Molecular population genetics of male accessory gland proteins in Drosophila

Drosophila seminal proteins have an unusually high rate of molecular sequence evolution, suggesting either a high rate of neutral substitution or rapid adaptive evolution. To further quantify patterns of polymorphism and divergence in genes encoding seminal proteins, also called accessory gland proteins (Acp's), we conducted a sequencing survey of 10 Acp genes in samples of Drosophila melanogaster and D. simulans (Acp29AB, Acp32CD, Acp33A, Acp36DE, Acp53Ea, Acp62F, Acp63F, Acp76A, Acp95EF, and Acp98AB). Mean heterozygosity at replacement sites in D. simulans was 0.0074 for Acp genes and 0.0013 for a set of 19 non-Acp genes, and mean melanogaster-simulans divergence at replacement sites was 0.0497 for Acp genes and 0.0107 at non-Acp genes. The elevated divergence of Acp genes is thus accompanied by elevated within-species polymorphism. In addition to the already-reported departures of Acp26A, Acp29AB, and Acp70A from neutrality, our data reject neutrality at Acp29AB and Acp36DE in the direction of excess replacements in interspecific comparisons.     

Genetics of sexual isolation in females of the Drosophila simulans species complex.

Genetic analysis of hybrids between Drosophila simulans and D. sechellia shows that sexual isolation in females is caused by at least two genes, one on each major autosome, while the X chromosome has no effect. These results are similar to those of a previous study of hybrids between D. simulans and another sibling species, D. mauritiana. In this latter hybridization, each arm of the second chromosome carries genes causing sexual isolation in females, implying a total divergence of at least three loci. The genetic similarity between the D. simulans/D. mauritiana and D. simulans/D. sechellia hybridizations probably results from independent evolution and not phylogenetic artifacts, because the dominance relationships and behavioural interactions differ between the two hybridizations. The lack of an X-chromosome effect on sexual isolation contrasts with genetic studies of post-zygotic reproductive isolation, which invariably show strong effects of this chromosome.     

The population genetics of the origin and divergence of the Drosophila simulans complex species

The origins and divergence of Drosophila simulans and close relatives D. mauritiana and D. sechellia were examined using the patterns of DNA sequence variation found within and between species at 14 different genes. D. sechellia consistently revealed low levels of polymorphism, and genes from D. sechellia have accumulated mutations at a rate that is approximately 50% higher than the same genes from D. simulans. At synonymous sites, D. sechellia has experienced a significant excess of unpreferred codon substitutions. Together these observations suggest that D. sechellia has had a reduced effective population size for some time, and that it is accumulating slightly deleterious mutations as a result. D. simulans and D. mauritiana are both highly polymorphic and the two species share many polymorphisms, probably since the time of common ancestry. A simple isolation speciation model, with zero gene flow following incipient species separation, was fitted to both the simulans/mauritiana divergence and the simulans/sechellia divergence. In both cases the model fit the data quite well, and the analyses revealed little evidence of gene flow between the species. The exception is one gene copy at one locus in D. sechellia, which closely resembled other D. simulans sequences. The overall picture is of two allopatric speciation events that occurred quite near one another in time.     

Adaptive evolution of recently duplicated accessory gland protein genes in desert Drosophila

The relationship between animal mating system variation and patterns of protein polymorphism and divergence is poorly understood. Drosophila provides an excellent system for addressing this issue, as there is abundant interspecific mating system variation. For example, compared to D. melanogaster subgroup species, repleta group species have higher remating rates, delayed sexual maturity, and several other interesting differences. We previously showed that accessory gland protein genes (Acp's) of Drosophila mojavensis and D. arizonae evolve more rapidly than Acp's in the D. melanogaster subgroup and that adaptive Acp protein evolution is likely more common in D. mojavensis/D. arizonae than in D. melanogaster/D. simulans. These findings are consistent with the idea that greater postcopulatory selection results in more adaptive evolution of seminal fluid proteins in the repleta group flies. Here we report another interesting evolutionary difference between the repleta group and the D. melanogaster subgroup Acp's. Acp gene duplications are present in D. melanogaster, but their high sequence divergence indicates that the fixation rate of duplicated Acp's has been low in this lineage. Here we report that D. mojavensis and D. arizonae genomes contain several very young duplicated Acp's and that these Acp's have experienced very rapid, adaptive protein divergence. We propose that rapid remating of female desert Drosophila generates selection for continuous diversification of the male Acp complement to improve male fertilization potential. Thus, mating system variation may be associated with adaptive protein divergence as well as with duplication of Acp's in Drosophila.     

