Genetic dissection of a key reproductive barrier between nascent species of house mice

Reproductive isolation between species is often caused by deleterious interactions among loci in hybrids. Finding the genes involved in these incompatibilities provides insight into the mechanisms of speciation. With recently diverged subspecies, house mice provide a powerful system for understanding the genetics of reproductive isolation early in the speciation process. Although previous studies have yielded important clues about the genetics of hybrid male sterility in house mice, they have been restricted to F1 sterility or incompatibilities involving the X chromosome. To provide a more complete characterization of this key reproductive barrier, we conducted an F2 intercross between wild-derived inbred strains from two subspecies of house mice, Mus musculus musculus and Mus musculus domesticus. We identified a suite of autosomal and X-linked QTL that underlie measures of hybrid male sterility, including testis weight, sperm density, and sperm morphology. In many cases, the autosomal loci were unique to a specific sterility trait and exhibited an effect only when homozygous, underscoring the importance of examining reproductive barriers beyond the F1 generation. We also found novel two-locus incompatibilities between the M. m. musculus X chromosome and M. m. domesticus autosomal alleles. Our results reveal a complex genetic architecture for hybrid male sterility and suggest a prominent role for reproductive barriers in advanced generations in maintaining subspecies integrity in house mice.     

Asymmetry and polymorphism of hybrid male sterility during the early stages of speciation in house mice

House mice offer a powerful system for dissecting the genetic basis of phenotypes that isolate species in the early stages of speciation. We used a series of reciprocal crosses between wild-derived strains of Mus musculus and M. domesticus to examine F(1) hybrid male sterility, one of the primary phenotypes thought to isolate these species. We report four main results. First, we found significantly smaller testes and fewer sperm in hybrid male progeny of most crosses. Second, in some crosses hybrid male sterility was asymmetric and depended on the species origin of the X chromosome. These observations confirm and extend previous findings, underscoring the central role that the M. musculus X chromosome plays in reproductive isolation. Third, comparisons among reciprocal crosses revealed polymorphism at one or more hybrid incompatibilities within M. musculus. Fourth, the spermatogenic phenotype of this polymorphic interaction appears distinct from previously described hybrid incompatibilities between these species. These data build on previous studies of speciation in house mice and show that the genetic basis of hybrid male sterility is fairly complex, even at this early stage of divergence.     

Genetics and evolution of hybrid male sterility in house mice

Comparative genetic mapping provides insights into the evolution of the reproductive barriers that separate closely related species. This approach has been used to document the accumulation of reproductive incompatibilities over time, but has only been applied to a few taxa. House mice offer a powerful system to reconstruct the evolution of reproductive isolation between multiple subspecies pairs. However, studies of the primary reproductive barrier in house mice-hybrid male sterility-have been restricted to a single subspecies pair: Mus musculus musculus and Mus musculus domesticus. To provide a more complete characterization of reproductive isolation in house mice, we conducted an F(2) intercross between wild-derived inbred strains from Mus musculus castaneus and M. m. domesticus. We identified autosomal and X-linked QTL associated with a range of hybrid male sterility phenotypes, including testis weight, sperm density, and sperm morphology. The pseudoautosomal region (PAR) was strongly associated with hybrid sterility phenotypes when heterozygous. We compared QTL found in this cross with QTL identified in a previous F(2) intercross between M. m. musculus and M. m. domesticus and found three shared autosomal QTL. Most QTL were not shared, demonstrating that the genetic basis of hybrid male sterility largely differs between these closely related subspecies pairs. These results lay the groundwork for identifying genes responsible for the early stages of speciation in house mice.     

The contribution of the y chromosome to hybrid male sterility in house mice

Hybrid sterility in the heterogametic sex is a common feature of speciation in animals. In house mice, the contribution of the Mus musculus musculus X chromosome to hybrid male sterility is large. It is not known, however, whether F(1) male sterility is caused by X-Y or X-autosome incompatibilities or a combination of both. We investigated the contribution of the M. musculus domesticus Y chromosome to hybrid male sterility in a cross between wild-derived strains in which males with a M. m. musculus X chromosome and M. m. domesticus Y chromosome are partially sterile, while males from the reciprocal cross are reproductively normal. We used eight X introgression lines to combine different X chromosome genotypes with different Y chromosomes on an F(1) autosomal background, and we measured a suite of male reproductive traits. Reproductive deficits were observed in most F(1) males, regardless of Y chromosome genotype. Nonetheless, we found evidence for a negative interaction between the M. m. domesticus Y and an interval on the M. m. musculus X that resulted in abnormal sperm morphology. Therefore, although F(1) male sterility appears to be caused mainly by X-autosome incompatibilities, X-Y incompatibilities contribute to some aspects of sterility.     

