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

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

Genetics and Lineage-Specific Evolution of a Lethal Hybrid Incompatibility Between Drosophila mauritiana and Its Sibling Species

The Dobzhansky–Muller model posits that intrinsic postzygotic reproductive isolation—the sterility or lethality of species hybrids—results from the evolution of incompatible epistatic interactions between species: favorable or neutral alleles that become fixed in the genetic background of one species can cause sterility or lethality in the genetic background of another species. The kind of hybrid incompatibility that evolves between two species, however, depends on the particular evolutionary history of the causative substitutions. An allele that is functionally derived in one species can be incompatible with an allele that is functionally derived in the other species (a derived-derived hybrid incompatibility). But an allele that is functionally derived in one species can also be incompatible with an allele that has retained the ancestral state in the other species (a derived-ancestral hybrid incompatibility). The relative abundance of such derived-derived vs. derived-ancestral hybrid incompatibilities is unknown. Here, we characterize the genetics and evolutionary history of a lethal hybrid incompatibility between Drosophila mauritiana and its two sibling species, D. sechellia and D. simulans. We show that a hybrid lethality factor(s) in the pericentric heterochromatin of the D. mauritiana X chromosome, hybrid lethal on the X (hlx), is incompatible with a factor(s) in the same small autosomal region from both D. sechellia and D. simulans, Suppressor of hlx [Su(hlx)]. By combining genetic and phylogenetic information, we infer that hlx-Su(hlx) hybrid lethality is likely caused by a derived-ancestral incompatibility, a hypothesis that can be tested directly when the genes are identified.     

EVOLUTIONARY GENETICS OF TWO SIBLING SPECIES,DROSOPHILA SIMULANS AND D. SECHELLIA

Drosophila simulans and D. sechellia are sibling species, the former cosmopolitan and the latter restricted to the Seychelles Islands. We used classical genetic analysis to measure the numbers and effects of genes responsible for reproductive isolation and morphological differences in male genitalia between these species. At least five loci are responsible for male sterility in hybrids, with the strongest effects produced by at least two genes on the X chromosome. At least three (and probably four) loci are responsible for the interspecific difference in the size of the posterior process of the male genital arch. These genetic results, as well as the pattern of morphological divergence between the species, show several parallels with the divergence between D. simulans and its other island relative, D. mauritiana. We also present the DNA sequence of a 4.5 kilobase region containing the alcohol dehydrogenase (Adh) locus of D. sechellia, and combine this with previous data to reconstruct the phylogenies of the three species and their more distant relative D. melanogaster. Both D. mauritiana and D. sechellia are very closely related to D. simulans. Although most phylogenies show the two island species to be independent offshoots of the D. simulans lineage (with D. sechellia the more recent), the branch points are too close to make this conclusion unambiguous. The genetic and evolutionary parallels between the simulans/mauritiana and the simulans/sechellia divergences may therefore represent either a striking evolutionary convergence or a close common ancestry of the island species. A comparison of Adh alleles within species shows that the divergence among them may be almost as large as among alleles from different species. We conclude that many of the nucleotide differences among these species actually represent polymorphisms within common ancestors. It may be difficult to build accurate phylogenies using only a single DNA sequence from each species.     

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.     

Variable post‐zygotic isolation in Drosophila melanogaster/D. simulans hybrids

The study of hybrid inviability reveals cryptic divergence between the genetic interactions that maintain stable phenotypes in the pure species . We characterized the effects of natural variation on the penetrance of hybrid inviability phenotypes in crosses between Drosophila melanogaster and two species of the D. simulans subcomplex, D. simulans and D. sechellia. Using a panel of wild‐caught lines, we studied the levels of genetic variance present in D. simulans and D. sechellia affecting prezygotic and post‐zygotic isolation in hybridizations with D. melanogaster females. We observed extensive variability in the viability of hybrid individuals, dependent on the genotype of the parents, suggesting that intraspecific natural variation manifests directly in hybrid phenotypes. Furthermore, we found that genetic background significantly affects the penetrance of a well‐studied determinant of hybrid inviability: the interaction between Hmr^mel–Lhr^sim. Our results suggest that hybrid inviability – and reproductive isolation generally – can be modified by polymorphisms at multiple loci segregating within the parental species. Just as the penetrance of most mutant phenotypes can be modified by the genetic background within the pure species, the penetrance of hybrid inviability phenotypes is highly influenced by the parental genotypes.     

