Genome-wide QTL mapping of saltwater tolerance in sibling species of Anopheles (malaria vector) mosquitoes.

Although freshwater (FW) is the ancestral habitat for larval mosquitoes, multiple species independently evolved the ability to survive in saltwater (SW). Here, we use quantitative trait locus (QTL) mapping to investigate the genetic architecture of osmoregulation in Anopheles mosquitoes, vectors of human malaria. We analyzed 1134 backcross progeny from a cross between the obligate FW species An. coluzzii, and its closely related euryhaline sibling species An. merus. Tests of 2387 markers with Bayesian interval mapping and machine learning (random forests) yielded six genomic regions associated with SW tolerance. Overlap in QTL regions from both approaches enhances confidence in QTL identification. Evidence exists for synergistic as well as disruptive epistasis among loci. Intriguingly, one QTL region containing ion transporters spans the 2Rop chromosomal inversion that distinguishes these species. Rather than a simple trait controlled by one or a few loci, our data are most consistent with a complex, polygenic mode of inheritance.

Widespread divergence between incipient Anopheles gambiae species revealed by whole genome sequences.

The Afrotropical mosquito Anopheles gambiae sensu stricto, a major vector of malaria, is currently undergoing speciation into the M and S molecular forms. These forms have diverged in larval ecology and reproductive behavior through unknown genetic mechanisms, despite considerable levels of hybridization. Previous genome-wide scans using gene-based microarrays uncovered divergence between M and S that was largely confined to gene-poor pericentromeric regions, prompting a speciation-with-ongoing-gene-flow model that implicated only about 3% of the genome near centromeres in the speciation process. Here, based on the complete M and S genome sequences, we report widespread and heterogeneous genomic divergence inconsistent with appreciable levels of interform gene flow, suggesting a more advanced speciation process and greater challenges to identify genes critical to initiating that process.

SNP genotyping defines complex gene-flow boundaries among African malaria vector mosquitoes.

Mosquitoes in the Anopheles gambiae complex show rapid ecological and behavioral diversification, traits that promote malaria transmission and complicate vector control efforts. A high-density, genome-wide mosquito SNP-genotyping array allowed mapping of genomic differentiation between populations and species that exhibit varying levels of reproductive isolation. Regions near centromeres or within polymorphic inversions exhibited the greatest genetic divergence, but divergence was also observed elsewhere in the genomes. Signals of natural selection within populations were overrepresented among genomic regions that are differentiated between populations, implying that differentiation is often driven by population-specific selective events. Complex genomic differentiation among speciating vector mosquito populations implies that tools for genome-wide monitoring of population structure will prove useful for the advancement of malaria eradication.

Genetic exchange in 2La inversion heterokaryotypes of Anopheles gambiae.

In the malaria vector Anopheles gambiae, alternative arrangements of chromosome 2 (2La and 2L+(a)) vary in relative frequency along clines of aridity, suggesting the action of natural selection on targets within the inversion. Our long term goal of detecting such targets depends in part on the level of genetic exchange between arrangements. Accordingly, we estimated recombination rates on 2L from the backcross progeny of 2La/+(a) heterokaryotypes and as a control, from 2L+(a) homokaryotypes. In homokaryotypes, the recombination rate was uniform at ~2.0 centimorgans per megabase (cM/Mb). In heterokaryotypes, recombination within the rearranged region was reduced to < 0.5 cM/Mb, with slightly higher but nevertheless reduced levels (< 1.0 cM/Mb) flanking the rearrangement. Yet, gene exchange was recorded between nearly all markers, including those very near the distal inversion breakpoint. These results suggest that reduced recombination is a necessary but not sufficient mechanism for genetic isolation between alternative arrangements, and that the targets of natural selection can be identified against the different chromosomal backgrounds.

Effective population size of Anopheles funestus chromosomal forms in Burkina Faso

Background: As Anopheles funestus is one of the principal Afro-tropical malaria vectors; a more complete understanding of its population structure is desirable. In West and Central Africa; An. funestus population structure is complicated by the coexistence of two assortatively mating chromosomal forms. Effective population size (Ne) is a key parameter in understanding patterns and levels of intraspecific variation; as it reflects the role of genetic drift. Here; Ne was estimated from both chromosomal forms; Kiribina and Folonzo; in Burkina Faso. Methods: Short-term Ne was estimated by evaluating variation at 16 microsatellite loci across temporal samples collected annually from 2000-2002. Estimates were based on standardized variance in allele frequencies or a maximum likelihood method. Long-term Ne was estimated from genetic diversity estimates using mtDNA sequences and microsatellites. Results: For both forms; short-term and long-term Ne estimates were on the order of 103 and 105; respectively. Long-term Ne estimates were larger when based on loci from chromosome 3R (both inside and outside of inversions) than loci outside of this arm. Conclusion: Ne values indicate that An. funestus is not subject to seasonal bottlenecks. Though not statistically different because of large and overlapping confidence intervals; short-term Ne estimates were consistently smaller for Kiribina than Folonzo; possibly due to exploitation of different breeding sites: permanent for Folonzo and intermittent for Kiribina. The higher long-term Ne estimates on 3R; the arm carrying the two inversions mainly responsible for defining the chromosomal forms; give natural selection broader scope and merit further study

