Genetic interactions underlying hybrid male sterility in the Drosophila bipectinata species complex

Understanding genetic mechanisms underlying hybrid male sterility is one of the most challenging problems in evolutionary biology especially speciation. By using the interspecific hybridization method roles of Y chromosome, Major Hybrid Sterility (MHS) genes and cytoplasm in sterility of hybrid males have been investigated in a promising group, the Drosophila bipectinata species complex that consists of four closely related species: D. pseudoananassae, D. bipectinata, D. parabipectinata and D. malerkotliana. The interspecific introgression analyses show that neither cytoplasm nor MHS genes are involved but X-Y interactions may be playing major role in hybrid male sterility between D. pseudoananassae and the other three species. The results of interspecific introgression analyses also show considerable decrease in the number of males in the backcross offspring and all males have atrophied testes. There is a significant positive correlation between sex - ratio distortion and severity of sterility in backcross males. These findings provide evidence that D. pseudoananassae is remotely related with other three species of the D. bipectinata species complex.     

Assessing the putative roles of X-autosome and X-Y interactions in hybrid male sterility of the Drosophila bipectinata species complex.

Interspecific F1 hybrid males of the Drosophila bipectinata species complex are sterile, while females are fertile, following Haldane's rule. A backcross scheme involving a single recessive visible marker on the X chromosome has been used to assess the putative roles of X-autosome and X-Y interactions in hybrid male sterility in the D. bipectinata species complex. The results suggest that X-Y interactions are playing the major role in hybrid male sterility in the crosses D. bipectinata x D. parabipectinata and D. bipectinata x D. pseudoananassae, while X-autosome interactions are largely involved in hybrid male sterility in the crosses D. malerkotliana x D. bipectinata and D. malerkotliana x D. parabipectinata. However, by using this single marker it is not possible to rule out the involvement of autosome-autosome interactions in hybrid male sterility. These findings also lend further support to the phylogenetic relationships among 4 species of the D. bipectinata complex.     

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.     

Genetic studies of two sister species in the Drosophila melanogaster subgroup, D. yakuba and D. santomea

We performed genetic analysis of hybrid sterility and of one morphological difference (sex-comb tooth number) on D. yakuba and D. santomea, the former species widespread in Africa and the latter endemic to the oceanic island of S?o Tomé, on which there is a hybrid zone. The sterility of hybrid males is due to at least three genes on the X chromosome and at least one on the Y, with the cytoplasm and large sections of the autosomes having no effect. F1 hybrid females carrying two X chromosomes from either species are perfectly fertile despite their genetic similarity to completely sterile F1 hybrid males. This implies that the appearance of Haldane's rule in this cross is at least partially due to the faster accumulation of genes causing male than female sterility. The larger effects of the X and Y chromosomes than of the autosomes, however, also suggest that the genes causing male sterility are recessive in hybrids. Some female sterility is also seen in interspecific crosses, but this does not occur between all strains. This is seen in pure-species females inseminated by heterospecific males (probably reflecting incompatibility between the sperm of one species and the female reproductive tract of the other) as well as in inseminated F1 and backcross females, probably reflecting genetically based incompatibilities in hybrids that affect the reproductive system. The latter 'innate' sterility appears to involve deleterious interactions between D. santomea chromosomes and D. yakuba cytoplasm. The difference in male sex-comb tooth number appears to involve fairly large effects of the X chromosome. We discuss the striking evolutionary parallels in the genetic basis of sterility, in the nature of sexual isolation, and in morphological differences between the D. santomea/D. yakuba divergence and two other speciation events in the D. melanogaster subgroup involving island colonization.     

Interspecific sexual isolation and phylogeny among different members of the Drosophila bipectinata species complex

The degree and pattern of sexual isolation among closely related species can be utilized for predicting the direction of evolution and deriving a phylogeny. The Drosophila bipectinata species complex is a group of four morphologically very similar species, belonging to the ananassae subgroup of the melanogaster species group. Sexual isolation among the members of this complex was studied by employing the male-choice technique. To test the difference between the homogamic and heterogamic matings, chi-square values were calculated under the assumption of random mating and the degree of sexual isolation was measured by estimating isolation index. To test the difference in proportions of heterogamic matings between reciprocal crosses, normal deviate (z) was calculated. The direction of evolution among these species has been discussed in the light of models proposed by different workers. Our results suggest that while D. bipectinata, Drosophila parabipectinata and Drosophila malerkotliana are closely related to each other, D. pseudoananassae is distantly related to these three species. The species pairs D. bipectinata and D. parabipectinata and D. parabipectinata and D. malerkotliana show asymmetrical sexual isolation. Based on this, it is suggested that D. bipectinata and D. malerkotliana share a common ancestor where as D. parabipectinata has been derived from D. bipectinata. Their phylogenetic relationship has been discussed in the light of phylogenies suggested by earlier workers.     

