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 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.     

The minimal interspecific introgression resulting in male sterility in Drosophila

Introgression of Drosophila simulans genes into the Drosophila melanogaster genome provides an ideal system for analysing genetic incompatibility between species. Females and males homozygous for the introgression Int(2L)S (cytologically, 30F3-31C5 to 36A2-7) are sterile. Genetic dissection of the proximal part of the introgression (34D1-3 to 36A2-7) has indicated that introgressions of 0.7-1.6 Mb size result in male sterility when homozygous. In the present analysis we examine the distal part of the introgression (30F3-31C to 34D1-3) and reveal that introgressions with similar DNA content (1.8-2.1 Mb) result in male sterility. Compared with introgressions between the more closely related species Drosophila mauritiana and D. simulans, the minimal introgression resulting in male sterility is smaller by several-fold.     

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.     

Genetic dissection of hybrid incompatibilities between Drosophila simulans and D. mauritiana. I. Differential accumulation of hybrid male sterility effects on the X and autosomes

The genetic basis of hybrid incompatibility in crosses between Drosophila mauritiana and D. simulans was investigated to gain insight into the evolutionary mechanisms of speciation. In this study, segments of the D. mauritiana third chromosome were introgressed into a D. simulans genetic background and tested as homozygotes for viability, male fertility, and female fertility. The entire third chromosome was covered with partially overlapping segments. Many segments were male sterile, while none were female sterile or lethal, confirming previous reports of the rapid evolution of hybrid male sterility (HMS). A statistical model was developed to quantify the HMS accumulation. In comparison with previous work on the X chromosome, we estimate that the X has approximately 2.5 times the density of HMS factors as the autosomes. We also estimate that the whole genome contains approximately 15 HMS "equivalents"-i.e., 15 times the minimum number of incompatibility factors necessary to cause complete sterility. Although some caveats for the quantitative estimate of a 2.5-fold density difference are described, this study supports the notion that the X chromosome plays a special role in the evolution of reproductive isolation. Possible mechanisms of a "large X" effect include selective fixation of new mutations that are recessive or partially recessive and the evolution of sex-ratio distortion systems.     

A novel system of fertility rescue in Drosophila hybrids reveals a link between hybrid lethality and female sterility

Hybrid daughters of crosses between Drosophila melanogaster females and males from the D. simulans species clade are fully viable at low temperature but have agametic ovaries and are thus sterile. We report here that mutations in the D. melanogaster gene Hybrid male rescue (Hmr), along with unidentified polymorphic factors, rescue this agametic phenotype in both D. melanogaster/D. simulans and D. melanogaster/D. mauritiana F(1) female hybrids. These hybrids produced small numbers of progeny in backcrosses, their low fecundity being caused by incomplete rescue of oogenesis as well as by zygotic lethality. F(1) hybrid males from these crosses remained fully sterile. Hmr(+) is the first Drosophila gene shown to cause hybrid female sterility. These results also suggest that, while there is some common genetic basis to hybrid lethality and female sterility in D. melanogaster, hybrid females are more sensitive to fertility defects than to lethality.     

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.     

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.     

The Broom of the Sorcerer''s Apprentice: The Fine Structure of a Chromosomal Region Causing Reproductive Isolation between Two Sibling Species of Drosophila

How many genes contribute to reproductive isolation between closely related species? We determined the number of genes located in the 9D-12B region of the Drosophila mauritiana X chromosome that cause hybrid male sterility in a D. simulans background. Previous low resolution studies suggested that a single hybrid sterility factor was associated with this region. In this study, by taking advantage of a cluster of visible and DNA markers, we identified three D. mauritiana factors in this region and then subjected one of them to detailed analysis. This factor again turned out to be comprised of three factors; one of which, mapped to within 200 kb, may in fact be two factors. The title refers to this exercise of splitting sterile introgressions into ever smaller ones, each of which retains partial or full sterility effects. In a region representing a mere 3% of the Drosophila genome, no fewer than six loci of hybrid sterility were identified between two sibling species that have not shown clear divergence at the molecular level. These results suggest that levels of genetic divergence between closely related species may be quite high for functionally important traits even when the opposite is true for randomly chosen loci.     

