Natural variation for a hybrid incompatibility between two species of Mimulus

Understanding the process by which hybrid incompatibility alleles become established in natural populations remains a major challenge to evolutionary biology. Previously, we discovered a two-locus Dobzhansky-Muller incompatibility that causes severe hybrid male sterility between two inbred lines of the incompletely isolated wildflower species, Mimulus guttatus and M. nasutus. An interspecific cross between these two inbred lines revealed that the M. guttatus (IM62) allele at hybrid male sterility 1 (hms1) acts dominantly in combination with recessive M. nasutus (SF5) alleles at hybrid male sterility 2 (hms2) to cause nearly complete hybrid male sterility. In this report, we extend these genetic analyses to investigate intraspecific variation for the hms1-hms2 incompatibility in natural populations of M. nasutus and M. guttatus, performing a series of interspecific crosses between individuals collected from a variety of geographic locales. Our results suggest that hms2 incompatibility alleles are common and geographically widespread within M. nasutus, but absent or rare in M. guttatus. In contrast, the hms1 locus is polymorphic within M. guttatus and the incompatibility allele appears to be extremely geographically restricted. We found evidence for the presence of the hms1 incompatibility allele in only two M. guttatus populations that exist within a few kilometers of each other. The restricted distribution of the hms1 incompatibility allele might currently limit the potential for the hms1-hms2 incompatibility to act as a species barrier between sympatric populations of M. guttatus and M. nasutus. Extensive sampling within a single M. guttatus population revealed that the hms1 locus is polymorphic and that the incompatibility allele appears to segregate at intermediate frequency, a pattern that is consistent with either genetic drift or natural selection.     

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.     

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.     

A simple genetic incompatibility causes hybrid male sterility in mimulus

Much evidence has shown that postzygotic reproductive isolation (hybrid inviability or sterility) evolves by the accumulation of interlocus incompatibilities between diverging populations. Although in theory only a single pair of incompatible loci is needed to isolate species, empirical work in Drosophila has revealed that hybrid fertility problems often are highly polygenic and complex. In this article we investigate the genetic basis of hybrid sterility between two closely related species of monkeyflower, Mimulus guttatus and M. nasutus. In striking contrast to Drosophila systems, we demonstrate that nearly complete hybrid male sterility in Mimulus results from a simple genetic incompatibility between a single pair of heterospecific loci. We have genetically mapped this sterility effect: the M. guttatus allele at the hybrid male sterility 1 (hms1) locus acts dominantly in combination with recessive M. nasutus alleles at the hybrid male sterility 2 (hms2) locus to cause nearly complete hybrid male sterility. In a preliminary screen to find additional small-effect male sterility factors, we identified one additional locus that also contributes to some of the variation in hybrid male fertility. Interestingly, hms1 and hms2 also cause a significant reduction in hybrid female fertility, suggesting that sex-specific hybrid defects might share a common genetic basis. This possibility is supported by our discovery that recombination is reduced dramatically in a cross involving a parent with the hms1-hms2 incompatibility.     

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.     

Leaf shape evolution has a similar genetic architecture in three edaphic specialists within the Mimulus guttatus species complex

Background and Aims The genetic basis of leaf shape has long interested botanists because leaf shape varies extensively across the plant kingdom and this variation is probably adaptive. However, knowledge of the genetic architecture of leaf shape variation in natural populations remains limited. This study examined the genetic architecture of leaf shape diversification among three edaphic specialists in the Mimulus guttatus species complex. Lobed and narrow leaves have evolved from the entire, round leaves of M. guttatus in M. laciniatus, M. nudatus and a polymorphic serpentine M. guttatus population (M2L).Methods Bulk segregant analysis and next-generation sequencing were used to map quantitative trait loci (QTLs) that underlie leaf shape in an M. laciniatus × M. guttatus F₂ population. To determine whether the same QTLs contribute to leaf shape variation in M. nudatus and M2L, F₂s from M. guttatus × M. nudatus and lobed M2L × unlobed M. guttatus crosses were genotyped at QTLs from the bulk segregant analysis.Key Results Narrow and lobed leaf shapes in M. laciniatus, M. nudatus and M. guttatus are controlled by overlapping genetic regions. Several promising leaf shape candidate genes were found under each QTL.Conclusions The evolution of divergent leaf shape has taken place multiple times in the M. guttatus species complex and is associated with the occupation of dry, rocky environments. The genetic architecture of elongated and lobed leaves is similar across three species in this group. This may indicate that parallel genetic evolution from standing variation or new mutations is responsible for the putatively adaptive leaf shape variation in Mimulus.     

