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.     

Mechanisms of conspecific sperm precedence in Drosophila

The postmating, prezygotic isolating mechanism known as conspecific sperm precedence (CSP) may play an important role in speciation, and understanding the mechanism of CSP is important in reconstructing its evolution. When a Drosophila simulans female mates with both a D. simulans male and a D. mauritiana male, the vast majority of her progeny are fathered by D. simulans, regardless of the order of mating. The dearth of hybrid progeny does not result from inviability of eggs fertilized by heterospecific sperm or from the relative inviability of heterospecific larvae. Instead, CSP apparently results from a prefertilization obstacle to heterospecific sperm. We identified two independent barriers to heterospecific fertilization, sperm displacement and incapacitation, whose action depends on the order of mating. When a D. simulans female mates first with a conspecific male, the seminal fluid from this mating incapacitates heterospecific sperm transferred two days later. This sperm incapacitation occurs with no change in the retention of stored sperm over time, but does not occur when the conspecific mating lasts for only 5 min. When the order of matings is reversed, the seminal fluid from the second mating physically displaces heterospecific sperm from storage, even if the conspecific copulation lasts only 5 min. Conspecific sperm are not susceptible to displacement by a second conspecific copulation, but are susceptible to interference by heterospecific sperm if the conspecific copulation is interrupted after 12 min. Curing the D. mauritiana males of their infection with the endosymbiont Wolbachia had no effect on CSP. Sperm displacement and incapacitation involve the same basic mechanisms seen in second-male sperm precedence within species, supporting the hypothesis that CSP is an evolutionary by-product of adaptations affecting sperm competition within species.     

The genetics of mating recognition between Drosophila simulans and D. sechellia

During courtship, visual and chemical signals are often exchanged between the sexes. The proper exchange of such signals ensures intraspecific recognition. We have examined the genetic basis of interspecific differences in male mating behaviour and pheromone concentration between Drosophila simulans and D. sechellia by using Drosophila simulans/D. sechellia introgression lines. Our results show a majority of quantitative trait loci (QTLs) explaining variation in both male mating behaviour and pheromone concentration to be located on the third chromosome. One QTL found on the third chromosome explains variation in time needed to start courtship and copulation as well as time spent courting. The position of such QTL (approximately 84A-88B) with effects on courtship and copulation aspects of mating includes the candidate sex determination gene doublesex (84E5-6) and Voila (86E1-2), a gene that affects male courtship in D. melanogaster. One additional third chromosome QTL explained variation in 7-tricosene pheromone concentrations among males. The interval mapping position of this QTL (approximately 68E-76E) did not overlap with the position detected for differences in mating behaviour and the intervals did not include candidate genes previously identified as having an effect on D. melanogaster cuticular hydrocarbon production. We did not detect any directionality of the effect of Drosophila sechellia allele introgressions in male mating recognition.     

Non-Mendelian segregation of sex chromosomes in heterospecific Drosophila males

Interspecific hybrids and backcrossed organisms generally suffer from reduced viability and/or fertility. To identify and genetically map these defects, we introgressed regions of the Drosophila sechellia genome into the D. simulans genome. A female-biased sex ratio was observed in 24 of the 221 recombinant inbred lines, and subsequent tests attributed the skew to failure of Y-bearing sperm to fertilize the eggs. Apparently these introgressed lines fail to suppress a normally silent meiotic drive system. Using molecular markers we mapped two regions of the Drosophila genome that appear to exhibit differences between D. simulans and D. sechellia in their regulation of sex chromosome segregation distortion. The data indicate that the sex ratio phenotype results from an epistatic interaction between at least two factors. We discuss whether this observation is relevant to the meiotic drive theory of hybrid male sterility.     

Genetics of Hybrid Male Sterility between Drosophila Sibling Species: A Complex Web of Epistasis Is Revealed in Interspecific Studies

To study the genetic differences responsible for the sterility of their male hybrids, we introgressed small segments of an X chromosome from Drosophila simulans into a pure Drosophila mauritiana genetic background, then assessed the fertility of males carrying heterospecific introgressions of varying size. Although this analysis examined less than 20% of the X chromosome (roughly 5% of the euchromatic portion of the D. simulans genome), and the segments were introgressed in only one direction, a minimum of four factors that contribute to hybrid male sterility were revealed. At least two of the factors exhibited strong epistasis: males carrying either factor alone were consistently fertile, whereas males carrying both factors together were always sterile. Distinct spermatogenic phenotypes were observed for sterile introgressions of different lengths, and it appeared that an interaction between introgressed segments also influenced the stage of spermatogenic defect. Males with one category of introgression often produced large quantities of motile sperm and were observed copulating, but never inseminated females. Evidently these two species have diverged at a large number of loci which have varied effects on hybrid male fertility. By extrapolation, we estimate that there are at least 40 such loci on the X chromosome alone. Because these species exhibit little DNA-sequence divergence at arbitrarily chosen loci, it seems unlikely that the extensive functional divergence observed could be due mainly to random genetic drift. Significant epistasis between conspecific genes appears to be a common component of hybrid sterility between recently diverged species of Drosophila. The linkage relationships of interacting factors could shed light on the role played by epistatic selection in the dynamics of the allele substitutions responsible for reproductive barriers between species.     

