A TEST FOR SEXUAL SELECTION ON HYBRIDS OF TWO SYMPATRIC STICKLEBACKS

In this study we assessed whether sexual selection against hybrids contributes to reproductive isolation between two sympatric stickleback species. The species are recently diverged and possibly in the final stages of speciation. Our aim was to find whether mating discrimination of the parental species selects against F₁ hybrids, and what conditions are necessary for such sexual selection to operate. We used conservative no-choice laboratory trials with reproductively naive, lab-reared fish to measure female mating preferences. Females exhibited ranked preferences, preferring in order: conspecific, hybrid, then heterospecific males. However, intermediate attractiveness does not necessarily imply selection against hybrids: two-way ANOVAs suggested that limnetic, benthic, and hybrid males were statistically equivalent when averaged across females. Thus, this experiment found no evidence for a hybrid mating disadvantage. Our interpretation is that if sexual selection against hybrids is present in the wild, then some factor that biases encounter rates between hybrids and parental species (e.g., habitat selection) is necessary to produce it.     

ECOLOGICAL SPECIATION IN STICKLEBACKS: ENVIRONMENT-DEPENDENT HYBRID FITNESS

“Ecological” speciation occurs when reproductive isolation evolves as a consequence of divergent selection between populations exploiting different resources or environments. We tested this hypothesis of speciation in a young stickleback species pair by measuring the direct contribution of ecological selection pressures to hybrid fitness. The two species (limnetic and benthic) are strongly differentiated morphologically and ecologically, whereas hybrids are intermediate. Fitness of hybrids is high in the laboratory, especially F₁ and F₂ hybrids (backcrosses may show some breakdown). We transplanted F₁ hybrids to enclosures in the two main habitats in the wild to test whether the distribution of resources available in the environment generates a hybrid disadvantage not detectable in the laboratory. Hybrids grew more slowly than limnetics in the open water habitat and more slowly than benthics in the littoral zone. Growth of F₁ hybrids was inferior to the average of the parent species across both habitats, albeit not significantly. The contrast between laboratory and field results supports the hypothesis that mechanisms of F₁ hybrid fitness in the wild are primarily ecological and do not result from intrinsic genetic incompatibilities. Direct selection on hybrids contributes to the maintenance of sympatric stickleback species and may have played an important role in their origin.     

Sexual Selection, Epistasis and Species Boundaries in Sympatric Hawaiian Picture-winged Drosophila

The Hawaiian picture-winged flies in the genus Drosophila are a spectacular example of rapid evolutionary diversification in which sexual selection is considered an important mechanism for reproductive isolation and speciation. We investigated the behavioral reproductive isolation of two closely related and sympatric Hawaiian picture-winged Drosophila species, D. silvestris and D. heteroneura, which are known to hybridize in nature and produce viable and fertile hybrids. We compared the mating success of parental, F1 and backcross males in pairings with D. heteroneura females. The F1 males were produced by mating D. heteroneura males with D. silvestris females, and the backcross males were produced by mating F1 females with D. heteroneura males. The mating success of backcross males paired with D. heteroneura females were significantly reduced relative to that of parental and F1 males. This reduced mating success occurred primarily at a late stage of courtship where female choice of mate may be important. Two- and three-gene models demonstrate that epistasis involving a few genes could account for the observed variation in male mating success. These results are consistent with negative epistasis in the backcross generation and support the importance of sexual selection and negative epistasis in the evolution and maintenance of these species.     