Analysis of ancestry heterozygosity suggests that hybrid incompatibilities in threespine stickleback are environment dependent

Hybrid incompatibilities occur when interactions between opposite ancestry alleles at different loci reduce the fitness of hybrids. Most work on incompatibilities has focused on those that are “intrinsic,” meaning they affect viability and sterility in the laboratory. Theory predicts that ecological selection can also underlie hybrid incompatibilities, but tests of this hypothesis using sequence data are scarce. In this article, we compiled genetic data for F₂ hybrid crosses between divergent populations of threespine stickleback fish (Gasterosteus aculeatus L.) that were born and raised in either the field (seminatural experimental ponds) or the laboratory (aquaria). Because selection against incompatibilities results in elevated ancestry heterozygosity, we tested the prediction that ancestry heterozygosity will be higher in pond-raised fish compared to those raised in aquaria. We found that ancestry heterozygosity was elevated by approximately 3% in crosses raised in ponds compared to those raised in aquaria. Additional analyses support a phenotypic basis for incompatibility and suggest that environment-specific single-locus heterozygote advantage is not the cause of selection on ancestry heterozygosity. Our study provides evidence that, in stickleback, a coarse—albeit indirect—signal of environment-dependent hybrid incompatibility is reliably detectable and suggests that extrinsic incompatibilities can evolve before intrinsic incompatibilities.

This study shows that hybrid incompatibilities between two independent pairs of hybridizing stickleback populations only appear under relevant ecological circumstances, implying that incompatibilities evolve before they can be detected in laboratory studies of speciation.     

Speciation genes in plants

Background

Analyses of speciation genes – genes that contribute to the cessation of gene flow between populations – can offer clues regarding the ecological settings, evolutionary forces and molecular mechanisms that drive the divergence of populations and species. This review discusses the identities and attributes of genes that contribute to reproductive isolation (RI) in plants, compares them with animal speciation genes and investigates what these genes can tell us about speciation.

Scope

Forty-one candidate speciation genes were identified in the plant literature. Of these, seven contributed to pre-pollination RI, one to post-pollination, prezygotic RI, eight to hybrid inviability, and 25 to hybrid sterility. Genes, gene families and genetic pathways that were frequently found to underlie the evolution of RI in different plant groups include the anthocyanin pathway and its regulators (pollinator isolation), S RNase-SI genes (unilateral incompatibility), disease resistance genes (hybrid necrosis), chimeric mitochondrial genes (cytoplasmic male sterility), and pentatricopeptide repeat family genes (cytoplasmic male sterility).

Conclusions

The most surprising conclusion from this review is that identities of genes underlying both prezygotic and postzygotic RI are often predictable in a broad sense from the phenotype of the reproductive barrier. Regulatory changes (both cis and trans) dominate the evolution of pre-pollination RI in plants, whereas a mix of regulatory mutations and changes in protein-coding genes underlie intrinsic postzygotic barriers. Also, loss-of-function mutations and copy number variation frequently contribute to RI. Although direct evidence of positive selection on speciation genes is surprisingly scarce in plants, analyses of gene family evolution, along with theoretical considerations, imply an important role for diversifying selection and genetic conflict in the evolution of RI. Unlike in animals, however, most candidate speciation genes in plants exhibit intraspecific polymorphism, consistent with an important role for stochastic forces and/or balancing selection in development of RI in plants.Key words: Speciation, reproductive isolation, mating system isolation, pollinator isolation, ecological isolation, unilateral incompatibility, hybrid necrosis, hybrid sterility, hybrid inviability, hybrid breakdown, cytoplasmic male sterility, restoration     

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.     

Genetic architecture of hybrid male sterility in Drosophila: analysis of intraspecies variation for interspecies isolation

Background

The genetic basis of postzygotic isolation is a central puzzle in evolutionary biology. Evolutionary forces causing hybrid sterility or inviability act on the responsible genes while they still are polymorphic, thus we have to study these traits as they arise, before isolation is complete.

