Plasmodium falciparum (PfMSP-119) constitutes the major component on the surface of merozoites and is considered as one of the leading candidates for asexual blood stage vaccines. Because the protein exhibits a level of sequence variation that may compromise the effectiveness of a vaccine, the global sequence diversity of PfMSP-119 has been subjected to extensive research, especially in malaria endemic areas. In Thailand, PfMSP-119 sequences have been derived from a single parasite population in Tak province, located along the Thailand-Myanmar border, since 1995. However, the extent of sequence variation and the spatiotemporal patterns of the MSP-119 haplotypes along the Thai borders with Laos and Cambodia are unknown.The 19-kDa C-terminal region of the merozoite surface protein-1 of the human malaria parasite P. falciparum from five geographically isolated populations along the Thai borders with Myanmar, Laos and Cambodia in three transmission seasons between 2002 and 2008 were collected and culture-adapted. The msp-1 gene block 17 was sequenced and analysed for the allelic diversity, frequency and distribution patterns of PfMSP-119 haplotypes in individual populations. The PfMSP-119 haplotype patterns were then compared between parasite populations to infer the population structure and genetic differentiation of the malaria parasite.Sixty-three isolates of PfMSP-119 were identified. Differences in the prevalence of PfMSP-119 haplotypes were detected in different geographical regions, with the highest levels of genetic diversity being found in the Kanchanaburi and Ranong provinces along the Thailand-Myanmar border and Trat province located at the Thailand-Cambodia border. Despite this variability, the distribution patterns of individual PfMSP-119 haplotypes seemed to be very similar across the country and over the three malarial transmission seasons, suggesting that gene flow may operate between parasite populations circulating in Thailand and the three neighboring countries.Five conserved polymorphic positions, which accounted for five distinct haplotypes, of 19 haplotypes of P. falciparum populations in all endemic populations during three transmission seasons in Thailand were identified, providing basic information on the common haplotypes of MSP-119 that is of use for malaria vaccine development and inferring the population structure of P. falciparum populations in Thailand.The major MSP-1 Plasmodium parasites [Because the clinical symptoms and pathology of malarial infections are caused by the erythrocytic stage cycles of the arasites -3, the darasites . The estarasites , mostly arasites . To datearasites ,8. Despiarasites -11, therPlasmodium falciparum spans ~5 kb in size on chromosome 9 and can be divided into 17 blocks, according to the presence or absence of 9 bp repeats and the type of repeating sequences [msp-1 gene are classified into two allelic types: K1 and MAD20, with the exception of the highly polymorphic block 2 that is represented by at least three allelic types  [83), 30 kDa (MSP-130), 38 kDa (MSP-138) and 42 kDa (MSP-142) [42 fragment is proteolytically processed to form the 33 kDa (MSP-133) and 19 kDa (MSP-119) fragments [19 fragment, containing two epidermal growth factor (EGF)-like domains, remains attached to the parasite’s surface [Components of blood stage malaria vaccines are generally derived from parasite ligands (antigens) expressed on the surface of merozoites, the only extracellular blood stage parasite. Among them is the merozoite surface protein-1 (MSP-1) that is currently used as a major component of anti-blood stage human malaria vaccines ,13. The equences ,15. The nd RO33) . The MSPMSP-142) ,19. Thes surface .msp-1 gene (blocks 2 and 17) have been identified to be prime targets of protective immunity. Previous studies have indicated that natural clinical immunity against blood malaria infections targets the N-terminal fragments encoded by block 2 of msp-1 gene in an allelic-type specific manner [19, encoded by block 17 of the msp-1 gene, could also inhibit erythrocyte invasion by preventing the secondary processing that released MSP-119 from the rest of the MSP-1 complex [19 have been detected in the sera from individuals living in areas that the highly endemic for malaria and were associated with protection from clinical malaria [19-based vaccines are currently in Phase I and Phase II clinical trials in humans and so far have been shown to be safe and immunogenic [Two coding regions of the c manner -24. Natu complex ,25. Inva malaria -33. As munogenic -36.P. falciparum msp-1 gene block 17 sequences has been fairly extensively researched and they have been classified into at least 10 MSP-119 haplotypes in natural populations of P. falciparum[19 haplotypes among different parasite populations revealed that the populations of P. falciparum are genetically diverse [PfMSP-119 haplotypes can also be utilized to infer the population structure of P. falciparum parasites. A recent study indicated the subdivision of two P. falciparum populations in China according to their variations in the distribution patterns of MSP-119 haplotypes, where the parasite populations in Yunnan (South China) appeared to be more closely related to the parasite populations in Southeast Asia (Thailand and Vietnam) than those in Hainan Island located on the South China Sea [Polymorphism in the alciparum-51. Of t diverse , consist diverse ,53. In ahina Sea . The shahina Sea .P. falciparum has been reported in Southeast Asia and has drawn the global attention to the need for improvement in malaria intervention [P. falciparum at the Thailand-Cambodian border in 2008 [P. falciparum isolates at these localities. In Thailand, much of the current knowledge of the msp-1 sequence diversity originated from the analyses of a single parasite population at Mea Sod, Tak province at the Thai-Myanmar border [msp-1 gene block 17 in five geographically isolated populations of P. falciparum in Thailand was performed in order to infer the genetic structure of the parasite populations from the distribution patterns of their MSP-119 haplotypes. The outcome of this work will shed light on the nature and genetic relationship within and between malaria populations and offer a chance to better understand the parasite’s evolution, which will be important for establishing an effective strategy for malaria control and intervention.Recently, the emergence of artemisinin resistance in rvention ,56. Afte in 2008 , artemis in 2008 . Becauser border ,39,51, bP. falciparum were collected from five localities (1-5), each in a different province, close to the borders of Thailand and three neighbouring countries. These were (1) Ubon Ratchathani, located at the Thailand-Laos border, (2) Trat, located at the Thailand-Cambodia border, and (3–5) Ranong, Kanchanaburi and Mae Hong Son, located at the Thailand-Myanmar border. The subjects were confirmed for P. falciparum mono-infection by microscopic examination of Giemsa-stained blood smears. The subjects were recruited in three transmission seasons between: (1) Jun 2002–Oct 2003, (2) Jun 2004–Dec 2005 but also including one subject in 2006 and (3) Jan 2008–Dec 2008  O2 and 90% (v/v) N2. The medium was changed daily, and the parasites were allowed to grow to a parasitaemia level of 5-10% and then harvested prior to genomic DNA preparation.Field isolates of calities -5, each eviously . Of theseviously   saponin solution in phosphate buffered saline  to release the blood stage malaria parasites from the human erythrocytes. The parasite pellets were collected by centrifugation at 10,000 g for 10 min and washed twice in PBS. The parasites were lysed in a lysis solution  sodium dodecyl sulfate, pH 8.0) containing 2 mg/mL proteinase K. The suspension was sequentially treated with an equal volume of phenol (pH 8.0), phenol/chloroform/isoamyl alcohol (25:24:1 (v/v/v), pH 8.0) and chloroform, harvesting the aqueous phase each time. The genomic DNA was then recovered by ethanol precipitation in the presence of 0.3 M sodium acetate and later dissolved in standard TE buffer  and stored at -20°C prior to PCR amplification.Procedures for preparation of the blood stage malaria parasites and genomic DNA were performed as previously described ,61. Briemsp-1 gene block 17 was amplified by the polymerase chain reaction (PCR) with the MM1/17F (5′-TCACAACACCAATGCGTAAAA-3′) and MM1/17R (5′-GAGTATTAATAAGAATGATATTCCTAAG-3′) primer pair, which correspond to nucleotide positions 4,825–4,845 and 5,136–5,109 of the coding sequence of the msp-1 gene of the P. falciparum strain 3D7  of iTaq™-DNA polymerase enzyme in 1X iTaq PCR buffer . Thermal cycling was performed with an optimized profile of an initial denaturation at 95°C for 5 min, followed by 30 cycles of 95°C for 40 s, 53°C for 40 s and 72°C for 40 s, and subsequently a final extension at 72°C for 5 min. PCR products were analysed by standard gel electrophoresis, stained with ethidium bromide and visualized by UV transillumination. All 63 samples generated single amplicons with an expected size of 220 bp, and were subjected to direct Sanger DNA sequencing in both the forward and reverse directions to ensure the accuracy of obtained sequences. Sequencing reactions were performed using the BigDye Terminator v1.1 kit  with an ABI3730 DNA analyser. DNA sequences were manually edited using Bioedit 7.0.0 software and aligned using the MUSCLE Sequence Alignment algorithm in the MEGA 5.2 program [A DNA fragment corresponding to the 1352134, ). The PCPfMSP-119 from the PCR sequenced genomic fragments were aligned with the corresponding sequence of the P. falciparum genome reference strain 3D7 using the MUSCLE Sequence Alignment algorithms as above. The extent of genetic diversity was estimated by the number and ratio of MSP-119 haplotypes prevalent in the populations. Divergences in the distribution pattern of PfMSP-119 haplotypes between two parasite populations was tested for using the Wright’s fixation index (Fst) with Arlequin suite version 3.5 [D*and Fu & Li’s F*indices) implement in the DnaSP 5.1 program [p value was less than 0.05 (p < 0.05).The predicted amino acid sequences of the C-terminal fragment of sion 3.5 . In addi program . ResultsP. falciparum msp-1 gene block 17  at nucleotide positions 4990 G/C, 5132 C/A, 5159 G/A, 5161 G/A and 5206 C/T were detected / Q (CAA), 1691 T (ACA)/K (AAA), 1700 S (AGC)/N (AAC), 1701 G (GGA)/ R (AGA) and 1716 L (CTT)/ F (TTT) . The frequency of amino acid substitutions at each site was E1664Q , T1691K , S1700N , R1701G  and L1716F . The PfMSP-119 fragment contained nine conserved Cys residues. The first amino acid substitution (E1664Q) was located between the second and the third Cys residues in the first putative EGF-like motif, while the other substitutions occurred in the second EGF-like motif of PfMSP-119  and Q/KNG/F, all of which have been reported previously [Nucleotide sequences of be et al.) encodiner et al.) were obeviously -51.PfMSP-119 haplotypes in the five geographical locations of Mae Hong Son (n = 9), Kanchanaburi (n = 15) and Ranong (n = 14), located at the Thailand-Myanmar border, Ubon Ratchathani (n = 12) located at the Thailand-Laos border, and Trat (n = 11), located at the Thailand-Cambodia