DNA Sequence Variation at the Period Locus within and among Species of the Drosophila Melanogaster Complex

A 1.9-kilobase region of the period locus was sequenced in six individuals of Drosophila melanogaster and from six individuals of each of three sibling species: Drosophila simulans, Drosophila sechellia and Drosophila mauritiana. Extensive genealogical analysis of 174 polymorphic sites reveals a complex history. It appears that D. simulans, as a large population still segregating very old lineages, gave rise to the island species D. mauritiana and D. sechellia. Rather than considering these speciation events as having produced ``sister' taxa, it seems more appropriate to consider D. simulans a parent species to D. sechellia and D. mauritiana. The order, in time, of these two phylogenetic events remains unclear. D. mauritiana supports a large number of polymorphisms, many of which are shared with D. simulans, and so appears to have begun and persisted as a large population. In contrast, D. sechellia has very little variation and seems to have experienced a severe population bottleneck. Alternatively, the low variation in D. sechellia could be due to recent directional selection and genetic hitchhiking at or near the per locus.     

Discord between the Phylogenies Inferred from Molecular versus Functional Data: Uneven Rates of Functional Evolution or Low Levels of Gene Flow?

According to measures of molecular divergence, the three species of the Drosophila simulans clade are closely related to and essentially equidistant from each other. We introgressed 10% of the D. sechellia X chromosome into a pure D. simulans genetic background and found that males carrying this introgressed region were consistently fertile; in contrast, males carrying the same segment from D. mauritiana are sterile and suffer from incompatibilities at a minimum of four loci. Together with other recent results, these data suggest that D. simulans and D. sechellia are much more closely related to each other than either is to D. mauritiana. How can we reconcile the phylogeny inferred from the density of hybrid sterility genes with that inferred from molecular divergence? If the molecular phylogeny is correct, the discrepancy might be explained by uneven rates of functional evolution, resulting in the uneven accumulation of substitutions with corresponding negative effects in hybrids. If the functional phylogeny is correct, then low levels of gene flow across nascent species boundaries, particularly for loci not tightly linked to a hybrid sterility gene, may have erased the original pattern of lineage splitting. We propose tests that will allow us to discriminate between these hypotheses.     

Evolutionary History of the Sex-Peptide (Acp70a) Gene Region in Drosophila Melanogaster

In Drosophila the products of the seminal fluid stimulate oviposition and suppress remating in the female. Of all the accessory gland peptides (Acp's) involved in these two responses, the sex-peptide (coded by the Acp70A gene) is among the best characterized at the functional level. A 1.2-kb fragment encompassing the Acp70A gene of nine lines from a natural population of D. melanogaster and one allele of D. sechellia was sequenced to study the forces shaping nucleotide variation within and between species. The coding region of D. simulans and D. mauritiana was also sequenced. A Ser to Ala replacement polymorphism at the last position of the signal peptide was detected in D. melanogaster. The Ser and Ala alleles are at intermediate frequencies. The level of nucleotide variation is lower for the derived Ala allele, which is compatible with a recent origin and an increase in frequency due to positive selection. Variation at the 5' flanking region is structured in two major highly differentiated haplotypes, whose distribution does not conform to neutral expectations. Selective and/or historical factors could contribute to the observed overall patterning of nucleotide variation at the Acp70A region.     

Patterns of Microsatellite Variability in the Drosophila Melanogaster Complex

Forty-seven microsatellite loci were amplified in Drosophila melanogaster, Drosophila simulans, Drosophila mauritiana and Drosophila sechellia. The two cosmopolitan species D. melanogaster and D. simulans were found to be the most variable ones, followed by D. mauritiana and D. sechellia. A model based clustering algorithm was applied to the population samples of D. melanogaster, D. simulans and D. sechellia. No evidence for population substructure was detected within species--most likely due to insufficient power. A Markov chain Monte Carlo method developed for demographic inference based on microsatellites provided unambiguous evidence for population contraction in D. melanogaster, D. simulans and D. sechellia, despite that the D. melanogaster and D. simulans population samples were of non-African origin and represented recently expanded populations.     