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.     

Haldane's rule: hybrid sterility affects the heterogametic sex first because sexual differentiation is on the path to species differentiation

Prevention of recombination is needed to preserve both phenotypic differentiation between species and sexual phenotypic differentiation within species. For species differentiation (speciation), isolating barriers preventing recombination may be pre-zygotic (gamete transfer barriers), or post-zygotic (either a developmental barrier resulting in hybrid inviability, or a chromosomal-pairing barrier resulting in hybrid sterility). The sterility barrier is usually the first to appear and, although often initially only manifest in the heterogametic sex (Haldane's rule), is finally manifest in both sexes. For sexual differentiation, the first and only barrier is chromosomal-pairing, and always applies to the heterogametic sex. For regions of sex chromosomes affecting sexual differentiation there must be something analogous to the process generating the hybrid sterility seen when allied species cross. Explanations for Haldane's rule have generally assumed that the chromosomal-pairing barrier initiating evolutionary divergence into species is due to incompatibilities between gene products ("genic), or sets of gene products ("polygenic), rather than between chromosomes per se ("chromosomal"). However, if chromosomal incompatibilities promoting incipient sexual differentiation could also contribute to the process of incipient speciation, then a step towards speciation would have been taken in the heterogametic sex. Thus, incipient speciation, manifest as hybrid sterility when "varieties" are crossed, would appear at the earliest stage in the heterogametic sex, even in genera with homomorphic sex chromosomes (Haldane's rule for hybrid sterility). In contrast, it has been proposed that Haldane's rule for hybrid inviability needs differences in dosage compensation, so could not apply to genera with homomorphic sex chromosomes.     

A genomic approach to identify hybrid incompatibility genes

Uncovering the genetic and molecular basis of barriers to gene flow between populations is key to understanding how new species are born. Intrinsic postzygotic reproductive barriers such as hybrid sterility and hybrid inviability are caused by deleterious genetic interactions known as hybrid incompatibilities. The difficulty in identifying these hybrid incompatibility genes remains a rate-limiting step in our understanding of the molecular basis of speciation. We recently described how whole genome sequencing can be applied to identify hybrid incompatibility genes, even from genetically terminal hybrids. Using this approach, we discovered a new hybrid incompatibility gene, gfzf, between Drosophila melanogaster and Drosophila simulans, and found that it plays an essential role in cell cycle regulation. Here, we discuss the history of the hunt for incompatibility genes between these species, discuss the molecular roles of gfzf in cell cycle regulation, and explore how intragenomic conflict drives the evolution of fundamental cellular mechanisms that lead to the developmental arrest of hybrids.     

The Population Genetics of Speciation: The Evolution of Hybrid Incompatibilities

Speciation often results from the accumulation of ``complementary genes,' i.e., from genes that, while having no deleterious effect within species, cause inviability or sterility when brought together with genes from another species. Here I model speciation as the accumulation of genic incompatibilities between diverging populations. Several results are obtained. First, and most important, the number of genic incompatibilities between taxa increases much faster than linearly with time. In particular, the probability of speciation increases at least as fast as the square of the time since separation between two taxa. Second, as Muller realized, all hybrid incompatibilities must initially be asymmetric. Third, at loci that have diverged between taxa, evolutionarily derived alleles cause hybrid problems far more often than ancestral alleles. Last, it is ``easier' to evolve complex hybrid incompatibilities requiring the simultaneous action of three or more loci than to evolve simple incompatibilities between pairs of genes. These results have several important implications for genetic analyses of speciation.     