Differential patterns of introgression across the X chromosome in a hybrid zone between two species of house mice

A complete understanding of the speciation process requires the identification of genomic regions and genes that confer reproductive barriers between species. Empirical and theoretical research has revealed two important patterns in the evolution of reproductive isolation in animals: isolation typically arises as a result of disrupted epistatic interactions between multiple loci and these disruptions map disproportionately to the X chromosome. These patterns suggest that a targeted examination of natural gene flow between closely related species at X-linked markers with known positions would provide insight into the genetic basis of speciation. We take advantage of the existence of genomic data and a well-documented European zone of hybridization between two species of house mice, Mus domesticus and M. musculus, to conduct such a survey. We evaluate patterns of introgression across the hybrid zone for 13 diagnostic X-linked loci with known chromosomal positions using a maximum likelihood model. Interlocus comparisons clearly identify one locus with reduced introgression across the center of the hybrid zone, pinpointing a candidate region for reproductive isolation. Results also reveal one locus with high frequencies of M. domesticus alleles in populations on the M. musculus side of the zone, suggesting the possibility that positive selection may act to drive the spread of alleles from one species on to the genomic background of the other species. Finally, cline width and cline center are strongly positively correlated across the X chromosome, indicating that gene flow of the X chromosome may be asymmetrical. This study highlights the utility of natural populations of hybrids for mapping speciation genes and suggests that the middle of the X chromosome may be important for reproductive isolation between species of house mice.     

The genomics of speciation: investigating the molecular correlates of X chromosome introgression across the hybrid zone between Mus domesticus and Mus musculus

Understanding the genetic details of reproductive isolation is a key goal in the study of speciation. Hybrid zones, geographical regions where two species meet and exchange genes, can provide insight into the genetic basis of reproductive isolation. This is especially true in species with mapped molecular markers because patterns of gene flow can be compared among different genomic regions. Even greater insight can be obtained in species with complete genome sequences because gene identity, gene number and other features of interest can be assessed for genomic regions with different patterns of introgression. Here, we review recent studies on the well-characterized hybrid zone between Mus domesticus and M. musculus , including a detailed survey of patterns of introgression for 13 markers on the X chromosome. We then compare levels of introgression for these 13 regions to a number of genomic attributes inferred from the complete sequence of the X chromosome, with two purposes. First, we identify candidate genes for reproductive isolation by finding genes that map to an X-linked region of reduced introgression and that are only expressed in the male germ line or that show high rates of protein evolution in comparison with rat. Second, we ask whether patterns of gene flow are correlated with recombination rate, gene density, base composition, CpG island density, mutation rate and the rate of protein evolution, as might be expected if many genes contribute to reproductive isolation. We identify seven candidate genes for reproductive isolation between M. domesticus and M. musculus , and our analyses reveal no general correlations between levels of introgression and other measured sequence characteristics. These results underline the utility of the house mouse as a model system for the study of speciation.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 523–534.     

Signatures of reproductive isolation in patterns of single nucleotide diversity across inbred strains of mice

Reproductive isolation is often caused by the disruption of genic interactions that evolve in geographically separate populations. Identifying the genomic regions and genes involved in these interactions, known as "Dobzhansky-Muller incompatibilities," can be challenging but is facilitated by the wealth of genetic markers now available in model systems. In recent years, the complete genome sequence and thousands of single nucleotide polymorphisms (SNPs) from laboratory mice, which are largely genetic hybrids between Mus musculus and M. domesticus, have become available. Here, we use these resources to locate genomic regions that may underlie reproductive isolation between these two species. Using genotypes from 332 SNPs that differ between wild-derived strains of M. musculus and M. domesticus, we identified several physically unlinked SNP pairs that show exceptional gametic disequilibrium across the lab strains. Conspecific alleles were associated in a disproportionate number of these cases, consistent with the action of natural selection against hybrid gene combinations. As predicted by the Dobzhansky-Muller model, this bias was differentially attributable to locus pairs for which one hybrid genotype was missing. We assembled a list of potential Dobzhansky-Muller incompatibilities from locus pairs that showed extreme associations (only three gametic types) among conspecific alleles. Two SNPs in this list map near known hybrid sterility loci on chromosome 17 and the X chromosome, allowing us to nominate partners for disrupted interactions involving these genomic regions for the first time. Together, these results indicate that patterns produced by speciation between M. musculus and M. domesticus are visible in the genomes of lab strains of mice, underscoring the potential of these genetic model organisms for addressing general questions in evolutionary biology.     