Methodology/Principal Findings

Isofemale strains of D. mojavensis vary significantly in their production of sterile F₁ sons when females are crossed to D. arizonae males. We took advantage of the intraspecific polymorphism, in a novel design, to perform quantitative trait locus (QTL) mapping analyses directly on F₁ hybrid male sterility itself. We found that the genetic architecture of the polymorphism for hybrid male sterility (HMS) in the F₁ is complex, involving multiple QTL, epistasis, and cytoplasmic effects.

Conclusions/Significance

The role of extensive intraspecific polymorphism, multiple QTL, and epistatic interactions in HMS in this young species pair shows that HMS is arising as a complex trait in this system. Directional selection alone would be unlikely to maintain polymorphism at multiple loci, thus we hypothesize that directional selection is unlikely to be the only evolutionary force influencing postzygotic isolation.     

Postzygotic isolation involves strong mitochondrial and sex-specific effects in Tigriopus californicus,a species lacking heteromorphic sex chromosomes

Detailed studies of the genetics of speciation have focused on a few model systems, particularly Drosophila. The copepod Tigriopus californicus offers an alternative that differs from standard animal models in that it lacks heteromorphic chromosomes (instead, sex determination is polygenic) and has reduced opportunities for sexual conflict, because females mate only once. Quantitative trait loci (QTL) mapping was conducted on reciprocal F₂ hybrids between two strongly differentiated populations, using a saturated linkage map spanning all 12 autosomes and the mitochondrion. By comparing sexes, a possible sex ratio distorter was found but no sex chromosomes. Although studies of standard models often find an excess of hybrid male sterility factors, we found no QTL for sterility and multiple QTL for hybrid viability (indicated by non-Mendelian adult ratios) and other characters. Viability problems were found to be stronger in males, but the usual explanations for weaker hybrid males (sex chromosomes, sensitivity of spermatogenesis, sexual selection) cannot fully account for these male viability problems. Instead, higher metabolic rates may amplify deleterious effects in males. Although many studies of standard speciation models find the strongest genetic incompatibilities to be nuclear–nuclear (specifically X chromosome–autosome), we found the strongest deleterious interaction in this system was mito–nuclear. Consistent with the snowball theory of incompatibility accumulation, we found that trigenic interactions in this highly divergent cross were substantially more frequent (>6 × ) than digenic interactions. This alternative system thus allows important comparisons to studies of the genetics of reproductive isolation in more standard model systems.     

Adaptive basis of geographic variation: genetic,phenotypic and environmental differences among beach mouse populations

A major goal in evolutionary biology is to understand how and why populations differentiate, both genetically and phenotypically, as they invade a novel habitat. A classical example of adaptation is the pale colour of beach mice, relative to their dark mainland ancestors, which colonized the isolated sandy dunes and barrier islands on Florida''s Gulf Coast. However, much less is known about differentiation among the Gulf Coast beach mice, which comprise five subspecies linearly arrayed on Florida''s shoreline. Here, we test the role of selection in maintaining variation among these beach mouse subspecies at multiple levels—phenotype, genotype and the environments they inhabit. While all beach subspecies have light pelage, they differ significantly in colour pattern. These subspecies are also genetically distinct: pair-wise F_st-values range from 0.23 to 0.63 and levels of gene flow are low. However, we did not find a correlation between phenotypic and genetic distance. Instead, we find a significant association between the average ‘lightness’ of each subspecies and the brightness of the substrate it inhabits: the two most genetically divergent subspecies occupy the most similar habitats and have converged on phenotype, whereas the most genetically similar subspecies occupy the most different environments and have divergent phenotypes. Moreover, allelic variation at the pigmentation gene, Mc1r, is statistically correlated with these colour differences but not with variation at other genetic loci. Together, these results suggest that natural selection for camouflage—via changes in Mc1r allele frequency—contributes to pigment differentiation among beach mouse subspecies.     