GENETICS OF STERILITY IN HYBRIDS BETWEEN TWO SUBSPECIES OF DROSOPHILA

Hybrids between D. pseudoobscura bogotana and D. pseudoobscura pseudoobscura are fertile except for males produced in one of the two reciprocal crosses. As there is no premating isolation between these subspecies, nonreciprocal male sterility represents the first step in speciation. Genetic analysis reveals two causes of hybrid F₁ sterility: a maternal effect and incompatibilities between chromosomes within males. The maternal effect appears to play the greatest role in hybrid sterility. The X chromosome has the largest effect on fertility of any chromosome, a ubiquitous result in analyses of hybrid sterility and inviability in Drosophila. This effect is entirely attributable to a region comprising less than 30% of the X chromosome. These results are compared to those from a similar study of D. pseudoobscura-D. persimilis hybrids, an older and more reproductively isolated species pair in the same lineage. Such comparisons may allow one to identify the genetic changes characterizing the early versus late stages of speciation.     

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

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

Genetics of food preference inDrosophila sechellia

To reveal the genetic mechanism of host selection in a monophagous fruit flyDrosophila sechellia, olfactory responses and oviposition preferences of this species were compared with those of closely related polyphagous species,D. simulans andD. melanogaster. Adult flies ofD. sechellia were strongly attracted to the ripe fruit ofMorinda citrifolia which is known to be the sole breeding site of this species. They were also attracted to the odor ofn-caproic acid which is contained in the ripe fruit ofM. citrifolia and is presumably responsible for the characteristic odor of the fruit. In contrast,D. simulans andD. melanogaster showed a strong repulsion ton-caproic acid. In parallel with the olfactory responses,D. sechellia females laid eggs preferentially on a medium containingn-caproic acid, to which the other two species showed an aversion. Genetic analyses using the hybrid progeny betweenD. sechellia andD. simulans suggested that the species differences in these behaviors are controlled by gene(s) located on the second chromosome.     

The Evolutionary History of DROSOPHILA BUZZATII. Xii. the Genetic Basis of Sterility in Hybrids between D. BUZZATII and Its Sibling D. SERIDO from Argentina

The genetic basis of hybrid sterility has been investigated in backcross segmental hybrids between two sibling species, Drosophila buzzatii and D. serido. Asynapsis of homologous bands in hybrid polytene chromosomes has been used to identify the D. serido chromosome segments introgressed into the D. buzzatti genome. All the investigated chromosomes contain male sterility factors. For autosomes, sterility is produced when an introgressed D. serido chromosome segment, or combination of segments, reaches a minimum size. On the other hand, any introgressed X chromosome segment from D. serido, irrespective of its size, produces either male hybrid sterility or inviability.     

EPISTASIS MODIFIES THE DOMINANCE OF LOCI CAUSING HYBRID MALE STERILITY IN THE DROSOPHILA PSEUDOOBSCURA SPECIES GROUP

Speciation, the evolution of reproductive isolation between populations, serves as the driving force for generating biodiversity. Postzygotic barriers to gene flow, such as F ₁ hybrid sterility and inviability, play important roles in the establishment and maintenance of biological species. F ₁ hybrid incompatibilities in taxa that obey Haldane's rule, the observation that the heterogametic sex suffers greater hybrid fitness problems than the homogametic sex, are thought to often result from interactions between recessive-acting X-linked loci and dominant-acting autosomal loci. Because they play such prominent roles in producing hybrid incompatibilities, we examine the dominance and nature of epistasis between alleles derived from Drosophila persimilis that confer hybrid male sterility in the genetic background of its sister species, D. pseudoobscura bogotana . We show that epistasis elevates the apparent dominance of individually recessive-acting QTL such that they can contribute to F ₁ hybrid sterility. These results have important implications for assumptions underlying theoretical models of hybrid incompatibilities and may offer a possible explanation for why, to date, identification of dominant-acting autosomal "speciation genes" has been challenging.     