Chromosomal evidence of incipient speciation in the Afrotropical malaria mosquito Anopheles funestus.

The analysis of chromosomal polymorphism of paracentric inversions in anopheline mosquitoes has often been instrumental to the discovery of sibling species complexes and intraspecific genetic heterogeneities associated with incipient speciation processes. To investigate the population structure of Anopheles funestus Giles (Diptera: Culicidae), one of the three most important vectors of human malaria in sub-Saharan Africa, a three-year survey of chromosomal polymorphism was carried out on 4,638 karyotyped females collected indoors and outdoors from two villages of central Burkina Faso. Large and temporally stable departures from Hardy-Weinberg equilibrium due to significant deficits of heterokaryotypes were found irrespective of the place of capture, and of the spatial and temporal units chosen for the analysis. Significant linkage disequilibrium was observed among inversion systems on independently assorting chromosomal arms, indicating the existence of assortative mating phenomena. Results were consistent with the existence of two chromosomal forms characterized by contrasting degrees of inversion polymorphism maintained by limitations to gene flow. This hypothesis was supported by the reestablishment of Hardy-Weinberg and linkage equilibria when individual specimens were assigned to each chromosomal form according to two different algorithms. This pattern of chromosomal variability is suggestive of an incipient speciation process in An. funestus populations from Burkina Faso.

Rangewide population genetic structure of the African malaria vector Anopheles funestus.

Anopheles funestus is a primary vector of malaria in Africa south of the Sahara. We assessed its rangewide population genetic structure based on samples from 11 countries, using 10 physically mapped microsatellite loci, two per autosome arm and the X (N = 548), and 834 bp of the mitochondrial ND5 gene (N = 470). On the basis of microsatellite allele frequencies, we found three subdivisions: eastern (coastal Tanzania, Malawi, Mozambique and Madagascar), western (Burkina Faso, Mali, Nigeria and western Kenya), and central (Gabon, coastal Angola). A. funestus from the southwest of Uganda had affinities to all three subdivisions. Mitochondrial DNA (mtDNA) corroborated this structure, although mtDNA gene trees showed less resolution. The eastern subdivision had significantly lower diversity, similar to the pattern found in the codistributed malaria vector Anopheles gambiae. This suggests that both species have responded to common geographic and/or climatic constraints. The western division showed signatures of population expansion encompassing Kenya west of the Rift Valley through Burkina Faso and Mali. This pattern also bears similarity to A. gambiae, and may reflect a common response to expanding human populations following the development of agriculture. Due to the presumed recent population expansion, the correlation between genetic and geographic distance was weak. Mitochondrial DNA revealed further cryptic subdivision in A. funestus, not detected in the nuclear genome. Mozambique and Madagascar samples contained two mtDNA lineages, designated clade I and clade II, that were separated by two fixed differences and an average of 2% divergence, which implies that they have evolved independently for approximately 1 million years. Clade I was found in all 11 locations, whereas clade II was sampled only on Madagascar and Mozambique. We suggest that the latter clade may represent mtDNA capture by A. funestus, resulting from historical gene flow either among previously isolated and divergent populations or with a related species.

SINE insertion polymorphism on the X chromosome differentiates Anopheles gambiae molecular forms.

Polymorphic SINE insertions can be useful markers for assessing population structure and differentiation. Maque is a family of SINE elements which, based on bioinformatic analysis, was suggested to have been active recently in Anopheles gambiae, the major vector of malaria. Here, we report the development of polymorphic Maque insertions as population genetic markers in A. gambiae, and the use of these markers to better characterize divergence on the X chromosome between A. gambiae M and S molecular forms in populations from Burkina Faso and Mali. Our data are consistent with the recent activity of Maque. Phylogenetic analysis suggests that at least two recently active lineages may have a role in mediating genome evolution. We found differences in element insertion frequency and sequence between the M and S populations analysed. Significant differentiation was observed between these two groups across a 6 Mb region at the proximal (centromeric) end of the X chromosome. Locus-specific F(ST) values ranged from 0.14 to 1.00 in this region, yet were not significantly different from zero in more distal locations on the X chromosome; the trend was consistent in populations from both geographical locales suggesting that differentiation is not due to local adaptation. Strong differentiation between M and S at the proximal end of the X chromosome, but not outside this region, suggests the action of selection counteracting limited gene flow between these taxa and supports their characterization as incipient species.