The genetics of reproductive isolation and the potential for gene exchange between Drosophila pseudoobscura and D. persimilis via backcross hybrid males

Hybrid male sterility, hybrid inviability, sexual isolation, and a hybrid male courtship dysfunction reproductively isolate Drosophila pseudoobscura and D. persimilis. Previous studies of the genetic bases of these isolating mechanisms have yielded only limited information about how much and what areas of the genome are susceptible to interspecies introgression. We have examined the genetic basis of these barriers to gene exchange in several thousand backcross hybrid male progeny of these species using 14 codominant molecular genetic markers spanning the five chromosomes of these species, focusing particularly on the autosomes. Hybrid male sterility, hybrid inviability, and the hybrid male courtship dysfunction were all associated with X-autosome interactions involving primarily the inverted regions on the left arm of the X-chromosome and the center of the second chromosome. Sexual isolation from D. pseudoobscura females was primarily associated with the left arm of the X-chromosome, although both the right arm and the center of the second chromosome also contributed to it. Sexual isolation from D. persimilis females was primarily associated with the second chromosome. The absence of isolating mechanisms being associated with many autosomal regions, including some large inverted regions that separate the strains, suggests that these phenotypes may not be caused by genes spread throughout the genome. We suggest that gene flow between these species via hybrid males may be possible at loci spread across much of the autosomes.     

A Combined Classical Genetic and High Resolution Two-Dimensional Electrophoretic Approach to the Assessment of the Number of Genes Affecting Hybrid Male Sterility in Drosophila Simulans and Drosophila Sechellia

We have attempted to estimate the number of genes involved in postzygotic reproductive isolation between two closely related species, Drosophila simulans and Drosophila sechellia, by a novel approach that involves the use of high resolution two-dimensional gel electrophoresis (2DE) to examine testis proteins in parents, hybrids and fertile and sterile backcross progenies. The important results that have emerged from this study are as follows: (1) about 8% of about 1000 proteins examined showed divergence (presence/absence) between the two species; (2) by tracing individual proteins in parental, hybrid and backcross males, we were able to associate the divergent proteins with different chromosomes and found that most divergent proteins are associated with autosomes and very few with X chromosome, Y chromosome and cytoplasm; (3) when proteins showing both quantitative and qualitative differences between the two species were examined in F(1) hybrid males, most (97.4%) proteins were expressed at levels between the two parents and no sign of large scale changes in spot density was observed. All the proteins observed in the two parental species were present in F(1) hybrid males except two species-specific proteins that may be encoded (or regulated) by sex chromosomes; (4) when different fertile and sterile backcross male testes were compared, a few D. sechellia-specific proteins were identified to be consistently associated with male sterility. These results along with the observation that a large proportion (23.6%) of first generation backcross males were fertile show that hybrid male sterility between D. simulans and D. sechellia involves a relatively small number of genes. Role of large scale genetic changes due to general genome incompatibility is not supported. The results also suggest that the large effect of X chromosome on hybrid male sterility is not due to higher divergence of X chromosome than autosomes.     

Genetics of Male and Female Sterility in Hybrids of Drosophila pseudoobscura and D. persimilis

The genetic basis of male and female sterility in hybrids of Drosophila pseudoobscura-Drosophila persimilis was studied using backcross analysis. Previous studies indirectly assessed male fertility by measuring testis size; these studies concluded that male sterility results from an X chromosome-autosome imbalance. By directly scoring for the production of motile sperm, male sterility is shown to be largely due to an incompatibility between genes on the X and Y chromosomes of these two species. These species have diverged at a minimum of nine loci affecting hybrid male fertility. Semisterility of hybrid females appears to result from an X chromosome-cytoplasm interaction; the X chromosome thus has the largest effect on sterility in both male and female hybrids. This is apparently the first analysis of the genetic basis of female sterility, or of sterility/inviability affecting both sexes, in an animal hybridization.     

