African history has been significantly influenced by the Sahara, which has represented a barrier for migrations of all living beings, including humans. Major exceptions were the gene flow events that took place between North African and sub-Saharan populations during the so-called African Humid Periods, especially in the Early Holocene (11.5 to 5.5 thousand years ago), and more recently in connection with trans-Saharan commercial routes. In this study, we describe mitochondrial DNA (mtDNA) diversity of human populations from both sides of the Sahara Desert, i.e., both from North Africa and the Sahel/Savannah belt. The final dataset of 7213 mtDNA sequences from 134 African populations encompasses 470 newly collected and 6743 previously published samples, which were analyzed using descriptive methods and Bayesian statistics. We completely sequenced 26 mtDNAs from sub-Saharan samples belonging to the Eurasian haplogroup N1. Analyses of these N1 mitogenomes revealed their possible routes to the Sahel, mostly via Bab el-Mandab. Our results indicate that maternal gene flow must have been important in this circum-Saharan space, not only within North Africa and the Sahel/Savannah belt but also between these two regions. The out-of-Africa event, during which a relatively small group of anatomically modern humans spread from East Africa into Eurasia , was a dThat, however, does not mean that after the out-of-Africa event migration had stopped. The genetic structure of inhabitants of the Sahel/Savannah belt was analyzed by researchers with respect to their linguistic affiliation, subsistence structure, and geographic localization of local populations ,9,10, anInterestingly, while some migrations may have had an ethnic association, others did not. For instance, the origin of the Chadic-speaking peoples living in the Lake Chad Basin was traced to East Africa based on linguistic evidence . AccordiSahelian populations also carry Eurasian mtDNA haplogroups. They are found more frequently in the nomadic pastoralists than in sedentary farmers  and a suIt may therefore seem that the pastoralist food-production strategy did not spread to sub-Saharan Africa by demic diffusion from the Near Eastern domestication center via northeastern Africa, but through the ancestors of Berbers from the Maghreb. In this context, it should be noted that the genetic architecture of the circum-Mediterranean space had undergone substantial changes since the Neolithic. For instance, ancient Near Eastern farmers are genetically better represented by the current populations of central and western Mediterranean, such as the Sardinians and the Basques ,23, thanThe importance of post-Neolithic gene flow from northwestern Africa to the western part of the Sahel/Savannah was also suggested by research on lactase persistence. In fact, the Fulani pastoralists from Burkina Faso share with Europeans the extended haplotype carrying Eurasian variant −13,910 × T. It was suggested that their ancestors received this haplotype via admixture with the Eurasian population two times . The firLast but not least, it was shown that Sahelian pastoralists tend to represent several mutually similar mtDNA haplotypes, which indicates either more recent origins of their diversity, isolation of their demes, lower gene flow, or lower effective size of the population . InteresThe above-mentioned studies show that inclusion of newly collected local populations, especially from the Sahel/Savannah belt, has significantly contributed to our knowledge of the peopling of Africa north of the equator by discoveries of not only new variants—which happens quite commonly when a new dataset of a sub-Saharan population is presented —but evenBecause sub-Saharan Africa is still underrepresented in population genetic and genomic studies , we compn = 470) sequences enriched the Sahelian dataset with four populations from Mali (n = 135), three from Mauritania (n = 191), and three from Sudan (n = 144). During the sampling, we focused on populations that have not been previously included in any genetic study, such as the Bella from Mali, Imraghen from Mauritania, Daju and Zaghawa from Sudan, but also on populations whose sampling coverage was insufficient due to their large geographic dispersals, such as the Fulani from Mali and Mauritania, Moors and Soninke from Mauritania, and Tuareg, Songhai, and Arabs from Mali.We built a large dataset of 7213 mtDNA hypervariable segment 1 (HVS-1) sequences from 134 African populations inhabiting the circum-Saharan space. It contains both published and newly acquired samples. All samples were collected with the help of African colleagues and appropriate informed consent was procured prior to sampling from all participants. Newly collected and here for the first time presented mtDNA (The population dataset  was furtn = 701) belonging to the same N1 haplogroup. Our total N1 dataset thus includes 727 mitogenomes  was carried out using primer pairs previously published in Gonder et al. [New samples published here for the first time were collected by the Oragene DNA Collection saliva kit . DNA was extracted according to supplier’s protocol. PCR amplification of all newly sequenced samples  method. Quantity of the extracted DNA was determined by Plexor HY (Promega) and the samples were diluted to the total DNA template of 15 ng per reaction. Whole-genome mtDNA MPS data were generated using commercially available QIAseqTM Targeted DNA Panel (Qiagen), which contains 222 overlapping primers, and then sequenced on MiSeq Fgx Sequencing system (Verogen) in the “Research Use Only” mode through the “Generate FASTQ” with “FASTQ Only” application and “Paired End Read”. MPS libraries and final sequencing reactions were prepared according to the manufacturer’s protocol to a final loading concentration of 12 pM. Sequencing reaction was performed using MiSeq v2 Reagent Illumina kit (300 cycles per kit).We computed both the standard and molecular diversity indices, such as haplotype diversity, nucleotide diversity, and the mean number of pairwise differences for each population using Arlequin software ver. 3.5.2.2 . Differep-value should be below 2% . Additionally, we evaluated demographic history by Harpending’s raggedness index (Hri) and the sum of squared deviations (SSD) while considering a model of demographic expansion as implemented in Arlequin [p-values was evaluated using Kruskal–Wallis one-way tests.To investigate the demographic history of our samples, we calculated tests of selective neutrality such as Tajima’s D , Fu’s FsArlequin . StatistInterpopulation comparisons were assessed via pairwise Reynolds’ genetic distances based on haplotype frequencies ,44 usingCT index) and/or relationships between populations within groups (for ΦSC index). The significance of fixation indices was tested using a hierarchical framework and 10,000 iterations of the random permutation procedure implemented in Arlequin [Analyses of molecular variance (AMOVA) assessed the levels of genetic variation within and between different groups of populations structured, as noted above, by several factors, namely language affiliation, geographical location, and subsistence strategy. The settings were: language grouping (Afro-Asiatic vs. Nilo-Saharan vs. Niger-Congo); geographic grouping as Region1 (Sahel/Savannah belt vs. North Africa) and Region2 (eastern North Africa vs. western North Africa vs. eastern Sahel/Savannah belt vs. western Sahel/Savannah belt), subsistence grouping as Lifestyle1  and Lifestyle2 . By inspecting the fixation indices, we were able to describe the structure between groups  using a Bayesian Markov chain Monte Carlo inference model to generate a posterior probability density distribution . This meIn terms of treatment of whole mtDNA sequences, we analyzed the raw FASTQ files using NextGENe software  and the resulting sequences were read with BioEdit version 7.0.9.1 . MutatioTo estimate time to the most recent common ancestor (TMRCA) for specific clades in the phylogeny, we used the ρ statistic  and maxiGene diversity, nucleotide diversity, and the mean number of pairwise differences of each population are shown in p-values of SSD and Hri—revealed demographic expansion in several North African populations. As far as sedentary farmers are concerned, demographic contraction can be deduced for the Bedik and Mandenka, but also for some groups of Moroccan Berbers such as the Kesra and Zriba. No signals of demographic expansion (based on Fu’s Fs) were detected also in several nomadic Fulani groups from the western Sahel/Savannah