A test of hybrid growth disadvantage in wild, free-ranging species pairs of threespine stickleback (Gasterosteus aculeatus) and its implications for ecological speciation

Ecological speciation is the evolution of reproductive isolation as a direct or indirect consequence of divergent natural selection. Reduced performance of hybrids in nature is thought to be an important process by which natural selection can favor the evolution of assortative mating and drive speciation. Benthic and limnetic sympatric species of threespine stickleback (Gasterosteus aculeatus) are adapted to alternative trophic niches (bottom browsing vs. open water planktivory, respectively) and reduced feeding performance of hybrids is thought to have contributed to the evolution of reproductive isolation. We tested this “hybrid‐disadvantage hypothesis” by inferring growth rates from otoliths sampled from wild, free‐ranging benthic, limnetic, and hybrid sticklebacks in two lakes. There were significant differences in growth rate between lakes, life‐history stages, and among years (maximum P = 0.02), as well as interactions between most factors, but not between hybrid and parental species sticklebacks in most comparisons. Our results provide little evidence of a growth disadvantage in hybrid sticklebacks when free‐ranging in nature. Although trophic ecology per se may contribute less to ecological speciation than envisioned, it may act in concert with other aspects of stickleback biology, such as interactions with parasites, predators, competitors, and/or sexual selection, to present strong multifarious selection against hybrids.     

Disruptive sexual selection against hybrids contributes to speciation between Heliconius cydno and Heliconius melpomene

Understanding the fate of hybrids in wild populations is fundamental to understanding speciation. Here we provide evidence for disruptive sexual selection against hybrids between Heliconius cydno and Heliconius melpomene. The two species are sympatric across most of Central and Andean South America, and coexist despite a low level of hybridization. No-choice mating experiments show strong assortative mating between the species. Hybrids mate readily with one another, but both sexes show a reduction in mating success of over 50% with the parental species. Mating preference is associated with a shift in the adult colour pattern, which is involved in predator defence through Müllerian mimicry, but also strongly affects male courtship probability. The hybrids, which lie outside the curve of protection afforded by mimetic resemblance to the parental species, are also largely outside the curves of parental mating preference. Disruptive sexual selection against F(1) hybrids therefore forms an additional post-mating barrier to gene flow, blurring the distinction between pre-mating and post-mating isolation, and helping to maintain the distinctness of these hybridizing species.     

Behavioral reproductive isolation inDrosophila silvestris,D. heteroneura,and their F1 hybrids (Diptera: Drosophilidae)

We investigated the role that courtship and aggressive interactions may have for the maintenance of reproductive isolation betweenDrosophila silvestris andD. heteroneura. We examined the behavioral bases of reproductive isolation between the parental species and we examined the courtship success of each sex of both reciprocal F₁ hybrids when paired with the parental species. We found reduced copulation success among heterotypic parental pairs compared to homotypic pairs, which was primarily due to the lack of courtship initiation betweenD. silvestris males andD. heteroneura females. When hybrid males from both reciprocal crosses were paired with parental females their copulation successes were not significantly different from that of parental males. In contrast, hybrid females from both crosses had reduced copulation success withD. silvestris males, which in turn was primarily due to a reduced success of reaching later stages of courtship. The time spent in copulation by hybrid males was intermediate between the two parental males. We studied aggression by observing the interactions of males of heterotypic pairs, both between the parental species and between the hybrids and parental males. A lack of aggressive interactions betweenD. silvestris males andD. heteroneura males in addition to the lack of courtship suggests thatD. silvestris males do not respond toD. heteroneura individuals of either sex. Hybrid males were equally successful in winning fights with bothD. silvestris andD. heteroneura males. These results indicate that the behavioral isolation betweenD. silvestris andD. heteroneura may be largely a consequence of the earliest stages of interactions. The two species may differ either in activity levels or in morphological or chemical traits that are important for species and mate recognition. The relatively high copulation and aggressive success of hybrids indicates that sexual selection against hybrids alone is unlikely to be a sufficient force to reduce gene flow and maintain species distinctions.     