Molecular differentiation between chromosomally defined incipient species of Anopheles funestus.

Anopheles funestus Giles is one of the most important vectors of malaria in sub-Saharan Africa. The population structure of this mosquito in Burkina Faso, West Africa based on chromosomal inversion data led to the description of two chromosomal forms, Kiribina and Folonzo. Because both forms co-occur in the same locales yet differ significantly, both in the frequency of inverted arrangements on chromosome arms 3R and 2R and in vectorial capacity, they were hypothesized to be emerging species with at least partial barriers to gene flow. This hypothesis would be strengthened by molecular evidence of differentiation between Kiribina and Folonzo at loci outside chromosomal inversions. We surveyed molecular variation in sympatric populations of the two forms using sequences from the mitochondrial ND5 gene and genotypes at sixteen microsatellite loci distributed across the genome. Both classes of marker revealed slight but significant differentiation between the two forms (mtDNA F(ST) = 0.023, P < 0.001; microsatellite F(ST) = 0.004, P < 0.001; R(st) = 0.009, P = 0.002). Locus-by-locus analysis of the microsatellite data showed that significant differentiation was not genome-wide, but could be attributed to five loci on chromosome 3R (F(ST) = 0.010, P < 0.001; R(st) = 0.016, P = 0.002). Importantly, three of these loci are outside of, and in linkage equilibrium with, chromosomal inversions, suggesting that differentiation between chromosomal forms extends beyond the inversions themselves. The slight overall degree of differentiation indicated by both marker classes is likely an underestimate because of recent population expansion inferred for both Folonzo and Kiribina. The molecular evidence from this study is consistent with the hypothesis of incipient speciation between Kiribina and Folonzo.

A microsatellite map of the African human malaria vector Anopheles funestus.

Microsatellite markers and chromosomal inversion polymorphisms are useful genetic markers for determining population structure in Anopheline mosquitoes. In Anopheles funestus (2N = 6), only chromosome arms 2R, 3R, and 3L are known to carry polymorphic inversions. The physical location of microsatellite markers with respect to polymorphic inversions is potentially important information for interpreting population genetic structure, yet none of the available marker sets have been physically mapped in this species. Accordingly, we mapped 32 polymorphic A. funestus microsatellite markers to the polytene chromosomes using fluorescent in situ hybridization (FISH) and identified 16 markers outside of known polymorphic inversions. Here we provide an integrated polytene chromosome map for A. funestus that includes the breakpoints of all known polymorphic inversions as well as the physical locations of microsatellite loci developed to date. Based on this map, we suggest a standard set of 16 polymorphic microsatellite markers that are distributed evenly across the chromosome complement, occur predominantly outside of inversions, and amplify reliably. Adoption of this set by researchers working in different regions of Africa will facilitate metapopulation analyses of this primary malaria vector.

Revisiting the role of introgression vs shared ancestral polymorphisms as key processes shaping genetic diversity in the recently separated sibling species of the Anopheles gambiae complex.

The role of interspecific hybridisation in the evolution of pest species is poorly understood. In mosquito disease vectors this is of particular importance due to the evolution of insecticide resistance and the proposed release of transgenic strains that are refractory to the malaria parasite. In this study, we apply population genetic methods in a novel manner to determine whether mitochondrial DNA sequences have introgressed between the closely related African malaria vectors Anopheles gambiae and A. arabiensis. Our results suggest that speciation was geologically recent and ancestral haplotypes at the ND5 locus are retained in both species. In addition, comparing haplotype frequencies in allopatric and sympatric populations, suggest locale specific unidirectional introgression of mitochondria from A. arabiensis into A. gambiae.

Semipermeable species boundaries between Anopheles gambiae and Anopheles arabiensis: evidence from multilocus DNA sequence variation.