border  identified. In these three sites, the E/KNG/L, Q/KNG/L and E/TSR/L haplotypes constituted more than 80% of the total haplotypes investigated. Thus, the level of genetic diversity varied according to the geographical location of the parasite populations, which might reflect variation in the transmission intensity and multiplicity of human malaria infections between sampling sites [These results also revealed that there were slight differences in the levels of genetic diversity, based upon the number of s Figure . P. falcng sites .st). In this test, the distribution patterns of PfMSP-119 haplotypes in Tak province (collected in 1995), which is located between Kanchanaburi (the region in which the parasite genetic diversity was highest) and Mae Hong Son (the region in which the parasite genetic diversity was lowest), from two previous studies were also included. One consisted of 8 E/TSR/L, 25 E/KNG/L, 12 Q/KNG/L and 3 Q/KNG/F haplotypes  [st values from the pairs of all the six parasite populations were low and non-significant (p > 0.05). Accordingly, the P. falciparum parasite populations (or at least the PfMSP-119) circulating in this region are genetically homogeneous, suggesting gene flow between allopatric populations of the malaria parasites near the borders of Thailand with Myanmar, Laos and Cambodia.To further determine whether the geographically isolated parasite populations are genetically isolated, pair-wise inter-population comparisons were performed for each parasite population using the Wright’s fixation index  , while tal = 48) . As show19 haplotypes in three transmission seasons is shown in Figure msp-1 sequences analysed, 21 sequences were obtained in a 15-month period between Jun 2002–Oct 2003 and contained three haplotypes ; 25 sequences were obtained in an 18-month period between Jun 2004–Dec 2005 (plus one sample collected in early 2006) and contained all five haplotypes; and 15 sequences were collected in a 12-month period between Jan–Dec 2008. As seen in Figure The prevalence of MSP-1PfMSP-119 haplotypes between years was compared using the Wright’s fixation index (Fst), and this revealed low and non-significant Fst values (p > 0.05)  Table , suggest in 1995 ,39) was P. falciparum in Thailand were found to not be significantly heterogeneous at the PfMSP-119 locus, suggesting that the different PfMSP-119 haplotypes may be maintained by balancing selection. Therefore, intra-population based analyses  were used to evaluate the selection signature on the PfMSP-119 locus. No significant positive D value  was found for the entire block 17 coding region. In addition, although a sliding window analyses of Tajima’s D test, Fu & Li’s D* test and Fu & Li’s F* test revealed positive D values for the EGF-1 and EGF-2 domains, they were not statistically significant  was not analysed because the number of synonymous differences (Ks) was too low (zero). Overall, the polymorphisms at the C-terminal fragment of the msp-1 gene block 17 were less likely to be under strong immune selection, suggesting a functional constraint that limits the allelic diversity of the sequences. Taken together, the polymorphisms detected in PfMSP-119 were neutral and highly conserved in the geographically isolated, but genetically homogenous P. falciparum populations in Thailand.The geographically separate and so potentially isolated populations of PfMSP-1 is considered the major target component for the developmetn of human blood stage malaria vaccines [msp-1 gene block 17 that encodes PfMSP-119 has gained considerable attention in the global malaria research community, leading to the identification of its polymorphism level and haplotypes [PfMSP-119 sequences have been primarily classified into two prototypic allelic groups: (1) the MAD20 prototypic allelic group (E/TSR/L haplotype) and (2) the Wellcome prototype allelic group (Q/KNG/L haplotype) [P. falciparum worldwide [PfMSP-119 sequence diversity in Thailand was previously conducted in a single locality in Tak, located on the western border of Thailand and identified at least five distinct haplotypes  [PfMSP-119 sequence variation is common in other populations of P. falciparum in Thailand or whether the distribution pattern of these haplotypes is conserved. This question formed the basis of the present study, which aimed to screen for polymorphism and evaluate the haplotypes of the msp-1 gene block 17 from different geographical regions in Thailand.The 19-kDa antigenic domain of vaccines . Therefoplotypes -51. PfMSplotype) . In addiorldwide -51. Thesorldwide . The anaQ/KNG/F) . Howevermsp-1 gene block 17 was performed on 61 Thai P. falciparum isolates originating from five endemic areas; Mea Hong Son, Kanchanaburi and Ranong, along the borders of Thailand and Myanmar, Trat at the border of Thailand and Cambodia, and Ubon Ratchathani, at the border of Thailand and Laos. These areas were among the top 15 provinces with the highest numbers of P. falciparum malaria cases in Thailand [PfMSP-119 sequences  previously identified in Tak [msp-1 gene block 17 in Thai P. falciparum populations was limited.Nucleotide sequence analysis of the Thailand . The fivd in Tak ,39,51 and in Tak ,40-50, wmsp-1 sequences in Thai populations varied according to the geographical locations. The lowest genetic diversity was detected in P. falciparum populations in Mae Hong Son and Ubon Ratchathani, with only three PfMSP-119 haplotypes of being found, compared to four and five in Kanchanaburi and Ranong, respectively. Variation in the levels of genetic diversity in geographically isolated populations may be due to variation in the multiplicity of infection, malaria incidence, transmission intensity and levels of gene flow between the parasite populations [P. falciparum were highly prevalent at the Thailand-Myanmar border region in Kanchanaburi compared to at the Thailand-Laos borders [msp-1 alleles in the mosquito and subsequently a high diversity of msp-1 would be maintained in Thai populations [The levels of genetic diversity of the ulations ,66. Malaulations . In addi borders . Elevate19 haplotypes in Thai populations in the present study were compared with results from a previous study based upon microsatellite typing [When the diversity and distribution patterns of MSP-1e typing , no genePfMSP-119 in populations in Thailand, as detected using F-statistics (Fst), revealed no significant differences between the P. falciparum populations located at the Thai borders with Myanmar, Laos and Cambodia. Accordingly, there may be no barriers that limit gene flow between geographically isolated P. falciparum populations in Thailand. This would likely reflect that there are numerous host (human) movements within and into Thailand from neighboring countries from immigrants who live across the borders [P. falciparum occurred around the Thai-Cambodia border in 2009 and now has recently been found at the Thai-Myanmar border in 2011. The results from this study suggest that it is likely that the artemisinin resistant strains of P. falciparum could also spread to other regions of Thailand.The level of genetic differentiation of  borders ,70. For  borders . A cross borders . The maj19 haplotypes in four transmission seasons (from 1995 to 2008) revealed that the overall frequency of individual PfMSP-119 haplotypes was relatively stable, although the two major haplotypes (E/KNG/L and Q/KNG/L) inversely fluctuated over time. This result was also seen in a previous longitudinal survey in Mali [PfMSP-119 haplotypes in endemic regions worldwide indicated a dramatic distribution pattern and frequency of PfMSP-119 haplotypes. The high prevalence of the E/KNG/L and Q/KNG/L haplotypes reported in Thailand has also been reported in the Yunan and Hainan provinces in China and Vietnam [P. falciparum populations in the Greater Mekong Sub-region (GMS)  could be genetically related. Whether the parasite populations in GMS are genetically homogeneous will need to be evaluated by large-scale genotyping approaches using genome-wide genetic markers  appeared to be the dominant haplotypes in other regions, such as South Asia (India and Iran), South America (Brazil and Peru), Vanatu and the Solomon Islands [PfMSP-119 haplotypes, this study could help inform choices about which PfMSP-119 haplotypes to formulate future vaccines from and to allow a more accurate interpretation of the efficacy of current formulations of PfMSP-119 based vaccines being tested in clinical trials. Currently, MSP-1 sequences from P. falciparum K1 (Q/KNG/L) are being formulated as components of the multivalent blood stage vaccine PfCP-2.9 [PfCP-2.9 vaccine would be advantageous for populations in the GMS and in Africa, but not in South America and the Asian Pacific Islands.The longitudinal survey of MSP-1 in Mali . Compari Vietnam ,46, suggNPs, see ). Similaand Mali -44. In c Islands -41,50. BPfCP-2.9 . If the 19 sequences may be due to positive selection or balancing selection, as has been demonstrated for other regions of MSP-1 [D* test and Fu & Li’s F* test all failed to reveal any statistically significant evidence for strong positive or balancing selection of the C-terminal fragments of PfMSP-119 in these Thai samples. The most probable explanation for the limited diversity in PfMSP-119 is the functional constraint of the protein. MSP-1 is considered to play an important role in erythrocyte entry by the merozoite [PfMSP-1 to PfMSP-119 is a prerequisite for merozoite invasion [PfMSP-119 fragment possesses different epitopes and variation in the motifs alters the conformational structure markedly [19 amino acid sequences.It is possible that the limited allelic diversity in the MSP-1of MSP-1 . Howevererozoite . Secondainvasion . The PfMmarkedly ,75. It mmarkedly . Thus, sPfMSP-119 polymorphisms in natural populations of P. falciparum in Thailand. The data supports the view that P. falciparum populations in the GMS region are genetically diverse. Analysis of the spatiotemporal patterns of MSP-1 haplotypes indicated that E/KNG/L and Q/KNG/L were the major haplotypes in Thailand, which were also highly prevalent in populations from other Southeast Asian countries, China and Africa. This study provides the basis for the accurate measurement and interpretation of the population structure and dynamics of malaria parasites that is critical for monitoring the population responses to MSP-1 based vaccines in clinical trials.This study extends the understanding and current knowledge of the variation and prevalence of The authors declare that they have no competing interests.PS carried out the molecular genetic work, interpretation of data and drafted the manuscript. SP performed data analysis and wrote the manuscript. NS and AS were responsible for parasite cultivation. MK and RB carried out the statistical analysis and guidance for data interpretation. PH provided conceptual framework for the project, guidance for the interpretation of data, participated in the manuscript preparation, revision and coordination. All authors read and approved the final manuscript.The origin and date of collection of the human malaria parasite Plasmodium falciparum used in the present study. Asterisks (*) indicate samples collected between 2002 and 2006 that were previously genotyped by 12 microsatellite loci and confirmed to be independent clones [t clones .Click here for fileNucleotide sequence alignment of the Plasmodium falciparum merozoite surface protein-1 (msp-1) gene block 17. Data shows the representative five variants  of the 61 samples analysed. The nucleotide positions are presented on top of the P. falciparum 3D7 sequence . Nucleo1352134; . AsterisClick here for file


