The peopling of Sardinia (Italy): history and effects

Over the last ten years the population of the Mediterranean island of Sardinia has been object of numerous studies in the fields of anthropology and population genetics. Its insularity, central position in the Mediterranean area and rich historical past have made the island a veritable laboratory for the study and understanding of those interacting evolutionary mechanisms which determine a population's genetic structure. Indeed, from work performed at different levels on genetic structure analysis there emerges an extremely complex picture of the relationships between Sardinian and other Italian and Mediterranean populations, but also of relationships within the Sardinian population itself The diversification from Mediterranean and Italian populations can be explained by Sardinia's historical and demographic past. Internal heterogeneity can be attributed, in part, to strict isolation and the accompanying high levels of endogamy and inbreeding, and in part to the endemic presence of malaria which exerted a strong selective pressure on some characteristics; determining, for example, the differentiation between the plains and the mountain areas. Finally, an influence on Sardinia's biological history not to be neglected could be attributed to the demographic events, which triggered off phenomena of genetic drift and to cultural factors.     

The HLA system in Italy

4,902 Italians were typed for HLA-A antigens, 4,721 for HLA-B and 1,503 for HLA-C. The samples, which were composed of unrelated, healthy individuals born in Italy, were used for estimating HLA-A, HLA-B and HLA-C gene frequencies with the maximum-likelihood method. Different Italian regions showed significant differences in the HLA alleles, providing further evidence for the genetic heterogeneity of the Italian population. HLA gene frequencies place continental Italy and Sicily in a position which is similar to that of other Mediterranean populations, whereas the genetic isolation of Sardinia is quite evident. The most significant linkage disequilibrium values found in the Italian population (except for Sardinia) were in agreement with those observed in other Caucasian populations. The difference between Northern and Southern Italy and between continental Italy and Sardinia was emphasized by the linkage disequilibrium values and by the principal-component analysis as well.     

Understanding biological conservation strategies: a molecular-genetic approach to the case of myrtle (<Emphasis Type="Italic">Myrtus communis</Emphasis> L.) in two Italian regions: Sardinia and Calabria

Myrtle (Myrtus communis L.), a shrub widespread in the Mediterranean area, is the only species belonging to the Myrtaceae family growing in Europe. The pharmacological and aromatic properties of myrtle have caused a growing interest in this plant. The use of myrtle as an aromatic plant is traditionally established in the Italian regions of Sardinia and Calabria, where it is subjected to an ecological stress due to the large biomass removal for liquor production and environmental damage. The reduction in size and number of wild populations is often related to a loss of genetic variation and reproductive potential. In this study fluorescent amplified fragment length polymorphisms (fAFLPs) were employed to assess the genetic variation within and among natural populations of myrtle from Sardinia and Calabria to gain new insights into their fitness and survival potential. AMOVA analysis indicated that genetic variation was greater within populations (51.86%) than among populations (16.99%), as previously reported for outcrossing species. A significant amount of variation (31.15%) was attributed to variation between Sardinia and Calabria population groups, suggesting a genotypic differentiation between the myrtle populations of these two regions. Intra-population genetic variation, assessed by estimating expected unbiased heterozygosity (H _E), ranged from 0.0595 to 0.2595. These values resulted correlated with population extension (r = 0.918; P < 0.01) and with two reproductive parameters: seed germinability (r = 0.793; P < 0.01) and number of seeds per fruit (r = 0.631; P < 0.05). A moderate gene flow among Sardinia myrtle populations and Calabria populations (1.2719 and 1.0478, respectively) counteracts the low level of genetic variation observed in some␣populations and avoids their differentiation and isolation.     

Does genetic population structure of Ambrosina bassii L. (Araceae,Ambrosineae) attest a post-Messinian land-bridge between Sicily and Africa?