belt. The exact values of all indices are specified in p-values with a Kruskal–Wallis one-way test and significant differences were detected only in the Hri index between linguistic groups ) with a stress value of 0.112 imply differentiation of populations in a 2D scale. Four subplots show the populations colored according to geography  were lower than for variation between populations within these groups (ΦSC). On the other hand, we were able to confirm the structure when considering languages or regionally divided populations (Region1 and Region2), or when the farmers/pastoralists in North Africa were contrasted with farmers in the Sahel/Savannah belt. Our results show rather balanced indices indicating no significant structure among pastoralists in North Africa and the Sahel/Savannah belt . All values were statistically highly significant (p < 0.05) except that for variance between the groups of farmers vs. pastoralists in the North African division.AMOVA was applied to evaluate genetic differentiation among populations grouped according to geography, language, and subsistence strategy. The results are presented in e in these groups. Moreover, we calculated the immigration rates and the numbers of immigrants into specific groups. Surprising results are seen in North African populations , who seem to have received the least number of immigrants from other regions. On the other hand, we ought to consider the differences in sample sizes of these groups: for eastern North African pastoralists, we had just 195 samples, while for western North African farmers we had 2343. The results also suggest a higher gene flow into farmer populations in general, with the exception of the eastern Sahel/Savannah belt where it was even lower than in other pastoralist groups. When evaluating signals of gene flow obtained from the Bayesian coalescent approach in Migrate-n software see , we deteThe phylogeny of haplogroup N1, together with its derived clade I, is presented in An mtDNA dataset containing 7213 mtDNA sequences in 134 African populations—which is much more than used in a previous studies ,56—and cThe lower level of differentiation among the populations of North Africa than among the populations of the Sahel/Sudan belt supports the idea of higher migration activity homogenizing the gene pool and eroding the population structure in the southern Mediterranean space. It is well possible that there was a long-range influx of population(s) from the Near East to the Maghreb already in preagricultural times . In factOur results which suggest that the eastern part of North African populations received immigrants from the Sahel/Savannah belt do not correspond with research on autosomal SNP variants, which had shown that the populations of Egypt and Libya are composed predominantly of a Near Eastern genetic component with very low input from sub-Saharan Africa . HoweverMigrations in the Sahel/Savannah belt were probably less important than in North Africa. When we look at the continent-wide African mtDNA diversity, the Sahel/Savannah belt can be viewed as a corridor between the Sahara and tropical rainforests which connects eastern and western Africa but also has—especially in the Lake Chad Basin—some distinctive genetic features . Food-prThe cultivation of cereals and tubers, which is autochthonous in the Sahel/Savannah belt  ancestry and received gene flow from sub-Saharan Africans , the FulFinally, we found that the N1 haplogroup, which diversified in southwestern Asia some 55 ka  and its


Convergent phenotypic evolution provides some of the strongest evidence for adaptation. However, the extent to which recurrent phenotypic adaptation has arisen via parallelism at the molecular level remains unresolved, as does the evolutionary origin of alleles underlying such adaptation. Here, we investigate genetic mechanisms of convergent highland adaptation in maize landrace populations and evaluate the genetic sources of recurrently selected alleles. Population branch excess statistics reveal substantial evidence of parallel adaptation at the level of individual single-nucleotide polymorphism (SNPs), genes, and pathways in four independent highland maize populations. The majority of convergently selected SNPs originated via migration from a single population, most likely in the Mesoamerican highlands, while standing variation introduced by ancient gene flow was also a contributor. Polygenic adaptation analyses of quantitative traits reveal that alleles affecting flowering time are significantly associated with elevation, indicating the flowering time pathway was targeted by highland adaptation. In addition, repeatedly selected genes were significantly enriched in the flowering time pathway, indicating their significance in adapting to highland conditions. Overall, our study system represents a promising model to study convergent evolution in plants with potential applications to crop adaptation across environmental gradients. FLC locus have evolved multiple times independently in Capsella rubella populations, conferring variation in flowering time  is unknown . Study oConvergence through molecular parallelism, when it occurs, can originate in a number of ways. Genetic variation underlying convergent traits can arise as independent mutations, be derived from existing standing variation in a shared ancestral population, or be transferred between populations via migration . EmpiricGiven its history, maize is an ideal model system to study parallel adaptation. Maize was first domesticated in the warm lowlands of the Balsas River Valley approximately 9,000 years ago , and subZea mays ssp. mexicana, hereafter mexicana) is thought to have played an important role during highland adaptation in Mexico  showing evidence of selection in both populations, but concluded that maize highland adaptation arose largely independently in the two populations . One conn Mexico , but main Mexico . Nonethemexicana , suggestHere, we evaluate evidence for trait convergence through parallel molecular means using resequencing data from four highland populations of maize  and two lowland populations (Mexico and South America). We assess the prevalence of molecular parallelism at the SNP, gene, and pathway levels, comparing models of de novo mutation, standing variation, and gene flow. Finally, our analysis of parallelism in pathways focuses on the well-characterized flowering time genes in maize and assesses signatures of polygenic adaptation for this trait.http://www.worldclim.org/) revealed that highland and lowland accessions were differentiated along PC1 (comprising 51.8% of variation); whereas PC2 (comprising 22.8% of variation) reflected latitudinal differences across highland populations  and two reference lowland populations  and South America ) were sampled to investigate highland adaptation in maize landraces  online. ulations  online. ulations  online.PBE) statistic  exhibited the highest PBE value in the Mexican Highland population and was also detected as a target of selection in all other highland populations , but not the Andes  in a subset of the neutral SNP set, and recalculated the number of codirectional and antidirectional SNPs. The same pattern was revealed  and Guatemalan (41.9%) highland populations, a result that could be partially explained by the substantial shared ancestry of these two populations and direct maize colonization from the Mexican highlands to the highlands of both the Southwestern US and Guatemala. In summary, results for outlier SNPs shared across all four highland regions suggest gene flow among highland populations and standing variation may have been the major sources of adaptive variation.To determine whether shared chanisms . We contchanisms B. The stchanisms  specifieWe further explored the source of shared adaptive variation across pairwise samples of highland populations. As seen in four-way comparisons after correction, the standing variant with a source population model , the standing variation model , and the migration model  were most commonly supported B. The stSTF, between pairwise highland populations to assess how this varies based on the assigned model. Repeatedly selected SNPs consistent with a migration model exhibited weaker divergence P3),O) shaped phylogenetic tree, the Previous studies have suggested that migration between the Andes and Mesoamerican populations is unlikely . We thertatistic  in 10-kbulations  online. ulations , the twoulations .PBE outlier SNPs within the gene or 10 kb upstream or downstream . To further evaluate