Pseudorasbora parva), was introduced into Europe in the 1960s. A highly invasive freshwater fish, it is currently found in at least 32 countries outside its native range. Here we analyse a 700 base pair fragment of the mitochondrial cytochrome b gene to examine different models of colonisation and spread within the invasive range, and to investigate the factors that may have contributed to their invasion success. Haplotype and nucleotide diversity of the introduced populations from continental Europe was higher than that of the native populations, although two recently introduced populations from the British Isles showed low levels of variability. Based on coalescent theory, all introduced and some native populations showed a relative excess of nucleotide diversity compared to haplotype diversity. This suggests that these populations are not in mutation-drift equilibrium, but rather that the relative inflated level of nucleotide diversity is consistent with recent admixture. This study elucidates the colonisation patterns of P. parva in Europe and provides an evolutionary framework of their invasion. It supports the hypothesis that their European colonisation was initiated by their introduction to a single location or small geographic area with subsequent complex pattern of spread including both long distance and stepping-stone dispersal. Furthermore, it was preceded by, or associated with, the admixture of genetically diverse source populations that may have augmented its invasive-potential.The Asian cyprinid fish, the topmouth gudgeon ( Population genetic studies of invasive species have become an instrumental component in the study of biological invasions Pseudorasbora parva . This small cyprinid species originating from East Asia was accidentally introduced into Europe in the 1960s in several countries around the Black Sea as part of contingents of Chinese carps for aquaculture One of the most compelling fish invasions in the world today is arguably the topmouth gudgeon P. parva invasion has been traced from the initial point of introduction towards the northern and western parts of Europe, as well as the south towards Turkey and Iran et al. (2010) Gambusia holbrooki) was introduced into Europe multiple times from USA. Some P. parva populations have also been identified as healthy carriers of pathogens, such as Anguillicola crassusSphaerothecum destruensWhilst this P. parva appear to be a model fish well suited to studying the evolution of ecologically significant traits, disease resistance and the role of genetic diversity in establishment success. Thus, we perform a population genetic analysis of P. parva across their native and introduced ranges in order to test different models of colonisation and to determine levels of genetic variation across the invasive range of the species . This will provide a first population genetic framework for further evolutionary studies on the species.Consequently, Samples were collected at a total of 22 sites, 14 in Europe and 8 in Asia . Sample P. parva in Europe: i) ‘multiple source-sink’ model where several independent introduction events from genetically differentiated native source populations to separate European locations would have occurred without involving admixture; ii) ‘stepping-stone’ model Thus, the aim was to test three non-mutually exclusive models that were proposed to explain the spread of F-statistics were used to quantify the degree of differentiation between populations and nucleotide diversity, and haplotype diversity at a standard sample size was used to estimate within population variability. These analyses were complemented by coalescent simulations and a Bayesian estimation of effective population size. The results were then compared with theoretical expectations from the various models and scenarios:Population genetic theory predicts that these demographic processes will result in different patterns of genetic population structure and therefore molecular approaches can be used to test the likelihood of alternative models. Therefore a number of phylogenetic and population genetic analyses were carried out in order to test the results against the theoretical expectations for the scenarios outlined above. Note that some of these tests assume that a relatively clear phylogeographic subdivision exists in the native range. Therefore the first step was to carry out a network analysis in order test this assumption. Genetic distances and ‘multiple-source-sink’ model: genetic differentiation among invasive populations is high and similar to that found in the native range;‘stepping stone’ model: genetic differentiation in the invasive range is lower than that in the native range, and there is a significant pattern of isolation-by-distance;‘long-distance’ model: genetic differentiation in the invasive range is lower than that in the native range, and there is no pattern of isolation-by-distance;‘genetic bottleneck’ scenario: genetic diversity of invasive populations, in particular haplotype diversity, is lower than that of the source populations; and‘genetic admixture’: genetic scenario: genetic variation expressed in nucleotide diversity is higher than that of the source population. Furthermore, recent admixture increases the nucleotide diversity above that expected under equilibrium conditions.cytochrome b gene was amplified applying standard PCR techniques using Verity Thermal Cycler. Primers L15267 and_H15891Ph, previously described by Briolay et al. (1998) 4, 1.5 µL 100 mM dNTPs, 0.4 µL 50 mM MgCl2, 0.075 µL Taq polymerase, 0.3 µL each of primers L15267 and H15891Ph, 9.425 µL PCR water and 1.5 µL of template DNA. Cycle parameters were as follows: 2 min at 95°C; 45 s at 94°C, 45 s at 48°C, 1 min at 72°C; 10 min at 72°C. PCR products were directly sequenced in both directions using the PCR primers by Macrogen Inc. Forward and reverse sequences were aligned and edited using CodonCode Aligner The fish were collected and stored in 98% ethanol. Genomic DNA was extracted from the caudal fin tissue using the HotShot method Phylogenetic relationships of haplotypes were reconstructed using the maximum composite likelihood method P. parva individuals et al. (2010) et al (2010) In order to increase the geographic coverage, GenBank sequences from five P. parva.A haplotype network was constructed using a median-joining algorithm in Network v. 4.5.10 Gobio gobio. Coalescent based simulations as implemented in DNaSP were used to predict the expected relationship between haplotype diversity (H) and nucleotide diversity (π) under drift-mutation equilibrium and constant population size DNaSP v. 4.5 FST and DXY   using a Mantel test (1000 permutations) as implemented in the software IBDWS v. 3.16 FST values were log-transformed to achieve a normal distribution. Second, a general linear model (GLM) was used to test the relationship between the geographic distance and genetic differentiation from the putative site of introduction. Third, a GLM was used to test the relationship the geographic distance from the putative site of introduction and genetic diversity of populations. Under a ‘stepping stone’ model, genetic diversity is expected to decrease with geographic distance to the original site of introduction, and hence, the genetic distance is expected to increase. The putative site of introduction was Nucet-Dombovita, Romania in the early 1960s Pairwise genetic differentiation among samples was computed as et al. 2008). DIY ABC is a computationally intensive approach and therefore only three simplified scenarios where chosen, which appeared most feasible after the initial population genetic analysis. An explicit rationale for choosing specific models will therefore be given in the 4 individuals, covering the full range of biologically feasible values and the Kimura 2 parameters (1980) 6 datasets were simulated with the parameter values drawn from the prior distribution  P. parva haplotypes (sequences divergence ∼5–6%). One highly divergent haplotype found in the invasive Polish populations clustered closely to a sequence of G. gobio. This haplotype and the Japanese haplotypes were therefore not included in the network analysis. Thirteen haplotypes were found in the invasive populations, five of which were found in more than one invasive population and will be subsequently referred to as common haplotypes. Three of the common haplotypes and two of the rare haplotypes were also found in at least one native population. Three main lineages of P. parva haplotypes can be recognised outside of Japan  et al (2010) are restricted to lineage 1 and these populations also share a common haplotype with most introduced populations. Furthermore the Pearl River samples from Liu et al (2010) A total of 30 haplotypes were identified using 310 sequences from 8 native and 14 introduced populations . The phyof Japan  and 4; aH = 0.34, 0.76; π = 0.006, 0.010), the populations from other drainages of mainland China and Taiwan where much less variable .  and invasive populations  was not significantly different . However, a more detailed analysis revealed that there were significant differences among certain groups of native and invasive populations. When the recently introduced UK populations were excluded from the analysis, both haplotype diversity and nucleotide diversity and were significantly higher in the invasive populations than native .After bootstrapping to account for differences in sample size, the genetic diversity of the native populations varied widely among geographical regions. Whereas the two populations of the Hai He drainage showed relatively high diversity (–0.002). . GeneticNe), and we compared this to the empirical data  and haplotype (H) diversity expected in a population that is in mutation-drift equilibrium with constant effective population size , using Migrate-n, differ by several orders of magnitude among native populations, ranging from 0.00006 for population TT to 0.01847 for population CRH  ranged from 0 to 0.02715 and pairwise genetic differentiation (FST) ranged from 0 to 1  was only marginally lower than that among native populations (median DXY = 0.012), (P>0.3). Similarly, the native-invasive pairwise comparison expressed in DXY (median DXY = 0.012)  nor from that among invasive populations (P>0.3).Pairwise genetic distance (FST) was considerably lower among the invasive populations (median FST = 0.21) than among the native populations (median FST = 0.58) (P<0.001). Furthermore, the FST between the native-invasive pairwise comparison (median FST = 0.53) was not significantly different from the genetic distance among native populations (P = 0.27), but it was significantly higher than that among invasive populations (P<0.001). This result is inconsistent with the ‘multiple-source-sink’ model, and supports both the ‘long-distance’ and ‘stepping stone’ models.In contrast, genetic differentiation . However, when the recently introduced English (EB) and Welsh (SWS) populations were removed, there was no significant genetic isolation-by-distance relationship . Regression analysis revealed no significant relationship between distance from source and genetic differentiation  or genetic diversity  respectively. This reveals that the English and Welsh populations are bottlenecked, resulting in the spurious isolation-by-distance signal obtained when including these samples in the Mantel test. However, across continental Europe, topmouth gudgeon does not show evidence of isolation-by-distance and so we conclude that the ‘long-distance’ model is most consistent with these data.A Mantel-test showed a significant relationship between genetic and geographic distance among the European populations  popScenario 1: The source of the invasive population (Pop 3) is the admixed Chinese population (Pop 2) which originates from an admixture of Pop 1 and a ghost population which split from Pop 4 at time t3.Scenario 2: The invasive population (Pop 3) is a result of an admixture between pop 1 and an unsampled ghost population which split from pop 4 at time t4. Pop 2 evolved as in scenario 1. Scenario 3: same as Scenario 2 but the admixture of the pop 1 and GH2 populations took place before the admixture of Pop 1 and GH1.t1) agrees with historical records  = 30–60). Full table of posterior distributions are given in the Appendix, A comparison of posterior probabilities of the three scenarios using local linear regression , Table 3P. parva) in the native range, two of which contributed to the colonisation of Europe; ii) most invasive populations have a higher genetic diversity than their native counterparts and a higher genetic diversity than expected under equilibrium conditions; iii) most native populations have a low genetic diversity typical for riverine fishes, an exception being samples from the Hai He river system which showed very high levels of genetic diversity, which under equilibrium conditions predict extremely high effective population sizes; and iv) the differentiation among invasive populations is much lower than among native populations.The outputs of these analyses revealed that i) there are three evolutionary lineages of the topmouth gudgeon  are several orders of magnitude lower; for example, other cyprinid fishes range around 500 to 1000 individuals The existence of four highly divergent haplotype lineages indicates a long isolation among geographic populations of Hemibarbus lameoSalanx ariakensisMugil cephalus) It is possible that the geographic area around the Hai He River basin represents a natural secondary contact zone between divergent phylogeographic lineages. Although the literature on this subject is relatively limited, it seems clear that high tectonic activity and sea level changes during the Pleistocene have created a complex phylogeographic pattern with little concordance among species et al (2010b) P. parva translocations have occurred in China prior to introduction in Europe. These cyprinid translocations coincided with the end of the Chinese civil war and the need for additional sources of animal proteins Alternatively, recent human translocations associated with aquaculture might have caused such an admixture; this may not be considered surprising given that freshwater aquaculture in this area of China is intense Our data showed that all invasive populations shared at least one of the four common haplotypes and that levels of genetic differentiation were low compared to native populations. Such a pattern would be expected if the invasive populations had spread from a single source. The alternative explanation of high levels of gene flow among initially differentiated invasive populations is extremely unlikely given that this would involve regular gene flow across watersheds. Therefore we reject the possibility that different European populations were independently colonised from divergent source populations (‘multiple-source-sink model’). However, a number of results indicated that the introduced populations represented an admixture of divergent source populations. First, the levels of nucleotide diversity of populations in continental Europe were, on average, higher when compared with native populations. Second, the nucleotide diversity of invasive populations was higher than expected from coalescent theory. Finally, the population structure analysis showed that the majority of the invasive populations and two (admixed) native populations occupied central positions in the MDS plot between divergent native populations. The main exceptions to this pattern were the two British populations, which showed a low genetic diversity and high levels of differentiation from other invasive populations, but were fixed for one or two common invasive haplotypes. These populations were founded relatively recently, most likely from sources in Germany P. parva were initially translocated to Romania and Hungary from the Yangtze River at Wuhan which is geographically close to our CY sample Although the sampling coverage in the native range was not comprehensive enough to pinpoint the exact location(s) which acted as a source of invasive European populations, some more general inferences can be drawn. The data outputs suggest that the invasive populations originate from mainland China rather than Taiwan or Japan. The haplotype distribution of invasive populations and populations from northern China raise the possibility that this area is the source of introduction. However, the DIY ABC analysis suggests that this is much less likely than a scenario where the invasive populations in Europe originate from an admixture between populations from lineage 1 (such as the Yangtze) and an unsampled population from lineage 2. Anecdotal reports suggest that P. parva in Europe. The weak but significant pattern of IBD across the whole data set was mainly caused by the highly bottlenecked British populations at the margin of the distribution. After excluding these two populations, none of the tests was significant. The ‘stepping-stone’ colonisation is therefore not likely to be the predominant process for the spread of the species in Europe. We suggest that long-distance dispersal must have played a major role, possibly as a consequence of fish transport associated with aquaculture. This is in agreement with Gozlan et al. (2010b) P. parva dispersal model showing dispersal distances of approximately 250 km from the 1970s to the end of the 1990s, followed by shorter dispersal of 20 km on average since 2000. Additional genetic analyses at the country level with greater resolution of the geographical pattern of haplotypes are likely to confirm this two-stepped invasion process.The isolation-by-distance analysis indicated that both ‘stepping-stone’ and ‘long-distance’ processes might have contributed to the spread of G. gobio. The genus Gobio belongs to the same cyprinid subfamily as Pseudorasbora, the Gobioninae and is a close European relative of P. parvaP. parva is able to hybridise with at least one native European species. This raises further concerns about the threat which P. parva poses to native European fish fauna and corroborates experimental evidence that hybrids between P. parva and another European cyprinid Leucaspius delineatus are possible A single individual from the Polish population contained a highly divergent haplotype. The phylogenetic analysis revealed that the sequence is very closely related to a published GenBank sequence of P. parva resulted from accidental releases from a human-induced faunal translocation P. parva, that potentially will lead to devastating consequences for native fishes The European introduction of Table S1FST values of pairwise genetic comparisons between all populations.Matrix of (DOC)Click here for additional data file.Table S2DXY values of pairwise genetic comparisons between all populations.Matrix of (DOC)Click here for additional data file.Table S3Set of prior distributions based on historical and demographic data. Posterior probabilities of the selected scenarios in DIY ABC: mean, median and mode values and four quantiles of the posterior distribution. Prior and posterior values of mutation rate used in DIY ABC.(DOC)Click here for additional data file.


Arabidopsis thaliana diversity across its geographic range provides the bases for elucidating the demographic history of this model plant. Despite the unique A. thaliana genomic resources currently available, its history in North Africa, the extreme southern limit in the biodiversity hotspot of the Mediterranean Basin, remains virtually unknown.Deciphering the genetic structure of A. thaliana evolutionary history in North Africa, we have analysed the genetic diversity and structure of 151 individuals collected from 20 populations distributed across Morocco. Genotyping of 249 genome-wide SNPs indicated that Morocco contains substantially lower diversity than most analyzed world regions. However, IBD, STRUCTURE and PCA clustering analyses showed that genetic variation is strongly geographically structured. We also determined the genetic relationships between Morocco and the closest European region, the Iberian Peninsula, by analyses of 201 populations from both regions genotyped with the same SNPs. These analyses detected four genetic groups, but all Moroccan accessions belonged to a common Iberian/Moroccan cluster that appeared highly differentiated from the remaining groups. Thus, we identified a genetic lineage with an isolated demographic history in the south-western Mediterranean region. The existence of this lineage was further supported by the study of several flowering genes and traits, which also found Moroccan accessions similar to the same Iberian group. Nevertheless, genetic diversity for neutral SNPs and flowering genes was higher in Moroccan than in Iberian populations of this lineage. Furthermore, we analyzed the genetic relationships between Morocco and other world regions by joint analyses of a worldwide collection of 337 accessions, which detected an additional weak relationship between North Africa and Asia.To approach A. thaliana in Morocco show that North Africa is part of the species native range and support the occurrence of a glacial refugium in the Atlas Mountains. In addition, the identification of a genetic lineage specific of Morocco and the Iberian Peninsula indicates that the Strait of Gibraltar has been an A. thaliana migration route between Europe and Africa. Finally, the genetic relationship between Morocco and Asia suggests another migration route connecting north-western Africa and Asia.The patterns of genetic diversity and structure of  Arabidopsis thaliana is a wild, annual, self-fertilizing plant with a broad geographic range as a native species in the Eurasian continent [A. thaliana diversity across its geographic range is now a major aim because it explains its current ecological distribution, it reflects its demographic history, and it enables the precise design and analysis of experimental populations used to determine the molecular mechanisms of adaptive traits [ontinent ,2. In thontinent  