Impact of experimental design on Drosophila sexual isolation studies: direct effects and comparison to field hybridization data

Abstract Many studies of speciation rely critically on estimates of sexual isolation obtained in the laboratory. Here we examine the sensitivity of sexual isolation to alterations in experimental design and mating environment in two sister species of Drosophila, D. santomea and D. yakuba. We use a newly devised measure of mating frequencies that is able to disentangle sexual isolation from species differences in mating propensity. Variation in fly density, presence or absence of a quasi‐natural environment, degree of starvation, and relative frequency of species had little or no effect on sexual isolation, but one factor did have a significant effect: the possibility of choice. Designs that allowed flies to choose between conspecific and heterospecific mates showed significantly more sexual isolation than other designs that did not allow choice. These experiments suggest that sexual isolation between these species (whose ranges overlap on the island of STo Tomé) is due largely to discrimination against D. yakuba males by D. santomea females. This suggestion was confirmed by direct observations of mating behavior. Drosophila santomea males also court D. yakuba females less ardently than conspecific females, whereas neither males nor females of D. yakuba show strong mate discrimination. Thus, sexual isolation appears to be a result of evolutionary changes in the derived island endemic D. santomea. Surprisingly, as reported in a companion paper (Llopart et al. 2005), the genotypes of hybrids found in nature do not accord with expectations from these laboratory studies: all F₁ hybrids in nature come from matings between D. santomea females and D. yakuba males, matings that occur only rarely in the laboratory.     

Female mate preferences for male body size and shape promote sexual isolation in threespine sticklebacks

Female mate preferences for ecologically relevant traits may enhance natural selection, leading to rapid divergence. They may also forge a link between mate choice within species and sexual isolation between species. Here, we examine female mate preference for two ecologically important traits: body size and body shape. We measured female preferences within and between species of benthic, limnetic, and anadromous threespine sticklebacks (Gasterosteus aculeatus species complex). We found that mate preferences differed between species and between contexts (i.e., within vs. between species). Within species, anadromous females preferred males that were deep bodied for their size, benthic females preferred larger males (as measured by centroid size), and limnetic females preferred males that were more limnetic shaped. In heterospecific mating trials between benthics and limnetics, limnetic females continued to prefer males that were more limnetic like in shape when presented with benthic males. Benthic females showed no preferences for size when presented with limnetic males. These results show that females use ecologically relevant traits to select mates in all three species and that female preference has diverged between species. These results suggest that sexual selection may act in concert with natural selection on stickleback size and shape. Further, our results suggest that female preferences may track adaptation to local environments and contribute to sexual isolation between benthic and limnetic sticklebacks.     

Predator experience overrides learned aversion to heterospecifics in stickleback species pairs

Predation risk can alter female mating decisions because the costs of mate searching and selecting attractive mates increase when predators are present. In response to predators, females have been found to plastically adjust mate preference within species, but little is known about how predators alter sexual isolation and hybridization among species. We tested the effects of predator exposure on sexual isolation between benthic and limnetic threespine sticklebacks (Gasterosteus spp.). Female discrimination against heterospecific mates was measured before and after females experienced a simulated attack by a trout predator or a control exposure to a harmless object. In the absence of predators, females showed increased aversion to heterospecifics over time. We found that predator exposure made females less discriminating and precluded this learned aversion to heterospecifics. Benthic and limnetic males differ in coloration, and predator exposure also affected sexual isolation by weakening female preferences for colourful males. Predator effects on sexual selection were also tested but predators had few effects on female choosiness among conspecific mates. Our results suggest that predation risk may disrupt the cognitive processes associated with mate choice and lead to fluctuations in the strength of sexual isolation between species.     

GENETICS OF SEXUAL ISOLATION AND COURTSHIP DYSFUNCTION IN MALE HYBRIDS OF DROSOPHILA PSEUDOOBSCURA AND DROSOPHILA PERSIMILIS

Despite the importance of sexual isolation to speciation, few studies have analyzed the genetic basis of interspecific mating discrimination, particularly using hybrid males. In this study, I investigated the genetic basis of sexual isolation using male hybrids of Drosophila pseudoobscura and D. persimilis. Hybrid male mating success was caused by interactions between the X-chromosome and autosomes (or Y-chromosome), and different arms of the X-chromosome contributed to mating success with females of each species. Further, although there was an X-chromosome component to mating success, its magnitude was not disproportionately large when compared with the proportion of the genome contained on this chromosome. Some hybrid males courted with an anomalously low intensity, so I simultaneously mapped the genetic basis of this “courtship dysfunction.” The courtship dysfunction was caused by an interaction between the left arm of the X-chromosome in D. persimilis with the autosomes or Y-chromosome from D. pseudoobscura. Anomalous courtship behavior in interspecific hybrids can obscure the conclusions of studies of the genetics of sexual isolation, so courtship intensity should be evaluated in all such investigations.     