The Genetic Basis of Haldane''s Rule and the Nature of Asymmetric Hybrid Male Sterility among Drosophila Simulans, Drosophila Mauritiana and Drosophila Sechellia

Haldane's rule (i.e., the preferential hybrid sterility and inviability of heterogametic sex) has been known for 70 years, but its genetic basis, which is crucial to the understanding of the process of species formation, remains unclear. In the present study, we have investigated the genetic basis of hybrid male sterility using Drosophila simulans, Drosophila mauritiana and Drosophila sechellia. An introgression of D. sechellia Y chromosome into a fairly homogenous background of D. simulans did not show any effect of the introgressed Y on male sterility. The substitution of D. simulans Y chromosome into D. sechellia, and both reciprocal Y chromosome substitutions between D. simulans and D. mauritiana were unsuccessful. Introgressions of cytoplasm between D. simulans and D. mauritiana (or D. sechellia) also did not have any effect on hybrid male sterility. These results rule out the X-Y interaction hypothesis as a general explanation of Haldane's rule in this species group and indicate an involvement of an X-autosome interaction. Models of symmetrical and asymmetrical X-autosome interaction have been developed which explain the Y chromosome substitution results and suggest that evolution of interactions between different genetic elements in the early stages of speciation is more likely to be of an asymmetrical nature. The model of asymmetrical X-autosome interaction also predicts that different sets of interacting genes may be involved in different pairs of related species and can account for the observation that hybrid male sterility in many partially isolated species is often nonreciprocal or unidirectional.     

Inter‐island divergence within Drosophila mauritiana,a species of the D. simulans complex: Past history and/or speciation in progress?

Speciation with gene flow may be more common than generally thought, which makes detailed understanding of the extent and pattern of genetic divergence between geographically isolated populations useful. Species of the Drosophila simulans complex provide a good model for speciation and evolutionary studies, and hence understanding their population genetic structure will increase our understanding of the context in which speciation has occurred. Here, we describe genetic diversity and genetic differentiation of two distant populations of D. mauritiana (Mauritius and Rodrigues Islands) at mitochondrial and nuclear loci. We surveyed the two populations for their mitochondrial haplotypes, eight nuclear genes and 18 microsatellite loci. A new mitochondrial type is fixed in the Rodrigues population of D. mauritiana. The two populations are highly differentiated, their divergence appears relatively ancient (100,000 years) compared to the origin of the species, around 0.25MYA, and they exhibit very limited gene flow. However, they have similar levels of divergence from their sibling, D. simulans. Both nuclear genes and microsatellites revealed contrasting demographic histories between the two populations, expansion for the Mauritius population and stable population size for the Rodrigues Island population. The discovery of pronounced geographic structure within D. mauritiana combined to genetic structuring and low gene flow between the two island populations illuminates the evolutionary history of the species and clearly merits further attention in the broad context of speciation.     

The Effects of Interspecific Y Chromosome Replacements on Hybrid Sterility within the Drosophila Simulans Clade

We attempted to introgress Y chromosomes between three sibling species of Drosophila: D. simulans, D. sechellia and D. mauritiana. Four D. sechellia Y chromosomes were introgressed into D. simulans without loss of fertility whereas the four reciprocal introgressions (D. simulans Y introgressed into D. sechellia) all result in sterility. Both reciprocal Y introgressions of D. simulans and D. mauritiana (four of each) also result in sterility. Compared with D. simulans males, the males with the D. sechellia Y chromosome in D. simulans background had lower productivity but only after multiple matings with virgin females. These males also were inferior compared with pure species males in sperm displacement and/or remating ability. The two different Y genotype males, however, were comparable in viability, longevity and mating success in female choice tests. We also use our results to estimate the effective number of autosomal loci interacting with X-linked genes to produce hybrid male sterility.     

Molecular population genetics of accessory gland protein genes and testis-expressed genes in Drosophila mojavensis and D. arizonae

Molecular population genetic investigation of Drosophila male reproductive genes has focused primarily on melanogaster subgroup accessory gland protein genes (Acp's). Consistent with observations from male reproductive genes of numerous taxa, Acp's evolve more rapidly than nonreproductive genes. However, within the Drosophila genus, large data sets from additional types of male reproductive genes and from different species groups are lacking. Here we report findings from a molecular population genetics analysis of male reproductive genes of the repleta group species, Drosophila arizonae and D. mojavensis. We find that Acp's have dramatically higher average pairwise Ka/Ks (0.93) than testis-enriched genes (0.19) and previously reported melanogaster subgroup Acp's (0.42). Overall, 10 of 19 Acp's have Ka/Ks > 1 either in nonpolarized analyses or in at least one lineage of polarized analyses. Of the nine Acp's for which outgroup data were available, average Ka/Ks was considerably higher in D. mojavensis (2.08) than in D. arizonae (0.87). Contrasts of polymorphism and divergence suggest that adaptive protein evolution at Acp's is more common in D. mojavensis than in D. arizonae.     

