FEMALE HETEROGAMETY AND SPECIATION: REDUCED INTROGRESSION OF THE Z CHROMOSOME BETWEEN TWO SPECIES OF NIGHTINGALES

Several lines of evidence suggest that the X chromosome plays a large role in intrinsic postzygotic isolation. The role of the Z chromosome in speciation is much less understood. To explore the role of the Z chromosome in reproductive isolation, we studied nucleotide variation in two closely related bird species, the Thrush Nightingale ( Luscinia luscinia ) and the Common Nightingale ( L. megarhynchos ). These species are isolated by incomplete prezygotic isolation and female hybrid sterility. We sequenced introns of four Z-linked and eight autosomal loci and analyzed patterns of polymorphism and divergence using a divergence-with-gene flow framework. Our results suggest that the nightingale species diverged approximately 1.8 Mya. We found strong evidence of gene flow after divergence in both directions, although more introgression occurred from L. megarhynchos into L. luscinia . Gene flow was significantly higher on the autosomes than on the Z chromosome. Our results support the idea that the Z chromosome plays an important role in intrinsic postzygotic isolation in birds, although it may also contribute to the evolution of prezygotic isolation through sexual selection. This highlights the similarities in the genetic basis of reproductive isolation between organisms with heterogametic males and organisms with heterogametic females during the early stages of speciation.     

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.     

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.     

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.     

Genetic analysis of female mating recognition between Drosophila ananassae and Drosophila pallidosa: application of interspecific mosaic genome lines

Drosophila ananassae and Drosophila pallidosa are closely related species that can produce viable and fertile hybrids of both sexes, although strong sexual isolation exists between the two species. Females are thought to discriminate conspecific from heterospecific males based on their courtship songs. The genetic basis of female discrimination behavior was analyzed using isogenic females from interspecific mosaic genome lines that carry homozygous recombinant chromosomes. Multiple regression analysis indicated a highly significant effect of the left arm of chromosome 2 (2L) on the willingness of females to mate with D. ananassae males. Not only 2L but also the left arm of chromosome X (XL) and the right arm of chromosome 3 (3R) had significant effects on the females' willingness to mate with D. pallidosa males. All regions with strong effects on mate choice have chromosome arrangements characterized by species-specific inversions. Heterospecific combinations of 2L and 3R have previously been suggested to cause postzygotic reproductive isolation. Thus, genes involved in premating as well as postmating isolation are located in or near chromosomal inversions. This conclusion is consistent with the recently proposed hypothesis that "speciation genes" accumulate at a higher rate in non-recombining genome regions when species divergence occurs in the presence of gene flow.     

The genetic basis of prezygotic reproductive isolation between Drosophila santomea and D. yakuba due to mating preference

Sexual isolating mechanisms that act before fertilization are often considered the most important genetic barriers leading to speciation in animals. While 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 santomea and D. yakuba. We mapped at least three QTL affecting discrimination of D. santomea females against D. yakuba males: one X-linked and one autosomal QTL affected the likelihood of copulation, and a second X chromosome QTL affected copulation latency. Three autosomal QTL also affected mating success of D. yakuba males with D. santomea. No epistasis was detected between QTL affecting sexual isolation. The QTL do not overlap between males and females and are not disproportionately concentrated on the X chromosome. There was some overlap in map locations of QTL affecting sexual isolation between D. santomea and D. yakuba with QTL affecting sexual isolation between D. simulans and D. mauritiana and with QTL affecting differences in pigmentation between D. santomea and D. yakuba. Future high-resolution mapping and, ultimately, positional cloning, will reveal whether these traits do indeed have a common genetic basis.     

Genetic linkage between a sexually selected trait and X chromosome meiotic drive

Previous studies on the stalk-eyed fly, Cyrtodiopsis dalmanni, have shown that males with long eye-stalks win contests and are preferred by females, and artificial selection on male relative eye span alters brood sex-ratios. Subsequent theory proposes that X-linked meiotic drive can catalyse the evolution of mate preferences when drive is linked to ornament genes. Here we test this prediction by mapping meiotic drive and quantitative trait loci (QTL) for eye span. To map QTL we genotyped 24 microsatellite loci using 1228 F2 flies from two crosses between lines selected for long or short eye span. The crosses differed by presence or absence of a drive X chromosome, X(D), in the parental male. Linkage analysis reveals that X(D) dramatically reduces recombination between X and X(D) chromosomes. In the X(D) cross, half of the F2 males carried the drive haplotype, produced partially elongated spermatids and female-biased broods, and had shorter eye span. The largest QTL mapped 1.3cM from drive on the X chromosome and explained 36% of the variation in male eye span while another QTL mapped to an autosomal region that suppresses drive. These results indicate that selfish genetic elements that distort the sex-ratio can influence the evolution of exaggerated traits.     