Isolation mechanisms in closely related grasshopper species <Emphasis Type="Italic">Chorthippus albomarginatus</Emphasis> and <Emphasis Type="Italic">Ch. oschei</Emphasis> (Orthoptera: Acrididae)

Two closely related grasshopper species Chorthippus albomarginatus and Ch. oschei are known to hybridize in the narrow contact zone at the territory of Ukraine and Moldova. Different isolaton mechanisms providing reproductive isolation between the two species were studied. In choice mating experiments, females of the both species demonstrated a strong assortative mating (80–90% preference for the conspecific males). Comparison of the parental and hybrid viability revealed a reduced hatching and increased larval mortality in F₁ and F₂ hybrids. In choice mating experiments, the hybrid females mated less assortatively than the parental females. An assymmetry was found in mating preferences and in viability of hybrids. The results demonstrate the existence of pre-and post-mating isolation between Ch. albomarginatus and Ch. oschei. A possible fate of the hybrid zone is discussed.     

Both Geography and Ecology Contribute to Mating Isolation in Guppies

Local adaptation to different environments can promote mating isolation – either as an incidental by-product of trait divergence, or as a result of selection to avoid maladaptive mating. Numerous recent empirical examples point to the common influence of divergent natural selection on speciation based largely on evidence of strong pre-mating isolation between populations from different habitat types. Accumulating evidence for natural selection''s influence on speciation is therefore no longer a challenge. The difficulty, rather, is in determining the mechanisms involved in the progress of adaptive divergence to speciation once barriers to gene flow are already present. Here, we present results of both laboratory and field experiments with Trinidadian guppies (Poecilia reticulata) from different environments, who do not show complete reproductive isolation despite adaptive divergence. We investigate patterns of mating isolation between populations that do and do not exchange migrants and show evidence for both by-product and reinforcement mechanisms depending on female ecology. Specifically, low-predation females discriminate against all high-predation males thus implying a by-product mechanism, whereas high-predation females only discriminate against low-predation males from further upstream in the same river, implying selection to avoid maladaptive mating. Our study thus confirms that mechanisms of adaptive speciation are not necessarily mutually exclusive and uncovers the complex ecology-geography interactions that underlie the evolution of mating isolation in nature.     

THE GENETIC BASIS OF SEXUAL ISOLATION BETWEEN DROSOPHILA MELANOGASTER AND D. SIMULANS

The genetic analysis of sexual isolation between the closely-related species Drosophila melanogaster and Drosophila simulans involved two experiments with no-choice tests. The efficiency of sexual isolation was measured by the frequency of courtship initiation and interspecific mating. We first surveyed the variation in sexual isolation between D. melanogaster strains and D. simulans strains of different geographic origin. Then, to investigate variation in sexual isolation within strains, we made F₁ diallel sets of reciprocal crosses within strains of D. melanogaster and D. simulans. The F₁ diallel progeny of one sex were paired with the opposite sex of the other species. The first experiment showed significant differences in the frequency of interspecific mating between geographic strains. There were more matings between D. simulans females and D. melanogaster males than between D. melanogaster females and D. simulans males. The second experiment uncovered that the male genotypes in the D. melanogaster diallel significantly differed in interspecific mating frequency, but not in courtship initiation frequency. The female genotypes in the D. simulans diallel were not significantly different in courtship initiation and interspecific mating frequency. Genetic analysis reveals that in D. melanogaster males sexual isolation was not affected by either maternal cytoplasmic effects, sex-linked effects, or epistatic interaction. The main genetic components were directional dominance and overdominance. The F₁ males achieved more matings with D. simulans females than the inbred males. The genetic architecture of sexual isolation in D. melanogaster males argues for a history of weak or no selection for lower interspecific mating propensity. The behavioral causes of variation in sexual isolation between the two species are discussed.     