A test of genetic models for the evolutionary maintenance of same-sex sexual behaviour

The evolutionary maintenance of same-sex sexual behaviour (SSB) has received increasing attention because it is perceived to be an evolutionary paradox. The genetic basis of SSB is almost wholly unknown in non-human animals, though this is key to understanding its persistence. Recent theoretical work has yielded broadly applicable predictions centred on two genetic models for SSB: overdominance and sexual antagonism. Using Drosophila melanogaster, we assayed natural genetic variation for male SSB and empirically tested predictions about the mode of inheritance and fitness consequences of alleles influencing its expression. We screened 50 inbred lines derived from a wild population for male–male courtship and copulation behaviour, and examined crosses between the lines for evidence of overdominance and antagonistic fecundity selection. Consistent variation among lines revealed heritable genetic variation for SSB, but the nature of the genetic variation was complex. Phenotypic and fitness variation was consistent with expectations under overdominance, although predictions of the sexual antagonism model were also supported. We found an unexpected and strong paternal effect on the expression of SSB, suggesting possible Y-linkage of the trait. Our results inform evolutionary genetic mechanisms that might maintain low but persistently observed levels of male SSB in D. melanogaster, but highlight a need for broader taxonomic representation in studies of its evolutionary causes.     

Sex Ratio Meiotic Drive as a Plausible Evolutionary Mechanism for Hybrid Male Sterility

Biological diversity on Earth depends on the multiplication of species or speciation, which is the evolution of reproductive isolation such as hybrid sterility between two new species. An unsolved puzzle is the exact mechanism(s) that causes two genomes to diverge from their common ancestor so that some divergent genes no longer function properly in the hybrids. Here we report genetic analyses of divergent genes controlling male fertility and sex ratio in two very young fruitfly species, Drosophila albomicans and D. nasuta. A majority of the genetic divergence for both traits is mapped to the same regions by quantitative trait loci mappings. With introgressions, six major loci are found to contribute to both traits. This genetic colocalization implicates that genes for hybrid male sterility have evolved primarily for controlling sex ratio. We propose that genetic conflicts over sex ratio may operate as a perpetual dynamo for genome divergence. This particular evolutionary mechanism may largely contribute to the rapid evolution of hybrid male sterility and the disproportionate enrichment of its underlying genes on the X chromosome – two patterns widely observed across animals.     

EVIDENCE FOR DOBZHANSKY-MULLER INCOMPATIBILITES CONTRIBUTING TO THE STERILITY OF HYBRIDS BETWEEN MIMULUS GUTTATUS AND M. NASUTUS

Abstract Both chromosomal rearrangements and negative interactions among loci (Dobzhansky‐Muller incompatibilities) have been advanced as the genetic mechanism underlying the sterility of interspecific hybrids. These alternatives invoke very different evolutionary histories during speciation and also predict different patterns of sterility in artificial hybrids. Chromosomal rearrangements require drift, inbreeding, or other special conditions for initial fixation and, because heterozygosity per se generates any problems with gamete formation, F1 hybrids will be most infertile. In contrast, Dobzhansky‐Muller incompatibilities may arise as byproducts of adaptive evolution and often affect the segregating F2 generation most severely. To distinguish the effects of these two mechanisms early in divergence, we investigated the quantitative genetics of hybrid sterility in a line cross between two members of the Mimulus guttatus species complex (M. guttatus and M. nasutus). Hybrids showed partial male and female sterility, and the patterns of infertility were not consistent with the action of chromosomal rearrangements alone. F2 and F1 hybrids exhibited equal decreases in pollen viability (> 40%) relative to the highly fertile parental lines. A large excess of completely pollen‐sterile F2 genotypes also pointed to the segregation of Dobzhansky‐Muller incompatibility factors affecting male fertility. Female fertility showed a pattern similarly consistent with epistatic interactions: F2 hybrids produced far fewer seeds per flower than F1 hybrids (88.0 ± 2.8 vs. 162.9 ± 8.5 SE, respectively) and either parental line, and many F2 genotypes were completely female sterile. Dobzhansky‐Muller interactions also resulted in the breakdown of several nonreproductive characters and appear to contribute to correlations between male and female fertility in the F2 generation. These results parallel and contrast with the genetics of postzygotic isolation in model animal systems and are a first step toward understanding the process of speciation in this well‐studied group of flowering plants.     