Aim of the present work is the analysis (through the study of enzyme polymorphism) of Sicilian and African (Tunisian) populations of Ambrosina bassii, a small perennial endemic to the Central-Western Mediterranean basin, in order to verify if the complex geological history of this part of the Mediterranean area left its mark in the present-day genetic structure of this taxon. Starch gel allozyme electrophoresis of seven putative loci of A. bassii was employed to estimate genetic diversity, genetic structure and gene flow. Populations from Sicily, Tunisia and Sardinia (as outgroup) were sampled. Results show that Sicily populations have 4 private alleles, Sardinia 3, Tunisia just one. One allele is private to both Sardinia and Tunisia, another one to Sardinia and Sicily. Even if there are no alleles private to Sicily and Tunisia, “cluster analysis” (based on Nei's genetic distances), “non-metric multidimensional scaling” (computed on the basis of a matrix with F_ST values between populations) and Bayesian analysis point out a clear isolation of Sardinian populations, and a greater similarity between Sicilian and Tunisian populations compared to Sardinian ones. The strong genetic affinity between populations from Sicily and Tunisia, considering the very low dispersal ability of the species, gives evidence of a recent continuity between the populations as well as between the two areas. Considering also the estimates of divergence times, a post-Messinian terrestrial connection between the two landmasses can be hypothesized.     

Chitotriosidase,a prematurely orphan enzyme

Human Chitotriosidase (CHIT) is a member of the chitinase family and is synthesized by activated macrophages. Recently, a genetic polymorphism was found to be responsible for the common deficiency in CHIT activity, frequently encountered in different population. We analyzed the CHIT gene in some ethnic groups from Mediterranean and frican area, to evaluate whether the CHIT gene polymorphism correlates with the changes in environmental features and the disappearance of parasitic diseases. We evaluate the plasma CHIT activity and analyzed, by PCR, the Chit gene polymorphism in 100 Sicilian, 107 Sardinian and 99 Sub-Saharan subjects. We found an heterozygote frequency for a duplication of 24 base pair in exon 10 of 44,54% in Sicily and 32,71 % in Sardinia, whereas homozygous chit deficient were 5,45 % and 3,73%, respectively. On the contrary in Burkina Faso, a mesoendemic regio nforPlasmodium falciparum malaria and other intestinal parasites, a low incidence of CHIT mutation was found (heterozygous 2%) and any subject was homozygous for CHIT deficiency. Our results suggest that in sub-Saharan population the intact CHIT gene seems essential for sustaining resistance against chitin-coated parasitic disease, whereas the presence of CHIT gene polymorphism in Mediterranean population could be the result of a recent positive selection due to improved environmental conditions, which makes prematurely orphan this enzyme.     

Genetic isolation of insular populations of the Maghrebian bat,Myotis punicus,in the Mediterranean Basin

Aim We investigate the population genetic structure of the Maghrebian bat, Myotis punicus, between the mainland and islands to assess the island colonization pattern and current gene flow between nearby islands and within the mainland. Location North Africa and the Mediterranean islands of Corsica and Sardinia. Methods We sequenced part of the control region (HVII) of 79 bats across 11 colonies. The phylogeographical pattern was assessed by analysing molecular diversity indices, examining differentiation among populations and estimating divergence time. In addition, we genotyped 182 bats across 10 colonies at seven microsatellite loci. We used analysis of molecular variance and a Bayesian approach to infer nuclear population structure. Finally, we estimated sex‐specific dispersal between Corsica and Sardinia. Results Mitochondrial analyses indicated that colonies between Corsica, Sardinia and North Africa are highly differentiated. Within islands there was no difference between colonies, while at the continental level Moroccan and Tunisian populations were highly differentiated. Analyses with seven microsatellite loci showed a similar pattern. The sole difference was the lack of nuclear differentiation between populations in North Africa, suggesting a male‐biased dispersal over the continental area. The divergence time of Sardinian and Corsican populations was estimated to date back to the early and mid‐Pleistocene. Main conclusions Island colonization by the Maghrebian bats seems to have occurred in a stepping‐stone manner and certainly pre‐dated human colonization. Currently, open water seems to prevent exchange of bats between the two islands, despite their ability to fly and the narrowness of the strait of Bonifacio. Corsican and Sardinian populations are thus currently isolated from any continental gene pool and must therefore be considered as different evolutionarily significant units (ESU).     