but alsoontinent -6. Decipe traits .A. thaliana from different world regions have been collected. Studies of the genetic diversity and structure, at global (worldwide) and regional scales, have proposed several major events in A. thaliana demographic history in Europe [A. thaliana centre of origin [A. thaliana history in East Asia and in regions outside the Eurasian continent. Nuclear and chloroplast analyses of A. thaliana populations from China have also shown substantial genetic variation and geographic structure, hence suggesting a rapid A. thaliana west–east expansion from Central Asia [Currently, more than 6000 wild genotypes (accessions) of n Europe -14. In pn Europe ,16. Furtf origin . Howeverf origin ,12,16,17f origin -20. In aral Asia ,22. Moreral Asia ,23,24.A. thaliana, the scarcity of accessions from many regions located outside of Europe has hampered our knowledge of its demographic history in Africa and Asia. For instance, the centre of origin of A. thaliana is still unknown, since conclusive evidence for one of the two classical hypothetical locations, Europe-North Africa and Middle Asia, requires further samples and studies from those regions [A. thaliana distribution in the Mediterranean Basin, and it is assumed to be part of the native range [A. thaliana in this region remains virtually ignored since less than a handful of African accessions have been studied until now [Despite the unique resources available for  regions ,15. In pve range . Howeverntil now ,14,15.A. thaliana, have focused in the northern limit of species from the North hemisphere [The relevance of northern Africa in structuring biodiversity, at global and regional scales, has been well documented for several plant species (reviewed in ). This imisphere -29. TherA. thaliana in North Africa by developing a new collection of accessions derived from populations distributed throughout the species range in Morocco. In particular, we aim to determine, first, if North Africa has been recently colonized, or if it might contain genetically isolated and structured populations supporting the occurrence of A. thaliana as a native species. Second, we aim to detect genetic relationships between Morocco and other geographic regions, which might provide information about the demographic history in North Africa. To approach these questions, we have analysed this Moroccan collection together with a collection from the nearest European region, the Iberian Peninsula, and a worldwide collection. Population genetic analyses of genome-wide neutral markers and potentially adaptive flowering genes and traits, demonstrate a shared history in southern Europe and northern Africa. Together, these analyses identified the Strait of Gibraltar as a migration route between Europe and Africa, and suggest that the Atlas area was a Pleistocene glacial refugium for A. thaliana.In this study we have addressed the genetic diversity and structure of A. thaliana in North Africa we sampled 151 individuals in 20 populations distributed across the major mountain ranges of Morocco  and allelic richness (RS) per population, which showed up to a tenfold range of variation among populations. The amount of genetic variation per population displayed a weak geographic pattern since the percentage of polymorphic loci (PL) correlated negatively with latitude , and RS showed similar negative but marginal correlation . In agreement, four geographic subregions established according to latitude differed slightly for PL, RS and HS (0.08 > P > 0.04), populations from the Riff containing less genetic variation than populations from the High Atlas (Table To determine the genetic diversity of FST range: 0.26-1) and only 18.4% occurring within populations (P < 0.001). Mantel tests of pair-wise geographic distances and genetic distances, measured as FST or as percentage of allele differences among populations, detected highly significant correlations  geographic pattern across Morocco.The genetic relationship among Moroccan populations was first analysed by constructing Neighbor-joining (NJ) trees of the 65 haplotypes  analyses. STRUCTURE analysis detected four major clusters that closely corresponded to the four geographic subregions  in agreement with the early distinction of this cluster in the first (K = 2) analysis of STRUCTURE  and low mean allelic differences (12.3%) were estimated between Iberian and Moroccan groups of accessions belonging to cluster 3. Genetic diversity measurements also showed that group 3 contained the lowest amount of variation, although Morocco accessions contained higher diversity than Iberian accessions of this group . Overall, flowering genes showed larger diversity in Iberia than Morocco as estimated by the private allelic richness . This CRY2 haplogroup appeared at very low frequency in Iberia (1.1%) and explained the larger Moroccan CRY2 variation. Therefore, A. thaliana populations from Morocco showed a similar amount and pattern of diversity to the Iberian Peninsula for FLC and PHYC. However, Moroccan populations displayed higher and lower amounts of potentially functional diversity at CRY2 and FRI, respectively.The genetic diversity of ss Table . Genes ses Table . Howeverco Table . Furtherla Table . InteresFRI loss-of-function alleles and for the major CRY2 haplogroups .The genetic differentiation between Morocco and the Iberian Peninsula was further analysed by measuring three quantitative traits related with flowering induction: vernalization requirement, flowering time and leaf number Figure . Four ofA. thaliana flowering variation has been involved in climatic adaptation [Since aptation  we analyA. thaliana genetic relationships between the south-western Mediterranean region and the rest of world was also studied by analyzing the 201 Moroccan and Iberian accessions together with 136 genotypes representing populations from eleven additional world regions  and the lowest genetic diversity  the wide geographic distribution of this group in Iberia and Morocco; ii) the substantial diversity of group 3 for some potentially adaptive haplogroups described for flowering genes such as CRY2 and PHYC; iii) the higher climatic similarity of Moroccan populations with the geographic range of southern Iberia occupied by genetic group 4 than with that occupied by genetic group 3.The Moroccan/Iberian specific distribution of this lineage provides new insights into ibraltar -34. HoweA. thaliana during the Pleistocene. Consequently, we speculate that this genetic lineage might have colonized northern Morocco and southern Spain during the postglacial period. Latitudinal declines in genetic diversity have been associated with recent northward colonization in temperate species [CRY2, FLC and PHYC) showed higher private allelic richness in Moroccan than Iberian populations assigned to this genetic lineage. Interestingly, it has been described for several forest plants that colonizations from glacial refugia do not necessarily involve a gradual diversity decrease away from the source populations [A. thaliana is also compatible with the large genetic diversity and complex structure found in the Iberian Peninsula, since additional A. thaliana refugia in this region probably accounted for its overall larger intrapopulation and regional variation. In fact, previous analyses of Iberian intrapopulation variation using the same set of SNPs [On the other hand, the strongly isolated and structured distribution of this genetic lineage in the western Mediterranean region suggests that it was maintained as a refugium during the last glaciations. As reported for several plant and animal species , we hypo species ,35-37. Iulations . In cont of SNPs  shows thA. thaliana migration route connecting north-western Africa and Asia. Accordingly, a colonization event of Morocco is hypothesized to have occurred from Asia through this northern African route, which could predate the latest glaciations or could have occurred later during the African humid period documented between 15,000 and 5,000 years BP [The worldwide genetic and geographic structures detected in this study are in agreement with the previously proposed postglacial colonization routes of Europe during the Pleistocene ,12,14,17years BP .A. thaliana displays low genetic diversity in Morocco but the genetic variation is strongly geographically structured, hence supporting a native history in North Africa. In addition, Morocco appears genetically related mainly to the Iberian Peninsula, which indicates a shared demographic history between south-western Europe and North Africa. Our work illustrates the relevance of systematic population genetic analyses of unknown geographic regions to infer the history of A. thaliana. Further comparative studies including underrepresented regions from the rest of North Africa, the Middle East and Central Asia are needed to test the propossed historical hypotheses. In particular, it is likely that the speculated A. thaliana refugia trace back to different Pleistocene dates as consequence of strong climatic fluctuations and occurrence of several species retraction/expansion episodes during this period [A. thaliana’s evolutionary history.The research presented here shows that s period . Eventuas period ,40 will A. thaliana populations were sampled and geo-referenced during 2005–2009, in the main mountain ranges of Morocco .Twenty ariables . Seeds oA. thaliana accessions collected from different previously described populations [A set of 181 Iberian ulations ,30 and ahttp://www.cegen.org). A total of 249 SNPs showing less than 25% missing data (average of 3.9%) were used for population genetic analysis. For the analysis of Morocco genetic diversity, 130 SNPs  segregating among the 151 samples were used after removing monomorphic (106 SNPs) or SNPs missing within entire Moroccan population samples (13 SNPs). For analyses of the 201 Moroccan and Iberian populations, 237 polymorphic SNP loci  were used after removal of monomorphic loci. For analyses of the 337 Moroccan, Iberian, and worldwide populations, all 249 polymorphic SNPs  were included. Genotypic data of all accessions are given in Additional file Accessions were genotyped for a genome-wide set of 343 SNP loci previously selected as frequent polymorphisms in Central Europe , the IbAnalyses were done with all SNPs and with the three sets of markers separately. Similar results were observed for all sets of markers, indicating that there was no substantial effect on our analyses derived from potential SNP bias ascertainment. Therefore, only results obtained with the total SNP set are shown.NH), percentage of polymorphic loci (PL) and gene diversity (HS) using GenAlex v6.41 [RS) per locus and mean private allelic richness (Rp) were estimated with rarefaction to a common sample population size using HP-RARE [Genetic diversity was measured as number of haplotypes per population  using Arlequin v3.5.1.2 with multilocus genotypes , which cIsolation by distance (IBD) was tested using Mantel tests for correlation between paired population matrices of geographic distances and genetic distances using Arlequin v3.5.1.2  with 10,Genetic relationships among genotypes were determined by neighbor-joining (NJ) analysis. Genetic distances