Pervasive genetic associations between traits causing reproductive isolation in Heliconius butterflies

Ecological speciation proceeds through the accumulation of divergent traits that contribute to reproductive isolation, but in the face of gene flow traits that characterize incipient species may become disassociated through recombination. Heliconius butterflies are well known for bright mimetic warning patterns that are also used in mate recognition and cause both pre- and post-mating isolation between divergent taxa. Sympatric sister taxa representing the final stages of speciation, such as Heliconius cydno and Heliconius melpomene, also differ in ecology and hybrid fertility. We examine mate preference and sterility among offspring of crosses between these species and demonstrate the clustering of Mendelian colour pattern loci and behavioural loci that contribute to reproductive isolation. In particular, male preference for red patterns is associated with the locus responsible for the red forewing band. Two further colour pattern loci are associated, respectively, with female mating outcome and hybrid sterility. This genetic architecture in which ‘speciation genes’ are clustered in the genome can facilitate two controversial models of speciation, namely divergence in the face of gene flow and hybrid speciation.     

Hitchhiking to Speciation

The modern evolutionary synthesis codified the idea that species exist as distinct entities because intrinsic reproductive barriers prevent them from merging together. Understanding the origin of species therefore requires understanding the evolution and genetics of reproductive barriers between species. In most cases, speciation is an accident that happens as different populations adapt to different environments and, incidentally, come to differ in ways that render them reproductively incompatible. As with other reproductive barriers, the evolution and genetics of interspecific hybrid sterility and lethality were once also thought to evolve as pleiotripic side effects of adaptation. Recent work on the molecular genetics of speciation has raised an altogether different possibility—the genes that cause hybrid sterility and lethality often come to differ between species not because of adaptation to the external ecological environment but because of internal evolutionary arms races between selfish genetic elements and the genes of the host genome. Arguably one of the best examples supporting a role of ecological adaptation comes from a population of yellow monkey flowers, Mimulus guttatus, in Copperopolis, California, which recently evolved tolerance to soil contaminants from copper mines and simultaneously, as an incidental by-product, hybrid lethality in crosses with some off-mine populations. However, in new work, Wright and colleagues show that hybrid lethality is not a pleiotropic consequence of copper tolerance. Rather, the genetic factor causing hybrid lethality is tightly linked to copper tolerance and spread to fixation in Copperopolis by genetic hitchhiking.New species arise when populations gradually evolve intrinsic reproductive barriers to interbreeding with other populations [1]–[3]. Two species can be reproductively isolated from one another in ways that prevent the formation of interspecific hybrids—the species may, for instance, have incompatible courtship signals or occupy different ecological habitats. Two species can also be reproductively isolated from one another if interspecific hybrids are formed but are somehow unfit—the hybrids may be sterile, inviable, or may simply fall between parental ecological niches. All forms of reproductive isolation limit the genetic exchange between species, preventing their fusion and facilitating their further divergence. Understanding the genetic and evolutionary basis of speciation—a major cause of biodiversity—therefore involves understanding the genetics and evolutionary basis of the traits that mediate reproductive isolation.Most reproductive barriers arise as incidental by-products of selection—either ecological adaptation or sexual selection. For these cases, the genetic basis of speciation is, effectively, the genetics of adaptation. But hybrid sterility and lethality have historically posed two special problems. Darwin [4] devoted an entire chapter of his Origin of Species to the first problem: as the sterility or lethality of hybrids provides no advantage to parents, how could the genetic factors involved possibly evolve by natural selection? The second problem was recognized much later [5], after the rediscovery of Mendelian genetics: if two species (with genotypes AA and aa) produce, say, sterile hybrids (Aa) due to an incompatibility between the A and a alleles, then how could, e.g., the AA genotype have evolved from an aa ancestor in the first place without passing through a sterile intermediate genotype (Aa)? Not only does natural selection not directly favor the evolution of hybrid sterility or lethality, but there is reason to believe natural selection positively prevents its evolution.Together these problems stymied evolutionists and geneticists for decades. T.H. Huxley [6] and William Bateson [5], writing decades apart, each branded the evolution of hybrid sterility one of the most serious