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.     

Rapid evolution of postzygotic reproductive isolation in stalk-eyed flies

We test the relative rates of evolution of pre- and postzygotic reproductive isolation using eight populations of the sexually dimorphic stalk-eyed flies Cyrtodiopsis dalmanni and C. whitei. Flies from these populations exhibit few morphological differences yet experience strong sexual selection on male eyestalks. To measure reproductive isolation we housed one male and three female flies from within and between these populations in replicate cages and then recorded mating behavior, sperm transfer, progeny production, and hybrid fertility. Using a phylogeny based on partial sequences of two mitochondrial genes, we found that premating isolation, postmating isolation prior to hybrid eclosion, and female hybrid sterility evolve gradually with respect to mitochondrial DNA sequence divergence. In contrast, male hybrid sterility evolves much more rapidly-at least twice as fast as any other form of reproductive isolation. Hybrid sterility, therefore, obeys Haldane's rule. Although some brood sex ratios were female biased, average brood sex ratio did not covary with genetic distance, as would be expected if hybrid inviability obeyed Haldane's rule. The likelihood that forces including sexual selection and intra- and intergenomic conflict may have contributed to these patterns is discussed.     

Tests of reproductive isolation among species in the <Emphasis Type="Italic">Fundulus notatus</Emphasis> (Cyprinodontiformes: Fundulidae) species complex

We assessed prezygotic (probability of spawning) and postzygotic (hatching success) reproductive isolation among the three ecologically and morphologically similar species in the Fundulus notatus species complex. We employed a multi-generation breeding experiment to test the hypotheses that karyotypic differences, body size differences, or geographic isolation among populations will increase pre or postzygotic reproductive barriers. Overall, prezygotic barriers were strong and postzygotic barriers weak in crosses of non-hybrid heterospecifics (F1 hybrid crosses) while prezygotic barriers were weaker and postzygotic barriers stronger in crosses involving hybrid individuals (F2 hybrid crosses and backcrosses). Prezygotic barriers among the two smaller species (Fundulus notatus and F. euryzonus) broke down rapidly; first generation hybrids spawned (F2 hybrid crosses and backcrosses) as frequently as parental forms in intraspecific crosses. There was no increase in postzygotic barriers among species with cytogenetic differences. There were increased prezygotic, but not postzygotic, barriers among geographically isolated populations of one species. While pure males and females were just as likely to spawn with hybrids, some types of hybrid females suffered from increased sterility, but not inviability, over hybrid males. Female sterility was only seen in hybrids with a Fundulus euryzonus parent, while other female hybrids produced viable eggs.     

A genomic map of clinal variation across the European rabbit hybrid zone

Speciation is a process proceeding from weak to complete reproductive isolation. In this continuum, naturally hybridizing taxa provide a promising avenue for revealing the genetic changes associated with the incipient stages of speciation. To identify such changes between two subspecies of rabbits that display partial reproductive isolation, we studied patterns of allele frequency change across their hybrid zone using whole‐genome sequencing. To connect levels and patterns of genetic differentiation with phenotypic manifestations of subfertility in hybrid rabbits, we further investigated patterns of gene expression in testis. Geographic cline analysis revealed 253 regions characterized by steep changes in allele frequency across their natural region of contact. This catalog of regions is likely to be enriched for loci implicated in reproductive barriers and yielded several insights into the evolution of hybrid dysfunction in rabbits: (i) incomplete reproductive isolation is likely governed by the effects of many loci, (ii) protein–protein interaction analysis suggest that genes within these loci interact more than expected by chance, (iii) regulatory variation is likely the primary driver of incompatibilities, and (iv) large chromosomal rearrangements appear not to be a major mechanism underlying incompatibilities or promoting isolation in the face of gene flow. We detected extensive misregulation of gene expression in testis of hybrid males, but not a statistical overrepresentation of differentially expressed genes in candidate regions. Our results also did not support an X chromosome‐wide disruption of expression as observed in mice and cats, suggesting variation in the mechanistic basis of hybrid male reduced fertility among mammals.     