Long-term changes in population genetic features of a rapidly expanding marine invader: implication for invasion success

Large blooms of Rhopilema nomadica, a highly venomous rhizostamatid scyphozoan species introduced to the Mediterranean through the Suez Canal, have become ubiquitous in the summer and winter months along the Israeli coasts since the mid-1980s. This species has since spread across the eastern Mediterranean and was sighted as far west as Tunisia and Sardinia. For the past 12 years, we have studied changes in the mitochondrial COI haplotypes diversity of R. nomadica to investigate small scale fluctuactions of genetic diversity and to reveal possible genetic structuring of the fast spreading invader in the Eastern Mediterranean. The 1091 COI sequences analysed, revealed a highly diverse population displaying 89 haplotypes, 46 of which appeared as singletons, low frequency haplotypes. All the specimens analysed throughout the period belong to a single unstructured population. Though lacking data from the source population in the Red Sea, the high within-population diversity and the high diversity of COI haplotypes support the hypothesis of multiple introductions events, or an open corridor with a continuous influx of propagules. Tajima’s D and Fu’s Fst negative values and the increased numbers of COI singletons from early to late sampling periods, have verified that the Israeli population is characterized by a rapid expanding population. Further research is needed for the evaluation of COI diversity and patterns in R. nomadica populations across the eastern Mediterranean Sea and Red Sea, as well as any correlation of the high variability between COI locus and phenotypic diversity.

     

Environment change, geographic migration and sickle cell anaemia

The classic model describing the interaction between sickle cell anaemia and malaria is one of the most notable achievements of population genetics. Nevertheless, only panmictic populations in steady environments have been studied theoretically to date. In this paper, environment change and geographic inhomogeneity are introduced. The rate of decrease of mutation after environment improvement is obtained. The kinetics of the spread of disease after the initial mutation, together with the genetic composition profile near the borders of malaria areas, are calculated. The results are compared with the empirical data on the mutation level in African and African-American populations. It is shown that the spread of disease and decrease in mutation are highly asymmetric: the mutation level increases exponentially and decreases much more slowly (as a power function). The mathematical and biological reasons for this behaviour are discussed.     

Genetic structure of insular Mediterranean populations of the house mouse

The genetic structure of six western Mediterranean island populations of Mus musculus domesticus were investigated by means of an electrophoretic analysis at 34 loci and compared to that of five neighbouring mainland populations. No reduction in variability (H = 0.09 for both island and mainland samples) was observed in the island populations except for a very small island (6 ha), in which one-third of the variability was lost. Patterns of colonization inferred from a Wagner cluster analysis and the distribution of rare variants suggest that, although these island populations are clearly related to European mainland mice, their genetic structure is the result of multiple founding events from sources dispersed throughout the Mediterranean Basin. Furthermore, the presence of the same rare alleles in Sardinia, Corsica and Piana, suggest that the three islands share a common history of colonization.
Estimates of genetic distance and gene flow indicate that the level of genie differentiation is greater between island and mainland populations that between the latter due to geographic isolation. Multiple founder events and post-colonization evolution are the factors that best explain the observed levels of genie variability and differentiation in these Mediterranean islands.     

Are captive wild boar more introgressed than free-ranging wild boar? Two case studies in Italy

Hybridization between wild boar (Sus scrofa) and domestic pig occurred in the past and still occurs today, having great evolutionary and management implications. In fact, genetic introgression from the domestic form may alter traits like behavior, reproduction rate, and immunology in wild populations, with likely demographic impacts. Thus, it is crucial to understand under what conditions hybridization occurs in S. scrofa. Captive crosses with domestic pigs (released or escaped) have been suggested to constitute the major source of the spread of domestic genes into wild boar populations. However, to date, few studies have assessed the degree of admixture in farmed animals in comparison to the surrounding wild populations. With this purpose, we analyzed microsatellite loci in wild boar sampled in breeding stations and in the local wild population in two Italian regions (Sardinia and Piedmont). Both captive populations had lower allelic richness than the corresponding wild population, but a similar expected heterozygosity. In Piedmont, introgression from the domestic form into the wild population seems to be extremely low, while there are significant signs of admixture in the sampled breeding stations. In Sardinia, instead, the captive sample did not differ significantly from the wild population, which showed moderate signs of introgression. We conclude that hybridization in nature seems to play the key role in Sardinia, while intentional hybridization in captivity is the major source of introgression in Piedmont. Our findings emphasize the need for a routine genetic monitoring of wild boar captive populations, coupled with reference data on the neighboring wild populations.     