Drosophila bipectinata species complex: study of phylogenetic relationship among four members through the analysis of morphology of testes and seminal vesicles

Species maintain their identity through reproductive isolating mechanisms, which are broadly classified into prezygotic and postzygotic isolating mechanisms. In the Drosophila bipectinata species complex, investigations were made on the degree of crossability (a prezygotic isolating mechanism) and the causes of hybrid male sterility (a postzygotic isolating mechanism) to analyse the phylogenetic relationship. Among the four species, D. bipectinata crosses with Drosophila parabipectinata freely in one direction and both of them also cross with Drosophila malerkotliana easily but it is difficult to cross all the three species with Drosophila pseudoananassae . In the hybrids involving D. pseudoananassae , no sperm were observed indicating high degree of perturbance during spermatogenesis while in the other hybrids immotile sperm were present indicating comparatively less disturbance during spermatogenesis. Testis size, which is an indicator of degree of perturbance during spermatogenesis and used as a proxy for sterility was measured in the four species and their hybrids. It was of same size in D. bipectinata , D. parabipectinata and D. malerkotliana but larger in D. pseudoananassae . In the hybrids involving D. pseudoananassae , testes were atrophied while in other hybrids it was larger. Since, the size of testis does not exhibit uniform pattern of variation in hybrids, it cannot be used as a good indicator for sterility. Therefore, we also measured the size of seminal vesicles (storing organ of sperm) in the four species and their hybrids. Interestingly, the size of seminal vesicles was reduced uniformly in all the hybrids indicating its use as better proxy for sterility. Further, the seminal vesicle size in D. pseudoananassae was smaller than that in the other three species. These observations provide evidence for phylogenetic proximity of D. bipectinata , D. parabipectinata and D. malerkotliana and their remote relationships with D. pseudoananassae .     

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.     

A complex genetic basis to X-linked hybrid male sterility between two species of house mice

The X chromosome plays a central role in the evolution of reproductive isolation, but few studies have examined the genetic basis of X-linked incompatibilities during the early stages of speciation. We report the results of a large experiment focused on the reciprocal introgression of the X chromosome between two species of house mice, Mus musculus and M. domesticus. Introgression of the M. musculus X chromosome into a wild-derived M. domesticus genetic background produced male-limited sterility, qualitatively consistent with previous experiments using classic inbred strains to represent M. domesticus. The genetic basis of sterility involved a minimum of four X-linked factors. The phenotypic effects of major sterility QTL were largely additive and resulted in complete sterility when combined. No sterility factors were uncovered on the M. domesticus X chromosome. Overall, these results revealed a complex and asymmetric genetic basis to X-linked hybrid male sterility during the early stages of speciation in mice. Combined with data from previous studies, we identify one relatively narrow interval on the M. musculus X chromosome involved in hybrid male sterility. Only a handful of spermatogenic genes are within this region, including one of the most rapidly evolving genes on the mouse X chromosome.     

Evolutionary history of the Drosophila bipectinata species complex

Groups of recently diverged species offer invaluable glimpses into the history and genetic basis of speciation and phenotypic evolution. In this report, we combine phylogenetic and population-genetic approaches to reconstruct the evolutionary history of the Drosophila bipectinata species complex. This complex is a group of four closely related, largely sympatric species--D. bipectinata, D. parabipectinata, D. malerkotliana and D. pseudoananassae. Using the sequences of one mitochondrial and six nuclear loci, we show that D. bipectinata and D. parabipectinata are the two most closely related species, and that together with D. malerkotliana they form a monophyletic clade to which D. pseudoananassae is a relatively distant outgroup. Genetic divergence among D. bipectinata, D. parabipectinata and D. malerkotliana is extremely low, and we estimate that these species diverged only 283,000-385,000 years ago. We also find that mitochondrial DNA shows evidence of recent gene flow across species boundaries. Despite the low genetic divergence, species of the bipectinata complex show an unusually high degree of morphological differentiation. This contrast underscores the importance of understanding the genetic basis of functional differentiation among closely related species.     

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.     

