOP v. 3.451, and P values were adjusted using the Sequential Bonferroni procedure52. Null alleles were identified using Micro-Checker v. 2.2.353. T-tests were used to evaluate the significance of differences in diversity estimates haplotype diversity (H), nucleotide diversity (π) and number of alleles (A) of the populations using JMP v.12.The number of alleles (A), allelic richness (AR), inbreeding index (Fis), and observed and expected heterozygosity (Ho and H54. The most likely number of clusters (K) was determined by repeating the analysis with K ranging from 2 to 6 and selecting the subdivision scheme associated with the highest FCT. The clustering of individuals from all populations was determined using Structure v. 2.055. The most supported number of clusters (K) was obtained based on multilocus microsatellite data. We used an admixture ancestry model and the correlated allele frequency model with a burn-in period of 100,000 iterations and 1,000,000 Markov chain Monte Carlo (MCMC) repetitions to calculate the most likely number of genetic clusters. We performed 10 independent runs for each K to confirm consistency across runs, tested K from 1 to 8, and used the ΔK method of Evanno et al.56 to choose the most supported value of K.Population groups were identified using concatenated sequences of the three mtDNA genes; information on longitude and latitude were included, and the data were analyzed using SAMOVA v. 1.057. The definition of groups in AMOVA was determined by SAMOVA and Structure analysis for mtDNA sequences and microsatellite data, respectively. Pairwise FST (differentiation index) was analyzed by ARLEQUIN v. 3.057.Analysis of molecular variance (AMOVA) was implemented by ARLEQUIN v. 3.0ST/(1 − FST)] and geographic distance matrices in log scale58 was performed using the Isolation by Distance Web Service (IBDWS)59 with 10,000 randomizations.The correlation between genetic [F60. We used a Bayesian search strategy with the following parameters: three long chains  with 10,000 trees discarded in the initial “burn-in”; replicates = YES, 5; randomtree = YES; and heating = ADAPTIVE: 1 {1.0 1.2 1.5 3.0}. The MIGRATE program was conducted for four cycles, and the seed number was changed for each run.The gene flow between pairs of populations and groups was estimated by MIGRATE v. 3.2.162.Median-joining (MJ) networks of haplotypes for the three mtDNA genes were constructed using Network v. 4.630 as implemented in DIY ABC v. 2.1.031. Gene flow analyses revealed that migrants moved from the eastern regions  to western China (G1). Therefore, to simplify scenarios for ABC analysis, we first combined the three groups from eastern regions and then tested two alternative scenarios for potential sources in China ina Fig. . A totalons Fig. .D and Fu’s Fs tests were implemented to analyze neutrality. Effective population size before expansion (Θ0), effective population size after expansion (Θ1), population expansion time (τ), and the sum of squared deviation (SSD) between observed and expected mismatches were also calculated. The significance of these statistics was tested by parametric bootstrapping  in ARLEQUIN v. 3.0.The mismatch distribution in ARLEQUIN v. 3.0 was used for demographic analysis, and Tajima’s 63. Analyses were based on the frequency of each allele with the following three models: the infinite allele model (IAM)64; the step-wise mutation model (SMM)65, and the two-phased model of mutation (TPM)66. The number of iterations for Bottleneck was 1,000, and the Wilcoxon sign-rank test was implemented.Bottleneck effects were detected using Bottleneck v. 3.452 was applied to adjust the nominal significance level.In all instances where multiple tests were conducted simultaneously, the sequential Bonferroni procedureST values for populations were calculated using ARLEQUIN v. 3.0 with 1,000 permutations. NJ phylogenetic trees were generated using PHYLIP v. 3.267 and based on Nei’s genetic distance. The NJ tree was constructed using COI sequences from the 23 populations listed in Table Pairwise FC. ciliata invasion, we built a species distribution model (SDM) based on field observations of C. ciliata occurrences coupled with environmental maps. Data from sites known to be infested with C. ciliata in China were entered into MaxEnt32. Nineteen bioclimatic data layers were acquired from the WorldClim dataset (http://www.worldclim.org/)68 at a spatial resolution of 2.5 arc minutes. The random test percentage was set to 25%, and the Jackknife procedure was used to estimate the contribution of each variable based on performance of the model. The area under the curve (AUC) value was calculated for model validation; AUC reflects the model’s ability to distinguish between present records and random background points. AUC values ranged from 0.5 (not different from a randomly-selected predictive distribution) to 1 (with perfect predictive ability). Models having AUC values > 0.9 were considered to have very good, >0.8 good, and >0.7 useful discrimination abilities69. The final map was visualized and processed using the ArcGIS platform (http://www.esri.com/software/arcgis).To estimate the environmental suitability of areas for supplementary materials


