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.     

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.     

Characterization of defects in adult germline development and oogenesis of sterile and rescued female hybrids in crosses between Drosophila simulans and Drosophila melanogaster

Crosses between Drosophila melanogaster and D. simulans normally result in progeny that are either inviable or sterile. Recent discovery of strains that rescue these inviability and sterility phenotypes has made it possible to study the developmental basis of reproductive isolation between these two species in greater detail. By producing both rescued and unrescued hybrids and examining the protein product staining patterns of genes known to be involved in early germline development and gametogenesis, we have found that in crosses between D. simulans and D. melanogaster, hybrid female sterility results from the improper control of primordial germline proliferation, germline stem cell maintenance, and cystoblast formation and differentiation during early oogenesis. Rescued hybrid females are fertile, yet they generally have lower amounts of adult germline from the outset and show a premature degeneration of adult germline cells with age. In addition, older rescued hybrid females also exhibit mutant egg phenotypes associated with defects in dorso-ventral patterning which may result from the improper partitioning of cytoplasmic factors during early oogenesis that could stem from the early defect. Although a variety of germline and oogenic defects are described for the hybrid females, all of them can potentially result from the same underlying primary defect. Hybrid males from these same crosses, on the other hand, have no detectable germline in adult reproductive tissues, even when hybrid sterility rescue strains are used, indicating that male sterility and female sterility stem from distinctly different developmental defects.     

Genome-Wide Survey of Hybrid Incompatibility Factors by the Introgression of Marked Segments of Drosophila Mauritiana Chromosomes into Drosophila Simulans

In hybrids between Drosophila simulans and D. mauritiana, males are sterile and females are fertile, in compliance with HALDANE's rule. The genetic basis of this phenomenon was investigated by introgression of segments of the mauritiana genome into a simulans background. A total of 87 positions throughout the mauritiana genome were marked with P-element insertions and replicate introgressions were made by repeated backcrossing to simulans for 15 generations. The fraction of hemizgyous X chromosomal introgressions that are male sterile is ~50% greater than the fraction of homozygous autosomal segments. This result suggests that male sterility factors have evolved at a higher rate on the X, but chromosomal differences in segment length cannot be ruled out. The fraction of homozygous autosomal introgressions that are male sterile is several times greater than the fraction that are either female sterile or inviable. This observation strongly indicates that male sterility factors have evolved more rapidly than either female sterility or inviability factors. These results, combined with previous work on these and other species, suggest that HALDANE's rule has at least two causes: recessivity of incompatibility factors and differential accumulation of sterility factors affecting males and females.     

Hybrid Lethal Systems in the Drosophila Melanogaster Species Complex. II. the Zygotic Hybrid Rescue (Zhr) Gene of D. Melanogaster

Hybrid females from Drosophila simulans females X Drosophila melanogaster males die as embryos while hybrid males from the reciprocal cross die as larvae. We have recovered a mutation in melanogaster that rescues the former hybrid females. It was located on the X chromosome at a position close to the centromere, and it was a zygotically acting gene, in contrast with mhr (maternal hybrid rescue) in simulans that rescues the same hybrids maternally. We named it Zhr (Zygotic hybrid rescue). The gene also rescues hybrid females from embryonic lethals in crosses of Drosophila mauritiana females X D. melanogaster males and of Drosophila sechellia females X D. melanogaster males. Independence of the hybrid embryonic lethality and the hybrid larval lethality suggested in a companion study was confirmed by employing two rescue genes, Zhr and Hmr (Hybrid male rescue), in doubly lethal hybrids. A model is proposed to explain the genetic mechanisms of hybrid lethalities as well as the evolutionary pathways.     

A Genetic Basis for the Inviability of Hybrids between Sibling Species of Drosophila

A mutation of Drosophila melanogaster whose only known effect is the rescue of otherwise lethal interspecific hybrids has been characterized. This mutation, Hmr, maps to 1-31.84 (9D1-9E4). Hmr may be the consequence of a P element insertion. It rescues hybrid males from the cross of D. melanogaster females to males of its three sibling species, D. simulans, D. mauritiana and D. sechellia. This rescue is recessive, since hybrid males that carry both Hmr and a duplication expected to be Hmr+ are not rescued. Hmr also rescues the otherwise inviable female hybrids from the cross of compound-X D. melanogaster females to males of its sibling species. This rescue is also recessive, since a compound-X heterozygous for Hmr does not rescue. Another mutation, discovered on the In(1)AB chromosome of D. melanogaster, is also found to rescue normally inviable species hybrids: unlike Hmr, however, In(1)AB rescues hybrid females from the cross of In(1)AB/Y males to sibling females, as well as hybrid males from the cross of In(1)AB females to sibling males. These data are interpreted on the basis of a model for the genetic basis of hybrid inviability of complementary genes.     