Incompatibility between X chromosome factor and pericentric heterochromatic region causes lethality in hybrids between Drosophila melanogaster and its sibling species

The Dobzhansky-Muller model posits that postzygotic reproductive isolation results from the evolution of incompatible epistatic interactions between species: alleles that function in the genetic background of one species can cause sterility or lethality in the genetic background of another species. Progress in identifying and characterizing factors involved in postzygotic isolation in Drosophila has remained slow, mainly because Drosophila melanogaster, with all of its genetic tools, forms dead or sterile hybrids when crossed to its sister species, D. simulans, D. sechellia, and D. mauritiana. To circumvent this problem, we used chromosome deletions and duplications from D. melanogaster to map two hybrid incompatibility loci in F(1) hybrids with its sister species. We mapped a recessive factor to the pericentromeric heterochromatin of the X chromosome in D. simulans and D. mauritiana, which we call heterochromatin hybrid lethal (hhl), which causes lethality in F(1) hybrid females with D. melanogaster. As F(1) hybrid males hemizygous for a D. mauritiana (or D. simulans) X chromosome are viable, the lethality of deficiency hybrid females implies that a dominant incompatible partner locus exists on the D. melanogaster X. Using small segments of the D. melanogaster X chromosome duplicated onto the Y chromosome, we mapped a dominant factor that causes hybrid lethality to a small 24-gene region of the D. melanogaster X. We provide evidence suggesting that it interacts with hhl(mau). The location of hhl is consistent with the emerging theme that hybrid incompatibilities in Drosophila involve heterochromatic regions and factors that interact with the heterochromatin.     

Rapid Male-Specific Regulatory Divergence and Down Regulation of Spermatogenesis Genes in Drosophila Species Hybrids

In most crosses between closely related species of Drosophila, the male hybrids are sterile and show postmeiotic abnormalities. A series of gene expression studies using genomic approaches have found significant down regulation of postmeiotic spermatogenesis genes in sterile male hybrids. These results have led some to suggest a direct relationship between down regulation in gene expression and hybrid sterility. An alternative explanation to a cause-and-effect relationship between misregulation of gene expression and male sterility is rapid divergence of male sex regulatory elements leading to incompatible interactions in an interspecies hybrid genome. To test the effect of regulatory divergence in spermatogenesis gene expression, we isolated 35 fertile D. simulans strains with D. mauritiana introgressions in either the X, second or third chromosome. We analyzed gene expression in these fertile hybrid strains for a subset of spermatogenesis genes previously reported as significantly under expressed in sterile hybrids relative to D. simulans. We found that fertile autosomal introgressions can cause levels of gene down regulation similar to that of sterile hybrids. We also found that X chromosome heterospecific introgressions cause significantly less gene down regulation than autosomal introgressions. Our results provide evidence that rapid male sex gene regulatory divergence can explain misexpression of spermatogenesis genes in hybrids.     

Haldane''s Rule and X-Chromosome Size in Drosophila

The ``dominance theory' of HALDANE's rule postulates that hybrids of the heterogametic sex are more likely to be inviable or sterile than the homogametic sex because some of the epistatic incompatibilities contributing to postzygotic isolation behave as X-linked partial recessives. When this is true, pairs of taxa with relatively large X chromosomes should require less divergence time, on average, to produce HALDANE's rule than pairs with smaller Xs. Similarly, if the dominance theory is correct and if the X chromosome evolves at a similar rate to the autosomes, the size of the X should not influence the rate at which homogametic hybrids become inviable or sterile. We use Drosophila data to examine both of these predictions. As expected under the dominance theory, pairs of taxa with large X chromosomes (~40% of the nuclear genome) show HALDANE's rule for sterility at significantly smaller genetic distances than pairs with smaller X chromosomes (~20% of the genome). As also predicted, the genetic distances between taxa that exhibit female inviability/sterility show no differences between ``large X' vs. ``small X' pairs. We present some simple mathematical models to relate these data to the dominance theory and alternative hypotheses involving faster evolution of the X vs. the autosomes and/or faster evolution of incompatibilities that produce male-specific vs. female-specific sterility. Although the data agree qualitatively with the predictions of the dominance theory, they depart significantly from the quantitative predictions of simple models of the dominance theory and the other hypotheses considered. These departures probably stem from the many simplifying assumptions needed to tractably model epistatic incompatibilities and to analyze heterogeneous data from many taxa.     