were measured as pairwise allele differences among Moroccan haplotypes and 10,000 bootstrap permutations were applied to determine the significance of groups using MEGA v5.05 .K number of ancestral populations and the ancestry membership proportions of each individual in each population, the algorithm was run 20 times for each defined number of groups  from 1 to 10. The number of distinct genetic clusters was determined by testing the differences between the data likelihood for successive K values using Wilcoxon tests for two related samples. The final K number was estimated as the largest K value with significantly higher likelihood than that of K-1 runs (two-sided P < 0.005). This was supported by a high similarity among the ancestry membership matrices from different runs of the same K value (H’ > 0.84). The average symmetric similarity coefficient H’ among runs and the average matrix of ancestry membership proportions, derived from the 20 runs, were calculated with CLUMPP v1 [Discrete clustering of genetic structure was analyzed in three sets of accessions: the 65 different Moroccan haplotypes, the 201 Moroccan and Iberian accessions, and all 337 genotypes of the worldwide collection. Since we were mainly interested in detecting the major genetic groups that were consistently differentiated in each geographic region, we compared two distinct methods. First, genetic structure was inferred using the Bayesian model-based clustering algorithm implemented in STRUCTURE v2.3.3 . Model sLUMPP v1 .H' between the STRUCTURE matrix of majority membership proportions and the matrix of cluster assignments derived after PCA.Population structure was also determined by principal component analysis (PCA) as implemented in the smartpca module of EigenSoft v4.2 . Allele The average membership proportions of each accession to the different genetic groups and the geographic distribution of the majority population assignments of accessions were plotted using R v2.13 and the R package maps .A. thaliana were sequenced in one individual per Moroccan population: FRI and FLC involved in the vernalization pathway; CRY2 and PhyC affecting the photoperiod pathway. These genes have been previously studied in world-wide and Iberian collections, where several haplotypes have been associated with flowering and with climatic factors [FRI gene (3.5 kb), 0.7 kb of the first intron of FLC, 1.5 kb of CRY2 coding region and 0.9 kb of PHYC promoter. Sequencing was done as previously described [FST values using Arlequin v3.5.1.2. Genbank accession numbers of sequences generated in this study are KF275035-KF275114.Four flowering time genes known to contribute to the natural variation for flowering traits in  factors ,54-56. T factors , includiescribed  using anescribed  and nuclescribed . The subQST values estimated from analysis of variance. Between groups (VB) and within groups (VW) variances were estimated by the REML method of variance component analysis, and QST was calculated as VB/(VB + VW).Fifteen plants of one selected accession from each of the 20 Moroccan populations were grown in a glasshouse without vernalization using a randomized experimental design. Flowering initiation was quantified as flowering time (FT) and leaf number (LN) following ref. . FT was The authors declare that they have no competing interests.ACB, JJM-Z, FXP and CA-B conceived the study. AH, FXP, JMM-Z and CA-B collected the samples. BM-V and CA-B generated the laboratory data. ACB, BM-V, FXP and CA-B analysed the data. ACB, FXP and CA-B wrote the paper with the input of all authors. All authors read and approved the final manuscript.Figure S1. Moroccan populations surveyed in this study. Figure S2. Population structure of A. thaliana in Morocco. Figure S3. Isolation by distance structure of A. thaliana in the south-western Mediterranean region. Figure S4. Population structure of A. thaliana in the south-western Mediterranean region. Figure S5. Geographic distribution of 337 A. thaliana populations analysed in this study. Figure S6. Population structure of A. thaliana at a worldwide scale. Figure S7. Genetic and geographic structure of A. thaliana at global scale established by PCA.Click here for fileTable S1. Geographical and ecological information of Moroccan populations surveyed in this study. Table S2. Comparison of STRUCTURE and PCA analyses. Table S3. Genetic differentiation between Morocco and Iberian genetic groups for flowering genes. Table S4. Worldwide A. thaliana accessions analysed in this work. Table S5. SNP markers genotyped in this study. Table S6. Genotypic data of the 468 accessions for the 249 SNPs analysed in this study.Click here for file


Anopheles culicifacies sensu lato is an important vector of malaria in Southeast Asia contributing to almost 70% of malaria cases in India. It exists as morphologically similar sibling species A, B, C, D and E with varied geographical distribution patterns. Vector control measures have been difficult for this important vector as the sibling species have developed varying levels of resistance to the currently used insecticides. In view of the importance of this vector, we developed and validated a set of microsatellite markers and the same were used to analyze the population genetic structure of five different geographical populations of An. culicifacies A.Anopheles culicifacies A samples were collected from different localities across India, and genotyping was performed using eight microsatellite markers on ABI Prism 310 Genetic Analyzer. Several statistical analyses were performed to ascertain the genetic diversity that exists within and between the populations.The markers were found to be moderately polymorphic in the populations. Genetic analysis indicated significant genetic differentiation between the majority of the population pairs analyzed and was not found to be related to the geographical distances between populations.An. culicifacies A. Host feeding and breeding habits of species A suggest that factors other than ecological and geographical barriers were responsible for the genetic differentiation that has been observed between the populations.This is the first and successful attempt to test the microsatellite markers developed for population genetic analysis of  Anopheles culicifacies sensu lato has a wide distribution in India and extends into the west to Ethiopia, Yemen, Iran, Afghanistan and Pakistan, and in the east to Bangladesh, Myanmar, Thailand, Cambodia and Vietnam and is also found in Nepal and southern China in the north and extends to Sri Lanka in the south , , Anophelhe south  and E   . To invewas used .An. culicifacies specimens were collected from five different localities in India that were selected to best represent the diversity of Indian geography and its ecology , expected and observed heterozygosity , deficiency of number of heterozygotes (D), etc. were analyzed  .A total of 104 samples were analyzed for eight microsatellite markers, in order to understand various population genetic parameters that might be influencing the present genetic make-up of ed Table . Among tet al. [The genotype frequencies at several loci did not conform to HWE. For the majority of markers, the observed number of heterozygotes was less than the expected number and this deficiency was statistically significant (P < 0.05). A total of 40 tests  in species A for conformance to HWE at the locus level within the populations were performed. Of these tests, 29 (72.5%) tests showed a significant deviation from HWE in species A. Furthermore, in all the cases heterozygote deficiency was observed  against the natural logarithm of pair-wise geographical distance  than that among the populations (13%) , which exhibited 17 alleles, suggesting that these markers are moderately polymorphic in the populations. Except for AcAVIIB46 and AcAVB93A with the least number of alleles, the other markers did not exhibit all the alleles in any of the five populations. Significant deviations from the Hardy-Weinberg expectations for these markers were observed in the populations studied. The deficiency of heterozygotes observed in these populations could be due to population sub-structuring or due to the presence of null alleles, i.e. mutation/s in the primer sites that may prevent annealing of primers to the DNA samples. This results in either total failure of amplification , or most commonly one strand will be amplified and the sample will be scored as a homozygote, which in fact is a heterozygote [ST values have shown a great genetic differentiation between the pairs of populations analyzed. Low Nm values (<1) between Haryana and Uttar Pradesh, and Rajasthan and Haryana suggest a limited gene flow. The geographical distances of these two population pairs were less than those observed for Gujarat and Haryana, and Gujarat and Karnataka which had maximum Nm values (>5 and 7 respectively).All the eight microsatellite markers examined in rozygote . During rozygote . The D-vAn. culicifacies being a rural mosquito and agriculture being practiced extensively in these areas indicate free availability of cattle for blood feeding. Irrigation channels are preferred breeding habitat for species A. This suggests that other than geographical or ecological, some other barriers are playing a role for differential genetic differentiation levels observed between these populations. Residual sprays with effective insecticides alter and interfere with population sizes. At the time of collection of mosquitoes, no spray operations were going on in sites in Uttar Pradesh, Haryana and Rajasthan states. In Gujarat and Karnataka, malathion was being sprayed in the study sites, which even at present is effective on An. culicifacies populations in these areas. Therefore, the effect of insecticide sprays on the mosquito population sampling cannot be ruled out.Vindhyachal Mountain ranges which pass through central India (Maharashtra and Madhya Pradesh States) separate northern and southern parts of India. Karnataka is located in the southern part of India. There are no known geographical barriers that exist between the other four populations studied. Furthermore, anopheline species in general are known to have limited flight range. For species A, which is predominantly zoophagic (maximum anthropophilic index observed was 3-4%), An. culicifacies using microsatellite markers. Genetic analysis of five different populations of species A was carried out with eight microsatellite markers. FST values indicated significant genetic differentiation between the majorities of the population pairs analyzed. We hope that these results may add a further step in understanding the dynamics of the vector species for planning effective vector control activities based on population genetic structure.This is the first and successful attempt made to study the population genetic structure of The authors declare they have no competing interests.SKS conceived and RK, OPS and SKS planned the study. RK made the field collections and NN identified all mosquito samples used in study. SS was involved in setting up all experiments. OPS and NR performed data analysis. SS, NR and SKS drafted the manuscript. All authors read and approved the final version of the manuscript.