Inviability of hybrids between D. melanogaster and D. simulans results from the absence of simulans X not the presence of simulans Y chromosome.

Interspecific crosses between D. melanogaster and D. simulans or its sibling species result in unisexual inviability of the hybrids. Mostly, crosses of D. melanogaster females x D. simulans males produce hybrid females. On the other hand, only hybrid males are viable in the reciprocal crosses. A classical question is the cause of the unisexual hybrid inviability on the chromosomal level. Is it due to the absence of a D. simulans X chromosome or is it due to the presence of a D. simulans Y chromosome? A lack of adequate chromosomal rearrangements available in D. simulans has made it difficult to answer this question. However, it has been assumed that the lethality results from the absence of the D. simulans X rather than the presence of the D. simulans Y. Recently I synthesized the first D. simulans compound-XY chromosome that consists of almost the entire X and Y chromosomes. Males carrying the compound-XY and no free Y chromosome are fertile. By utilizing the compound-XY chromosome, the viability of hybrids with various constitutions of cytoplasm and sex chromosomes has been examined. The results consistently demonstrate that the absence of a D. simulans X chromosome in hybrid genome, and not the presence of the Y chromosome, is a determinant of the hybrid inviability.     

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 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.     

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 Drosophila melanogaster hybrid male rescue gene causes inviability in male and female species hybrids

The Drosophila melanogaster mutation Hmr rescues inviable hybrid sons from the cross of D. melanogaster females to males of its sibling species D. mauritiana, D. simulans, and D. sechellia. We have extended previous observations that hybrid daughters from this cross are poorly viable at high temperatures and have shown that this female lethality is suppressed by Hmr and the rescue mutations In(1)AB and D. simulans Lhr. Deficiencies defined here as Hmr(-) also suppressed lethality, demonstrating that reducing Hmr(+) activity can rescue otherwise inviable hybrids. An Hmr(+) duplication had the opposite effect of reducing the viability of female and sibling X-male hybrid progeny. Similar dose-dependent viability effects of Hmr were observed in the reciprocal cross of D. simulans females to D. melanogaster males. Finally, Lhr and Hmr(+) were shown to have mutually antagonistic effects on hybrid viability. These data suggest a model where the interaction of sibling species Lhr(+) and D. melanogaster Hmr(+) causes lethality in both sexes of species hybrids and in both directions of crossing. Our results further suggest that a twofold difference in Hmr(+) dosage accounts in part for the differential viability of male and female hybrid progeny, but also that additional, unidentified genes must be invoked to account for the invariant lethality of hybrid sons of D. melanogaster mothers. Implications of our findings for understanding Haldane's rule-the observation that hybrid breakdown is often specific to the heterogametic sex-are also discussed.     

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.     

Mapping and characterization of a 'speciation gene' in Drosophila.

Almost nothing is known about the identity of the genes causing reproductive isolation between species. As a first step towards molecular isolation of a 'speciation gene', I mapped and partly characterized a gene causing hybrid male sterility in Drosophila. This analysis shows that sterility of D. melanogaster males who carry the 'dot' fourth chromosome from D. simulans is due entirely to a very small region of the D. simulans chromosome (including only about 5 salivary gland bands or approximately 250 kb of DNA). Thus the hybrid sterility effect of the D. simulans fourth chromosome is almost surely due to a single gene of very large effect (here named hms, hybrid male sterile). Hms is zygotically acting, and the D. simulans allele of hms is completely recessive. Furthermore, complementation tests suggest that hms is not an allele of any known locus in D. melanogaster.     