Drosophila suzukii is a highly destructive pest species, causing substantial economic losses in soft fruit production. To better understand migration patterns, gene flow and adaptation in invaded regions, we studied the genetic structure of D. suzukii collected across Italy, where it was first observed in 2008. In particular, we analysed 15 previously characterised Simple Sequence Repeat (SSR) markers to estimate genetic differentiation across the genome of 278 flies collected from nine populations.FIS) values in seven of the populations indicated a low level of inbreeding, as suggested by the high number of alleles. STRUCTURE, Principal Coordinate and Neighbour Joining analysis also revealed that the Sicilian population was fairly divergent compared to other Italian populations. Moreover, migration was present across all populations, with the exception of the Sicilian one, confirming its isolation relative to the mainland.The nine populations showed high allelic diversity, mainly due to very high heterozygosity. The high Polymorphism Information Content (PIC) index values (ranging from 0.68 to 0.84) indicated good discrimination power for the markers. Negative fixation index  contains supplementary material, which is available to authorized users. Drosophila suzukii Matsumura (Diptera: Drosophilidae), is a pest species which has spread from its original range in Asia to a number of western countries in the past decade, including the Mediterranean basin .Results from the individual population analysis using the TPM model did not support an expansion scenario. On the contrary, population bottleneck, defined by significant heterozygote deficiency was present in the Trentino2 population . This could be the consequence of a steady introduction of new alleles due to migration, possibly associated with human-mediated transport [This research investigated the genetic structure of in Italy . The higin Italy , 58. Bahin Italy . Washing alleles . This coransport .Moderate genetic differentiation between most of the groups was in evidence for the nine populations, while the Sicilian population was the most differentiated from the others. This is supported by the NJ tree, PCoA data and structural analysis. At the same time, low differentiation between the other populations may be due to gene flow, which can homogenize gene frequency across populations. Data concerning the reduction or expansion of the studied populations indicated that Trentino2 was the only group having indices of genetic bottleneck.D. suzukii. [Fst) does not clearly decline (increase) from the hypothetical source population  and that there is a high migration rate among localities, suggests that D. suzukii moves extensively across most of the Italian peninsula. Most of the Sicilian production of vegetables and fruit, including high D. suzukii susceptible hosts, is frequently exported to central and northern Italy. While this could suggest a high probability of flies being transported between Sicily and the rest of the peninsula, our results indicate that there was no gene flow from Sicily to other regions. This is probably due to the fact that ripe fruits are exported from Sicily mostly during the cold season, when moderate temperatures allow the production of berry fruit in Sicily, but not in the rest of Italy. Therefore, any D. suzukii accidentally moving from Sicily to the rest of Italy would arrive at a time when the local population is made up of a few individuals in winter diapause [Human transportation is the most probable explanation for the extensive spread of suzukii. , 59. Whesuzukii. , 61. In suzukii. . This sidiapause .A second interesting piece of information revealed by our results is related to the scenario in Sardinia. To satisfy local demand for berry fruit, this region imports fruit from Italy and northern Europe, Spain, the USA and South America. The flies used in this study were collected in Arborea, a town 13 km away from the port of Oristano, one of the most important commercial ports in Italy. Thus, it is likely that the Sardinian population is made up of immigrants from other regions, as suggested by the low differentiation between this population and those on the mainland.D. suzukii following its introduction to Italy. Defining the population structure of a species, in particular of an invasive species, it is necessary not only to improve our knowledge of the genetic architecture, but also to apply knowledge. Indeed, understanding the current genetic structure of D. suzukii has significant implications in relation to geographical and economic impact. The evaluation of the genetic status of the D. suzukii populations in newly invaded areas and their expansion or reduction phases during defined periods of the year, may thus provide valuable information for predicting population spread, outbreaks, and improve integrated pest management programmes. Proper genetic management practices for D. suzukii and constant monitoring are therefore critical for maintaining populations under control.This research represents the first study investigating the pattern of genetic variability for The information obtained can be applied in particular to the management of coastal areas; one important action could be to increase monitoring control with the use of traps and other early warning tools in order to limit either multiple reintroductions of the same species or new introductions of exotic organisms.