Genetic structure of the killifish Aphanius fasciatus, Nardo 1827 (Teleostei, Cyprinodontidae), results of mitochondrial DNA analysis

Aphanius fasciatus is a cyprinodont distributed in the salty coastal water of the central and eastern Mediterranean Sea and occasionally in internal fresh water. In this work, the authors have investigated the genetic structure of eight populations of the killifish A. fasciatus from Sardinia and Sicily. The comparison of the mtDNA control region of 237 individuals revealed a total of 49 haplotypes. Several unique haplotypes were present in each population, and no common haplotype was found among Sicilian and Sardinian populations. Almost all Sardinian populations shared a common haplotype, and indeed the four Sicilian populations examined did not share any as determined by the parsimony network analysis. The analysis of molecular variance showed that the percentage of variation among populations is much higher than within each population of A. fasciatus . The overall F _ST value is very high (0·78) and supports an extensive genetic structure of the populations. The observed genetic differentiations of A. fasciatus populations were discussed taking into account the palaeogeographic and palaeoclimatic events that interested the Mediterranean area from Miocenic to Pleistocenic age. The results provide new insight into the knowledge of the pattern of genetic structure and of evolutionary processes occurring in this species.     

Inverse Medea as a novel gene drive system for local population replacement: a theoretical analysis

One strategy to control mosquito-borne diseases, such as malaria and dengue fever, on a regional scale is to use gene drive systems to spread disease-refractory genes into wild mosquito populations. The development of a synthetic Medea element that has been shown to drive population replacement in laboratory Drosophila populations has provided encouragement for this strategy but has also been greeted with caution over the concern that transgenes may spread into countries without their consent. Here, we propose a novel gene drive system, inverse Medea, which is strong enough to bring about local population replacement but is unable to establish itself beyond an isolated release site. The system consists of 2 genetic components--a zygotic toxin and maternal antidote--which render heterozygous offspring of wild-type mothers unviable. Through population genetic analysis, we show that inverse Medea will only spread when it represents a majority of the alleles in a population. The element is best located on an autosome and will spread to fixation provided any associated fitness costs are dominant and to very high frequency otherwise. We suggest molecular tools that could be used to build the inverse Medea system and discuss its utility for a confined release of transgenic mosquitoes.     

Main roads and land cover shaped the genetic structure of a Mediterranean island wild boar population

Patterns of genetic differentiation within and among animal populations might vary due to the simple effect of distance or landscape features hindering gene flow. An assessment of how landscape connectivity affects gene flow can help guide management, especially in fragmented landscapes. Our objective was to analyze population genetic structure and landscape genetics of the native wild boar (Sus scrofa meridionalis) population inhabiting the island of Sardinia (Italy), and test for the existence of Isolation‐by‐Distance (IBD), Isolation‐by‐Barrier (IBB), and Isolation‐by‐Resistance (IBR). A total of 393 Sardinian wild boar samples were analyzed using a set of 16 microsatellite loci. Signals of genetic introgression from introduced non‐native wild boars or from domestic pigs were revealed by a Bayesian cluster analysis including 250 reference individuals belonging to European wild populations and domestic breeds. After removal of introgressed individuals, genetic structure in the population was investigated by different statistical approaches, supporting a partition into five discrete subpopulations, corresponding to five geographic areas on the island: north‐west (NW), central west (CW), south‐west (SW), north‐central east (NCE), and south‐east (SE). To test the IBD, IBB, and IBR hypotheses, we optimized resistance surfaces using genetic algorithms and linear mixed‐effects models with a maximum likelihood population effects parameterization. Landscape genetics analyses revealed that genetic discontinuities between subpopulations can be explained by landscape elements, suggesting that main roads, urban settings, and intensively cultivated areas are hampering gene flow (and thus individual movements) within the Sardinian wild boar population. Our results reveal how human‐transformed landscapes can affect genetic connectivity even in a large‐sized and highly mobile mammal such as the wild boar, and provide crucial information to manage the spread of pathogens, including the African Swine Fever virus, endemic in Sardinia.     

Isolation and characterization of microsatellite loci in the striped mullet,Mugil cephalus

Forty‐three microsatellites were isolated from grey mullet (Mugil cephalus) of Mediterranean origin using the standard enrichment methods followed by enhanced chemiluminescence for the rapid identification of clones containing microsatellites. Eleven microsatellites were characterized to assess genetic diversity of different populations surrounding the island of Sardinia in Italy. The versatility of these markers was also assessed using an Australian population. All 11 microsatellites were highly polymorphic with a mean heterozygosity of 0.834. The number of alleles ranged from 14 to 38 in the Sardinian populations and from 11 to 25 in the Australian samples.     