Quantitative trait loci for sexual isolation between Drosophila simulans and D. mauritiana

Sexual isolating mechanisms that act before fertilization are often considered the most important genetic barriers leading to speciation in animals. While recent progress has been made toward understanding the genetic basis of the postzygotic isolating mechanisms of hybrid sterility and inviability, little is known about the genetic basis of prezygotic sexual isolation. Here, we map quantitative trait loci (QTL) contributing to prezygotic reproductive isolation between the sibling species Drosophila simulans and D. mauritiana. We mapped at least seven QTL affecting discrimination of D. mauritiana females against D. simulans males, three QTL affecting D. simulans male traits against which D. mauritiana females discriminate, and six QTL affecting D. mauritiana male traits against which D. simulans females discriminate. QTL affecting sexual isolation act additively, are largely different in males and females, and are not disproportionately concentrated on the X chromosome: The QTL of greatest effect are located on chromosome 3. Unlike the genetic components of postzygotic isolation, the loci for prezygotic isolation do not interact epistatically. The observation of a few QTL with moderate to large effects will facilitate positional cloning of genes underlying sexual isolation.     

High-resolution genome-wide dissection of the two rules of speciation in Drosophila

Postzygotic reproductive isolation is characterized by two striking empirical patterns. The first is Haldane's rule—the preferential inviability or sterility of species hybrids of the heterogametic (XY) sex. The second is the so-called large X effect—substitution of one species's X chromosome for another's has a disproportionately large effect on hybrid fitness compared to similar substitution of an autosome. Although the first rule has been well-established, the second rule remains controversial. Here, we dissect the genetic causes of these two rules using a genome-wide introgression analysis of Drosophila mauritiana chromosome segments in an otherwise D. sechellia genetic background. We find that recessive hybrid incompatibilities outnumber dominant ones and that hybrid male steriles outnumber all other types of incompatibility, consistent with the dominance and faster-male theories of Haldane's rule, respectively. We also find that, although X-linked and autosomal introgressions are of similar size, most X-linked introgressions cause hybrid male sterility (60%) whereas few autosomal introgressions do (18%). Our results thus confirm the large X effect and identify its proximate cause: incompatibilities causing hybrid male sterility have a higher density on the X chromosome than on the autosomes. We evaluate several hypotheses for the evolutionary cause of this excess of X-linked hybrid male sterility.     

Genetic complexity underlying hybrid male sterility in Drosophila

Recent genetic analyses of closely related species of Drosophila have indicated that hybrid male sterility is the consequence of highly complex synergistic effects among multiple genes, both conspecific and heterospecific. On the contrary, much evidence suggests the presence of major genes causing hybrid female sterility and inviability in the less-related species, D. melanogaster and D. simulans. Does this contrast reflect the genetic distance between species? Or, generally, is the genetic basis of hybrid male sterility more complex than that of hybrid female sterility and inviability? To clarify this point, the D. simulans introgression of the cytological region 34D-36A to the D. melanogaster genome, which causes recessive male sterility, was dissected by recombination, deficiency, and complementation mapping. The 450-kb region between two genes, Suppressor of Hairless and snail, exhibited a strong effect on the sterility. Males are (semi-)sterile if this region of the introgression is made homozygous or hemizygous. But no genes in the region singly cause the sterility; this region has at least two genes, which in combination result in male sterility. Further, the males are less fertile when heterozygous with a larger introgression, which suggests that dominant modifiers enhance the effects of recessive genes of male sterility. Such an epistatic view, even in the less-related species, suggests that the genetic complexity is special to hybrid male sterility.     