Attempts to reconstruct the phylogenetic history of the Anopheles gambiae cryptic species complex have yielded strongly conflicting results. In particular, An. gambiae, the primary African malaria vector, is variously placed as a sister taxon to either Anopheles arabiensis or Anopheles merus. The recent divergence times for members of this complex complicate phylogenetic analysis, making it difficult to unambiguously implicate interspecific gene flow, versus retained ancestral polymorphism, as the source of conflict. Using sequences at four unlinked loci, which were determined from multiple specimens within each of five species in the complex, we found contrasting patterns of sequence divergence between the X chromosome and the autosomes. The isolation model of speciation assumes a lack of gene flow between species since their separation. This model could not be rejected for An. gambiae and An. arabiensis, although the data fit the model poorly. On the other hand, evidence from gene trees supports genetic introgression of chromosome 2 inversions between An. gambiae and An. arabiensis, and also points to more broad scale genetic exchange of autosomal sequences between this species pair. That such exchange has been relatively recent is suggested not only by the lack of fixed differences at three autosomal loci but also by the sharing of full haplotypes at two of the three loci, which is in contrast to several fixed differences and considerably deeper divergence on the X. The proposed acquisition by An. gambiae of sequences from the more arid-adapted An. arabiensis may have contributed to the spread and ecological dominance of this malaria vector.

Speciation within Anopheles gambiae--the glass is half full.

Restrictions to gene flow among molecular forms of the mosquito Anopheles gambiae sensu stricto reveal an ongoing speciation process affecting the epidemiology of malaria in sub-Saharan Africa.

Evolution of mitochondrial and ribosomal gene sequences in anophelinae (Diptera: Culicidae): implications for phylogeny reconstruction.

In this study, two mitochondrial genes, cyt b and ND5, and the D2 expansion segment of the 28S nuclear ribosomal gene were used to reconstruct a phylogeny of the mosquito subfamily Anophelinae. The ingroup consisted of all three genera of Anophelinae and five of six subgenera of Anopheles. Six genera of Culicinae were used as the outgroup. Extreme conservation at the protein level coupled with rapid saturation of synonymous positions probably accounted for the lack of meaningful phylogenetic signal in the cyt b gene. In contrast, abundant variation at all codon positions of the ND5 gene allowed recovery of the basal and most of the recent relationships. Phylogenetic analysis of D2 produced results consistent with those of ND5. Combined analysis indicated well-supported monophyletic Anophelinae (with Chagasia basal), Anopheles + Bironella, and subgeneric clades within the genus Anopheles. Moreover, subgenera Nyssorhynchus and Kerteszia were supported as a monophyletic lineage. The Kishino-Hasegawa test could not reject the monophyly of Anopheles, whereas the recently proposed hypothesis of close affinity of Bironella to the subgenus Anopheles was rejected by the analyses of ND5 and combined data sets. The lack of resolution of Bironella and Anopheles clades, or basal relationships among subgeneric clades within Anopheles, suggests their rapid diversification. Recovery of relationships consistent with morphology and previous molecular studies provides evidence of substantial phylogenetic signal in D2 and ND5 genes at levels of divergence from closely related species to subfamily in mosquitoes.

Patterns of DNA sequence variation in chromosomally recognized taxa of Anopheles gambiae: evidence from rDNA and single-copy loci.

Patterns of DNA sequence variation in the ribosomal DNA (rDNA) second internal transcribed spacer (ITS2) and five unlinked single-copy nuclear loci were examined for evidence of reproductive isolation among four chromosomally recognized taxa of Anopheles gambiae from West Africa: Savanna, Bamako, Mopti and Forest, as well as sibling species An. arabiensis and An. merus. Included among the single-copy loci were three sequence-tagged random amplified polymorphic DNA (RAPD) loci, two of which (R15 and R37) had been reported as discriminating between Mopti and other chromosomal forms. Each of the five single-copy sequences were highly polymorphic in most samples. However, the R15 and R37 loci had no diagnostic value, and therefore are not recommended as tools in recognition of field-collected An. gambiae chromosomal forms. Although pairwise comparisons between species generally revealed significant levels of differentiation at all five loci, variation was not partitioned by chromosomal form within An. gambiae at any single-copy locus examined. The few exceptions to these trends appear related to a location either inside or nearby chromosomal inversions. At the tryptophan oxygenase locus inside inversion 2Rb, variation was structured only by inversion orientation and not by taxonomic designation even between An. gambiae and An. arabiensis, providing the first molecular evidence that the 2Rb inversion was transferred between species by introgressive hybridization. By contrast, the rDNA showed fixed differences between species and a difference diagnostic for Mopti, consistent with effective, if not complete, reproductive isolation. The apparent disagreement between the data from this locus and multiple single-copy loci within An. gambiae may be explained by the much lower effective population size of rDNA, owing to concerted evolution, which confers increased sensitivity at much shorter divergence times. Taken together with the accompanying reports by della Torre et al. (2001), Favia et al. (2001) and Gentile et al. (2001), our data suggest that neutral molecular markers may not have the sensitivity required to detect isolation between these recently established taxa.