A Reanalysis of Protein Polymorphism in Drosophila Melanogaster, D. Simulans, D. Sechellia and D. Mauritiana: Effects of Population Size and Selection

Comparison of synonymous and nonsynonymous variation/substitution within and between species at individual genes has become a widely used general approach to detect the effect of selection versus drift. The sibling species group comprised of two cosmopolitan (Drosophila melanogaster and Drosophila simulans) and two island (Drosophila mauritiana and Drosophila sechellia) species has become a model system for such studies. In the present study we reanalyzed the pattern of protein variation in these species, and the results were compared against the patterns of nucleotide variation obtained from the literature, mostly available for melanogaster and simulans. We have mainly focused on the contrasting patterns of variation between the cosmopolitan pair. The results can be summarized as follows: (1) As expected the island species D. mauritiana and D. sechellia showed much less variation than the cosmopolitan species D. melanogaster and D. simulans. (2) The chromosome 2 showed significantly less variation than chromosome 3 and X in all four species which may indicate effects of past selective sweeps. (3) In contrast to its overall low variation, D. mauritiana showed highest variation for X-linked loci which may indicate introgression from its sibling, D. simulans. (4) An average population of D. simulans was as heterozygous as that of D. melanogaster (14.4% v.s. 13.9%) but the difference was large and significant when considering only polymorphic loci (37.2% v.s. 26.1%). (5) The species-wise pooled populations of these two species showed similar results (all loci = 18.3% v.s. 20.0%, polymorphic loci = 47.2% v.s. 37.6%). (6) An average population of D. simulans had more low-frequency alleles than D. melanogaster, and the D. simulans alleles were found widely distributed in all populations whereas the D. melanogaster alleles were limited to local populations. As a results of this, pooled populations of D. melanogaster showed more polymorphic loci than those of D. simulans (48.0% v.s. 32.0%) but the difference was reduced when the comparison was made on the basis of an average population (29.1% v.s. 21.4%). (7) While the allele frequency distributions within populations were nonsignificant in both D. melanogaster and D. simulans, melanogaster had fewer than simulans, but more than expected from the neutral theory, low frequency alleles. (8) Diallelic loci with the second allele with a frequency less than 20% had similar frequencies in all four species but those with the second allele with a frequency higher than 20% were limited to only melanogaster the latter group of loci have clinal (latitudinal) patterns of variation indicative of balancing selection. (9) The comparison of D. simulans/D. melanogaster protein variation gave a ratio of 1.04 for all loci and 1.42 for polymorphic loci, against a ratio of approximately 2-fold difference for silent nucleotide sites. This suggests that the species ratios of protein and silent nucleotide polymorphism are too close to call for selective difference between silent and allozyme variation in D. simulans. In conclusion, the contrasting levels of allozyme polymorphism, distribution of rare alleles, number of diallelic loci and the patterns of geographic differentiation between the two species suggest the role of natural selection in D. melanogaster, and of possibly ancient population structure and recent worldwide migration in D. simulans. Population size differences alone are insufficient as an explanation for the patterns of variation between these two species.     

Evidence for Complex Genic Interactions between Conspecific Chromosomes Underlying Hybrid Female Sterility in the Drosophila Simulans Clade

F(1) hybrid females between the sibling species Drosophila simulans, Drosophila mauritiana and Drosophila sechellia are completely fertile. However, we have found that female sterility can be observed in F(2) backcross females who are homozygous for D. simulans X chromosomes and homozygous for autosomal regions from either D. mauritiana or D. sechellia. Our results indicate that neither D. mauritiana autosome (2 or 3) can cause complete female sterility in a D. simulans background. The simultaneous presence of homozygous regions from both the second and third chromosomes of D. mauritiana, however, causes nearly complete female sterility which cannot be accounted for by their individual effects. The two autosomes of D. sechellia may show a similar pattern. From the same crosses, we also obtained evidence against a role for cytoplasmic or maternal effects in causing hybrid male sterility between these species. Taken with the results presented elsewhere, these observations suggest that epistatic interactions between conspecific genes in a hybrid background may be the prevalent mode of hybrid sterility between recently diverged species.     