Development of unique house mouse resources suitable for evolutionary studies of speciation

Two house mouse subspecies, Mus musculus domesticus and Mus musculus musculus, form a hybrid zone in Europe and represent a suitable model for inferring the genes contributing to isolation barriers between parental taxa. Despite long-term intensive studies of this hybrid zone, we still know relatively little about the causes and mechanisms maintaining the 2 taxa as separate subspecies; therefore, to gain insight into this process, we developed 8 wild-derived inbred house mouse strains. In order to produce strains as pure domesticus or musculus genomes as possible, the individuals used to establish the breeding colony for the 3 domesticus and 2 of the musculus strains were captured in the Czech Republic from wild populations at extreme western and eastern edges of the subspecific contact zone, respectively. The remaining 3 musculus strains were bred from mice captured about 250 km east of the hybrid zone. Genetic analysis based on 361 microsatellite loci showed that 82% of these markers are diagnostic for either the musculus or the domesticus strains. In order to demonstrate the potential utility of this genetic differentiation in such strains, phenotypic variation was scored for 2 strains from opposite edges of the hybrid zone and significant differences in morphology, reproductive performance, in vitro immune responses, mate choice based on urinary signals, and aggressiveness were found. In addition, the 3 strains derived from musculus populations far from the hybrid zone display significant differences in polymorphism in hybrid male sterility when crossed with the laboratory strains C57BL/6 or C57BL/10, which have a predominantly domesticus genome. Although further studies will be necessary to demonstrate intersubspecific differences, all analyses presented here indicate that these newly developed house mouse strains represent a powerful tool for elucidating the genetic basis of isolation barriers in hybrid zones and for studying speciation in general.     

A Novel Mouse Chromosome 17 Hybrid Sterility Locus: Implications for the Origin of T Haplotypes

The effects of heterospecific combinations of mouse chromosome 17 on male fertility and transmission ratio were investigated through a series of breeding studies. Animals were bred to carry complete chromosome 17 homologs, or portions thereof, from three different sources-Mus domesticus, Mus spretus and t haplotypes. These chromosome 17 combinations were analyzed for fertility within the context of a M. domesticus or M. spretus genetic background. Two new forms of hybrid sterility were identified. First, the heterospecific combination of M. spretus and t haplotype homologs leads to complete male sterility on both M. spretus and M. domesticus genetic backgrounds. This is an example of symmetrical hybrid sterility. Second, the presence of a single M. domesticus chromosome 17 homolog within a M. spretus background causes sterility, however, the same combination of chromosome 17 homologs does not cause sterility within the M. domesticus background. This is a case of asymmetrical hybrid sterility. Through an analysis of recombinant chromosomes, it was possible to map the M. domesticus, M. spretus and t haplotype alleles responsible for these two hybrid sterility phenotypes to the same novel locus (Hybrid sterility-4). Previous structural studies had led to the hypothesis that the ancestral t haplotype originated through an introgression event from M. spretus or a related species. If this were true, one might expect that (1) M. spretus homologs would be transmitted at a non-Mendelian ratio within the M. domesticus background, and (2) t haplotypes would be transmitted at a ratio closer to Mendelian within the M. spretus background.(ABSTRACT TRUNCATED AT 250 WORDS)     

Disruption of genetic interaction between two autosomal regions and the X chromosome causes reproductive isolation between mouse strains derived from different subspecies

Reproductive isolation that initiates speciation is likely caused by incompatibility among multiple loci in organisms belonging to genetically diverging populations. Laboratory C57BL/6J mice, which predominantly originated from Mus musculus domesticus, and a MSM/Ms strain derived from Japanese wild mice (M. m. molossinus, genetically close to M. m. musculus) are reproductively isolated. Their F1 hybrids are fertile, but successive intercrosses result in sterility. A consomic strain, C57BL/6J-ChrX(MSM), which carries the X chromosome of MSM/Ms in the C57BL/6J background, shows male sterility, suggesting a genetic incompatibility of the MSM/Ms X chromosome and other C57BL/6J chromosome(s). In this study, we conducted genomewide linkage analysis and subsequent QTL analysis using the sperm shape anomaly that is the major cause of the sterility of the C57BL/6J-ChrX(MSM) males. These analyses successfully detected significant QTL on chromosomes 1 and 11 that interact with the X chromosome. The introduction of MSM/Ms chromosomes 1 and 11 into the C57BL/6J-ChrX(MSM) background failed to restore the sperm-head shape, but did partially restore fertility. This result suggests that this genetic interaction may play a crucial role in the reproductive isolation between the two strains. A detailed analysis of the male sterility by intracytoplasmic sperm injection and zona-free in vitro fertilization demonstrated that the C57BL/6J-ChrX(MSM) spermatozoa have a defect in penetration through the zona pellucida of eggs.     