constraint in parallel adaptation, we tested an alternative null model which assumes the set of genes selected in at least one population represents all genes that can possibly affect the trait. We then tested whether there was evidence of excessive sharing beyond the expectations under physiological constraint . Our finding of an excess of shared candidates across regions within a large set of known genes that affect flowering time suggests physiological constraint alone cannot explain patterns of convergence, but instead that variation at only a subset of genes can affect flowering without deleterious pleiotropic consequences.Given this support for selection on flowering time during highland adaptation, we explored the overlap between our selected genes and known flowering time candidates. First, we utilized a list of 904 maize flowering time candidate genes aggregated by .8−100%)  online. .8−100%) . We alsoP = 0.005, hypergeometric test). Although common outlier genes between the Andes and other highland populations were not significantly enriched in this smaller list .We then narrowed our focus to a subset of core flowering time genes  with higPBE values of outlier SNPs within or in the 10-kb flanking regions of flowering time genes were in the extreme tail of the distribution of all genes (ZCN8 (GRMZM2G179264), which is orthologous to FLOWERING LOCUS T (FT) in Arabidopsis, has been shown to play a significant role in maize adaptation to high latitudes (ZMM4 (GRMZM2G032339), which is under selection in all but the Southwestern U.S. highlands. VGT1  was identified as an outlier in all four highland populations, with the same nonsynonymous SNP  targeted in the Mexican and Guatemalan highlands  was a synonymous mutation near the 3′ UTR  genes, CONZ1 and ZmCCT, were selected in both the Mexican and Guatemalan highland populations. We further found Gigantea2 (GRMZM5G844173) was selected in all but the Guatemalan highlands and FKF2 (GRMZM2G106363) in all but the Mexican highlands. Among circadian clock genes, LUX (GRMZM2G067702) , RVE2 (GRMZM2G145041) and PRR5 (GRMZM2G179024) were selected in all four highland populations. Finally, the light transduction gene PHYA1 (GRMZM2G157727) was targeted by selection in all but the Southwestern U.S. highland populations.The ll genes B, with sll genes C. For exatitudes  and was ighlands  online. G067702)  online wThe prevalence of parallel evolution is a long-standing question in the field of evolution. Through a comprehensive evaluation of parallel adaptation among independent maize highland populations, we found that, whereas most adaptation is independent, parallelism is more common than expected. We observed a particularly high level of parallelism among Mesoamerican highland populations relative to comparisons including Andean maize. The vast majority of parallel adaptive alleles we discovered have risen in frequency in the highlands from migration and standing variation, and only a small percentage represent de novo mutations. However, the proportion of repeatedly selected SNPs from de novo mutations is the highest in pairs of populations that include the Andes. Our analyses further reveal that adaptive routes from genotype to phenotype have been canalized by both physiological  and pleiotropic  processes. For example, we observe that only a subset of possible genes related to flowering time contribute to highland adaptation, with many of these loci showing evidence of selection in multiple highland regions, particularly in Mesoamerica.PBE statistic allowed for detection of selection specifically in the highlands and reduced the extent of false positives, a limitation of studies that focus solely on population differentiation outliers. Our polygenic adaptation analyses also helped to clarify specific phenotypes targeted by selection during adaptation, showing clear evidence of selection on flowering time. We have also uncovered a pervasive role of migration in transferring adaptive alleles across highland regions. In fact, our analyses revealed a moderate proportion of parallel adaptation through migration even between the Andes and other highland populations, in contrast to previous studies that appeared to exclude this possibility based on a simple population genetic model (Approximately one quarter (mean 24.0%) of highland adaptation SNPs showed a pattern of molecular parallelism in Mesoamerica. A smaller percentage also showed this pattern in Andean comparisons (mean 10.7%), but this represents a slightly higher proportion than previously reported (7–8% between the Andes and the Mexican Highlands) . We haveic model . This fiic model , allowinDrosophila hydei contained more adaptive alleles than D. melanogaster. Second, the recent timing of expansion and divergence in maize has likely affected the extent of parallelism. The probability of parallelism will decrease when populations diverge over a greater period of time, as less shared standing variation would be expected to be the source of adaptation. Increased divergence time would also allow for novel beneficial mutations to arise, potentially in distinct genes and pathways  likely occurred as other crops expanded from their centers of origin . Howevermexicana introgression regions (primarily in low-recombination regions such as pericentromeres) were removed from analyses due to nonindependence of SNPs within these regions and their enrichment for highly differentiated loci. The filtered SNP data set was shared through the CyVerse platform . Nineteen environmental variables were obtained from the WorldClim data set  and were utilized to perform a PCA with the vegan package in R PBE=PBE is expected to be strongly positive when selection acts specifically on the focal population.Here, PBE for filtered SNPs among one highland population, its corresponding lowland population  and the parviglumis Palmar Chico population  with the outlier SNP set and sampled the same amount of SNPs in each allele frequency bin. Last, we tabulated the directionality of the randomly sampled neutral SNPs to check how ancestral allele frequency influenced the proportion of codirectional SNPs.In order to exclude the bias of genetic drift on the directionality of changes in allele frequency, we approximated the two-dimensional site frequency spectrum (2dSFS) of outlier SNPs using a subset of the neutral SNP set. We divided the nonreference allele frequency in Mexican Lowland maize and t generations later, is selected. The details of these four models can be found in Following the method developed and applied in P values and compared the differences.Following the method in We followed the methods developed by To identify loci associated with traits that could be under selection, we conducted GWAS using a maize panel developed for GWAS, often referred to as “the 282” or “the Major Goodman panel” . SNPs foP value < 0.1 that had been pruned down to the strongest hit per 1 cM window using a linkage map from M loci associated with a trait, each with effect size mα, imp is the allele frequency of the mth allele in line i, which will either be 0, 0.5, or 1, then we can calculate the polygenic score for the ith line as iZ whereWe used the GWAS hits to generate polygenic scores for each individual in the landrace panel using all loci with a Shared population structure between the GWAS panel and the test panel can lead to false positive signals of selection . To testiZ for the 31 highland and lowland landraces discussed in this paper and iZ for the individuals used in the GWAS. We model the combined vector of polygenic scores in both panels asμ is the mean of the combined vector K11 and K22 are the kinship matrices of the genotyping and GWAS panels, and K12 is the set of relatedness coefficients between lines in the genotyping panel (rows) and GWAS panel (columns).In light of this shared structure, we conducted a conditional test, following The conditional multivariate null model for our polygenic scores in the landraces conditional on the GWAS panel is thenTo test for selection, we calculate the difference between observed polygenic scores and conditional means as C) using the following equation (shown here for After calculating AV using the allele frequencies and effect sizes of the GWAS loci, using the following formula. If mth locus across all individuals and mα is the effect size of that locus,We estimate We then test for a linear relationship between ion in R  and contion in R .Molecular Biology and Evolution online.msab119_Supplementary_DataClick here for additional data file.


