An introgression approach to mapping differences in mating success and sperm competitive ability in Drosophila simulans and D. sechellia

The progeny of Drosophila females doubly-mated to males from the same and a closely related species are mostly sired by conspecific males. We examined the genetic basis for conspecific mating preference and sperm precedence by using 186 Drosophila lines in which random chromosomal fragments of D. sechellia were introgressed into D. simulans. Sperm competition was measured for each of these lines by crossing ebony D. simulans female with ebony D. simulans males followed by wild-type males from the introgressed lines. Variation in sperm competition (proportion of progeny sired by the second male), mating discrimination (proportion of introgressed males that failed to remate), and male fecundity (proportion of progeny sired by introgressed males) were scored. The introgressed lines exhibited highly significant heterogeneity in the three phenotypes scored, motivating an analysis to locate quantitative trait loci (QTLs) responsible for the differences. Applying composite interval mapping, we found eight QTLs that explain a significant level of variation among introgressed lines in the phenotypes scored. Cytological position overlapped among some QTLs suggesting possible pleiotropic effects. Analysis of the joint effects of simulans/sechellia genetic composition at different QTLs and markers suggests that complex interactions among alleles are partially responsible for interspecific differences in sexual traits.     

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.     

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.     

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.     

Comparative analysis of transposable elements in the melanogaster subgroup sequenced genomes

Transposable elements (TEs) are indwelling components of genomes, and their dynamics have been a driving force in genome evolution. Although we now have more information concerning their amounts and characteristics in various organisms, we still have little data from overall comparisons of their sequences in very closely-related species. While the Drosophila melanogaster genome has been extensively studied, we have only limited knowledge regarding the precise TE sequences in the genomes of the related species Drosophila simulans, Drosophila sechellia and Drosophila yakuba. In this study we analyzed the number and structure of TE copies in the sequenced genomes of these four species. Our findings show that, unexpectedly, the number of TE insertions in D. simulans is greater than that in D. melanogaster, but that most of the copies in D. simulans are degraded and in small fragments, as in D. sechellia and D. yakuba. This suggests that all three species were invaded by numerous TEs a long time ago, but have since regulated their activity, as the present TE copies are degraded, with very few full-length elements. In contrast, in D. melanogaster, a recent activation of TEs has resulted in a large number of almost-identical TE copies. We have detected variants of some TEs in D. simulans and D. sechellia, that are almost identical to the reference TE sequences in D. melanogaster, suggesting that D. melanogaster has recently been invaded by active TE variants from the other species. Our results indicate that the three species D. simulans, D. sechellia, and D. yakuba seem to be at a different stage of their TE life cycle when compared to D. melanogaster. Moreover, we show that D. melanogaster has been invaded by active TE variants for several TE families likely to come from D. simulans or the ancestor of D. simulans and D. sechellia. The numerous horizontal transfer events implied to explain these results could indicate introgression events between these species.     

The Genetics of Reproductive Isolation in the Drosophila Simulans Clade: X Vs. Autosomal Effects and Male Vs. Female Effects

A strong effect of homozygous autosomal regions on reproductive isolation was found for crosses between the species in the Drosophila simulans clade. Second chromosome regions were introgressed from D. mauritiana and D. sechellia into D. simulans and tested for their homozygous effects on hybrid male and hybrid female sterility and inviability. Most introgressions are fertile as heterozygotes, yet produce sterile male offspring when made homozygous. The density of homozygous autosomal factors contributing to hybrid male sterility is comparable to the density of X chromosome factors for this level of resolution. Female sterility was also revealed, yet the disparity between male and female levels of sterility was great, with male sterility being up to 23 times greater than female sterility. Complete hybrid inviability was also associated with some regions of the second chromosome, yet there were no strong sex differences. In conclusion, we find no evidence to support a strong X chromosome bias in the evolution of hybrid sterility or inviability but do find a very strong sex bias in the evolution of hybrid sterility. In light of these findings, we reevaluate the current models proposed to explain the genetic pattern of reproductive isolation.     

Genetics of Reproductive Isolation in the Drosophila Simulans Clade: Complex Epistasis Underlying Hybrid Male Sterility

We have analyzed the sterility associated with introgressions of the distal one-fourth of the X chromosome from either Drosophila mauritiana or Drosophila sechellia into the genome of Drosophila simulans using a series of visible and DNA markers. Because in Drosophila hybrids, male sterility is usually complete and is often tightly linked with each of several markers used in crosses, a simple genetic basis has generally been assumed. In our low resolution mapping experiment, we were not able to reject the null hypothesis that a single gene, introgressed from either D. mauritiana or D. sechellia, is the cause of male sterility. High resolution mapping, however, reveals a much more complex picture. At least three distinct factors from D. mauritiana, or two from D. sechellia, were identified that need to be jointly present to confer full sterility. Each individual factor by itself is relatively ineffective in causing sterility, or even a partial spermatogenic defect. Moreover, there appear to be more sterility factors on comparable introgressions from D. mauritiana than from D. sechellia. On the basis of these observations, we propose a model which suggests that multilocus weak allele interactions are a very common cause of reproductive incompatibility between closely related species. We also present theoretical argument and empirical evidence against extrapolating the results of within-species analysis to interpret the genetic basis of species differences. The implications of this model on the theories of evolution of species differences and the attempt to understand the mechanisms of hybrid sterility/inviability at the molecular level are discussed.     