Hybrid breakdown caused by substitution of the X chromosome between two mouse subspecies

Hybrid breakdown is a type of reproductive failure that appears after the F2 generation of crosses between different species or subspecies. It is caused by incompatibility between interacting genes. Genetic analysis of hybrid breakdown, particularly in higher animals, has been hampered by its complex nature (i.e., it involves more than two genes, and the phenotype is recessive). We studied hybrid breakdown using a new consomic strain, C57BL/6J-X(MSM), in which the X chromosome of C57BL/6J (derived mostly from Mus musculus domesticus) is substituted by the X chromosome of the MSM/Ms strain (M. m. molossinus). Males of this consomic strain are sterile, whereas F1 hybrids between C57BL/6J and MSM/Ms are completely fertile. The C57BL/6J-X(MSM) males showed reduced testis weight with variable defects in spermatogenesis and abnormal sperm head morphology. We conducted quantitative trait locus (QTL) analysis for these traits to map the X-linked genetic factors responsible for the sterility. This analysis successfully detected at least three distinct loci for the sperm head morphology and one for the testis weight. This study revealed that incompatibility of interactions of X-linked gene(s) with autosomal and/or Y-linked gene(s) causes the hybrid breakdown between the genetically distant C57BL/6J and MSM/Ms strains.     

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.     

Low Diversity of T Haplotypes in the Eastern Form of the House Mouse, Mus Musculus L

In previous studies, 13 different recessive embryonic lethal genes have been associated with t haplotypes in the wild mice of the species Mus domesticus. In this communication we have analyzed five populations of Mus musculus for the presence and identity of t haplotypes. The populations occupy geographically distant regions in the Soviet Union: Altai Mountains, western and eastern Siberia, Azerbaijan and Turkmenistan. No t haplotypes were found in mice from eastern Siberia. In the remaining four populations, t haplotypes occurred with frequencies ranging from 0.07 to 0.21. All the t haplotypes extracted from these populations and analyzed by the genetic complementation test were shown to carry the same lethal gene tcl-w73. In one population (that of western Siberia), another lethal gene (tcl-w5) was found to be present on the same chromosome as tcl-w73. This situation is in striking contrast to that found in the populations of the western form of the house mouse, M. domesticus. In the latter species, tcl-w73 has not been found at all and the different populations are characterized by the presence of several different lethal genes. The low diversity of t haplotypes in M. musculus is consistent with lower genetic variability of other traits and indicates a different origin and speciation mode compared to M. domesticus. Serological typing for H-2 antigenic determinants suggests that most, if not all, of the newly described t haplotypes might have arisen by recombination of tw73 from M. musculus with t haplotypes from M. domesticus either in the hybrid zone between the two species or in regions where the two species mixed accidentally.     

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.     

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.     

The Genetics of Postzygotic Isolation in the Drosophila Virilis Group

In a genetic study of postzygotic reproductive isolation among species of the Drosophila virilis group, we find that the X chromosome has the largest effect on male and female hybrid sterility and inviability. The X alone has a discernible effect on postzygotic isolation between closely related species. Hybridizations involving more distantly related species also show large X-effects, although the autosomes may also play a role. In the only hybridization yet subjected to such analysis, we show that hybrid male and female sterility result from the action of different X-linked loci. Our results accord with genetic studies of other taxa, and support the view that both Haldane's rule (heterogametic F1 sterility or inviability) and the large effect of the X chromosome on reproductive isolation result from the accumulation by natural selection of partially recessive or underdominant mutations. We also describe a method that allows genetic analysis of reproductive isolation between species that produce completely sterile or inviable hybrids. Such species pairs, which represent the final stage of speciation, cannot be analyzed by traditional methods. The X chromosome also plays an important role in postzygotic isolation between these species.     

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.