Schistosoma haematobium (Sh) and S. bovis (Sb) have been found in several African countries as well as in Europe. Since the consequences of this hybridization are still unknown, this study aims to verify the presence of such hybrids in Cameroonian humans, to describe the structure of S. haematobium populations on a large geographic scale, and to examine the impact of these hybrids on genetic diversity and structure of these populations.Hybrids between Cox1 (RD-PCR) and ITS2 gene (PCR-RFLP). Population genetic diversity and structure was assessed by genotyping each miracidium with a panel of 14 microsatellite markers. Gene diversity was measured using both heterozygosity and allelic richness indexes, and genetic structure was analyzed using paired Fst, PCA and Bayesian approaches.From January to April 2019, urine from infected children was collected in ten geographically distinct populations. Miracidia were collected from eggs in this urine. To detect the presence of hybrids among these miracidia we genotyped both S. haematobium (Sh_Sh/Sh) while the remaining 11.3% were hybrids . No miracidium has been identified as a pure genotype of S. bovis. Allelic richness ranged from 5.55 (Loum population) to 7.73 (Matta-Barrage) and differed significantly between populations. Mean heterozygosity ranged from 53.7% (Loum) to 59% (Matta Barrage) with no significant difference. The overall genetic differentiation inferred either by a principal component analysis or by the Bayesian approach shows a partial structure. Southern populations (Loum and Matta Barrage) were clearly separated from other localities but genetic differentiation between northern localities was limited, certainly due to the geographic proximity between these sites.Of the 1327 miracidia studied, 88.7% were identified as pure genotypes of S. haematobium and S. bovis were identified in 11.3% of miracidia that hatched from eggs present in the urine of Cameroonian schoolchildren. The percentages of these hybrids are correlated with the genetic diversity of the parasite, indicating that hybridization increases genetic diversity in our sampling sites. Hybridization is therefore a major biological process that shapes the genetic diversity of S. haematobium.Hybrids between The online version contains supplementary material available at 10.1186/s40249-022-00958-0. Hybridization is defined as the interbreeding between individuals or populations that differ by one or more inheritable characters , 2. SuchSchistosoma mansoni with S. mansoni (a species infecting humans) and S. rodhaini (a species infecting rodents) and (2) the clade S. haematobium with two species infecting humans  and six species that mainly infect ruminants and/or rodents   results for the genetic structureof the parasite populations : (A) mean likelihood (with their variance over the 10 replicates) and (B) Delta K valueper number of simulated genetic clusters K (from 1 to 12). These figures were made using StructureHarvester software, version 0.6.1 [43].


Aedes albopictus is a very invasive mosquito, which has recently colonized tropical and temperate regions worldwide. Of concern is its role in the spread of emerging or re-emerging mosquito-borne diseases. Ae. albopictus from south-western Europe and Brazil were studied to infer genetic and phenetic diversity at intra-individual, intra-population and inter-population levels, and to analyse its spread.cox1 sequencing to assess haplotype and nucleotide diversity, genetic distances and phylogenetic networks. Male and female phenotyping included combined landmark-and outlined-based geometric morphometrics of wing size and shape.Genotyping was made by rDNA 5.8S-ITS-2 and mtDNA cox1 and 162 5.8S-ITS-2 haplotypes, with great genetic variability difference between both markers (0.9% vs 31.2%). Five cox1 haplotypes were shared with other countries, mainly Italy, USA and China, but none was shared between Europe and Brazil. The 5.8S-ITS-2 showed 2–7 intra-individual (mean 4.7) and 16–34 intra-/inter-population haplotypes (24.7), including haplotypes shared between Spain, France and Brazil. A 4.3% of ITS-2 haplotypes were shared, mainly with Italy, USA and Thailand, evidencing worldwide spread and introductions from areas where recent outbreaks of Ae. albopictus-transmitted pathogens occurred. Wing size showed sex differences. Wing shape distinguished between Brazilian and European specimens. Both genetic and morphometric markers showed differences between insular Spain and continental Spain, France and Brazil.Specimens from seven populations from Spain, France and Brazil provided 12 Ae. albopictus spread, providing pronouncedly more information than cox1, including intra-individual, intra-population and inter-population levels, furnishing a complete overview of the evolutionary exchanges followed by this mosquito. Wing morphometry proves to be a useful phenotyping marker, allowing to distinguish different populations at the level of both male and female specimens. Results indicate the need for periodic surveillance monitorings to verify that no Ae. albopictus with high virus transmission capacity is introduced into Europe.ITS-2 proves to be a useful marker to assess The online version contains supplementary material available at 10.1186/s13071-021-04829-9. One of the most important current health alerts is the risk of spread of vector-borne diseases due to the effects of climate change and global change. Vector-borne diseases are human illnesses caused by parasites, viruses and bacteria that are transmitted by vectors and cause every year more than 700,000 deaths . Over thAedes (Stegomyia) albopictus  is one of the most invasive species which has successfully colonized most tropical and temperate regions around the world [The Asian tiger mosquito he world . This sphe world , especiaAe. albopictus was restricted to the islands of the Indian Ocean and the islands in the Pacific Ocean until the 1970s, when an expansion of its geographical distribution rapidly occurred throughout all continents, except the Antarctica [The geographic distribution of tarctica . Its adaAe. albopictus is known to be spreading in several European countries such as the Balkan countries, Greece, France, and Spain, where this species established along the Mediterranean coast [Ae. albopictus has been recorded in 27 European countries and has become established in 19 of them [In Europe, the first record of this species was in 1979 in Albania [an coast –12. At p of them –16.Ae. albopictus in the Balearic Islands [Aedes mosquitoes in these ecosystems. In 2017, Ae. albopictus was reported for the first time in northern Portugal [Ae. albopictus having been dispersed by vehicles, with Barcelona as the largest source of inter-province tiger mosquito transfers [Ae. albopictus in Europe [Since the arrival of the tiger mosquito to Spain in 2004 , the numPortugal  and recePortugal . Althougransfers . Similarransfers . Human tn Europe .Ae. albopictus was first recorded in Brazil in 1986, in the states of Rio de Janeiro and Minas Gerais [The distribution of this invader in South America follows a similar pattern of rapid expansion. Detected in Brazil during the 1990s, it subsequently spread to Venezuela, Colombia, Argentina, Bolivia, Paraguay, Uruguay and recently Ecuador . Ae. albs Gerais  and now s Gerais .Ae. albopictus, several genetic markers  have been used to study population genetics. Everything seems to indicate the existence of a global chaotic pattern of Ae. albopictus distribution, with several introductions and admixture between unrelated genomes [Molecular markers generate a large amount of information about genetic diversity and phylogenetic relationships of the organisms. In insects, DNA markers prove to be useful for this endeavour in general, although disadvantages and limitations of each marker should be taken into account , 30. In  genomes , and a g genomes . The few genomes , 33–35.Internal transcribed spacers (ITSs) of the nuclear ribosomal DNA (rDNA) have proved to be useful for the classification of species, subspecies, hybrids, and populations of different groups of insects and