Nup96-dependent hybrid lethality occurs in a subset of species from the simulans clade of Drosophila

The cross of Drosophila melanogaster females to D. simulans males typically produces lethal F(1) hybrid males. F(1) male lethality is suppressed when the D. simulans Lhr(1) hybrid rescue strain is used. Viability of these F(1) males carrying Lhr(1) is in turn substantially reduced when the hybrids are heterozygous for some mutant alleles of the D. melanogaster Nup96 gene. I show here that similar patterns of Nup96-dependent lethality occur when other hybrid rescue mutations are used to create F(1) males, demonstrating that Nup96 does not reduce hybrid viability by suppressing the Lhr(1) rescue effect. The penetrance of this Nup96-dependent lethality does not correlate with the penetrance of the F(1) hybrid rescue, arguing that these two phenomena reflect genetically independent processes. D. simulans, together with two additional sister species, forms a clade that speciated after the divergence of their common ancestor from D. melanogaster. I report here that Nup96(-) reduces F(1) viability in D. melanogaster hybrids with one of these sister species, D. sechellia, but not with the other, D. mauritiana. These results suggest that Nup96-dependent lethality evolved after the speciation of D. melanogaster from the common ancestor of the simulans clade and is caused by an interaction among Nup96, unknown gene(s) on the D. melanogaster X chromosome, and unknown autosomal gene(s), at least some of which have diverged in D. simulans and D. sechellia but not in D. mauritiana. The genetic properties of Nup96 are also discussed relative to other hybrid lethal genes.     

The impact of shared ancestral variation on hybrid male lethality--a 16 codon indel in the Drosophila simulans Lhr gene

The Lethal hybrid rescue (Lhr) gene causes hybrid male lethality in crosses between Drosophila simulans and D. melanogaster. Lhr(2) is a D. simulans allele, which rescues hybrid males. It has been recently proposed that a 16 codon insertion, which distinguishes the D. melanogaster and the canonical D. simulans allele, and is lacking in Lhr(2), may be responsible for the functional divergence of D. melanogaster and D. simulans Lhr alleles. Here, we show that the Lhr(2) allele lacking the insertion represents an ancestral polymorphism segregating at a moderate frequency in D. simulans. Crosses of D. melanogaster females to males from two D. simulans strains carrying this deletion showed a severe deficiency of viable hybrid males. Our results suggest that the absence of this insertion alone is not sufficient to explain functional differences between D. melanogaster and D. simulans Lhr alleles.     

Genetic dissection of Nucleoporin 160 (Nup160), a gene involved in multiple phenotypes of reproductive isolation in Drosophila

Previous reports have suggested that the Nucleoporin 160 (Nup160) gene of Drosophila simulans (Nup160(sim)) causes the hybrid inviability, female sterility, and morphological anomalies that are observed in crosses with D. melanogaster. Here we have confirmed this observation by transposon excision from the P{EP}Nup160(EP372) insertion mutation of D. melanogaster. Null mutations of the Nup160 gene resulted in the three phenotypes caused by Nup160(sim), but revertants of the gene did not. Interestingly, several mutations produced by excision partially complemented hybrid inviability, female sterility, or morphological anomalies. In the future, these mutations will be useful to further our understanding of the developmental mechanisms of reproductive isolation. Based on our analyses with the Nup160(sim) introgression line, the lethal phase of hybrid inviability was determined to be during the early pupal stage. Our analysis also suggested that homozygous Nup160(sim) in D. melanogaster leads to slow development. Thus, Nup160(sim) is involved in multiple aspects of reproductive isolation between these two species.     

The Developmental Genetics of Hybrid Inviability: A Mitotic Defect in Drosophila Hybrids

We report studies of the developmental basis of hybrid inviability in the Drosophila melanogaster complex. The pathology of these hybrids closely resembles that of mitotic mutants in D. melanogaster. We use mosaic and cytological analyses to show that hybrid male inviability is associated with, and probably caused by, a defect in mitotic cell division. In the mosaic study, we find that male clones produced in otherwise female hybrids are not cell lethal but are very small, probably reflecting defects in mitotic proliferation. Cytological inspection of larval neuroblasts reveals a profound mitotic defect in hybrids: chromosomes show a near-complete failure to condense even after 2 hr of incubation in colchicine. Both the defect in clonal proliferation and in chromatin condensation are rescued by mutations known to rescue normally inviable hybrid males. We present a simple model in which hybrid inviability is partly or entirely caused by a mitotic defect; this defect is, in turn, caused by an interaction between the Hybrid male rescue (Hmr) locus of D. melanogaster and autosomal gene(s) from D. melanogaster's sister species.     

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

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