Ecological zones rather than molecular forms predict genetic differentiation in the malaria vector Anopheles gambiae s.s. in Ghana

The malaria mosquito Anopheles gambiae s.s. is rapidly becoming a model for studies on the evolution of reproductive isolation. Debate has centered on the taxonomic status of two forms (denoted M and S) within the nominal taxon identified by point mutations in the X-linked rDNA region. Evidence is accumulating that there are significant barriers to gene flow between these forms, but that the barriers are not complete throughout the entire range of their distribution. We sampled populations from across Ghana and southern Burkina Faso, West Africa, from areas where the molecular forms occurred in both sympatry and allopatry. Neither Bayesian clustering methods nor F(ST)-based analysis of microsatellite data found differentiation between the M and S molecular forms, but revealed strong differentiation among different ecological zones, irrespective of M/S status and with no detectable effect of geographical distance. Although no M/S hybrids were found in the samples, admixture analysis detected evidence of contemporary interform gene flow, arguably most pronounced in southern Ghana where forms occur sympatrically. Thus, in the sampled area of West Africa, lack of differentiation between M and S forms likely reflects substantial introgression, and ecological barriers appear to be of greater importance in restricting gene flow.     

Evidence for subdivision within the M molecular form of Anopheles gambiae

The principal vector of malaria in sub-Saharan Africa, Anopheles gambiae is subdivided into two molecular forms M and S. Additionally, several chromosomal forms, characterized by the presence of various inversion polymorphisms, have been described. The molecular forms M and S each contain several chromosomal forms, including the Savanna, Mopti and Forest forms. The M and S molecular forms are now considered to be the reproductive units within A. gambiae and it has recently been argued that a low recombination rate in the centromeric region of the X chromosome has facilitated isolation between these forms. The status of the chromosomal forms remains unclear however. Therefore, we studied genetic differentiation between Savanna S, Forest S, Forest M and Mopti M populations using microsatellites. Genetic differentiation between Savanna S and Forest S populations is very low (F(ST) = 0.0053 +/- 0.0049), even across large distances. In comparison, the Mopti M and Forest M populations show a relatively high degree of genetic differentiation (F(ST) = 0.0406 +/- 0.0054) indicating that the M molecular form may not be a single entity, but could be subdivided into at least two distinct chromosomal forms. Previously it was proposed that inversions have played a role in the origin of species within the A. gambiae complex. We argue that a possible subdivision within the M molecular form could be understood through this process, with the acquisition of inversions leading to the expansion of the M molecular form into new habitat, dividing it into two distinct chromosomal forms.     

Evidence of introgression of the ace-1(R) mutation and of the ace-1 duplication in West African Anopheles gambiae s. s

Background

The role of inter-specific hybridisation is of particular importance in mosquito disease vectors for predicting the evolution of insecticide resistance. Two molecular forms of Anopheles gambiae s.s., currently recognized as S and M taxa, are considered to be incipient sibling species. Hybrid scarcity in the field was suggested that differentiation of M and S taxa is maintained by limited or absent gene flow. However, recent studies have revealed shared polymorphisms within the M and S forms, and a better understanding of the occurrence of gene flow is needed. One such shared polymorphism is the G119S mutation in the ace-1 gene (which is responsible for insecticide resistance); this mutation has been described in both the M and S forms of A. gambiae s.s.

Methods and Results

To establish whether the G119S mutation has arisen independently in each form or by genetic introgression, we analysed coding and non-coding sequences of ace-1 alleles in M and S mosquitoes from representative field populations. Our data revealed many polymorphic sites shared by S and M forms, but no diversity was associated with the G119S mutation. These results indicate that the G119S mutation was a unique event and that genetic introgression explains the observed distribution of the G119S mutation within the two forms. However, it was impossible to determine from our data whether the mutation occurred first in the S form or in the M form. Unexpectedly, sequence analysis of some resistant individuals revealed a duplication of the ace-1 gene that was observed in both A. gambiae s.s. M and S forms. Again, the distribution of this duplication in the two forms most likely occurred through introgression.

Conclusions

These results highlight the need for more research to understand the forces driving the evolution of insecticide resistance in malaria vectors and to regularly monitor resistance in mosquito populations of Africa.     