Drosophila subobscura, repeatable across continents. Nevertheless, not much is known about the molecular variation associated with these polymorphisms. We characterized the genetic content of ca. 600 individuals from nine European populations following a latitudinal gradient by analysing 19 microsatellite loci from two autosomes (J and U) and the sex chromosome (A), taking into account their chromosomal inversions. Our results clearly demonstrate the molecular genetic uniformity within a given chromosomal inversion across a large latitudinal gradient, particularly from Groningen (Netherlands) in the north to Málaga (Spain) in the south, experiencing highly diverse environmental conditions. This low genetic differentiation within the same gene arrangement across the nine European populations is consistent with the local adaptation hypothesis for th evolutionof chromosomal polymorphisms. We also show the effective role of chromosomal inversions in maintaining different genetic pools within these inverted genomic regions even in the presence of high gene flow. Inversions represent thus an important barrier to gene flux and can help maintain specific allelic combinations with positive effects on fitness. Consistent patterns of microsatellite allele-inversion linkage disequilibrium particularly in loci within inversions were also observed. Finally, we identified areas within inversions presenting clinal variation that might be under selection.There is increasing evidence regarding the role of chromosomal inversions in relevant biological processes such as local adaptation and speciation. A classic example of the adaptive role of chromosomal polymorphisms is given by the clines of inversion frequencies in  Drosophila subobscura. In this species latitudinal clines for several chromosomal arrangements are well established in Europe D. melanogaster). These latitudinal clines of chromosomal inversion frequencies are widely maintained in natural populations despite the high dispersal ability and strong gene flow between populations of this species Chromosomal inversions are widespread in nature, being present in several animal and plant species. Increasing evidence has been mounting on the role of these mutations in relevant biological processes such as adaptation, speciation and the evolution of sex chromosomes Drosophila pseudoobscura as model, Dobzhansky Several mechanisms have been proposed to explain the maintenance of these polymorphisms in natural populations Drosophila species high levels of linkage disequilibrium (LD) have been found for genes located within inverted regions, with a LD decrease towards the middle of the inversion  in these regions nversion . This refect see .Drosophila melanogaster cline of alpha-Gpdh loci, located inside ln(2L)t, an inversion that also presents a clinal distribution et al.D. americana despite clinal variation in gene arrangements.Despite the abovementioned studies, not much is known about possible regions under selection within inverted segments but see . An impovs. outside inverted regions might help to differentiate between effects of gene flow versus selection on clinal variation Caution is needed when interpreting clinal variation as evidence of selection, due to the confounding effects of gene flow. The comparison of patterns obtained in loci located inside Drosophila subobscura populations, its underlying clinal variation at the molecular level has not yet been addressed. In this study we characterize the molecular genetic content in three chromosomes (two autosomes and the sex chromosome) associated with the most frequent chromosomal inversions along a wide geographic latitudinal cline. This approach may serve as a tool to detect regions under selection and provide insight into the different hypothesis to explain the maintenance and spread of chromosomal inversions.Despite the well-known latitudinal inversion clines in European ca. 600 individuals from nine populations of Drosophila subobscura ranging up to 24° degrees of latitude in the European continent. These populations were characterized in 19 microsatellite loci mapping inside and outside the most frequent arrangements in the J and U autosomes and the A sex chromosome. More specifically we aim to 1) assess differences in genetic content both within and among chromosomal inversions across a wide geographic gradient, 2) search for conserved Linkage Disequilibrium patterns between molecular markers and inversions, 3) test for molecular genetic clinal variation within inversions and finally 4) compare patterns of linkage disequilibrium and clinal variation to identify genomic regions under selection.Our study involves a total of We found clear impact of inversion polymorphisms in the patterns of molecular genetic variation, contributing to the existence of distinct gene pools in inverted regions even in individuals from the same natural location. We also found high levels of genetic differentiation between chromosomal inversions and low differentiation in the same inversion across populations, findings that agree with the local adaptation hypothesis. We also provide evidence on the maintenance of latitudinal clines for inversion frequencies in Western Europe and pinpoint specific regions located within inversions that might be under clinal selection.Drosophila subobscura samples were collected from nine European locations  were individually crossed with virgin females of the some see .ca 600 individuals: dsub11, dsub37, dsub76, dsub05, dsub21, dsub39, dsub70, dsub19 (for the A chromosome); dsub18, dsub59, dsub69, dsub74, dsub62, dsub27 (for the J chromosome); and dsub10, dsub03, dsub42, dsub64, dsub15 (for the U chromosome). These markers were chosen due to their localization on the chromosomes relative to inversions . Analyses were performed taking into account the location of the different loci (inside/outside inverted regions). The definition of loci inside/outside was done by comparison of chromosomes carrying different inversions/arrangements. As such, dsub59 and dsub69 were defined as “loci inside” since their position is inverted in J1 chromosomes relative to JST chromosomes and loci dsub42 and dsub64 were also defined as “inside” since they change position in U1+2 chromosomes relative to UST chromosomes. For the A chromosome, due to the combined effect of non-overlapping inversions which renders difficult the direct comparison explained above, the definition of three regions was used in order to allow a comparison between inverted vs. non-inverted chromosomes , 1.5 µl of primer mix (2 µM of each primer) and 1 µl of DNA. Locus dub64 (chromosome U) was amplified individually with the Amersham Taq polymerase. All reactions were performed on an AB GeneAmp PCR System 2700 machine using the following steps: 15 min at 94°C, then 30 cycles of 30 s at 94°C, 30 s at 50°C and 30 s at 72°C; and as final step of 30 min at 60°C. After amplification, the products were visualized in a 2% agarose gel, diluted correspondingly and loaded on an ABI PRISM 3700 automatic sequencer from the Scientific and Technical Services of the University of Barcelona, with CST ROX 70–500  used as internal molecular ladder. Allele sizes were assigned with GeneMapper™ version 3.7 .e, or gene diversity) using the FSTAT software package version 2.9.3.2 ST, A1 and A2; JST and J1; U1+2, U1+2+8and UST for each chromosome respectively) were defined as fixed factors and Locus as random factor. Interaction terms between factors were also included in the models. In all analyses, the arcsine of the square root heterozygosity was used as the dependent variable to meet the ANOVA assumptions of normality. ANOVA models were computed with the STATISTICA 8.0 software package.Levels of genetic variability in microsatellite loci were assessed by measuring the Expected heterozygosity  or only the same arrangement across different populations . Statistical significance was tested after 10000 permutations.Individuals carrying the same arrangement from a given population were grouped in the same study unit. Molecular genetic differentiation associated with chromosomal arrangements was visualized with a Principal Coordinate Analysis using D′-statistic, D′m = Σijpiqi|D′ij| P-values obtained after 10000 permutations and adjusted for multiple comparisons using the False Discovery Rate correction  between multiallelic markers implemented in MIDAS 2) with Yates correction. To avoid spurious patterns associations between microsatellite alleles and inversions were only considered as significant for cases with three or more observations.For each chromosome linkage disequilibrium (LD) between microsatellites and inversions was quantified with the multiallelic version of Lewontin’s STF values and the logarithm of the geographical distances (in Kms) between populations were carried out using data from each microsatellite locus independently and all loci combined. Statistical significance of these linear regressions was obtained through 10000 permutations using the SPAGeDi software v1.3 In order to search for an association of microsatellite variation with geographical distance, for each arrangement separately, linear regressions between pairwise population The two most common alleles for each locus, defined after averaging frequencies across populations, were analysed in order to detect specific alleles presenting clinal frequency variation. Linear regressions between the frequencies of the two most common alleles (arcsin transformed) for each locus and latitude were carried out. Regression analyses were computed with the STATISTICA 8.0 software package and FDR correction was applied to their significance levels.STF values with a neutral distribution of expected STF values, conditioned on heterozygosity, obtained from coalescent simulations http://popgen.eu/soft/lositan/) to generate a neutral distribution based on an island model of population structure, assuming an Infinite Allele mutation model, with 100 000 paired values of STF and heterozygosity.A test to detect selection on microsatellite loci was carried out to identify outlier loci through the comparison of observed 1. A2, J1, U1+2and U1+2+8 arrangements decreased in frequency with increasing latitude while all standard arrangements increased in frequency . Nevertheless, the populations in lower and higher latitudes (respectively Iberian and Scandinavian populations) presented consistently lower He values particularly in the A and J chromosomes . This is in accordance with the inverse latitudinal clinal frequency patterns of most inversions as previously mentioned, which results in populations from intermediate latitude presenting higher variability in chromosomal arrangements. However, for the U chromosome its inversion polymorphism did not follow the same trend due to the frequency of the U1+2arrangement which itself was better fit to a quadratic regression with latitude .ANOVAs were performed to search for differences in the variability of chromosomal arrangement frequencies across populations. No significant differences across populations were obtained in the heterozygosity for chromosomal arrangements, after arcsin transformation  in the individuals carrying the A2 inversion – see Trifactorial ANOVAs were performed per chromosome to search for differences in genetic variability between populations, arrangements, loci and interactions between the different factors . For theST individuals both loci located inside the inverted region – dsub69 and mostly dsub59– presented significantly lower genetic variability than other loci of the standard chromosome and also than all loci in J1 individuals . Interestingly, these differences disappear if the Scandinavian populations are excluded from the analysis, as these are the ones presenting consistently lower values of genetic diversity .2 inversion constituted one single analysis group; see Material and Methods). For each chromosome, between arrangements STF analyses indicated a much higher genetic differentiation than comparisons between individuals carrying the same arrangement across populations (within-arrangement differentiation). These differences are statistically significant in chromosomes A and U: A chromosome – STF between arrangements = 0.015, STF within arrangements = 0.007 (Wilcoxon P<0.02); J Chromosome – STF between = 0.037, STF within = 0.007 (Wilcoxon P<0.08); U Chromosome – STF between = 0.017, STF within = 0.007 (Wilcoxon P<0.05). Between arrangement STF values were statistically significant in all chromosomes (P<0.001), while the within arrangement STF values were statistically significant for A1 (P<0.01) and AST (P<0.001); JST (P<0.001); U1+2 (P<0.01) and UST (P<0.001) – see also STF values of each locus. Importantly, when excluding the Scandinavian populations from the analysis due to their consistently higher genetic differentiation relative to the other populations . The general trend of higher between than within-arrangement differentiation was also observed when excluding the Scandinavian populations although only statistically significant in the A chromosome . Heterogeneity across loci in STF values for the between arrangement comparisons may be responsible for the large P-value in the J chromosome; in fact, loci located inside inverted regions present much higher STF values than loci located outside , dsub59 and dsub69 (located within J1) and dsub42 (located within U1+2 and U1+2+8), which present high values of between arrangement differentiation – see To study the genetic differentiation within and among chromosomal arrangements across populations, individuals were grouped according to the population and chromosomal arrangement they carried  based on combined  as well combined .1 inversion, region II that includes the A2 inversion and region III with no inversion included (see 2 from different populations clustered together and were separated from the other two arrangements (A1 and AST) by the first axis explaining 44% of the variation, while the second axis (26% of variation) separated the