Sex-linked differentiation between incipient species of Anopheles gambiae.

Emerging species within the primary malaria vector Anopheles gambiae show different ecological preferences and significant prezygotic reproductive isolation. They are defined by fixed sequence differences in X-linked rDNA, but most previous studies have failed to detect large and significant differentiation between these taxa elsewhere in the genome, except at two other loci on the X chromosome near the rDNA locus. Hypothesizing that this pericentromeric region of the X chromosome may be accumulating differences faster than other regions of the genome, we explored the pattern and extent of differentiation between A. gambiae incipient species and a sibling species, A. arabiensis, from Burkina Faso, West Africa, at 17 microsatellite loci spanning the X chromosome. Interspecific differentiation was large and significant across the entire X chromosome. Among A. gambiae incipient species, we found some of the highest levels of differentiation recorded in a large region including eight independent loci near the centromere of the X chromosome. Outside of this region, no significant differentiation was detected. This pattern suggests that selection is playing a role in the emergence of A. gambiae incipient species. This process, associated with efficient exploitation of anthropogenic modifications to the environment, has public health implications as it fosters the spread of malaria transmission both spatially and temporally.

Variation in recombination rate across the X chromosome of Anopheles gambiae.

The M and S molecular forms of Anopheles gambiae are considered to be incipient species, despite residual gene exchange. Of the three small genome regions that are strongly differentiated between the molecular forms ("speciation islands"), two are located near centromeres, on the left arm of chromosome 2 and the X chromosome. To test the prediction of reduced recombination in these islands, we estimated recombination rates between microsatellite loci on the X chromosome using two M-form strains. Across most of the chromosome, recombination occurred at approximately 1 centimorgan per megabase (cM Mb(-1)), a value closely matching the genome-wide average estimated for A. gambiae and for other eukaryotes. Recombination was much higher at the telomeric end, > 7 cM Mb(-1). In the speciation island at the centromeric end, recombination was sharply reduced to approximately 0.2 cM Mb(-1), consistent with a role for reduced recombination in maintaining differentiation between nascent species despite gene flow.

Dynamics of the pyrethroid knockdown resistance allele in western Kenyan populations of Anopheles gambiae in response to insecticide-treated bed net trials.

Permethrin and DDT resistance in Anopheles gambiae s.s. associated with a leucine-serine knockdown resistance (kdr) mutation in the voltage-gated sodium channel gene was discovered recently in western Kenya where a large scale permethrin-impregnated bed net (ITN) program has been implemented. Collections of An. gambiae s.l. were made from intervention and control villages prior to and after onset of the program. The kdr genotypes were determined using allele-specific polymerase chain reaction diagnostic tests. In An. gambiae s.s., the frequency of the kdr mutation prior to ITN introduction was approximately 3-4% in western Kenya and zero in samples from the coast. After ITN introduction, the kdr mutation increased in ITN and neighboring villages from approximately 4% to approximately 8%, but remained unchanged in villages at least 20 km distant and was not detected in coastal Kenya. The identical leucine-serine mutation was found in a single An. arabiensis individual among 658 tested. The leucine-phenylalanine kdr mutation common in west African An. gambiae populations was not detected in An. gambiae s.l. from Kenya. Implications for the population structure and control of An. gambiae are discussed.

Selective constraint and the evolution of the RNA polymerase II C-Terminal Domain.

The C-Terminal Domain (CTD) of the large subunit (Rpb1) of RNA Polymerase II has a Tyrosine-Serine-Proline-Threonine-Serine-Proline-Serine repeat structure in many eukaryotes. Chemical modifications of these residues play a central role in the regulation and coordination of the events of transcription. However, substantial variability in the presence and regularity of repeat arrays exists between eukaryote taxa. Following a survey of CTD structure from diverse eukaryote species, two hypotheses were tested relating to repeat structure and the action of selection on the CTD. First, it was found that degenerated repeat structure is associated with lower serine and proline frequencies in some eukaryote taxa but not in others. Second, maximum likelihood models of the evolution of Rpb1 in a number of species groups found that purifying selection on the non-repetitive CTD of several Leishmania species was substantially lower than for the rest of Rpb1, whereas purifying selection in a number of species groups containing repeat arrays was usually as high or nearly as high as for the rest of Rpb1. Characterization of CTD structure for a larger number of species than has been completed previously also revealed a greater diversity of CTD structures in eukaryotes than previously known, along with loss of repeat structure in the animals and fungi, two taxa where it has not previously been known. These results suggest that loss of CTD repeat structure has been an important aspect of RNA Polymerase II evolution in diverse eukaryotes.