Divergence with gene flow in Anopheles funestus from the Sudan Savanna of Burkina Faso, West Africa

Anopheles funestus is a major vector of malaria across Africa. Understanding its complex and nonequilibrium population genetic structure is an important challenge that must be overcome before vector populations can be successfully perturbed for malaria control. Here we examine the role of chromosomal inversions in structuring genetic variation and facilitating divergence in Burkina Faso, West Africa, where two incipient species (chromosomal forms) of A. funestus, defined principally by rearrangements of chromosome 3R, have been hypothesized. Sampling across an approximately 300-km east-west transect largely contained within the Sudan-Savanna ecoclimatic zone, we analyzed chromosomal inversions, 16 microsatellite loci distributed genomewide, and 834 bp of the mtDNA ND5 gene. Both molecular markers revealed high genetic diversity, nearly all of which was accounted for by within-population differences among individuals, owing to recent population expansion. Across the study area there was no correlation between genetic and geographic distance. Significant genetic differentiation found between chromosomal forms on the basis of microsatellites was not genomewide but could be explained by chromosome 3R alone on the basis of loci inside and near inversions. These data are not compatible with complete reproductive isolation but are consistent with differential introgression and sympatric divergence between the chromosomal forms, facilitated by chromosome 3R inversions.     

Population structure, speciation, and introgression in the Anopheles gambiae complex

We review here what is known about the population structure and evolutionary dynamics of members of the Anopheles gambiae complex with emphasis on the situation in West Africa. First, the importance of the 2nd chromosome inversion polymorphism is demonstrated especially in adaptation to levels of aridity, a major environmental variable in Africa. This affects the distribution of karyotypes on both a macro- and micro-geographic scale as well as temporally. Such differentiation leads to karyotypes being differentially effective transmitters of malaria and differentially susceptible to indoor residual spraying of insecticides. Second, we review the evidence that cryptic taxa, especially in An. gambiae s.s., exist. This observation stems from both karyotype studies and molecular studies. It is abundantly clear that West African populations of An. gambiae s.s. are often not panmictic units, with premating factors evidently acting to maintain distinct genetic forms. Third, we review phylogenetic studies that have revealed the presence of introgression between the two most important vectors, An. gambiae and An. arabiensis. This is most evident for the 2nd chromosome inversions. This interpretation of phylogenetic data is consistent with a direct laboratory study indicating inversions in this chromosome are stably maintained in back-crossed populations. All of this information has led to the view that members of the An. gambiae complex are highly variable with an abundance of adaptive genetic variation. This presents a significant challenge to vector control programs designed to reduce malaria in sub-Saharan Africa.     

Genetic differentiation in the African malaria vector, Anopheles gambiae s.s., and the problem of taxonomic status

Of the seven recognized species of the Anopheles gambiae complex, A. gambiae s.s. is the most widespread and most important vector of malaria. It is becoming clear that, in parts of West Africa, this nominal species is not a single panmictic unit. We found that the internal transcribed spacer (ITS) of the X-linked rDNA has two distinct sequences with three fixed nucleotide differences; we detected no heterozygotes at these three sites, even in areas of sympatry of the two ITS types. The intergenic spacer (IGS) of this region also displays two distinct sequences that are in almost complete linkage disequilibrium with the distinct ITS alleles. We have designated these two types as S/type I and M/type II. These rDNA types correspond at least partly to the previously recognized chromosomal forms. Here we expand the geographic range of sampling to 251 individuals from 38 populations. Outside of West Africa, a single rDNA type, S/type I, corresponds to the Savanna chromosomal form. In West Africa, both types are often found in a single local sample. To understand if these findings might be due to unusual behavior of the rDNA region, we sequenced the same region for 46 A. arabiensis, a sympatric sibling species. No such distinct discontinuity was observed for this species. Autosomal inversions in one chromosome arm (2R), an insecticide resistance gene on 2L, and this single X-linked region indicate at least two genetically differentiated subpopulations of A. gambiae. Yet, rather extensive studies of other regions of the genome have failed to reveal genetic discontinuity. Evidently, incomplete genetic isolation exists within this single nominal species.     

Multilevel analyses of genetic differentiation in Anopheles gambiae s.s. reveal patterns of gene flow important for malaria-fighting mosquito projects

Malaria control projects based on the introduction and spread of transgenes into mosquito populations depend on the extent of isolation between those populations. On the basis of the distribution of paracentric inversions, Anopheles gambiae has been subdivided into five subspecific chromosomal forms. Estimating gene flow between and within these forms of An. gambiae presents a number of challenges. We compared patterns of genetic divergence (F(ST)) between sympatric populations of the Bamako and Mopti forms at five sites. We used microsatellite loci within the j inversion on chromosome 2, which is fixed in the Bamako form but absent in the Mopti form, and microsatellites on chromosome 3, a region void of inversions. Estimates of genetic diversity and F(ST)'s suggest genetic exchanges between forms for the third chromosome but little for the j inversion. These results suggest a role for the inversion in speciation. Extensive gene flow within forms among sites resulted in populations clustering according to form despite substantial gene flow between forms. These patterns underscore the low levels of current gene flow between chromosomal forms in this area of sympatry. Introducing refractoriness genes in areas of the genome void of inversions may facilitate their spread within forms but their passage between forms may prove more difficult than previously thought.     