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.     

Wolbachia segregation dynamics and levels of cytoplasmic incompatibility in Drosophila sechellia

In Drosophila sechellia, the endocellular bacterium Wolbachia induces cytoplasmic incompatibility (CI): in crosses involving infected males, a partial or complete embryonic mortality occurs unless the female bears the same Wolbachia. D. sechellia is known to harbour two Wolbachia variants, namely wSh and wSn, closely related to wHa and wNo, respectively, two strains infecting the populations of D. simulans from the Seychelles archipelago and New Caledonia. Strikingly, the two species show similar infection patterns: in D. sechellia, wSh can be present on its own or in double infection with wSn, but individuals carrying wSn only do not occur; in D. simulans, wHa can be present on its own or in double infection with wNo, but individuals carrying wNo only do not occur, or occur at very low frequency. Previous experiments on D. simulans showed that lines singly infected by wNo can be obtained by segregation, and stably maintained. Here we investigate this issue in D. sechellia through an 18 generation experiment, and show that wSn and wSh singly infected lines can arise by segregation. Using singly infected lines obtained in this experiment, we estimate the CI intensities of wSh and wSn in D. sechellia, and compare these to the CI intensities of the same Wolbachia injected into D. simulans. Our results do not suggest any consistent effect of the host species on the CI induced by wSh. On the contrary, it seems that wSn expression is repressed by host factors in D. sechellia.     

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.     

An interspecific QTL study of Drosophila wing size and shape variation to investigate the genetic basis of morphological differences

The Drosophila wing has been used as a model in studies of morphogenesis and evolution; the use of such models can contribute to our understanding of mechanisms that promote morphological divergence among populations and species. We mapped quantitative trait loci (QTL) affecting wing size and shape traits using highly inbred introgression lines between D. simulans and D. sechellia, two sibling species of the melanogaster subgroup. Eighteen QTL peaks that are associated with 12 wing traits were identified, including two principal components. The wings of D. simulans and D. sechellia significantly diverged in size; two of the QTL peaks could account for part of this interspecific divergence. Both of these putative QTLs were mapped at the same cytological regions as other QTLs for intraspecific wing size variation identified in D. melanogaster studies. In these regions, one or more loci could account for intra- and interspecific variation in the size of Drosophila wings. Three other QTL peaks were related to a pattern of interspecific variation in wing size and shape traits that is summarized by one principal component. In addition, we observed that female wings are significantly larger and longer than male wings and the second, fourth and fifth longitudinal veins are closer together at the distal wing area. This pattern was summarized by another principal component, for which one QTL was mapped.     

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.     

Variation in fiber number of a male-specific muscle between Drosophila species: a genetic and developmental analysis

We characterize a newly discovered morphological difference between species of the Drosophila melanogaster subgroup. The muscle of Lawrence (MOL) contains about four to five fibers in D. melanogaster and Drosophila simulans and six to seven fibers in Drosophila mauritiana and Drosophila sechellia. The same number of nuclei per fiber is present in these species but their total number of MOL nuclei differs. This suggests that the number of muscle precursor cells has changed during evolution. Our comparison of MOL development indicates that the species difference appears during metamorphosis. We mapped the quantitative trait loci responsible for the change in muscle fiber number between D. sechellia and D. simulans to two genomic regions on chromosome 2. Our data eliminate the possibility of evolving mutations in the fruitless gene and suggest that a change in the twist might be partly responsible for this evolutionary change.     

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.      

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.     

Wolbachia Infections and the Expression of Cytoplasmic Incompatibility in Drosophila Sechellia and D. Mauritiana

Various stocks of Drosophila mauritiana and D. sechellia were found to be infected with Wolbachia, a Rickettsia-like bacterium that is known to cause cytoplasmic incompatibility and other reproductive abnormalities in arthropods. Testing for the expression of cytoplasmic incompatibility in these two species showed partial incompatibility in D. sechellia but no expression of incompatibility in D. mauritiana. To determine whether absence of cytoplasmic incompatibility in D. mauritiana was due to either the bacterial or host genome, we transferred bacteria from D. mauritiana into an uninfected strain of D. simulans, a host species known to express high levels of incompatibility with endogenous Wolbachia. We also performed the reciprocal transfer of the natural D. simulans Riverside infection into a tetracycline-treated stock of D. mauritiana. In each case, the ability to express incompatibility was unaltered by the different host genetic background. These experiments indicate that in D. simulans and D. mauritiana expression of the cytoplasmic incompatibility phenotype is determined by the bacterial strain and that D. mauritiana harbors a neutral strain of Wolbachia.     