Interspecific divergence, intrachromosomal recombination, and phylogenetic utility of Y-chromosomal genes in Drosophila

Reconstruction of phylogenetic relationships among recently diverged species is complicated by three general problems: segregation of polymorphisms that pre-date species divergence, gene flow during and after speciation, and intra-locus recombination. In light of these difficulties, the Y chromosome offers several important advantages over other genomic regions as a source of phylogenetic information. These advantages include the absence of recombination, rapid coalescence, and reduced opportunity for interspecific introgression due to hybrid male sterility. In this report, we test the phylogenetic utility of Y-chromosomal sequences in two groups of closely related and partially inter-fertile Drosophila species. In the D. bipectinata species complex, Y-chromosomal loci unambiguously recover the phylogeny most consistent with previous multi-locus analysis and with reproductive relationships, and show no evidence of either post-speciation gene flow or persisting ancestral polymorphisms. In the D. simulans species complex, the situation is complicated by the duplication of at least one Y-linked gene region, followed by intrachromosomal recombination between the duplicate genes that scrambles their genealogy. We suggest that Y-chromosomal sequences are a useful tool for resolving phylogenetic relationships among recently diverged species, especially in male-heterogametic organisms that conform to Haldane's rule. However, duplication of Y-linked genes may not be uncommon, and special care should be taken to distinguish between orthologous and paralogous sequences.     

A cytonuclear incompatibility causes anther sterility in Mimulus hybrids

Multilocus interactions (also known as Dobzhansky-Muller incompatibilities) are thought to be the major source of hybrid inviability and sterility. Because cytoplasmic and nuclear genomes have conflicting evolutionary interests and are often highly coevolved, cytonuclear incompatibilities may be among the first to develop in incipient species. Here, we report the discovery of cytoplasm-dependent anther sterility in hybrids between closely related Mimulus species, outcrossing M. guttatus and selfing M. nasutus. A novel pollenless anther phenotype was observed in F2 hybrids with the M. guttatus cytoplasm (F2G) but not in the reciprocal F2N hybrids, F1 hybrids or parental genotypes. The pattern of phenotypic segregation in the F2G hybrids and two backcross populations fit a Mendelian single-locus recessive model, allowing us to map the underlying nuclear locus to a small region on LG7 of the Mimulus linkage map. Anther sterility was associated with a 20% reduction in flower size in backcross hybrids and we mapped a major cytoplasm-dependent corolla width QTL with its peak at the anther sterility locus. We argue that the cytonuclear anther sterility seen in hybrids reflects the presence of a cryptic cytoplasmic male sterility (CMS) and restorer system within the hermaphroditic M. guttatus population and therefore name the anther sterility locus restorer-of-male-fertility (RMF). The genetic mapping of RMF is a first step toward testing hypotheses about the molecular basis, individual fitness consequences, and ecological context of CMS and restoration in a system without stable CMS-restorer polymorphism (i.e., gynodioecy). The discovery of cryptic CMS in a hermaphroditic wildflower further suggests that selfish cytoplasmic evolution may play an important, but often undetected, role in shaping patterns of hybrid incompatibility and interspecific introgression in plants.     

The genetic basis of postzygotic reproductive isolation between Drosophila santomea and D. yakuba due to hybrid male sterility

A major unresolved challenge of evolutionary biology is to determine the nature of the allelic variants of "speciation genes": those alleles whose interaction produces inviable or infertile interspecific hybrids but does not reduce fitness in pure species. Here we map quantitative trait loci (QTL) affecting fertility of male hybrids between D. yakuba and its recently discovered sibling species, D. santomea. We mapped three to four X chromosome QTL and two autosomal QTL with large effects on the reduced fertility of D. yakuba and D. santomea backcross males. We observed epistasis between the X-linked QTL and also between the X and autosomal QTL. The X chromosome had a disproportionately large effect on hybrid sterility in both reciprocal backcross hybrids. However, the genetics of hybrid sterility differ between D. yakuba and D. santomea backcross males, both in terms of the magnitude of main effects and in the epistatic interactions. The QTL affecting hybrid fertility did not colocalize with QTL affecting sexual isolation in this species pair, but did colocalize with QTL affecting the marked difference in pigmentation between D. yakuba and D. santomea. These results provide the basis for future high-resolution mapping and ultimately, molecular cloning, of the interacting genes that contribute to hybrid sterility.     

DISSECTING THE GENETIC ARCHITECTURE OF F1 HYBRID STERILITY IN HOUSE MICE

Hybrid sterility as a postzygotic reproductive isolation mechanism has been studied for over 80 years, yet the first identifications of hybrid sterility genes in Drosophila and mouse are quite recent. To study the genetic architecture of F₁ hybrid sterility between young subspecies of house mouse Mus m. domesticus and M. m. musculus, we conducted QTL analysis of a backcross between inbred strains representing these two subspecies and probed the role of individual chromosomes in hybrid sterility using the intersubspecific chromosome substitution strains. We provide direct evidence that the asymmetry in male infertility between reciprocal crosses is conferred by the middle region of M. m. musculus Chr X, thus excluding other potential candidates such as Y, imprinted genes, and mitochondrial DNA. QTL analysis identified strong hybrid sterility loci on Chr 17 and Chr X and predicted a set of interchangeable autosomal loci, a subset of which is sufficient to activate the Dobzhansky–Muller incompatibility of the strong loci. Overall, our results indicate the oligogenic nature of F₁ hybrid sterility, which should be amenable to reconstruction by proper combination of chromosome substitution strains. Such a prefabricated model system should help to uncover the gene networks and molecular mechanisms underlying hybrid sterility.     