Mate choice in Drosophila melanogaster and D. sechellia: criteria and their variation depending on courtship song

Courtship song is one of the most important male signals in Drosophila mating. A female D. melanogaster copulates more readily when given a conspecific type of courtship song. Female D. melanogaster accepted winged D. sechellia more than wingless ones in a no choice test. Copulation frequencies varied between strains and were significantly correlated with male mating propensity of D. sechellia. Females from three of five strains of D. sechellia accepted winged D. melanogaster less than wingless ones in no choice tests, suggesting that D. melanogaster songs reduce mating in D. sechellia females. Multiple choice tests showed that males prefer conspecifics. In female choice tests, D. melanogaster females copulated with winged heterospecific D. sechellia males more than with wingless conspecific D. melanogaster males in the confined condition, suggesting that song differences between D. melanogaster and D. sechellia affect D. melanogaster females less than the absence of courtship song. Copyright 2000 The Association for the Study of Animal Behaviour.     

Cryptic reproductive isolation in the Drosophila simulans species complex

Forms of reproductive isolation that act after copulation but before fertilization are potentially important components of speciation, but are studied only infrequently. We examined postmating, prezygotic reproductive isolation in three hybridizations within the Drosophila simulans species complex. We allowed females to mate only once, observed and timed all copulations, dissected a subset of the females to track the storage and retention of sperm, examined the number and hatchability of eggs laid after insemination, counted all progeny produced, and measured the longevity of mated females. Each of the three hybridizations is characterized by a different set of cryptic barriers to heterospecific fertilization. When D. simulans females mate with D. sechellia males, few heterospecific sperm are transferred, even during long copulations. In contrast, copulations of D. simulans females with D. mauritiana males are often too short to allow sperm transfer. Those that are long enough to allow insemination, however, involve the transfer of many sperm, but only a fraction of these heterospecific sperm are stored by females, who also lay fewer eggs than do D. simulans females mated with conspecific males. Finally, when D. mauritiana females mate with D. simulans males, sperm are transferred and stored in abundance, but are lost rapidly from the reproductive tract and are therefore used inefficiently. These results add considerably to the list of reproductive isolating mechanisms in this well-studied clade and possibly to the list of evolutionary processes that could contribute to their reproductive isolation.     

Courtship song recognition in the Drosophila melanogaster complex: heterospecific songs make females receptive in D. melanogaster, but not in D. sechellia

Abstract. The courtship song emitted by male wing vibration has been regarded as one of the most important signals in sexual isolation in the species of the Drosophila melanogaster complex. Inter- and intraspecific crosses were observed using males whose wings were removed (mute) or females whose aristae were removed (deaf). Females of D. melanogaster, D. simulans , and D. mauritiana mated with heterospecific males in the song-present condition (cross between normal females and winged males) more often than in the no-song condition (cross between normal females and wingless males or between aristaless females and winged males) or they showed no preference between the two conditions. It is possible that in these females heterospecific courtship songs play a role as if they were conspecific. In contrast, the females of D. sechellia mated with D. melanogaster or D. simulans males in the no-song condition more often than in the song-present condition, suggesting that they reject males with heterospecific song. Female mate recognition depending on the courtship song in D. melanogaster, D. simulans , and D. mauritiana is considered to be relatively broader and that in D. sechellia narrower.     

Quantitative trait loci and interaction effects responsible for variation in female postmating mortality in Drosophila simulans and D. sechellia introgression lines

Mating appears to inflict a cost to Drosophila females, resulting in a reduction of their lifespan shortly after mating. Males from different chromosome extracted lines differ significantly in their detrimental effects on postmating female survival, and seminal fluid proteins produced in the male accessory glands are at least partially responsible for the effect. This suggests that there is a genetic basis underlying the male inflicted effect on female's postmating mortality. However, the genes responsible for this effect remain elusive. Using males from introgression lines between D. simulans and D. sechellia genomes and a quantitative trait locus (QTL) mapping approach, we identified chromosomal regions that affect postmating mortality of females. We found a second chromosome QTL with an effect on average female lifespan after mating and a third chromosome QTL with an effect on postmating female mortality rate. Under the general observation of a faster divergence of sex-related genes among closely related species, it is predicted that genes for reproductive traits other than hybrid sterility will show evidence of epistatic effects when brought into a heterospecific background. We detected a significant epistatic genetic effect on postmating female mortality rate that supports this prediction.     