for inferring their phylogenetic relationships . The ITSAedes mosquitoes.Opposite to the clonal maternal inheritance of mtDNA genes, ITS-2, together with the whole nuclear ribosomal DNA operon, follows Mendelian inheritance and is therefore of interest in biparental organisms such as mosquitoes. Moreover, it presents the peculiarity of following a concerted evolution. With sufficient time, this mechanism effectively homogenizes the many copies of nuclear rDNA among both homologous and non-homologous chromosomes containing rDNA clusters within the genome. This gives rise to a uniformity inside all individuals of a population and becomes extremely useful , 38. AltBecause of concerted evolution, when two different ITS-2 sequences are found in a specimen collected in nature, it indicates that it is a hybrid, either (i) the direct result of a crossbreeding between parental specimens belonging to two different populations (differing in haplotype profile), subspecies , or two close species (as in cryptic species living in sympatry), or (ii) a descendant of a viable hybrid lineage originated so recently that concerted evolution has not yet had the time needed for homogenisation . This kiAe. albopictus, ITS-2 has been also used as indicator of discontinuity between populations and thus used to address the presence of intraspecific differentiation (genetic barriers) among groups of Ae. albopictus populations from different ancestral and recently colonized geographic areas [In ic areas .Ae. albopictus [cytochrome oxidase I (cox1) gene is the most widely used and is the gold standard for mosquito species identification [cox1, leading to difficulties in comparisons of different populations [Different mtDNA regions or some complete mitogenomes have been used to study the genetic structure and analyse the phylogeography of bopictus , 40–42. fication . Howeverulations .Ae. albopictus from temperate regions of Western Mediterranean Europe and tropical regions of Brazil to assess their relationships. Research focused on haplotype and nucleotide diversity as well as genetic distances and phylogenetic networks combined with landmark- and outlined-based geometric morphometrics of wings. The aim was to infer genetic and phenetic diversity at intra-population and inter-population levels, as well as the genetic diversity at intra-individual level, of European and Brazilian populations of this vector to contribute to the understanding of vector spread and disease transmission risks.In the present work, we use rDNA/mtDNA genotyping and wing morphometry phenotyping to analyse populations of Ae. albopictus were collected between 2014 and 2018 in three countries, representing different localities in south-western Europe and Brazil, and analysed genetically and morphologically , and reared in laboratory colony under controlled conditions of 25 ± 1 °C, 75 ± 5% relative humidity and a 12 h/12 h light/darkness photoperiod [Ae. albopictus from Brazilian localities were obtained by spawning of females collected in oviposition traps in the aforementioned localities. The traps were brought to the laboratory under biosafety conditions required by current legislation. All material was sorted and the straws containing the eggs were transferred to vats containing water for hatching of the larvae. Emerging adults were identified and separated into breeding cages. The specimens were raised in the Laboratório de Mosquitos Transmissores de Hematozoários / IOC-Fiocruz, under controlled temperature and humidity.Adult mosquitoes from Spain were collected using oviposition traps , 44  of the mtDNA.A total of 111 cox1 gene was amplified with 2 sets of primers [2, 0.2 mM dNTPs, 10 pmol of each primer and 0.5 U of DNA Polymerase . Amplification procedures and thermal cycler conditions for each one of the DNA markers were carried out in a Mastercycle epgradient , as previously described [Each one of the DNA markers were PCR amplified independently for each specimen and each PCR product was sequenced for a bona-fide haplotype characterization. The complete rDNA ITS-2 was amplified using primers and PCR conditions previously described –50. The  primers . Each 25escribed , 51.Ae. albopictus specimens, the decision to go for ITS-2 cloning was taken already from the beginning, when double peaks were observed in many positions in the electropherograms of the ITS-2 sequences obtained by direct specimen sequencing. No one such situation of double peaks was observed in the cox1 sequences obtained. In the case of ITS-2, the PCR product obtained was cloned with pGEM-T Easy Vector System I  and introduced in Escherichia coli DH5α competent cells. After the growth of colonies, standard PCR of 2–8 different colonies per sample was performed and individually sequenced. This cloning allowed for the obtaining of a DNA sequence including half the length of the 5.8S gene followed by the complete ITS-2 sequence, in its turn followed by ~ 20 nucleotides of the initial 5' region of the 28S gene.When sequencing the PCR products were purified using the Ultra Clean™ PCR Clean-up DNA Purification System  according to the manufacturer's protocol and resuspended in 50 μl of 10 mM TE buffer (pH 7.6). The final DNA concentration (in μg/ml) and the absorbance at 260/280 nm were determined in a Eppendorf BioPhotometer  .Sequencing was performed on both strands by the dideoxy chain termination method carried out with the Taq dye-terminator chemistry kit on an Applied Biosystems 3730xl DNA Analyser  using the same amplification PCR primers.cox1). Minor corrections for a better fit of nucleotide or indel (insertion/deletion) correspondences were made in the cases of the ITS-2 spacer. Aligned sequences were collapsed to haplotypes, counting gaps as differences, using the ALTER web server [http://www.ncbi.nlm.nih.gov/BLAST). Genetic distances were measured using parameters provided by PAUP v.4.0a (build166) [Ae. albopictus downloaded from GenBank.Sequences were edited and assembled using Sequencher v5.4.6.  and aligned using CLUSTALW2 , using db server . Homologuild166) . Moleculuild166) . ComparaH), haplotype diversity (Hd), nucleotide diversity, expressed as the average number of nucleotide differences between two sequences by site (π), average number of nucleotide differences between sequences (k), number of polymorphisms and insertions/deletions (S). Deviations from selective neutrality were analysed using the statistics Fu’s Fs and Tajima’s D of the neutrality test by means of the DnaSP software. In FS, a negative value is evidence for an excess number of alleles, as would be expected from a recent population expansion or from genetic hitchhiking, whereas a positive value is evidence for a deficiency of alleles, as would be expected from a recent population bottleneck. In Tajima’s D, a negative value signifies an excess of low frequency polymorphisms relative to expectation, whereas a positive value signifies low levels of both low and high frequency polymorphisms. Hierarchical analysis of molecular variance (AMOVA) among the ITS-2 of south-western Europe and Brazil samples were computed in GenALEx 6.5 [DnaSP v.5.10.01  was usedALEx 6.5  accordinAe. albopictus populations from different geographical regions with PopART [http://www.fluxus-engineering.com/) software. Networks were constructed with the sequences obtained from each one of the molecular markers, using the TCS inference method in PopART, and using median-joining (MJ) network algorithm, with default parameters  in Network. Haplotypes were connected when the parsimony had a probability of at least 0.95 of being true, as established in the theory of coalescence, starting with the shortest distance to join all the haplotypes and until the distance exceeds the parsimony limit. Hypothetical median vectors (a hypothesized sequence that is required to connect existing sequences within the network with maximum parsimony) were added to the network for shortest connection between the data set.Haplotype networks were generated to depict relationship among h PopART  and NetwAe. albopictus populations. Both, the left and right wing of each specimen