Genetic data on Alghero population (Sardinia): Contrast between biological and cultural evidence

Data on 20 genetic polymorphisms (61 alleles) in the Algehero population on the northwestern coast of Sardinia are presented and discussed in relation to its linguistic peculiarity inside the island. Since the Aragonese (Spain) conquest of Sardinia in the 13th century, the Catalan language, the same as that spoken in Northeastern Spain and certain districts of Southern France, has been used in Alghero even until today. Analysis for heterogeneity of gene frequency distributions indicates that the genetic information obtained on Alghero is adequate to discriminate Sardinians from other neighbouring populations. Genetic variation between populations measured through genetic distances and principal-component analysis shows that the present-day population of Alghero is much closer genetically to Sardinians than to Catalonians. Our genetic results do not support any interpretation of the linguistic affinities between Alghero and Catalonia at present as indicative of biological kinship. © 1994 Wiley-Liss, Inc.     

Molecular population genetics of the NADPH cytochrome P450 reductase (CPR) gene in Anopheles minimus

Development of insecticide resistance (IR) in mosquito vectors is a primary huddle to malaria control program. Since IR has genetic basis, and genes constantly evolve with response to environment for adaptation to organisms, it is important to know evolutionary pattern of genes conferring IR in malaria vectors. The mosquito Anopheles minimus is a major malaria vector of the Southeast (SE) Asia and India and is susceptible to all insecticides, and thus of interest to know if natural selection has shaped variations in the gene conferring IR. If not, the DNA fragment of such a gene could be used to infer population structure and demography of this species of malaria vector. We have therefore sequenced a ~569 bp DNA segment of the NADPH cytochrome P450 reductase (CPR) gene (widely known to confer IR) in 123 individuals of An. minimus collected in 10 different locations (eight Indian, one Thai and one Vietnamese). Two Indian population samples were completely mono-morphic in the CPR gene. In general, low genetic diversity was found with no evidence of natural selection in this gene. The data were therefore analyzed to infer population structure and demography of this species. The 10 populations could be genetically differentiated into four different groups; the samples from Thailand and Vietnam contained high nucleotide diversity. All the 10 populations conform to demographic equilibrium model with signature of past population expansion in four populations. The results in general indicate that the An. minimus mosquitoes sampled in the two SE Asian localities contain several genetic characteristics of being parts of the ancestral population.     

Environmental gradients shape the genetic structure of two medicinal Salvia species in Jordan

• Environmental gradients, and particularly climatic variables, exert a strong influence on plant distribution and, potentially, population genetic diversity and differentiation. Differences in water availability can cause among‐population variation in ecological processes and can thus interrupt populations’ connectivity and isolate them environmentally. The present study examines the effect of environmental heterogeneity on plant populations due to environmental isolation unrelated to geographic distance.
• Using AFLP markers, we analyzed genetic diversity and differentiation among 12 Salvia spinosa populations and 13 Salvia syriaca populations from three phytogeographical regions (Mediterranean, Irano‐Turanian and Saharo‐Arabian) representing the extent of the species’ geographic range in Jordan. Differences in geographic location and climate were considered in the analyses.
• For both species, flowering phenology varied among populations and regions. Irano‐Turanian and Saharo‐Arabian populations had higher genetic diversity than Mediterranean populations, and genetic diversity increased significantly with increasing temperature. Genetic diversity in Salvia syriaca was affected by population size, while genetic diversity responded to drought in S. spinosa. For both species, high levels of genetic differentiation were found as well as two well‐supported phytogeographical groups of populations, with Mediterranean populations clustering in one group and the Irano‐Turanian and Saharo‐Arabian populations in another. Genetic distance was significantly correlated to environmental distance, but not to geographic distance.
• Our data indicate that populations from moist vs. arid environments are environmentally isolated, where environmental gradients affect their flowering phenology, limit gene flow and shape their genetic structure. We conclude that environmental heterogeneity may act as driver for the observed variation in genetic diversity.