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.     

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.     

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.     

Wolbachia infection reduces sperm competitive ability in an insect

The maternally inherited bacterium Wolbachia pipientis imposes significant fitness costs on its hosts. One such cost is decreased sperm production resulting in reduced fertility of male Drosophila simulans infected with cytoplasmic incompatibility (CI) inducing Wolbachia. We tested the hypothesis that Wolbachia infection affects sperm competitive ability and found that Wolbachia infection is indeed associated with reduced success in sperm competition in non-virgin males. In the second male role, infected males sired 71% of the offspring whereas uninfected males sired 82% of offspring. This is the first empirical evidence indicating that Wolbachia infection deleteriously affects sperm competition and raises the possibility that polyandrous females can utilize differential sperm competitive ability to bias the paternity of broods and avoid the selfish manipulations of Wolbachia. This suggests a relationship between Wolbachia infection and host reproductive strategies. These findings also have important consequences for Wolbachia population dynamics because the transmission advantage of Wolbachia is likely to be undermined by sperm competition.     

A QTL analysis of female variation contributing to refractoriness and sperm competition in Drosophila melanogaster

Sperm competition is an important fitness component in many animal groups. Drosophila melanogaster males exhibit substantial genetic variation for sperm competitive ability and females show considerable genetic variation for first versus second male sperm use. Currently, the forces responsible for maintaining genetic variation in sperm competition related phenotypes are receiving much attention. While several candidate genes contributing to the variation seen in male competitive ability are known, genes involved in female sperm use remain largely undiscovered. Without knowledge of the underlying genes, it will be difficult to distinguish between different models of sexual selection such as cryptic female choice and sexual conflict. We used quantitative trait locus (QTL) mapping to identify regions of the genome contributing to female propensity to use first or second male sperm, female refractoriness to re-mating, and early-life fertility. The most well supported markers influencing the phenotypes include 33F/34A (P2), 57B (refractoriness) and 23F/24A (fertility). Between 10% and 15% of the phenotypic variance observed in these recombinant inbred lines was explained by these individual QTLs. More detailed investigation of the regions detected in this experiment may lead to the identification of genes responsible for the QTLs identified here.     

Meiotic drive alters sperm competitive ability in stalk-eyed flies.

Meiotic drive results when sperm carrying a driving chromosome preferentially survive development. Meiotic drive should therefore influence sperm competition because drive males produce fewer sperm than non-drive males. Whether meiotic drive also influences the competitive ability of sperm after ejaculation is unknown. Here we report the results from reciprocal crosses that are designed for estimating the sperm precedence of male stalk-eyed flies (Cyrtodiopsis whitei) with or without X-linked meiotic drive. We find that nearly half of all sex-ratio males, as compared with 14% of non-sex-ratio males, fail to produce young in a reciprocal cross. Furthermore, the proportion of progeny sired by a sex-ratio male in a female jointly inseminated by a non-sex-ratio male was less than expected from the number of sperm transferred. These effects are not due to differential sperm storage by females because, after a single mating with a sex-ratio male, all females stored sperm and because two sex-ratio males share paternity after jointly mating with a female. In addition to demonstrating a new mechanism of sperm competition, these results provide insight into the maintenance of sex-ratio polymorphisms. Sex-ratio males have less than one-half the fertility of non-sex-ratio males, as is required in order for frequency-dependent selection on males to produce a stable sex-ratio polymorphism.     

Direct visualization of sperm competition and sperm storage in Drosophila.

Drosophila females engage in multiple matings [1] [2] [3] [4] even though they can store sperm in specialized organs for most of their life [5]. The existence of sperm competition in Drosophila has been inferred from the proportion of progeny sired by the second male in double-mating experiments [6] [7] [8]. Investigators have used this approach to quantify genetic variation underlying sperm competition [8] [9] [10], to elucidate its genetic basis [11], to identify the dependence of different male competitive ability on the genotype of the females with which they mate [12] and to discern the potential role of sperm competition in species isolation [13] [14]. This approach assumes that the sperm from two males stored in a female compete to fertilize the eggs. The mechanism by which sperm competition is accomplished is still unknown, however. Here, fluorescence microscopy, cytometry, and differently labeled sperm were used to analyze the fate of sperm inside the female's sperm storage organs, to quantify sperm competition, and to assess how closely paternity success corresponds to the appearance and location of the sperm. The results show that the first male's sperm is retained for a shortened period if the female remates, and that the second males that sire more progeny either induce females to store and use more of their sperm or strongly displace resident sperm.     