were dissected and mounted using STARMount® medium  on standard microscope slides. All wings were photographed with a Nikon Digital Sight DS-L1 (Nikon Corporation) camera linked to a Nikon ECLIPSE E800 microscope. A total of 103 wing pictures of the seven populations of Ae. albopictus was analysed for landmark and outline-based techniques. Left wings were used unless damaged, in which case right wings were used instead. For Ae. albopictus phenotyping, populations were divided in males and females. Furthermore, in order to improve the sample size, populations were analysed according to the following grouping criteria: Brazil , continental Spain , insular Spain , and France (Perpignan).Of the 111 specimens molecularly analysed, male and female mosquitoes with at least one suitable wing for morphometric analyses , were selected for phenotyping  comparison and discriminant analysis on partial warps or shape variables, using the MOG module of the CLIC package version 99 , 62, whiThe outline considered for population comparisons was the external contour of the wing Fig. b. Wing sIn order to perform a shape-based discrimination analysis, the same FOG module was used. Each pairwise Mahalanobis distance was computed between the normalized elliptic Fourier coefficients .cox1 gene sequences obtained provided 12 different haplotypes among all localities from south-western Europe and Brazil. Seven of them, were new haplotypes for Ae. albopictus and the other five haplotypes were detected shared with other countries, mainly with USA and Italy . Their length was of 1433 bp, with a biased AT content of 69.7–70.1% . The haplotype Ae. albopictus cox1-H1 was the most abundant, present in Valencia, Barcelona, and Mallorca Island, Spain, and in Perpignan, France. In the three distant localities analysed from Brazil , only one haplotype was detected (cox1-H12), which was not present in south-western Europe, but already reported in Brazil .cox1 haplotypes of Ae. albopictus, a total of 13 variable positions were detected, including 10 parsimony informative (p-info) positions and 3 singleton sites. Therefore, a comparative sequence analysis was made including these 12 Ae. albopictus haplotypes and other cox1 haplotypes and isolates of similar length  of the same species available in the GenBank database showing highly similar sequences according to BLASTN   and 0.0007 to 0.0083 (0.003), respectively. The highest values were obtained when comparing haplotypes from Philippines with haplotypes present in other countries  was 0.00108 (SD = 0.00009), the average number of nucleotide differences was 1.544, and the number of polymorphisms was 13. Tajima’s D and Fu’s Fs values were − 0.9953 (P > 0.10) and = − 3.363, respectively. A detailed analysis of the genetic diversity of each of the populations analysed is noted in Table In a first step, sequences obtained from 111 specimens, representing the 12 haplotypes here described from south-western Europe and Brazil, were analysed. The global haplotype diversity (Hd) was 0.752 (SD = 0.025), the nucleotide diversity , distant by only one mutational difference from haplotype cox1-H3, present in Barcelona and Mallorca , plus 42 sequences representing haplotypes/isolates from different localities and countries in Asia, Europe, and the Americas (haplotypes H13 to H27)  and proves to be the most common haplotype, present in 9 countries  , but also in Italy, China, and USA. The haplotype network shows the cox1-H1 haplotype to be highly connected, in a star distribution, by only one mutational step of distance with many haplotypes from Spain, but also with some ones from China  and, at two mutational steps, with one haplotype from USA (H26).A second network analysis was performed including 153 specimens representing our 12 haplotypes , one exclusive for France (cox1-H11), and five  are shared with other countries, mainly with Italy, USA and China. It is worth mentioning that the European haplotype more linked to Asiatic haplotypes is cox1-H2, present only in Barcelona and Mallorca Island  and subsequently a small last fragment of the initial at least 20 nucleotides of the 28S. After deleting the small 28S fragment, a total of 253 such 5.8S-ITS-2 sequences were obtained from the mosquitoes analysed. These sequences collapsed into 162 haplotypes (GenBank Acc. Nos. MW281865-MW282026).The pronounced length variability of the ITS-2 was assessed by means of the alignment of these 162 haplotypes. This alignment was 491-bp-long and contained 337 conserved positions and 153 variables sites, of which, 50 were p-info and 103 singleton sites. The ITS-2 length variability was seen to be caused by regions of insertions in consecutive positions which are unexpectedly long, i.e. concerning more than 10 consecutive positions. Such long regions involved 57–58 and/or 20–30 consecutive insertion/deletions (indels) located in the positions 302–361 and 333–389 of the total alignment, respectively. These correspond to positions 222–281 and 253–309 of the ITS-2. In Mallorca and Perpignan, two and three clone sequences showed such deletion regions in the alignment, respectively. In Manaus, such deletion regions were observed in up to six clone sequences. Only in Barcelona, in the aforementioned alignment no such clone sequence showing long deletion regions was detected in the long-sized ITS-2 of the specimens from this origin.The length and GC content of the 162 haplotypes of the 5.8S-ITS-2 rDNA varied between 429 and 487 bp in length (481.2 bp) and 52.8–56.8% (55.5%), respectively. Sequences were aligned according to their geographical locations and polymorphic sites were analysed. All populations presented some mutated positions in the conserved fragment of the 5.8S rRNA gene, ranging from 0–7 mutations (average 2.14), except in samples from Barcelona, whereas in the ITS-2 mutations ranged between 16 and 47 36.9). Except in Jurujuba, in all populations the majority of these mutations were due to singleton sites. Gaped positions, generated by indels, were observed in the alignments of each population, ranging from 11–83, with an average of 49.2 indels  and 1–6 (3.3) in European and Brazilian specimens, respectively. The number of haplotypes found in a population was also very high, ranging 16–34 (27.6) haplotypes in Spain , 30 haplotypes in France, and 14–38 (24.7) in Brazil.When comparing the sequences of these 162 haplotypes, the pairwise 5.8S-ITS-2 evolutionary divergence matrix obtained with MEGA 7 shows that the total number of absolute differences (gaps not included) in pairwise comparisons ranged between 1 and 14 (average 4.8). The highest values were obtained when comparing haplotypes from Valencia with haplotypes from Manaus (Table not shown).Ae. albopictus haplotypes and other haplotypes of similar length  of the same species available in the GenBank and showing highly similar sequences according to BLASTN. The first data set included 258 sequences, of which 162 from the present study and 96 from GenBank . Among these 258 sequences, haplotypes of Ae. albopictus sensu stricto and Aedes sp. cryptic species [Ae. albopictus cryptic species were present in our samples.A comparative ITS-2 sequence analysis was made including our 162  species , 66 wereA second analysis was performed retrieving those cryptic haplotypes. The data set resulted in 230 sequences, 162 from our samples plus 68 from GenBank, representing the same seven aforementioned countries. Due to differences in length among the samples included in this data set, the region corresponding to the 5.8S gene was deleted and sequences were adjusted to the same length of the ITS-2 in a 421 bp-long alignment. This alignment contains 244 variable positions of which, 117 were p-info and 127 singleton sites. These 230 sequences collapsed into 185 haplotypes according to ALTER. Among these 185 haplotypes, only 12 GenBank entries showed identity with 8 of our haplotypes. Italy was the country sharing most haplotypes (6) with Valencia (H89), France (H151), Goiania , and Manaus (H49). Two of the haplotypes shared between our European and Brazilian populations (H12 and H25)  from