Origin of Reproductive Isolation in the Absence of Apparent Genic Differentiation in a Geographic Isolate of DROSOPHILA PSEUDOOBSCURA

F(1) males obtained from the cross of D. pseudoobscura females from Bogotá (Colombia) x males of this species from mainland, i.e. populations from various locations in the United States and from Guatemala, are sterile. This sterility is due to genes located on the X chromosome and the autosomes; the Y chromosome is not involved. The percentage of sterile males in backcrosses can be explained by assuming an interaction between two loci on the Bogotá X chromosome and probably two loci, one each on two of the mainland autosomes. The role of founder events, inbreeding and geographic isolation in the development of reproductive isolation and the magnitude of gene differences responsible for the origin of reproductive isolation is discussed. It is concluded that founder events, inbreeding and geographic isolation play a major role in the development of reproductive isolation and that major adaptive incorporation of new alleles at a large number of structural loci is not necessary for the origin of reproductive isolation.     

Male eyespan size is associated with meiotic drive in wild stalk-eyed flies (Teleopsis dalmanni)

This study provides the first direct evidence from wild populations of stalk-eyed flies to support the hypothesis that male eyespan is a signal of meiotic drive. Several stalk-eyed fly species are known to exhibit X-linked meiotic drive. A recent quantitative trait locus analysis in Teleopsis dalmanni found a potential link between variation in male eyespan, a sexually selected ornamental trait, and the presence of meiotic drive. This was based on laboratory populations subject to artificial selection for male eyespan. In this study, we examined the association between microsatellite markers and levels of sex ratio bias (meiotic drive) in 12 wild T. dalmanni populations. We collected two data sets: (a) brood sex ratios of wild-caught males mated to standard laboratory females and (b) variation in a range of phenotypic traits associated with reproductive success of wild-caught males and females. In each case, we typed individuals for eight X-linked microsatellite markers, including several that previously were shown to be associated with male eyespan and meiotic drive. We found that one microsatellite marker was very strongly associated with meiotic drive, whereas a second showed a weaker association. We also found that, using both independent data sets, meiotic drive was strongly associated with male eyespan, with smaller eyespan males being associated with more female-biased broods. These results suggest that mate preference for exaggerated male eyespan allows females to avoid mating with males carrying the meiotic drive gene and is thus a potential mechanism for the maintenance and evolution of female mate preference.     

Gametic selection,developmental trajectories,and extrinsic heterogeneity in Haldane's rule

Deciphering the genetic and developmental causes of the disproportionate rarity, inviability, and sterility of hybrid males, Haldane's rule, is important for understanding the evolution of reproductive isolation between species. Moreover, extrinsic and prezygotic factors can contribute to the magnitude of intrinsic isolation experienced between species with partial reproductive compatibility. Here, we use the nematodes Caenorhabditis briggsae and C. nigoni to quantify the sensitivity of hybrid male viability to extrinsic temperature and developmental timing, and test for a role of mito‐nuclear incompatibility as a genetic cause. We demonstrate that hybrid male inviability manifests almost entirely as embryonic, not larval, arrest and is maximal at the lowest rearing temperatures, indicating an intrinsic‐by‐extrinsic interaction to hybrid inviability. Crosses using mitochondrial substitution strains that have reciprocally introgressed mitochondrial and nuclear genomes show that mito‐nuclear incompatibility is not a dominant contributor to postzygotic isolation and does not drive Haldane's rule in this system. Crosses also reveal that competitive superiority of X‐bearing sperm provides a novel means by which postmating prezygotic factors exacerbate the rarity of hybrid males. These findings highlight the important roles of gametic, developmental, and extrinsic factors in modulating the manifestation of Haldane's rule.     

Lineages of Silene nutans developed rapid,strong, asymmetric postzygotic reproductive isolation in allopatry

Reproductive isolation can rise either as a consequence of genomic divergence in allopatry or as a byproduct of divergent selection in parapatry. To determine whether reproductive isolation in gynodioecious Silene nutans results from allopatric divergence or from ecological adaptation following secondary contact, we investigated the pattern of postzygotic reproductive isolation and hybridization in natural populations using two phylogeographic lineages, western (W1) and eastern (E1). Experimental crosses between the lineages identified strong, asymmetric postzygotic isolation between the W1 and the E1 lineages, independent of geographic overlap. The proportion of ovules fertilized, seeds aborted, and seeds germinated revealed relatively little effect on the fitness of hybrids. In contrast, hybrid mortality was high and asymmetric: while half of the hybrid seedlings with western lineage mothers died, nearly all hybrid seedlings with E1 mothers died. This asymmetric mortality mirrored the proportion of chlorotic seedlings, and is congruent with cytonuclear incompatibility. We found no evidence of hybridization between the lineages in regions of co‐occurrence using nuclear and plastid markers. Together, our results are consistent with the hypothesis that strong postzygotic reproductive isolation involving cytonuclear incompatibilities arose in allopatry. We argue that the dynamics of cytonuclear gynodioecy could facilitate the evolution of reproductive isolation.     