Sequence Analysis of Active Mariner Elements in Natural Populations of Drosophila Simulans

Active and inactive mariner elements from natural and laboratory populations of Drosophila simulans were isolated and sequenced in order to assess their nucleotide variability and to compare them with previously isolated mariner elements from the sibling species Drosophila mauritiana and Drosophila sechellia. The active elements of D. simulans are very similar among themselves (average 99.7% nucleotide identity), suggesting that the level of mariner expression in different natural populations is largely determined by position effects, dosage effects and perhaps other factors. Furthermore, the D. simulans elements exhibit nucleotide identities of 98% or greater when compared with mariner elements from the sibling species. Parsimony analysis of mariner elements places active elements from the three species into separate groups and suggests that D. simulans is the species from which mariner elements in D. mauritiana and D. sechellia are most likely derived. This result strongly suggests that the ancestral form of mariner among these species was an active element. The two inactive mariner elements sequenced from D. simulans are very similar to the inactive peach element from D. mauritiana. The similarity may result from introgression between D. simulans and D. mauritiana or from selective constraints imposed by regulatory effects of inactive elements.     

Molecular population genetics of male accessory gland proteins in the Drosophila simulans complex

Accessory gland proteins are a major component of Drosophila seminal fluid. These proteins have a variety of functions and may be subject to sexual selection and/or antagonistic evolution between the sexes. Most population genetic data from these proteins are from D. melanogaster and D. simulans. Here, we extend the population genetic analysis of Acp genes to the other simulans complex species, D. mauritiana and D. sechellia. We sequenced population samples of seven Acp's from D. mauritiana, D. sechellia, and D. simulans. We investigated the population genetics of these genes on individual simulans complex lineages and compared Acp polymorphism and divergence to polymorphism and divergence from a set of non-Acp loci in the same species. Polymorphism and divergence data from the simulans complex revealed little evidence for adaptive protein evolution at individual loci. However, we observed a dramatically inflated index of dispersion for amino acid substitutions in the simulans complex at Acp genes, but not at non-Acp genes. This pattern of episodic bursts of protein evolution in Acp's provides the strongest evidence to date that the population genetic mechanisms driving Acp divergence are different from the mechanisms driving evolution at most Drosophila genes.     

Genetics of egg production in Drosophila sechellia

Drosophila sechellia, an island endemic that specializes on a single host plant, has a lower rate of egg production than its generalist sister species D. melanogaster, D. simulans, and D. mauritiana. Earlier work showed that part of this difference in egg production was due to a reduction in the number of ovarioles in D. sechellia relative to its sister species. Here, I extend this earlier work by genetically analyzing the difference in egg production between D. sechellia and D. simulans. In all, 10 genetic markers were used in several interspecific backcrosses to identify chromosome regions that affected the rate of egg production. While previously mapped factors affecting ovariole number appear to impact the rate of egg production, new, non-ovariole factors were also identified. Overall, the difference in egg production between D. sechellia and D. simulans appears to be a polygenic trait. The relationship between these factors and genes involved the adaptation of D. sechellia to its host plant is not yet clear. The data are consistent with the hypothesis that decline in egg production is, in part, a negative pleiotropic effect of genetic changes required for host specialization in D. sechellia, although finer-scale genetic analysis of both traits is needed to truly test this hypothesis.     

High level of divergence of male-reproductive-tract proteins, between Drosophila melanogaster and its sibling species, D. simulans

We compared male-reproductive-tract polypeptides of Drosophila melanogaster and D. simulans by using two-dimensional gel electrophoresis. Approximately 64% of male-reproductive-tract polypeptides were identical between two randomly chosen isofemale lines from these two species, compared with 83% identity for third-instar imaginal wing-disc polypeptides. Qualitatively similar differences were found between reproductive tracts and imaginal discs when D. sechellia was compared with D. melanogaster and with D. simulans. When genic polymorphism was taken into account, approximately 10% of male- reproductive-tract polypeptides were apparently fixed for different alleles between D. melanogaster and D. simulans; this proportion is the same as that found for soluble enzymes by one-dimensional gel electrophoresis. Strikingly, approximately 20% of male-reproductive- tract polypeptides of either D. melanogaster or D. simulans had no detectable homologue in the other species. We propose that proteins of the Drosophila male reproductive tract may have diverged more extensively between species than have other types of proteins and that much of this divergence may involve large changes in levels of polypeptide expression.