Genetics of Hybrid Inviability and Sterility in Drosophila: Dissection of Introgression of D. simulans Genes in D. melanogaster Genome

Interspecific crosses between Drosophila melanogaster and Drosophila simulans usually produce sterile unisexual hybrids. The barrier preventing genetic analysis of hybrid inviability and sterility has been taken away by the discovery of a D. simulans strain which produces fertile female hybrids. D. simulans genes in the cytological locations of 21A1 to 22C1-23B1 and 30F3-31C5 to 36A2-7 have been introgressed into the D. melanogaster genetic background by consecutive backcrosses. Flies heterozygous for the introgression are fertile, while homozygotes are sterile both in females and males. The genes responsible for the sterility have been mapped in the introgression. The male sterility is caused by the synergistic effect of multiple genes, while the female sterility genes have been localized to a 170 kb region (32D2 to 32E4) containing 20 open reading frames. Thus, the female sterility might be attributed to a single gene with a large effect. We have also found that the Lethal hybrid rescue mutation which prevents the inviability of male hybrids from the cross of D. melanogaster females and D. simulans males cannot rescue those carrying the introgression, suggesting that D. simulans genes maybe non-functional in this hybrid genotype. The genes responsible for the inviability have not been separated from the female sterility genes by recombination.     

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.     

Female sterility in hybrids between Anopheles gambiae and A. arabiensis, and the causes of Haldane's rule

Although F1 female hybrids between Anopheles gambiae and A. arabiensis are fully fertile, sterility is present in backcross females. Here we report the results of a study into the genetic basis of backcross female sterility. Using 23 markers, we performed quantitative trait loci (QTL) mapping analyses to identify chromosomal regions involved in hybrid female sterility. We found that female sterility in backcrosses in both directions is primarily caused by interspecific interactions between a heterozygous X chromosome and recessive autosomal factors. In addition, our data provide support for two theories implicated in Haldane's rule in a single taxon. A comparison with data from a previous study shows that male hybrid sterility QTL are present in higher numbers than female hybrid sterility QTL. Furthermore, autosomal female sterility factors tend to be recessive, supporting the dominance theory for female sterility. Finally, our data indicate a very large effect of the X chromosome from both species on hybrid female sterility, despite the fact that the X chromosome represents less than 9% of the genome. However, this could be the result of a lack of introgression of the X chromosome between A. gambiae and A. arabiensis, rather than a faster evolution of sterility factors on the X chromosome.     

Ephemeral Association Between Gene CG5762 and Hybrid Male Sterility in Drosophila Sibling Species

Interspecies divergence in regulatory pathways may result in hybrid male sterility (HMS) when dominance and epistatic interactions between alleles that are functional within one genome are disrupted in hybrid genomes. The identification of genes contributing to HMS and other hybrid dysfunctions is essential for understanding the origin of new species (speciation). Previously, we identified a panel of male-specific loci misexpressed in sterile male hybrids of Drosophila simulans and D. mauritiana relative to parental species. In the current work, we attempt to dissect the genetic associations between HMS and one of the genes, CG5762, a Drosophila-unique locus characterized by rapid sequence divergence within the genus, presumably driven by positive natural selection. CG5762 is underexpressed in sterile backcross males compared with their fertile brothers. In CG5762 heterozygotes, the D. mauritiana allele is consistently overexpressed on both the D. simulans and D. mauritiana backcross genomic background, suggesting a cis-acting regulation factor. There is a significant association between heterozygosity and HMS in hybrid males from early but not later backcross generations. Microsatellite markers spanning CG5762 fail to associate with HMS. These observations lead to a conclusion that CG5762 is not a causative factor of HMS. Although genetic linkage between CG5762 and a neighboring causative introgression cannot be ruled out, it seems that the pattern is most consistent with CG5762 participating in epistatic interactions that are disrupted in flies with HMS.