Population Structure of Anopheles gambiae in Africa

The population structure of Anopheles gambiae in Africa was studied using 11 microsatellite loci in 16 samples from 10 countries. All loci are located outside polymorphic inversions. Heterogeneity among loci was detected and two putative outlier loci were removed from analyses aimed at capturing genome-wide patterns. Two main divisions of the gene pool were separated by high differentiation (F(ST) > 0.1). The northwestern (NW) division included populations from Senegal, Ghana, Nigeria, Cameroon, Gabon, Democratic Republic of Congo (DRC), and western Kenya. The southeastern (SE) division included populations from eastern Kenya, Tanzania, Malawi, and Zambia. Inhospitable environments for A. gambiae along the Rift Valley partly separate these divisions. Reduced genetic diversity in the SE division and results of an analysis based on private alleles support the hypothesis that a recent bottleneck, followed by colonization from the NW populations shaped this structure. In the NW division, populations possessing the M rDNA genotype appeared to form a monophyletic clade. Although genetic distance increased with geographic distance, discontinuities were suggested between certain sets of populations. The absence of heterozygotes between sympatric M and S populations in the DRC and the high differentiation in locus 678 (F(ST)>0.28) contrasted with low differentiation in all other loci (-0.02相似文献   

Genomic Architecture of Rapid Parallel Adaptation to Fresh Water in a Wild Fish

Rapid adaptation to novel environments may drive changes in genomic regions through natural selection. However, the genetic architecture underlying these adaptive changes is still poorly understood. Using population genomic approaches, we investigated the genomic architecture that underlies rapid parallel adaptation of Coilia nasus to fresh water by comparing four freshwater-resident populations with their ancestral anadromous population. Linkage disequilibrium network analysis and population genetic analyses revealed two putative large chromosome inversions on LG6 and LG22, which were enriched for outlier loci and exhibited parallel association with freshwater adaptation. Drastic frequency shifts and elevated genetic differentiation were observed for the two chromosome inversions among populations, suggesting that both inversions would undergo divergent selection between anadromous and resident ecotypes. Enrichment analysis of genes within chromosome inversions showed significant enrichment of genes involved in metabolic process, immunoregulation, growth, maturation, osmoregulation, and so forth, which probably underlay differences in morphology, physiology and behavior between the anadromous and freshwater-resident forms. The availability of beneficial standing genetic variation, large optimum shift between marine and freshwater habitats, and high efficiency of selection with large population size could lead to the observed rapid parallel adaptive genomic change. We propose that chromosomal inversions might have played an important role during the evolution of rapid parallel ecological divergence in the face of environmental heterogeneity in C. nasus. Our study provides insights into the genomic basis of rapid adaptation of complex traits in novel habitats and highlights the importance of structural genomic variants in analyses of ecological adaptation.     

Gene flow between chromosomal forms of the malaria vector Anopheles funestus in Cameroon, Central Africa, and its relevance in malaria fighting

Knowledge of population structure in a major vector species is fundamental to an understanding of malaria epidemiology and becomes crucial in the context of genetic control strategies that are being developed. Despite its epidemiological importance, the major African malaria vector Anopheles funestus has received far less attention than members of the Anopheles gambiae complex. Previous chromosomal data have shown a high degree of structuring within populations from West Africa and have led to the characterization of two chromosomal forms, "Kiribina" and "Folonzo." In Central Africa, few data were available. We thus undertook assessment of genetic structure of An. funestus populations from Cameroon using chromosomal inversions and microsatellite markers. Microsatellite markers revealed no particular departure from panmixia within each local population and a genetic structure consistent with isolation by distance. However, cytogenetic studies demonstrated high levels of chromosomal heterogeneity, both within and between populations. Distribution of chromosomal inversions was not random and a cline of frequency was observed, according to ecotypic conditions. Strong deficiency of heterokaryotypes was found in certain localities in the transition area, indicating a subdivision of An. funestus in chromosomal forms. An. funestus microsatellite genetic markers located within the breakpoints of inversions are not differentiated in populations, whereas in An. gambiae inversions can affect gene flow at marker loci. These results are relevant to strategies for control of malaria by introduction of transgenes into populations of vectors.     