Evidence for Extensive Genetic Differentiation between the Sex-Ratio and the Standard Arrangement of DROSOPHILA PSEUDOOBSCURA and D. PERSIMILIS and Identification of Hybrid Sterility Factors

This study deals with sex-ratio genes tightly linked within the Sex-Ratio inversion. By taking advantage of the fact that the Sex-Ratio chromosome of Drosophila persimilis [SR(B)] is homosequential to the Standard chromosome of D. pseudoobscura [ST(A)], we carried out two reciprocal introgression experiments. Individual segments of SR(B) or ST(A) were introgressed into the genome of D. pseudoobscura or D. persimilis, respectively. Males possessing a hybrid SR(B)-ST(A) X chromosome and a genetic background derived from either of the two species were tested for fertility and sex-ratio expression.—It was found that, in terms of the meiotic drive genes, the Sex-Ratio chromosome differs extensively from the Standard chromosome. Because recombinations of these genes result in a complete loss of sex-ratio expression, this finding lends strong support to the hypothesis of gene coadaptation. Coadaptation, in this context, is the advantage of being transmitted preferentially. In light of this finding, the evolution of the sex-ratio system in these two sibling species is discussed.—Introgression experiments also yielded information about hybrid sterility. With reciprocal introgression, sterility interactions were found to be "asymmetric." The asymmetry is fully expected from the viewpoint of evolution of postmating reproductive isolation.     

A three-locus system of interspecific incompatibility underlies male inviability in hybrids between Drosophila buzzatii and D. koepferae

In hybrids between the sibling species D. buzzatii and D. koepferae, both sexes are more or less equally viable in the F₁: However, backcross males to D. buzzatii are frequently inviable, apparently because of interspecific genetic incompatibilities that are cryptic in the F₁. We have performed a genetic dissection of the effects of the X chromosome from D. koepferae. We found only two cytological regions, termed hmi-1 and hmi-2, altogether representing 9% of the whole chromosome, which when introgressed into D. buzzatii cause inviability of hybrid males. Observation of the pattern of asynapsis of polytene chromosomes (incomplete pairing, marking introgressed material) in females and segregation analyses were the technique used to infer the X chromosome regions responsible for this hybrid male inviability. The comparison of these results with those previously obtained with the same technique for hybrid male sterility in this same species pair indicate that in the X chromosome of D. koepferae there are at least seven times more regions that produce hybrid male sterility than hybrid male inviability. We have also found that the inviability brought about by the introgression of hmi-1 is suppressed by the cointrogression of two autosomal sections from D. koepferae. Apparently, these three regions conform to a system of species-specific complementary factors involved in an X-autosome interaction that, when disrupted in backcross hybrids by recombination with the genome of its sibling D. buzzatii, brings about hybrid male inviability.     

The genetics of hybrid male sterility between the allopatric species pair Drosophila persimilis and D. pseudoobscura bogotana: dominant sterility alleles in collinear autosomal regions

F(1) hybrid male sterility is thought to result from interactions between loci on the X chromosome and dominant-acting loci on the autosomes. While X-linked loci that contribute to hybrid male sterility have been precisely localized in many animal taxa, their dominant autosomal interactors have been more difficult to localize precisely and/or have been shown to be of relatively smaller effect. Here, we identified and mapped at least four dominant autosomal factors contributing to hybrid male sterility in the allopatric species pair Drosophila persimilis and D. pseudoobscura bogotana. Using these results, we tested predictions of reduced recombination models of speciation. Consistent with these models, three of the four QTL associated with hybrid male sterility occur in collinear (uninverted) regions of these genomes. Furthermore, these QTL do not contribute significantly to hybrid male sterility in crosses between the sympatric species D. persimilis and D. pseudoobscura pseudoobscura. The autosomal loci identified in this study provide the basis for introgression mapping and, ultimately, for molecular cloning of interacting genes that contribute to F(1) hybrid sterility.