challenges for a then-young evolutionary theory. Darwin had, in fact, offered a simple solution to the first problem. Namely, hybrid sterility and lethality are not advantageous per se but rather “incidental on other acquired differences" [4]. Then Bateson [5], in a few short, forgotten lines solved the second problem (see [7]). Later, Dobzhansky [2] and Muller [8] would arrive at the same solution, showing that hybrid sterility or lethality could evolve readily, unopposed by natural selection, under a two-locus model with epistasis. In particular, they imagined that separate populations diverge from a common ancestor (genotype aabb), with the A allele becoming established in one population (AAbb) and the B allele in the other (aaBB); while A and B alleles must function on their respective genetic backgrounds, there is no guarantee that the A and B alleles will be functionally compatible with one another. Hybrid sterility and lethality most likely result from incompatible complementary genetic factors that disrupt development when brought together in a common hybrid genome. Dobzhansky [2] and Muller [8] could point to a few supporting data in fish, flies, and plants. Notably, like Darwin, neither speculated on the forces responsible for the evolution of the genetic factors involved.Today, there is no doubt that the Dobzhansky-Muller model is correct, as the data for incompatible complementary genetic factors is now overwhelming [1],[9]. In the last decade, a fast-growing number of speciation genes involved in these genetic incompatibilities have been identified in mice, fish, flies, yeast, and plants [9]–[11]. Perhaps not surprisingly, these speciation genes often have histories of recurrent, adaptive protein-coding sequence evolution [10],[11]. The signature of selection at speciation genes has been taken by some as tacit evidence for the pervasive role of ecological adaptation in speciation, including the evolution of hybrid sterility and lethality [12]. What is surprising, however, from the modern molecular analysis of speciation genes is how often their rapid sequence evolution and functional divergence seems to have little to do with adaptation to external ecological circumstances. Instead, speciation genes often (but not always [9]–[11]) seem to evolve as by-products of evolutionary arms races between selfish genetic elements—e.g., satellite DNAs [13],[14], meiotic drive elements [15], cytoplasmic male sterility factors [16]—and the host genes that regulate or suppress them [9]–[11],[17]. The notion that selfish genes are exotic curiosities is now giving way to a realization that selfish genes are common and diverse, each generation probing for transmission advantages at the expense of their bearers, fueling evolutionary arms races and, not infrequently, contributing to the genetic divergence that drives speciation. Indeed, the case has become so strong that examples of hybrid sterility and lethality genes that have evolved in response to ecological challenges (other than pathogens) appear to be the exception [9],[11],[17].Perhaps the most clear-cut case in which a genetic incompatibility seems to have evolved as a by-product of ecological adaptation comes from populations of the yellow monkey flower, Mimulus guttatus, from Copperopolis (California, U.S.A.). In the last ∼150 years, the Copperopolis population has evolved tolerance to the tailings of local copper mines (Figure 1). These copper-tolerant M. guttatus plants also happen to be partially reproductively isolated from many off-mine M. guttatus plants, producing hybrids that suffer tissue necrosis and death. In classic work, Macnair and Christie showed that copper tolerance is controlled by a single major factor [18] and hybrid lethality, as expected under the Dobzhansky-Muller model, by complementary factors [19]. Surprisingly, in crosses between tolerant and nontolerant plants, hybrid lethality perfectly cosegregates with tolerance [19],[20]. The simplest explanation is that the copper tolerance allele that spread to fixation in the Copperopolis population also happens to cause hybrid lethality as a pleiotropic by-product. The alternative explanation is that the copper tolerance and hybrid lethality loci happen to be genetically linked; when the copper tolerance allele spread to fixation in Copperopolis, hybrid lethality hitchhiked to high frequency along with it [20]. But with 2n = 28 chromosomes, the odds that copper tolerance and hybrid lethality alleles happen to be linked would seem vanishingly small [20].Open in a separate windowFigure 1Yellow monkey flowers (Mimulus guttatus) growing in the heavy-metal contaminated soils of copper-mine tailings.In this issue, Wright and colleagues [21] revisit this classic case of genetic incompatibility as a by-product of ecological adaptation. They make two discoveries, one genetic and the other evolutionary. By conducting extensive crossing experiments and leveraging the M. guttatus genome sequence (www.mimulusevolution.org), Wright et al. [21] map copper tolerance and hybrid necrosis to tightly linked but genetically separable loci, Tol1 and Nec1, respectively. Hybrid lethality is not a pleiotropic consequence of copper tolerance. Instead, the tolerant Tol1 allele spread to fixation in