Genetic dissection of hybrid incompatibilities between Drosophila simulans and D. mauritiana. II. Mapping hybrid male sterility loci on the third chromosome

Hybrid male sterility (HMS) is a rapidly evolving mechanism of reproductive isolation in Drosophila. Here we report a genetic analysis of HMS in third-chromosome segments of Drosophila mauritiana that were introgressed into a D. simulans background. Qualitative genetic mapping was used to localize 10 loci on 3R and a quantitative trait locus (QTL) procedure (multiple-interval mapping) was used to identify 19 loci on the entire chromosome. These genetic incompatibilities often show dominance and complex patterns of epistasis. Most of the HMS loci have relatively small effects and generally at least two or three of them are required to produce complete sterility. Only one small region of the third chromosome of D. mauritiana by itself causes a high level of infertility when introgressed into D. simulans. By comparison with previous studies of the X chromosome, we infer that HMS loci are only approximately 40% as dense on this autosome as they are on the X chromosome. These results are consistent with the gradual evolution of hybrid incompatibilities as a by-product of genetic divergence in allopatric populations.     

A comprehensive study of genic variation in natural populations of Drosophila melanogaster. VII. Varying rates of genic divergence as revealed by two-dimensional electrophoresis.

Four sibling species from the melanogaster subgroup (Drosophila melanogaster, D. simulans, D. sechellia, and D. mauritiana) were studied for genetic divergence, by high-resolution two-dimensional protein electrophoresis (2DE) coupled with ultrasensitive silver staining. A total of eight tissues from larval and adult developmental stages representing both gonadal (germ-line) and nongonadal (somatic) tissues were analyzed for protein divergence between species. Close to 400 polypeptides (protein spots) were scored from each tissue and species, and protein divergence was measured on the basis of qualitative differences (presence/absence) of protein spots in pairwise species comparisons. The observed levels of genic divergence varied among tissues and among species. When larval hemolymph proteins (which are known to be highly polymorphic) were excluded, there was no evidence to suggest that either the larval or adult-stage proteins, as a whole, are more diverged than the other; variation between different tissues rather than between developmental stages appears to be the most significant factor affecting genetic divergence between species. The reproductive tissue (testis and accessory gland) showed more divergence than did the nonreproductive tissue; D. melanogaster testis (from both larvae and adult males) showed the highest level of divergence. In view of the previous observation that D. simulans, D. mauritiana, and D. sechellia show similar but significantly less reproductive isolation from each other than from D. melanogaster, the present results suggest a correlation between the levels of reproductive-tract-protein divergence and the degree of reproductive isolation in these species.     

Molecular evolution and population genetics of duplicated accessory gland protein genes in Drosophila

To investigate the potential importance of gene duplication in D. melanogaster accessory gland protein (Acp) gene evolution we carried out a computational analysis comparing annotated D. melanogaster Acp genes to the entire D. melanogaster genome. We found that two known Acp genes are actually members of small multigene families. Polymorphism and divergence data from these duplicated genes suggest that in at least four cases, protein divergence between D. melanogaster and D. simulans is a result of directional selection. One putative Acp revealed by our computational analysis shows evidence of a recent selective sweep in a non-African population (but not in an African population). These data support the idea that selection on reproduction-related genes may drive divergence of populations within species, and strengthen the conclusion that Acps may often be under directional selection in Drosophila.     

Population genetics and phylogenetics of DNA sequence variation at multiple loci within the Drosophila melanogaster species complex

Two regions of the genome, a 1-kbp portion of the zeste locus and a 1.1- kbp portion of the yolk protein 2 locus, were sequenced in six individuals from each of four species: Drosophila melanogaster, D. simulans, D. mauritiana, and D. sechellia. The species and strains were the same as those of a previous study of a 1.9-kbp region of the period locus. No evidence was found for recent balancing or directional selection or for the accumulation of selected differences between species. Yolk protein 2 has a high level of amino acid replacement variation and a low level of synonymous variation, while zeste has the opposite pattern. This contrast is consistent with information on gene function and patterns of codon bias. Polymorphism levels are consistent with a ranking of effective population sizes, from low to high, in the following order: D. sechellia, D. melanogaster, D.mauritiana, and D. simulans. The apparent species relationships are very similar to those suggested by the period locus study. In particular, D. simulans appears to be a large population that is still segregating variation that arose before the separation of D. mauritiana and D. sechellia. It is estimated that the separation of ancestral D. melanogaster from the other species occurred 2.5-3.4 Mya. The separations of D. sechellia and D. mauritiana from ancestral D. simulans appear to have occurred 0.58- 0.86 Mya, with D. mauritiana having diverged from ancestral D. simulans 0.1 Myr more recently than D. sechellia.