St. Denis in Reunion Island , presentWhen comparing the sequences of the 253 specimens representing these 162 haplotypes, the pairwise 5.8S-ITS-2 distance matrix obtained with MEGA 7 allowed us to analyse the overall mean distance at intra-individual, intra- and inter-populational groups, showing that the total number of differences (ts + tv) ranged 3.3–6.0 , 3.7–7.5 (5.6) and 4.6–8.9 (6.7), respectively. Substitutions per site (using Kimura 2-parameter model) ranged between 0.00964 and 0.01919 (average 0.01433) between the seven populations. The highest values were obtained when comparing intraindividual sequences and intrapopulation sequences from Goiania. At inter-population level, the greatest genetic distance (8.9) was obtained between Manaus and Jurujuba, while the shortest (4.6) were between Valencia, Barcelona and Perpignan populations  within and among . The global haplotype (gene) diversity (Hd) was 0.927 (SD = 0.013), the nucleotide diversity (π) was 0.01059 (SD = 0.00049), the average number of nucleotide differences was 4.225, and the number of polymorphisms was 153. Tajima’s D and Fu’s Fs values were − 2.422  and = − 180.685, respectively. At population level, the Hd estimates across the analysed sequences ranged from 0.862 ± 0.045 in Jurujuba (Brazil) to 0.971 ± 0.032 in Barcelona (Spain), and the nucleotide diversity (π) ranged between 0.00773 ± 0.00119 in Valencia (Spain) up to 0.01658 ± 0.00081 in Goiania (Brazil). A detailed analysis of the genetic diversity of each population analysed is listed in Table Haplotype network analysis revealed 162 unique haplotypes among the 253 sequences obtained from south-western Europe and BrazilThe haplotype ITS2-H12 was the most common, including samples from Goiania and Manaus (Brazil), Valencia and Mallorca (Spain) and Perpignan (France). A large portion of the haplotypes, namely 138/162 (mean 85.2%), proved to be unique, whereas 9/162 (5.6%) were shared only between specimens of the same population, and 15/162 (9.2%) were shared among populations. Among those 15 haplotypes, 3 (1.85%) were shared between Brazilian and European populations, 8 4.93%) were shared exclusively between Mediterranean populations of Europe , and the remaining 4 (2.46%) haplotypes appeared shared between only Brazilian populations  did not show noticeable differences. However, some landmark displacements were visible in the final part of the female wings and in the upper and the final part of the male wings Fig. a. In botil Table a. Landmail Table a. Summinil Table a.Fig. 6avs European and Brazilian specimens.Size was considered as the outline perimeter and yielded similar results to those obtained in the CS analysis Table b. The coFor the outline-based method, the factor maps of the discriminant analysis Fig. b showed Ae. albopictus diversity and relationships in south-western Europe and Brazil. In Europe, the climate is temperate, and the introduction of this mosquito is recent (first record in 1979 in Albania—[This is the first study that explores the combined use of rDNA/mtDNA and phenotypic markers to assess  Albania—), includ Albania—. In bothcox1 marker, different haplotype variability results have been obtained in numerous studies on Ae. albopictus in different geographical zones [cox1 fragments used in many of these studies [cox1 haplotyping based on a long fragment of 1433 bp  of populations from south-western Europe and Brazil to compare their potential genetic similitude and their phylogenetic relationships with other strains from Europe, Asia and the Americas. In this analysis, surprisingly no one cox1 haplotype appeared shared by Europe and Brazil, suggesting different genetic structure and possible different susceptibility to infectious agents.Regarding the al zones , 69, 70, studies . Accordi studies , 41, 51,cox1 haplotypes obtained in the present study is remarkably small (12 haplotypes), of which 6 are exclusive for Spain, 1 for France, and 5 shared (1 between Brazilian populations and 4 between European populations), and with very little genetic variability (0.90% of polymorphic sites). When this cox1 analysis was expanded including 153 samples representing 13 countries in Europe, Asia and the Americas, the number of haplotypes obtained was also scarce (27 haplotypes) and the genetic variability was low (2.0%). These results differ from the 66 haplotypes obtained when sequencing the same cox1 fragment (1433 bp) in 12 populations from several countries of 2 continents, covering a wider geographic region [cox1 polymorphism, or high genetic similarity, was also reported in other European populations, not allowing to resolve the structuring of the different populations [Ae. albopictus, but also to the recent invasion of this species into countries or the inclusion of samples from a relatively narrow geographical range [FS in the neutrality test indicate an excess of low frequency polymorphisms and a recent population expansion.Despite having studied seven populations, from three countries and two continents, the total number of c region . A lowerulations , 71. Thial range . The oveWolbachia found co-infecting almost all Ae. albopictus populations studied [cox1 gene when working with longer sequences of this gene posed a question mark on the Wolbachia impact hypothesis [The low diversity found in mtDNA markers was initially hypothesized to be the consequence of a mitochondrial hitchhiking caused by the co-infection by the two strains of the endosymbiotic bacteria  studied , 72. Howpothesis , 73.cox1  highly distributed not only in Spain , but also in Italy, China and USA.The median joining haplotype network of ox1 Fig. b shows tabundant , 51, 66.cox1 gene among the Brazilian populations, despite the long geographical distances between the populations studied , should be emphasized. This may be due to its recent introduction and spread, as suggested by its very recent first detection in this country [cox1 haplotype (cox1-H12). Reduced levels of genetic variation in Ae. albopictus from Manaus (Amazonas state), and in other four populations from south eastern Brazil, have been also detected, although based on the mtDNA gene coding for NADH dehydrogenase subunit 5 (nad5) [The absence of genetic variability in the  country . All of 5 (nad5) , 75.The rDNA ITS-2 is considered as relatively conserved and species-specific. Among the hugely considered molecular markers for mosquito taxonomy, ITS-2 have been widely used to infer relationships at species level, up to even closely related species, subspecies or populations .Ae. albopictus is recently increasing in GenBank, few studies have focused on this marker to infer intra-individual or intra- and inter-populational variability [Ae. albopictus based on 5.8S-ITS-2 and highlights the presence of high intraspecific variability among populations collected from different eco-regions. This genetic variability was revealed by:differences in length, ranging from 429 to 487 bp (average 481.2 bp);high mutation rate ;high indel number ;mutations in the conserved 5.8S fragment ;high number of haplotypes obtained, both within the same specimen and within the same population; than between individuals of the same population  and both slightly less than between populations .overall pairwise distances similar within individuals Although the number of ITS-2 sequences of iability . Most ofiability , 76–80 oiability , 66. OurAccording to AMOVA, most of the genetic variation was observed within individuals (73%) or within populations (88%) and not among populations (9%) or among groups (4%) at both singe population and at geographic region groups, respectively.cox1 (0.9%) for the same specimens and geographical origins. This high 5.8S-ITS-2 variability has allowed us to describe a high number of haplotypes within the same specimen: 2–7 haplotypes (mean 4.7) in south-western Europe, and 1–6 (3.3) in Brazil. The ITS-2 haplotype number is higher than the mean of 2 haplotypes per individual previously reported [The 253 5.8S-ITS-2 sequences here obtained provided 162 unique haplotypes, of which 66 haplotypes for Brazil, 73 for Spain and 23 for France. Their