WEEDY ADAPTATION IN SETARIA SPP. I. ISOZYME ANALYSIS OF GENETIC DIVERSITY AND POPULATION GENETIC STRUCTURE IN SETARIA VIRIDIS

Setaria viridis is an important self-pollinating, cosmopolitan weed of temperate regions worldwide. Allozyme markers were used to investigate genetic diversity and structure in 168 accessions (including four S. italica) collected mainly from North America and Eurasia. Genetic diversity in green foxtail, and its population genetic structure, provided important clues about this weed's evolutionary history. Genetic diversity was low, with marked population differentiation: the percentage of polymorphic loci was 25% (0.95 criterion); mean number of alleles per locus was 1.86; mean panmictic heterozygosity was 0.07; and the coefficient of population genetic differentiation was 0.65. A common genotype occurred in 25 accessions distributed in six countries from both the Old World and New World, in a wide variety of ecological situations. Relatively little genetic divergence occurred between Eurasia and North America, with Nei's unbiased genetic identity between the two regions equaling 1.0. Populations from these two continents also had equivalent genetic diversity. Within North America, regional differentiation was indicated by northern and southern groups separated at 43.5° N latitude. No geographic pattern in genetic diversity was found within Eurasia. The size of the geographic range from which populations were sampled was not an accurate indicator of the extent of genetic diversity found among populations from that region. These results suggest that present patterning among green foxtail populations in North America is the consequence of multiple introductions into the New World followed by local adaptation and regional differentiation. Finally, S. italica and several green foxtail varieties did not differ isozymatically from typical forms of green foxtail. This supports the view that S. italica and S. viridis are conspecific, that the former (foxtail millet) is a domesticated form of the latter, and also questions the taxonomic validity of formally recognizing morphological varieties within green foxtail.     

Morphological differentiation may mediate mate-choice between incipient species of Anopheles gambiae s.s

The M and S molecular forms of Anopheles gambiae s.s. have been considered incipient species for more than ten years, yet the mechanism underlying assortative mating of these incipient species has remained elusive. The discovery of the importance of harmonic convergence of wing beat frequency in mosquito mating and its relation to wing size have laid the foundation for exploring phenotypic divergence in wing size of wild populations of the two forms. In this study, wings from field collected mosquitoes were measured for wing length and wing width from two parts of the sympatric distribution, which differ with respect to the strength of assortative mating. In Mali, where assortative mating is strong, as evidenced by low rates of hybridization, mean wing lengths and wing widths were significantly larger than those from Guinea-Bissau. In addition, mean wing widths in Mali were significantly different between molecular forms. In Guinea-Bissau, assortative mating appears comparatively reduced and wing lengths and widths did not differ significantly between molecular forms. The data presented in this study support the hypothesis that wing beat frequency may mediate assortative mating in the incipient species of A. gambiae and represent the first documentation of a morphological difference between the M and S molecular forms.     

Multiple chromosomal inversions contribute to adaptive divergence of a dune sunflower ecotype

Both models and case studies suggest that chromosomal inversions can facilitate adaptation and speciation in the presence of gene flow by suppressing recombination between locally adapted alleles. Until recently, however, it has been laborious and time‐consuming to identify and genotype inversions in natural populations. Here we apply RAD sequencing data and newly developed population genomic approaches to identify putative inversions that differentiate a sand dune ecotype of the prairie sunflower (Helianthus petiolaris) from populations found on the adjacent sand sheet. We detected seven large genomic regions that exhibit a different population structure than the rest of the genome and that vary in frequency between dune and nondune populations. These regions also show high linkage disequilibrium and high heterozygosity between, but not within, arrangements, consistent with the behaviour of large inversions, an inference subsequently validated in part by comparative genetic mapping. Genome–environment association analyses show that key environmental variables, including vegetation cover and soil nitrogen, are significantly associated with inversions. The inversions colocate with previously described “islands of differentiation,” and appear to play an important role in adaptive divergence and incipient speciation within H. petiolaris.     