individuals from Scandinavian populations with inversions A1 and AST (2), with the two first axis explaining 77% of the variation while the loci on regions I and III explained lower percentages of variation  and did not show such a clear clustering of individuals . Locus dsub39 not only presented the highest global STF values, showing large differences between inversions across populations, but also significant differentiation within inversions A1 and AST across populations , with the JST inversions from the Scandinavian populations further differentiated from all others (second axis explaining 13%)  . This pahers see . PCoA anulations .1+2+8arrangement from those carrying the other two arrangements. When only the loci located inside U1+2(locus dsub42 and dsub64) were used the same pattern revealed with all loci combined was observed. However, when only the loci located outside the arrangements were used, the groups were more intermixed  of the PCoA analysis using all loci . The secixed see . When eangements .2 inversion . For locus dsub59, in the J chromosome allele 245 bp was in disequilibrium with JST in all populations. Similarly, for locus dsub69 the majority of the significant LD patterns obtained with the JST inversion involved the same allele .The D′m statistic - multiallelic version of Lewontin’s D′ nversion , allele ST inversion, non-random associations were detected between dsub19 and dsub39 and between dsub5 and dsub37 for both Sunne and Drøbak . Associations were also found between dsub19 and dsub37 and also between dsub5 and dsub19 for the Rasquera and Sunne populations . In general, no consistent associations across populations were found between loci in the other inversions of the sex chromosome or in the autosomes. To increase the sample size and thus also the statistical power to detect LD patterns, all individuals from different populations carrying the same inversion were grouped. In these analyses the Scandinavian populations were excluded, as they were the ones generating within-arrangement differentiation across populations . In the U chromosome LD was detected between loci dsub15 and dsub42  for the individuals carrying the U1+2+8 arrangement. Interestingly, multi-locus LD in AST chromosomes was also detected for loci dsub39-dsub70-dsub19, located in the distal part of the A chromosome .In order to detect regions within chromosomes presenting possible epistatic interactions, non-random associations between loci within the chromosomes carrying the same inversion were assessed. For chromosomes carrying the A2IS was always substantially higher than variance of LD within the overall population D´2ST (data not shown). This result points to different LD patterns between loci across the different populations, probably due to genetic drift or other stochastic events as selection would most likely generate similar LD patterns across populations Hierarquical analysis of Linkage Disequilibrium STF values between populations on the logarithm of geographical distances were calculated using information from all loci combined and each locus separately. For the A1 inversion only locus dsub5 presented a significant association with distance  after FDR correction. On the other hand, in the AST inversion significant associations of STF values with geographical distance were found for locus dsub39 and for all loci combined . For the JST inversion all loci combined  as well as several loci alone dsub18 , dsub69  and dsub62  presented significant association between genetic differentiation and geographical distance. For the U chromosome, there was only a significant association between genetic and geographic distances for the UST inversion when information from all loci was combined .Clinal variation in the genetic content of each chromosomal arrangement was analysed at a broad geographical scale ranging about 3700 km by comparing the genetic distance between populations and their geographic distance. Linear regressions of pairwise 2 inversion , and for locus dsub42 in individuals with U1+2arrangements  – The frequencies of the most common alleles of each locus were plotted against latitude to pinpoint specific microsatellite alleles presenting clinal variation. STF values, given their observed heterozygosity, as an indication of positive selection. Locus dsub39 was the only locus identified as being an outlier in individuals carrying the A1 and the AST inversions .In order to detect a signature of selection within inversions, we used a coalescent simulation model implemented in LOSITAN Drosophila subobscura and clearly shows the impact of chromosomal arrangements in shaping the molecular genetic patterns of the European populations of this species. We found large genetic differentiation between individuals carrying different arrangements and showed this was caused by loci mapping inside arrangements. Importantly, these patterns were consistent across chromosomes and populations at a wide geographical scale. The most likely explanation for our results is the reduction of recombination between chromosomal arrangements, with loci within and near breakpoints of inverted regions presenting restricted gene flux D. subobscura, which present a high number of inversions in the genome.This study is the widest geographical analysis performed on the molecular variation associated with clinal inversion polymorphism in 2 inversion  and dsub59 located inside J1 (in the middle of the inversion). In these loci, the same allele is in LD with the inversion in different populations indicating either a selective effect, association due to a bottleneck event or the origin of the inversion. In addition, variability patterns in these loci were also clearly influenced by their location inside inverted regions. The region within JST nearby the dsub59 locus might in fact mark a region subjected to selection (see below).The patterns of Linkage Disequilibrium reported in our study also support an important role of gene arrangements in shaping the molecular genetic content of the chromosome. The stronger LD levels were obtained for loci located inside arrangements see also . The mosD. subobscuraD. pseudoobscura across four American populations A clear finding of this study is the overall low levels of genetic differentiation in a given gene arrangement across a large number of European populations distributed along a latitudinal cline covering >3000 Km. Similarly, no geographic differentiation, based on nucleotide polymorphism, was observed within chromosomal arrangements of the O chromosome (Muller’s E element) in two south European populations of Drosophila populations collected in different years D. subobscura from northern Europe relative to more central European populations. Thus, the genetic differentiation detected in the present work relative to Scandinavian populations is more likely associated with restriction to gene flow due to the geographic barrier constituted by the North Sea. Also, lower effective size of the Scandinavian populations might be a factor, supported by the overall lower genetic variability of these northern populations.The low genetic differentiation within arrangements across populations is most likely due to the occurrence of important gene flow in this species ST individuals could be due to low recombination in the region concerned or physical constraints and not to any selective process. Evidence for epistasis interactions was reported in a study of 8 gene regions in D. pseudoobscura based on linkage disequilibrium among them Drosophila melanogasterThese findings do not agree with the expectation of the coadaptation model of differences in the genetic content of a given gene arrangement across populations as a result of contrasting selective scenarios in different environments Drosophila subobscura will be crucial in this regard.Alternatively, if few genes within inversions are involved in epistatic selection and the regions covered by our microsatellite loci are not located near selected regions we might be unable to detect associations among loci. Studies involving a higher number of markers, with a wider coverage of the inversions will allow higher resolution power to test if this indeed was the case and also if low within-arrangement genetic differentiation across populations is in fact the rule. Massive parallel sequencing methods, and the approaching possibility to obtain the entire genome sequence of ST individuals could be indicative of epistatic interactions and selection, although it might also be explained by low recombination in the region.Nonetheless different processes might affect different inversions, chromosomes and even species. In this context, the reported extensive LD between loci located in the distal part of the A chromosome in AST presented significantly lower variability than those carrying the J1 inversion. This could indicate a selective sweep event in the region nearby locus dsub59 thus leading to a decrease in variability, although other possible alternative explanations exist, such as (1) a recent bottleneck associated with the JST inversion or (2) an historical effect due to the possible recent origin of this inversion. Nevertheless these alternative explanations appear less likely since in (1) a bottleneck would most likely produce a reduction in variability in all loci within the inversion and not just around a particular locus; and in (2) an historical effect would be most susceptible to be observed in a derived inversion while JST is thought to be the ancestral arrangement, based on phylogenetic studies of inversions in the obscura group ST inversion are needed.Individuals carrying the JDespite the low genetic differentiation within arrangements across populations, there were some indications of within-arrangement variation in allele frequencies against latitude in our study. As discussed above, this was mainly due to the Scandinavian populations, as inversions from these populations were the ones presenting higher differentiation in their genetic content relative to others. This might also explain the fact that the overall patterns of clinal variation were higher in the “standard” chromosomes since these present higher frequencies in the Scandinavian populations. This overall pattern is most likely due to demographic factors, since this sign of isolation by distance is seen at several loci of different chromosomes, and particularly when information from all loci is taken together.2 inversion and dsub42 located inside the U1+2arrangement. Coincidentally, these two loci also presented the highest levels of LD with inversions. This pattern is suggestive of clinal selection in regions surrounding the aforementioned loci. Also, locus dsub39 presents indications of higher differentiation across populations than expected under the neutral model. The fact that these clinal patterns occur in loci that map inside inverted regions suggests these gene arrangements may protect specific combinations of alleles possibly under climatic selection from the effects of recombination 2 and dsub42 maps inside a region covered by multiple inversions . It will thus be interesting to analyse possible candidate genes associated with thermal adaptation located in the vicinity of the microsatellite loci described above.Interestingly, we also found specific alleles presenting changes in frequencies highly correlated with latitude which are not explained by the effect of Scandinavian populations. It is revealing that those presenting the stronger sign of clinal variation correspond to alleles of microsatellite loci located within inversions. This is the case of locus dsub39 within the AIn summary, our data indicates a general pattern of low within arrangement differentiation across populations and no consistent indications of epistasis. As proposed in D. subobscura European cline. Inversions represent an important barrier to gene flux and can thus contribute to maintain specific allelic combinations locally adapted with positive effects on fitness.Overall our study demonstrates the effective role of chromosomal inversions in maintaining different genetic pools even in the presence of high levels of gene flow along the Figure S1e) for chromosomal arrangements in the U, A and J chromosome plotted against latitude.Expected heterozygosity (H(DOCX)Click here for additional data file.Figure S2Genetic differentiation (STF) between and within chromosomal arrangements across populations. The first column represents the global differentiation for each locus, including both between and within-arrangement differentiation across populations (All); following columns represent differentiation within each of the different arrangements of the chromosome across all populations. Asterisks represent levels of significance at: P<001 (***); P<0.01 (**); P<0.05 (*).(DOCX)Click here for additional data file.Figure S3Principal Coordinate Analysis (PCoA) based on STF values .(DOCX)Click here for additional data file.Figure S4Microsatellite alleles presenting significant clinal variation within a given arrangement across populations.(DOCX)Click here for additional data file.Table S1Frequency of chromosomal arrangements in the nine studied populations.(XLSX)Click here for additional data file.Table S2Regression coefficients and significance of inversion frequencies plotted against latitude.(XLSX)Click here for additional data file.Table S3Levels of genetic variability (expected heterozygosity) of all loci for the populations and arrangements analysed.(XLSX)Click here for additional data file.Table S4ANOVA for each chromosome for the effects of population and arrangement as fixed factors and locus as random factor on the arcsin tranformed expected heterozygosity.(XLSX)Click here for additional data file.Table S5Microsatellite allele frequencies in individuals carrying different inversions. A) AST inversion; B) A1 inversion; C) A2 inversion; D) JST inversion; E) J1 inversion; F) UST arrangement; G) U1+2 arrangement; H) U1+2+8 arrangement.(XLS)Click here for additional data file.