     

Peopling of three Mediterranean islands (Corsica,Sardinia, and Sicily) inferred by Y-chromosome biallelic variability

An informative set of biallelic polymorphisms was used to study the structure of Y-chromosome variability in a sample from the Mediterranean islands of Corsica and Sicily, and compared with data on Sardinia to gain insights into the ethnogenesis of these island populations. The results were interpreted in a broader Mediterranean context by including in the analysis neighboring populations previously studied with the same methodology. All samples studied were enclosed in the comparable spectrum of European Y-chromosome variability. Pronounced differences were observed between the islands as well as in the percentages of haplotypes previously shown to have distinctive patterns of continental phylogeography. Approximately 60% of the Sicilian haplotypes are also prevalent in Southern Italy and Greece. Conversely, the Corsican sample had elevated levels of alternative haplotypes common in Northern Italy. Sardinia showed a haplotype ratio similar to that observed in Corsica, but with a remarkable difference in the presence of a lineage defined by marker M26, which approaches 35% in Sardinia but seems absent in Corsica. Although geographically adjacent, the data suggest different colonization histories and a minimal amount of recent gene flow between them. Our results identify possible ancestral continental sources of the various island populations and underscore the influence of founder effect and genetic drift. The Y-chromosome data are consistent with comparable mtDNA data at the RFLP haplogroup level of resolution, as well as linguistic and historic knowledge.     

The origin of clonal diversity and structure of Populus alba in Sardinia: evidence from nuclear and plastid microsatellite markers

Background and Aims

Populus alba is a thermophilic forest tree present in the Mediterranean basin. Its habitat is highly fragmented and its distribution range has been subject to long-term human interference, resulting in debate surrounding whether certain populations are native or exotic in origin. In particular, populations from the islands of Corsica and Sardinia are of uncertain origin. While populations of P. alba mainly reproduce sexually, clonal reproduction is also common. The aims of this study were to locate and molecularly characterize the poorly studied island populations of P. alba and compare these with samples from various spatial scales, in order to provide information on the genetic structure and phylogeography of this species. This information will provide evidence on whether the species is native to Sardinia, which is important for the development of conservation strategies.

Methods

DNA extracts were obtained from the following P. alba trees: 159 from Sardinia, 47 from Ticino regional park (northern Italy), 15 acquired from an Italian Germoplasm Bank (IRC; Italian Reference Collection) and 28 from the Mediterranean basin (MB). Genetic polymorphisms were revealed at nuclear and chloroplast DNA (cpDNA) microsatellite loci, both at the island scale (Sardinia) and at broader scales, for comparative assessment of the genetic and genotypic diversity and phylogeography.

Key Results

Based on nuclear microsatellite loci, Sardinian white poplar consists of a small number of genets (26), each of which is represented by several ramets. Despite the uniqueness of the Sardinian haplotypes and the very low value of genetic diversity at the cpDNA level (v_K = 0·15), the H_T (0·60) and the A_R (3·61) values, estimated at the nuclear level for Sardinia, were comparable with those of the other populations and collections.

Conclusions

The uniqueness of the cpDNA haplotypes, the prevalence of clonality and the restricted number of genets recorded suggest that Sardinian white poplar could be a floristic relict of the native flora of the island, which has spread through available habitats on the island mainly by means of vegetative propagation and human activities.Key words: Populus alba, Sardinia, genets, ramets, phylogeography, native forest species     

Environmental Heterogeneity Explains the Genetic Structure of Continental and Mediterranean Populations of Fraxinus angustifolia Vahl

Tree species with wide distributions often exhibit different levels of genetic structuring correlated to their environment. However, understanding how environmental heterogeneity influences genetic variation is difficult because the effects of gene flow, drift and selection are confounded. We investigated the genetic variation and its ecological correlates in a wind-pollinated Mediterranean tree species, Fraxinus angustifolia Vahl, within a recognised glacial refugium in Croatia. We sampled 11 populations from environmentally divergent habitats within the Continental and Mediterranean biogeographical regions. We combined genetic data analyses based on nuclear microsatellite loci, multivariate statistics on environmental data and ecological niche modelling (ENM). We identified a geographic structure with a high genetic diversity and low differentiation in the Continental region, which contrasted with the significantly lower genetic diversity and higher population divergence in the Mediterranean region. The positive and significant correlation between environmental and genetic distances after controlling for geographic distance suggests an important influence of ecological divergence of the sites in shaping genetic variation. The ENM provided support for niche differentiation between the populations from the Continental and Mediterranean regions, suggesting that contemporary populations may represent two divergent ecotypes. Ecotype differentiation was also supported by multivariate environmental and genetic distance analyses. Our results suggest that despite extensive gene flow in continental areas, long-term stability of heterogeneous environments have likely promoted genetic divergence of ashes in this region and can explain the present-day genetic variation patterns of these ancient populations.