Sex-ratio drive in Drosophila simulans: variation in segregation ratio of X chromosomes from a natural population

The sex-ratio trait that exists in a dozen Drosophila species is a case of naturally occurring X chromosome drive that causes males to produce female-biased progeny. Autosomal and Y polymorphism for suppressors are known to cause variation in drive expression, but the X chromosome polymorphism has never been thoroughly investigated. We characterized 41 X chromosomes from a natural population of Drosophila simulans that had been transferred to a suppressor-free genetic background. We found two clear-cut groups of chromosomes, sex-ratio and standard. The sex-ratio X chromosomes differed in their segregation ratio (81-96% females in the progeny), the less powerful drivers being less stable in their expression. A sib analysis, using a moderate driver, indicated that within-X variation in drive expression depended on genetic (autosomal) or epigenetic factors and that the age of the males also affected the trait. The other X chromosomes produced equal or roughly equal sex ratios, but again with significant variation. The continuous pattern of variation observed within both groups suggested that, in addition to a major sex-ratio gene, many X-linked loci of small effect modify the segregation ratio of this chromosome and are maintained in a polymorphic state. This was also supported by the frequency distribution of sex ratios produced by recombinant X chromosomes.     

The genetic basis of behavioral isolation between Drosophila mauritiana and D. sechellia

Understanding how species form is a fundamental question in evolutionary biology. Identifying the genetic bases of barriers that prevent gene flow between species provides insight into how speciation occurs. Here, I analyze a poorly understood reproductive isolating barrier, prezygotic reproductive isolation. I perform a genetic analysis of prezygotic isolation between two closely related species of Drosophila, D. mauritiana and D. sechellia. I first confirm the existence of strong behavioral isolation between D. mauritiana females and D. sechellia males. Next, I examine the genetic basis of behavioral isolation by (1) scanning an existing set of introgression lines for chromosomal regions that have a large effect on isolation; and (2) mapping quantitative trait loci (QTL) that underlie behavioral isolation via backcross analysis. In particular, I map QTL that determine whether a hybrid backcross female and a D. sechellia male will mate. I identify a single significant QTL, on the X chromosome, suggesting that few major-effect loci contribute to behavioral isolation between these species. In further work, I refine the map position of the QTL to a small region of the X chromosome.     

The Dominance Theory of Haldane''s Rule

``HALDANE's rule' states that, if species hybrids of one sex only are inviable or sterile, the afflicted sex is much more likely to be heterogametic (XY) than homogametic (XX). We show that most or all of the phenomena associated with HALDANE's rule can be explained by the simple hypothesis that alleles decreasing hybrid fitness are partially recessive. Under this hypothesis, the XY sex suffers more than the XX because X-linked alleles causing postzygotic isolation tend to have greater cumulative effects when hemizygous than when heterozygous, even though the XX sex carries twice as many such alleles. The dominance hypothesis can also account for the ``large X effect,' the disproportionate effect of the X chromosome on hybrid inviability/sterility. In addition, the dominance theory is consistent with: the long temporal lag between the evolution of heterogametic and homogametic postzygotic isolation, the frequency of exceptions to HALDANE's rule, puzzling Drosophila experiments in which ``unbalanced' hybrid females, who carry two X chromosomes from the same species, remain fertile whereas F(1) hybrid males are sterile, and the absence of cases of HALDANE's rule for hybrid inviability in mammals. We discuss several novel predictions that could lead to rejection of the dominance theory.     

THE GENETIC ARCHITECTURE OF REPRODUCTIVE ISOLATION IN LOUISIANA IRISES: HYBRID FITNESS IN NATURE

Negative epistasis in hybrid genomes commonly results in postzygotic isolation between divergent lineages. However, some genomic regions may be selectively neutral or adaptive in hybrids and thus may potentially cross species barriers. We examined postzygotic isolation between ecologically similar species of Louisiana Iris: Iris brevicaulis and I. fulva to determine the potential for adaptive introgression in nature. Line-cross analyses allowed us a general overview of the gene action responsible for fitness-related traits. We then used a QTL mapping approach to detect genomic regions responsible for variation in these traits. Although hybrid classes suffered reduced fitness for many traits, hybrid means were equivalent to at least one of the parental species in overall estimates of maternal and paternal fitness during the two years of the field study. The genetic architecture underlying the fitness-related traits varied across field site and year of the study, thus emphasizing the importance of the environment in determining the degree of postzygotic isolation and potential for introgression across natural hybrid zones.