Molecular evolution of the moonlighting protein SMN in metazoans.

The spinal muscular atrophy (SMA) associated protein survival of motor neuron (SMN) is known to be a moonlighting protein: having one primary, ancestral function (presumed to be involvement in U snRNP assembly) along with one or more secondary functions. One hypothesis for the evolution of moonlighting proteins is that regions of a structure under relatively weak negative selection could gain new functions without interfering with the primary function. To test this hypothesis, we investigated sequence conservation and dN/dS, which reflects the selection acting on a coding sequence, in SMN and a related protein, splicing factor 30 (SPF30), which is not currently known to be multifunctional. We found very different patterns of evolution in the two genes, with SPF30 characterized by strong sequence conservation and negative selection in most animal taxa investigated, and SMN with much lower sequence conservation, and much weaker negative selection at many sites. Evidence was found of positive selection acting on some sites in primate genes for SMN. SMN was also found to have been duplicated in a number of species, and with patterns that indicate reduced negative selection following some of these duplications. There were also several animal species lacking an SMN gene.

Distribution and mechanism of α-amanitin tolerance in mycophagous Drosophila (Diptera: Drosophilidae).

Many mycophagous species of Drosophila can tolerate the mushroom poison α-amanitin in wild mushrooms and in artificial diet. We conducted feeding assays with sixteen Drosophila species and α-amanitin in artificial diet to better determine the phylogenetic distribution of this tolerance. For eight tolerant and one related susceptible species, we sequenced the gene encoding the large subunit of RNA Polymerase II, which is the target site of α-amanitin. We found no differences in the gene that could account for differences in susceptibility to the toxin. We also conducted feeding assays in which α-amanitin was combined with chemical inhibitors of cytochrome P450s or glutathione S-transferases (GSTs) in artificial diet to determine if either of these enzyme families is involved in tolerance to α-amanitin. We found that an inhibitor of GSTs did not reduce tolerance to α-amanitin, but that an inhibitor of cytochrome P450s reduced tolerance in several species. It is possible that the same cytochrome P450 activity that produces tolerance of α-amanitin might produce tolerance of other mushroom toxins as well. If so, a general detoxification mechanism based on cytochrome P450s might answer the question of how tolerance to α-amanitin arose in mycophagous Drosophila when this toxin is found in relatively few mushrooms.

Centromere-proximal differentiation and speciation in Anopheles gambiae.

The M and S molecular forms of Anopheles gambiae are undergoing speciation as they adapt to heterogeneities in the environment, spreading malaria in the process. We hypothesized that their divergence despite gene flow is facilitated by reduced recombination at the centromeric (proximal) end of the X chromosome. We sequenced introns from 22 X chromosome genes in M and S from two locations of West Africa where the forms are sympatric. Generally, in both forms nucleotide diversity was high distally, lower proximally, and very low nearest the centromere. Conversely, differentiation between the forms was virtually zero distally and very high proximally. Pairwise comparisons to a close relative, the sibling species Anopheles arabiensis, demonstrated uniformly high divergence regardless of position along the X chromosome, suggesting that this pattern is not purely mechanical. Instead, the pattern observed for M and S suggests the action of divergent natural selection countering gene flow only at the proximal end of the X chromosome, where recombination is reduced. Comparison of sites with fixed differences between M and S to the corresponding sites in A. arabiensis revealed that derived substitutions had been fixed in both forms, further supporting the hypothesis that both have been under selection. These derived substitutions are fixed in the two West African samples and in samples of S from western and coastal Kenya, suggesting that selection occurred before the forms expanded to their current ranges. Our findings are consistent with a role for suppressed genetic recombination in speciation of A. gambiae.