Restricted gene flow between two social forms in the ant Formica truncorum

We studied genetic differentiation between two social forms (M-type: single queen, independent nest founding; P-type: multiple queens, dependent nest founding, building of colonial networks) of the ant Formica truncorum in a locality where the social types characterize two sympatric populations. The genetic results indicate restricted gene flow between the social forms. Female gene flow between the forms appears to be absent as they did not share mitochondrial haplotypes. Significant nuclear differentiation and the distribution of private alleles suggest that male gene flow between the forms is weak. However, the assignment analysis indicates some gene flow with P males mating with M females. The results have potentially important implications concerning social evolution within the forms but they need to be confirmed in other localities before they can be generalized. The colonies in the M-type population have earlier been shown to produce split sex ratios, depending on the mating frequency of the queens. The inferred gene flow from the P to the M type means that the split sex ratio is partly suboptimal, possibly because the P populations are not long-lived enough to influence the behavioural decisions in the M colonies.     

Variability and genetic basis for migratory behaviour in a spring population of the aphid, Aphis gossypii Glover in the Yangtze River Valley of China

The population dynamics, development of gonads, takeoff and flight behaviour of Aphis gossypii Glover were investigated in order to test whether there was variation of migratory ability in the spring population. Field surveys showed that not all the aphids overwintering on hibiscus migrated to the secondary host plants, and the host-alternating and host-specific life-cycle forms coexisted in Nanjing, China. Substantial variation in flight capacity of winged individuals, development of gonads and takeoff behaviour were found within the spring population. The frequency distribution of flight duration and the number of ovarioles per individual alatae exhibited two peaks, representing the migratory and sedentary genotypes, respectively. Significant response to directional selection on takeoff behaviour demonstrated the additive genetic component of this variation. Selection for 'takeoff' individuals caused a significant increase in takeoff angle from 39.8 degrees in the first selection to 68.7 degrees in the fifth; and, hence, screened out the migratory genotype (M), while selection for the sedentary individuals increased the rate of non-takeoffs significantly, and screened out the sedentary genotype (S). The reciprocal cross, M(female) x S(male), produced hybrid offspring performing significantly steeper takeoff angles compared with those from the cross S(female) x M(male), suggesting the presence of a maternal effect. On the other hand, takeoff rate was ranked as M(female) x S(male)=S(female) x M(male)>M>S, involving no sex-linkage and maternal effect. The coexistence of host-alternating and host-specific life-cycle forms of A. gossypii on the primary host has, as deduced from the present studies, a genetic basis.     

Reduced recombination rate and genetic differentiation between the M and S forms of Anopheles gambiae s.s

Genetic differentiation between the largely sympatric molecular forms M and S of Anopheles gambiae appears mostly limited to division 6 and part of division 5 of the X chromosome. This region is adjacent to the centromere and includes the rDNA that was used to define these forms. This localized differentiation between populations that experience gene flow strongly suggests that this region contains genes responsible for reproductive isolation. Regions adjacent to centromeres are known to experience less recombination in several species and it has recently been suggested that low recombination rates can facilitate the accumulation and maintenance of isolation genes in partially isolated populations. Therefore, we measured the recombination rate in division 5D/6 directly and estimate that it is at least 16-fold reduced across this region compared to the remainder of the X chromosome. Additionally, sequence data from four loci from field-collected mosquitoes from several West African countries show very strong differentiation between the molecular forms in division 5D/6, whereas none was observed in two loci elsewhere on the X chromosome. Furthermore, genetic variation was substantially lower in division 5D/6 compared to the two reference loci, and the inferred genealogies of the division 5D/6 genes show patterns consistent with selective sweeps. This suggests that the reduced recombination rate has increased the effect of selection on this region and that our data are consistent with the hypothesis that reduced recombination rates can play a role in the accumulation of isolation genes in the face of gene flow.     

Divergence between the Drosophila pseudoobscura and D. persimilis genome sequences in relation to chromosomal inversions

As whole-genome sequence assemblies accumulate, a challenge is to determine how these can be used to address fundamental evolutionary questions, such as inferring the process of speciation. Here, we use the sequence assemblies of Drosophila pseudoobscura and D. persimilis to test hypotheses regarding divergence with gene flow. We observe low differentiation between the two genome sequences in pericentromeric and peritelomeric regions. We interpret this result as primarily a remnant of the correlation between levels of variation and local recombination rate observed within populations. However, we also observe lower differentiation far from the fixed chromosomal inversions distinguishing these species and greater differentiation within and near these inversions. This finding is consistent with models suggesting that chromosomal inversions facilitate species divergence despite interspecies gene flow. We also document heterogeneity among the inverted regions in their degree of differentiation, suggesting temporal differences in the origin of each inverted region consistent with the inversions arising during a process of divergence with gene flow. While this study provides insights into the speciation process using two single-genome sequences, it was informed by lower throughput but more rigorous examinations of polymorphism and divergence. This reliance highlights the need for complementary genomic and population genetic approaches for tackling fundamental evolutionary questions such as speciation.     