Functional divergence caused by ancient positive selection of a Drosophila hybrid incompatibility locus

Interspecific hybrid lethality and sterility are a consequence of divergent evolution between species and serve to maintain the discrete identities of species. The evolution of hybrid incompatibilities has been described in widely accepted models by Dobzhansky and Muller where lineage-specific functional divergence is the essential characteristic of hybrid incompatibility genes. Experimentally tractable models are required to identify and test candidate hybrid incompatibility genes. Several Drosophila melanogaster genes involved in hybrid incompatibility have been identified but none has yet been shown to have functionally diverged in accordance with the Dobzhansky-Muller model. By introducing transgenic copies of the X-linked Hybrid male rescue (Hmr) gene into D. melanogaster from its sibling species D. simulans and D. mauritiana, we demonstrate that Hmr has functionally diverged to cause F1 hybrid incompatibility between these species. Consistent with the Dobzhansky-Muller model, we find that Hmr has diverged extensively in the D. melanogaster lineage, but we also find extensive divergence in the sibling-species lineage. Together, these findings implicate over 13% of the amino acids encoded by Hmr as candidates for causing hybrid incompatibility. The exceptional level of divergence at Hmr cannot be explained by neutral processes because we use phylogenetic methods and population genetic analyses to show that the elevated amino-acid divergence in both lineages is due to positive selection in the distant past—at least one million generations ago. Our findings suggest that multiple substitutions driven by natural selection may be a general phenomenon required to generate hybrid incompatibility alleles.     

Association of Misexpression with Sterility in Hybrids of <Emphasis Type="Italic">Drosophila simulans</Emphasis>and <Emphasis Type="Italic">D. mauritiana</Emphasis>

Recent studies have identified genes associated with hybrid sterility and other hybrid dysfunctions, but the consequences of introgressions of these speciation genes are often poorly understood. Previously, we identified a panel of genes that are underexpressed in sterile male hybrids of Drosophila simulans and D. mauritiana relative to pure species. Here, we build on this reverse-genetics approach to demonstrate that the underexpression of at least five of these genes in hybrids is associated with hybrid sterility and that these five genes are coordinately regulated. We map one upstream regulator of these genes to a region previously shown to harbor one or more factors causing hybrid sterility. Finally, we show that the genes underexpressed in hybrids are often highly conserved, as might be predicted for downstream targets of the genetic changes that cause hybrid sterility. This approach integrates forward genetics with reverse genetics to show a proximate consequence of the introgression of particular hybrid sterility-conferring regions between species: underexpression of genes necessary for normal spermatogenesis.[Reviewing Editor: Martin Kreitman]     

A Locus in Drosophila sechellia Affecting Tolerance of a Host Plant Toxin

Many insects feed on only one or a few types of host. These host specialists often evolve a preference for chemical cues emanating from their host and develop mechanisms for circumventing their host’s defenses. Adaptations like these are central to evolutionary biology, yet our understanding of their genetics remains incomplete. Drosophila sechellia, an emerging model for the genetics of host specialization, is an island endemic that has adapted to chemical toxins present in the fruit of its host plant, Morinda citrifolia. Its sibling species, D. simulans, and many other Drosophila species do not tolerate these toxins and avoid the fruit. Earlier work found a region with a strong effect on tolerance to the major toxin, octanoic acid, on chromosome arm 3R. Using a novel assay, we narrowed this region to a small span near the centromere containing 18 genes, including three odorant binding proteins. It has been hypothesized that the evolution of host specialization is facilitated by genetic linkage between alleles contributing to host preference and alleles contributing to host usage, such as tolerance to secondary compounds. We tested this hypothesis by measuring the effect of this tolerance locus on host preference behavior. Our data were inconsistent with the linkage hypothesis, as flies bearing this tolerance region showed no increase in preference for media containing M. citrifolia toxins, which D. sechellia prefers. Thus, in contrast to some models for host preference, preference and tolerance are not tightly linked at this locus nor is increased tolerance per se sufficient to change preference. Our data are consistent with the previously proposed model that the evolution of D. sechellia as a M. citrifolia specialist occurred through a stepwise loss of aversion and gain of tolerance to M. citrifolia’s toxins.     

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.