alignment contains 153 variable positions, which represent a variability of 31.2%, considerably greater (about 35 times higher) than those obtained with the reported .The number of ITS-2 haplotypes obtained for each one of the seven geographical populations was also high, ranging 16–34 (27.6) haplotypes in Spain , 31 haplotypes in France and 14–38 (24.7) in Brazil. The high number of haplotypes in the same population or the fact of sharing few haplotypes between populations, coincides with previous reports about native and introduced populations of this vector . OverallAnopheles mosquitoes, there are species as An. gambiae in which the ITS-2 shows no important length differences due to only one indel linked to species subtypes [An. barbirostris group a wide ITS-2 length variability was found as a consequence of different numbers of long tandem repeats [Ae. albopictus, the analysis of polymorphisms in ITS-2 confirms the existence of variable sites due to mutations (p-info + singleton sites), but also numerous indels, which generate consecutive regions with deletions underlying large differences in ITS-2 length both within the same individual and in different individuals of the same population. Interestingly, in the Barcelona population, considered the first Ae. albopictus population to arrive in Spain, none of its specimens presented the long regions of indels. In the remaining populations from Spain, France and Brazil we found specimens showing these anomalies in the length of this spacer.In regions of the rDNA operon with faster evolution, as is the case of ITS-2, the appearance of indels is common even in different populations of the same species. However, they are considered parsimony not-informative characters, although they may be useful for the distinguishing of populations . In Anopsubtypes , whereas repeats . In Ae. Ae. albopictus. In the literature there is only one case ;Italy shared at least 6 haplotypes with Valencia, France, Goiania and Manaus;Ae. albopictus around the world and mainly in areas where recent outbreaks of arboviruses and other pathogens have taken place.two of the 3 haplotypes shared between Europe and Brazil, were also detected in Italy, USA and Thailand, illustrating genetic relationships and spread of Compared analysis with other countries allowed us to confirm that no haplotypes corresponding to the subgroup of Aedes and Culex [Anopheles vectors [Ae. albopictus indicate multiple introduction events, evidenced by genetic differences with the founder population in Spain. Indeed, the Mallorca island population exhibits the highest cox1 haplotype diversity (Hd) and nucleotide diversity (π), typical of an origin population . From the founder population, the offspring expanded along the Mediterranean coast and the Balearic Islands. In these islands, they were affected by the introduction of other lineages due to recent anthropogenic activities, as e.g. tourism and commercial flow.Intra-individual variation in the sequence of the rDNA operon is unusual in animal genomes, although it has been reported in fishes and corals in relation to polyploidy, hybridization and a reduced rate of concerted evolution, and also in other insects . In mosqnd Culex  and in m vectors , 88. Thecox1, the overall negative values of Tajima’s D and FS in the neutrality test indicate an excess of low frequency polymorphisms and a recent population expansion.The large ITS-2 haplotype variability in populations and inside specimens may be interpreted as the consequence of successive introductory events. Moreover, the result furnished by the TCS network illustrating the absence of defined haplotype groups according to geography and the interconnections between all haplotypes Fig.  indicateAe. albopictus have a larger centroid wing size than males, in agreement with other studies in Ae. aegypti [Ae. albopictus was detected. The mosquitoes from insular Spain were significantly different from those of continental Spain/France and Brazil (P < 0.05). Although environmental factors, such as food quality and quantity, temperature and suitable habitat, have been evoked to underlie similar results in Mansonia species [cox1  and ITS-2  levels, and are in agreement with the phenotypic results.Morphometric studies have allowed to infer that females of  aegypti , 90. Fur species , the pos species , 93. MolRegarding wing shape variation, the factor map of the landmark-based discriminant analysis derived from final shape variables allowed for the detection of two zones: one corresponding to specimens from Brazil and another to specimens from Spain  and France.Ae. albopictus. Normally, living organisms, including mosquitoes, are adapted to their environment. The present analysis suggest that the geographical spread affected Ae. albopictus morphology. These results are consistent with those obtained in Ae. aegypti in Thailand [Ae. albopictus, distances were generally longer between males than between females, which is in agreement with other analyses on wing morphometrics of three Aedes vectors in Thailand [The factor map of the outline-based discriminant analysis derived from shape variables detected four separated areas corresponding to specimens from Brazil, France, continental and insular Spain, respectively, in the wing shape morpho space of male and female Thailand . InteresThailand .Ae. albopictus, providing pronouncedly more information than the conserved mtDNA cox1 usually used for this kind of studies on this mosquito species;the rDNA ITS-2 proves to be a useful marker for studies on the spread of the rDNA ITS-2 sequence variation allows for studies at intra-individual, intra-population and inter-population levels, thus furnishing a complete overview of the evolutionary exchanges followed by a mosquito species of such a high colonization power;Ae. albopictus also shows nucleotide differences between the clones of the highly conserved very short 155-bp-long rDNA gene 5.8S inside an individual, additional to intra-population and inter-population variability, according to an evolutionary rate lower that in the ITS-2 ;the mosquito species the wing morphometry proves to be a useful phenotyping marker, allowing to distinguish different populations at the level of both male and female specimens.Both rDNA and mtDNA marker sequencing has furnished very interesting results of high applied interest:The pronounced ITS-2 variability found in intra-individual clones suggests hybridizations with specimens of different origin and that these hybridizations were very recent. The presence of different haplotypes inside the same individual indicates haplotype accumulation at a very high speed, as for the rDNA concerted evolution to have not yet the time to operate towards uniformization.The large genetic diversity detected by the ITS-2 in the Spanish populations studied is surprising if it is considered that the spread of this mosquito species along the Mediterranean coast by truck transport  is belieAe. albopictus from Europe and Brazil should be highlighted. The existence of ITS-2 haplotypes shared by Brazil, Spain and France leaves the doors open for future tests of susceptibility and vector competence regarding strains of ZIKV and other viruses and pathogens. All in all, the results of this study indicate the need for periodic surveillance monitorings to verify that no Ae. albopictus with high virus transmission capacity has been introduced into Europe.The detection, for the first time, of a degree of kinship indicated by the similar genetic structure of populations of Additional file 1: Table S1. Distribution of the cox1 sequences analysed of Ae. albopictus and the corresponding 27 haplotypes they provided, according to their geographical origin.Additional file 2: Table S2. Pairwise distances between cox1 nucleotide sequences of Ae. albopictus populations analysed according to PAUP.Additional file 3: Table S3. Phenotypic diversity of Ae. albopictus from south-western Europe and Brazil. Landmark-based (LB) and outline based (OB) Mahalanobis distances (LB/OB) obtained with discriminant analysis between wing shapes of females and males.