Toward understanding Anophelinae (Diptera, Culicidae) phylogeny: insights from nuclear single-copy genes and the weight of evidence.

A phylogeny of the mosquito subfamily Anophelinae was inferred from fragments of two protein-coding nuclear genes, G6pd (462 bp) and white (801 bp), and from a combined data set (2,136 bp) that included a portion of the mitochondrial gene ND5 and the D2 region of the ribosomal 28S gene. Sixteen species from all three anopheline genera and six Anopheles subgenera were sampled, along with six species of other mosquitoes used as an outgroup. Each of four genes analyzed individually recovered the same well-supported clades; topological incongruence was limited to unsupported or poorly supported nodes. As assessed by the incongruence length difference test, most of the conflicting signal was contributed by third codon positions. Strong structural constraints, as observed in white and G6pd, apparently had little impact on phylogenetic inference. Compared with the other genes, white provided a superior source of phylogenetic information. However, white appears to have experienced accelerated rates of evolution in few lineages, the affinities of which are therefore suspect. In combined analyses, most of the inferred relationship were well-supported and in agreement with previous studies: monophyly of Anophelinae, basal position of Chagasia, monophyly of Anopheles subgenera, and subgenera Nyssorhynchus + Kerteszia as sister taxa. The results suggested also monophyletic origin of subgenera Cellia + Anopheles, and the white gene analysis supported genus Bironella as a sister taxon to Anopheles. The present data and other available evidence suggest a South American origin of Anophelinae, probably in the Mesozoic; a rapid diversification of Bironella and basal subgeneric lineages of Anopheles, potentially associated with the breakup of Gondwanaland; and a relatively recent and rapid dispersion of subgenus Anopheles.

Ribosomal DNA internal transcribed spacer (ITS2) sequences differentiate Anopheles funestus and An. rivulorum, and uncover a cryptic taxon.

Differentiation among the closely related Afrotropical species comprising the Funestus Group is difficult by traditional taxonomic measures. Anopheles rivulorum is the second most abundant and widespread species in the Funestus Group, and is occasionally collected indoors along with the dominant member and major malaria vector, An. funestus. The prospect of misidentification of An. rivulorum as An. funestus prompted the development of a rapid, polymerase chain reaction (PCR)-based method for identifying these two species. The ribosomal internal transcribed spacer 2 (ITS2) was amplified from thirty-five specimens of An. rivulorum collected from the extremes of its range: Eastern Africa (Kenya), Southern Africa (South Africa) and Western Africa (Burkina Faso). The ITS2 region of An. rivulorum ( approximately 380 bp) is sufficiently different in size from the ITS2 of An. funestus ( approximately 700 bp) that these species can be distinguished by agarose gel electrophoresis of PCR products without further manipulation. Comparison of the An. rivulorum and An. funestus ITS2 nucleotide sequences revealed such extensive divergence that meaningful alignment was impossible, except for a 25 bp island near the 5' end. Intraspecific sequence comparisons revealed no variation among An. rivulorum individuals collected from the same country. However, sequence divergence was 2% between specimens from South Africa and Kenya, and nearly tenfold higher ( approximately 19%) between specimens from Burkina Faso and either South Africa or Kenya, an unprecedented level of intraspecific ITS2 divergence in Anopheles. Taken together, these data suggest that the Burkina Faso sample is not An. rivulorum, but rather a cryptic taxon within the Funestus Group.

The Rift Valley complex as a barrier to gene flow for Anopheles gambiae in Kenya: the mtDNA perspective.

Descriptions of A. gambiae population structure based on microsatellite loci and mitochondrial DNA (mtDNA) were incongruent. High differentiation of populations was measured across the Rift Valley by microsatellites, but no differentiation was detected based on mtDNA. To resolve this conflict, we compared the old data to new mtDNA data using the same specimen previously genotyped in microsatellite loci. Analysis of a larger number of mtDNA sequences resulted in high and significant differentiation between populations across the Rift Valley. We developed a method to assess whether differentiation across the Rift Valley was generated by pure drift rather than mutation-drift, based on DNA sequence data. Applying this method to the mtDNA data suggested that pure drift was the primary force generating differentiation between the populations across the Rift, while mutation-drift generated differentiation across the continent. Given adequate sample size, mtDNA provided congruent results with microsatellite loci.