Female mate recognition and sexual isolation depending on courtship song in Drosophila sechellia and its siblings

In Drosophila sechellia, females accept males that sing heterospecific songs less than those that do not sing, whereas in D. melanogaster and D. simulans, females accept males that sing heterospecific song more than those that do not sing. Here we studied the sexual isolation of D. sechellia and its siblings using interspecific hybrids to reveal the mechanisms underlying female mate recognition. The females of hybrids mated more with winged males of the parent species than with wingless ones, suggesting that the discrimination against heterospecific songs by D. sechellia females is recessive. Female preference for courtship songs seems to be inherited additively or semidominantly. In addition, we examined female receptivity without the stimuli of courtship songs by comparing the mating frequencies between the crosses using wingless males and found that it is also inherited additively or semidominantly.     

Quantitative trait locus analysis of male mating success and sperm competition in Drosophila melanogaster

Much of sexual selection theory depends on assumptions about the genetic basis of variation in male mating success and sperm competitive ability. Despite intense interest in this topic, few genes have been identified that contribute to variation in these traits. Here we report the results of quantitative trait locus (QTL) analyses of mating success of male Drosophila melanogaster when exposed to virgin females, remating success of males with previously mated females, and both defense and offense components of sperm competition. We found two to four significant QTLs for remating success, but no QTLs for mating success, even though mating success was more genetically variable than remating success in the recombinant inbred lines used in this study. By combining these results with data from previous gene-expression experiments, we were able to identify three X-linked candidate genes for variation in remating ability. For two of these genes, QTL and expression data were completely concordant with respect to directionality of effects: high mating success was associated with high levels of gene expression and with beneficial QTL effects on the trait. We found equivocal evidence for genetic variation in sperm offense and defense in the recombinant inbred lines, and we did not find any significant QTLs for either sperm competition trait.     

Plastic male mating behavior evolves in response to the competitive environment*

Male reproductive phenotypes can evolve in response to the social and sexual environment. The expression of many such phenotypes may also be plastic within an individual's lifetime. For example, male Drosophila melanogaster show significantly extended mating duration following a period of exposure to conspecific male rivals. The costs and benefits of reproductive investment, and plasticity itself, can be shaped by the prevailing sociosexual environment and by resource availability. We investigated these ideas using experimental evolution lines of D. melanogaster evolving under three fixed sex ratios (high, medium, and low male‐male competition) on either rich or poor adult diets. We found that males evolving in high‐competition environments evolved longer mating durations overall. In addition, these males expressed a novel type of plastic behavioral response following exposure to rival males: they both significantly reduced and showed altered courtship delivery, and exhibited significantly longer mating latencies. Plasticity in male mating duration in response to rivals was maintained in all of the lines, suggesting that the costs of plasticity were minimal. None of the evolutionary responses tested were consistently affected by dietary resource regimes. Collectively, the results show that fixed behavioral changes and new augmentations to the repertoire of reproductive behaviors can evolve rapidly.     

Hidden effects of X chromosome introgressions on spermatogenesis in Drosophila simulans x D. mauritiana hybrids unveiled by interactions among minor genetic factors.

One of the most frequent outcomes of interspecific hybridizations in Drosophila is hybrid male sterility. Genetic dissection of this reproductive barrier has revealed that the number of responsible factors is very high and that these factors are frequently engaged in complex epistatic interactions. Traditionally, research strategies have been based on contrasting introgressions of chromosome segments that produce male sterility with those that allow fertility. Few studies have investigated the phenotypes associated with the boundary between fertility and sterility. In this study, we cointrogressed three different X chromosome segments from Drosophila mauritiana into D. simulans. Hybrid males with these three segments are usually fertile, by conventional fertility assays. However, their spermatogenesis shows a significant slowdown, most manifest at lower temperatures. Each of the three introgressed segments retards the arrival of sperm to the seminal vesicles. Other small disturbances in spermatogenesis are evident, which altogether lead to an overall reduction in the amount of motile sperm in their seminal vesicles. These results suggest that a delay in the timing of spermatogenesis, which might be brought about by the cumulative action of many different factors of minor segment, may be the primary cause of hybrid male sterility.