Copperopolis, and the tightly linked incompatible Nec1 allele spread with it by genetic hitchhiking. In a turn of bad luck, the loci happen to fall in a heterochromatic pericentric region, where genome assemblies are often problematic, putting identification of the Tol1 and Nec1 genes out of immediate reach. Wright et al. [21] were, however, able to identify linked markers within ∼0.3 cM of Tol1 and place Nec1 within a 10-kb genomic interval that contains a Gypsy3 retrotransposon, raising two possibilities. First, the Gypsy3 element is unlikely to cause hybrid lethality directly; instead, as transposable elements are often epigenetically silenced in plants, it seems possible that the Nec1-associated Gypsy3 is silenced with incidental consequences for gene expression on a gene (or genes) in the vicinity [22]. Second, although the Nec1 interval is 10-kb in the reference genome of M. guttatus, it could be larger in the (not-yet-sequenced) Copperopolis population, perhaps harboring additional genes.With Tol1 and Nec1 mapped near and to particular genomic scaffolds, respectively, Wright et al. were able to investigate the evolutionary history of the genomic region. Given the clear adaptive significance of copper tolerance in Copperopolis plants, we might expect to see the signatures of a strong selective sweep in the Tol1 region—a single Tol1 haplotype may have spread to fixation so quickly that all Copperopolis descendant plants bear the identical haplotype and thus show strongly reduced population genetic variability in the Tol1-Nec1 region relative to the rest of the genome [23],[24]. After the selective sweep is complete, variability in the region ought to recover gradually as new mutations arise and begin to fill out the mutation-drift equilibrium frequency spectrum expected for neutral variation in the Copperopolis population [25],[26]. Given that Tol1 reflects an adaptation to mine tailings established just ∼150 generations ago, there would have been little time for such a recovery. And yet, while Wright et al. find evidence of moderately reduced genetic variability in the Tol1-Nec1 genomic region, the magnitude of the reduction is hardly dramatic relative to the genome average.How, then, is it possible that the Tol1-Nec1 region swept to fixation in Copperopolis in fewer than ∼150 generations and yet left no strong footprint of a hitchhiking event? One possibility is that rather than a single, unique Tol1-Nec1 haplotype contributing to fixation, causing a “hard sweep," multiple Tol1-Nec1 haplotypes sampled from previously standing genetic variation contributed to fixation, causing a “soft sweep" [27]. A soft sweep would be plausible if Tol1 and Nec1 both segregate in the local off-mine ancestral population and if the two were, coincidentally, found on the same chromosome more often than expected by chance (i.e., in linkage disequilibrium). Then, after the copper mines were established, multiple plants with multiple Tol1 haplotypes (and, by association, Nec1) could have colonized the newly contaminated soils of the mine tailings. Tol1 segregates at ∼9% in surrounding populations, suggesting that standing genetic variation for copper tolerance may well have been present in the ancestral populations.Two big questions remain for the Tol1-Nec1 story, and both would be readily advanced by identification of Tol1 and Nec1. The first question concerns the history of Tol1 haplotypes in Copperopolis and surrounding off-mine populations. As Nec1-mediated hybrid lethality is incomplete, the ∼9% Tol1 frequency in surrounding populations could reflect its export via gene flow from the Copperopolis populations. Conversely, if there was a soft sweep from standing Tol1 variation in surrounding off-mine populations, then Tol1 and Nec1 may still be in linkage disequilibrium in those populations (assuming ∼150 years of recombination has not broken up the association). Resolving these alternative possibilities is a matter of establishing the history of movement of Tol1 haplotypes into or out of the Copperopolis population. The soft sweep scenario, if correct, presents a population genetics puzzle: during the historical time that mutations accumulated among the multiple tolerant but incompatible Tol1-Nec1 haplotypes in the ancestral off-mine populations, why did recombination fail to degrade the association, giving rise to tolerant but compatible haplotypes?The second question concerns the identity of Nec1 (or if it really is a Gypsy3 element, the identity of the nearby gene whose expression is disrupted as a consequence). The answer bears on one of the new emerging generalizations about genetic incompatibilities in plants [9]. Recently, Bomblies and Weigel [28] synthesized a century''s worth of observations on the commonly seen necrosis phenotype in plant hybrids and, based on their own genetic analyses in Arabidopsis [29], suggested that many of these cases may have a common underlying basis: incompatibilities between plant pathogen resistance genes can cause autoimmune responses that result in tissue necrosis and hybrid lethality. Hybrid necrosis, indeed, appears to involve pathogen resistance genes across multiple plants groups [9],[28]. It remains to be seen if the Nec1-mediated lethality provides yet another instance.