Environmental Factors Associated with the Distribution of Anopheles gambiae s.s in Ghana; an Important Vector of Lymphatic Filariasis and Malaria

Anopheles gambiae s.s mosquitoes are important vectors of lymphatic filariasis (LF) and malaria in Ghana. To better understand their ecological aspects and influence on disease transmission, we examined the spatial distribution of the An. gambiae (M and S) molecular forms and associated environmental factors, and determined their relationship with disease prevalence. Published and current data available on the An. gambiae species in Ghana were collected in a database for analysis, and the study sites were georeferenced and mapped. Using the An. gambiae s.s sites, environmental data were derived from climate, vegetation and remote-sensed satellite sources, and disease prevalence data from existing LF and malaria maps in the literature. The data showed that An. gambiae M and S forms were sympatric in most locations. However, the S form predominated in the central region, while the M form predominated in the northern and coastal savanna regions. Bivariate and multiple regression analyses identified temperature as a key factor distinguishing their distributions. An. gambiae M was significantly correlated with LF, and 2.5 to 3 times more prevalent in the high LF zone than low to medium zones. There were no significant associations between high prevalence An. gambiae s.s locations and malaria. The distribution of the An. gambiae M and S forms and the diseases they transmit in Ghana appear to be distinct, driven by different environmental factors. This study provides useful baseline information for disease control, and future work on the An. gambiae s.s in Ghana.     

Microsatellite genetic variation in small and isolated populations of Magnolia sieboldii ssp. japonica

Magnolia sieboldii ssp. japonica, distributed mainly in western Japan, is restricted to high elevation areas (1000-2000 m above sea level) and usually forms small isolated populations. Four microsatellite loci were assayed for 19 populations from six regions spanning the range of distribution, and the levels and distribution of genetic variation were estimated. All four loci were variable, with a total of 39 alleles, but the overall level of microsatellite genetic variation was low, especially compared with a related species, M. obovata. Genetic structure in M. sieboldii was characterised by low intrapopulational genetic variation (A = 3.74 and H(o) = 0.366 on average) and high genetic differentiation even among regional populations. Highly significant isolation-by-distance (IBD) models at the short distance were detected. Genetic drift and limited gene flow was considered to be important in determining the genetic structure within regions. Total genetic differentiation was remarkably high (F(ST) = 0.488 and R(ST) = 0.538), suggesting genetic barriers among regions. Neighbour-joining dendrograms relating the 19 populations, and further analysis on the IBD models, revealed that a stepwise mutation model was more suited than an infinite allele model to explain the genetic differentiation among regions. It is suggested that mutation at microsatellite loci might be influential in generating the genetic differentiation among regions. These results showed the potential of hypervariable microsatellite loci to evaluate the effects of genetic drift and population isolation within regions, and to detect genetic distinctiveness, in spite of the loss of overall genetic variation in M. sieboldii.     

Phylogeography of the black fly Simulium tani (Diptera: Simuliidae) from Thailand as inferred from mtDNA sequences

Intraspecific phylogeography has been used widely as a tool to infer population history. However, little attention has been paid to Southeast Asia despite its importance in terms of biodiversity. Here we used the cytochrome oxidase I gene of mitochondrial DNA (mtDNA) for a phylogeographic study of 147 individuals of the black fly Simulium tani from Thailand. The mtDNA revealed high genetic differentiation between the major geographical regions of north, east and central/south Thailand. Mismatch distributions indicate population expansions during the mid-Pleistocene and the late Pleistocene suggesting that current population structure and diversity may be due in part to the species' response to Pleistocene climatic fluctuations. The genealogical structure of the haplotypes, high northern diversity and maximum-likelihood inference of historical migration rates, suggest that the eastern and central/southern populations originated from northern populations in the mid-Pleistocene. Subsequently, the eastern region had had a largely independent history but the central/southern population may be largely the result of recent (c. 100,000 years ago) expansion, either from the north again, or from a relictual population in the central region. Cytological investigation revealed that populations from the south and east have two overlapping fixed chromosomal inversions. Since these populations also share ecological characteristics it suggests that inversions are involved in ecological adaptation. In conclusion both contemporary and historical ecological conditions are playing an important role in determining population genetic structure and diversity.