Assortative preferences and discrimination by females against hybrid male song in the grasshoppers Chorthippus brunneus and Chorthippus jacobsi (Orthoptera: Acrididae)

The grasshoppers Chorthippus brunneus and Chorthippus jacobsi are highly differentiated for male mating signals, and form a mosaic hybrid zone in northern Spain. At some sites within this zone, many hybrids are observed. At others, few hybrids are observed. Such bimodal sites may reflect recent contacts between parental genotypes, or local variation in levels of assortative mating or selection against hybrids. Playback of 12 parental and F1 male songs to 296 parental and hybrid females revealed positive assortative preferences in C. brunneus and C. jacobsi females, supporting a direct role of male mating signals in female choice. However, all female genotypic classes showed reduced responsiveness to F1 male songs. Such sexual selection against hybrids is consistent with the narrow cline width observed in the field for song characters relative to morphology. These results have implications for the genetic structure of the hybrid zone and for models of speciation by reinforcement.     

ESTIMATING THE MATING BEHAVIOR OF A PAIR OF HYBRIDIZING HELICONIUS SPECIES IN THE WILD

Premating isolation between incipient species is rarely studied in nature, even though mating tests in captivity may give an inaccurate picture of natural hybridization. We studied premating barriers between the warningly colored butterflies Heliconius erato and H. himera (Lepidoptera) in a narrow contact zone in Ecuador, where hybrids are found at low frequency. Eggs obtained from wild-mated females, supplemented with eggs and young larvae collected from the wild, were reared to adulthood. Adult color patterns of these progeny were then used to infer how their parents must have mated. Likelihood was used to estimate both the frequencies of potential parental genotypes from adult phenotypes collected in the wild, and the degree of assortative mating from the inferred parents. The frequencies of parental genotypes varied across the hybrid zone, but our statistical method allowed estimates of hybrid deficit and assortative mating to be integrated across all sites sampled. The best estimate of the frequency of F₁ and backcross hybrid adults in the center of the hybrid zone was 10%, with support limits (7.1%, 13.0%; support limits are asymptotically equivalent to 95% confidence limits). Mating was highly assortative: in the center of the hybrid zone the cross-mating probability between H. erato and H. himera was only 5% (0.3%, 21.4%). Wild hybrids themselves mated with both pure forms, and the probabilities that they mated in any direction were not significantly lower than those among conspecifics. These results are consistent with earlier laboratory studies on mate choice, and suggest that selection against hybrids must be strong to prevent formation of a hybrid swarm. Unfortunately, the wide support limits on mating behavior precluded a measure of the strength of selection from these data alone. Our statistical approach provides a useful general method for estimating mate choice in the wild.     

Male competition fitness landscapes predict both forward and reverse speciation

Speciation is facilitated when selection generates a rugged fitness landscape such that populations occupy different peaks separated by valleys. Competition for food resources is a strong ecological force that can generate such divergent selection. However, it is unclear whether intrasexual competition over resources that provide mating opportunities can generate rugged fitness landscapes that foster speciation. Here we use highly variable male F2 hybrids of benthic and limnetic threespine sticklebacks, Gasterosteus aculeatus Linnaeus, 1758, to quantify the male competition fitness landscape. We find that disruptive sexual selection generates two fitness peaks corresponding closely to the male phenotypes of the two parental species, favouring divergence. Most surprisingly, an additional region of high fitness favours novel hybrid phenotypes that correspond to those observed in a recent case of reverse speciation after anthropogenic disturbance. Our results reveal that sexual selection through male competition plays an integral role in both forward and reverse speciation.     

Divergent sexual selection via male competition: ecology is key

Sexual selection and ecological differences are important drivers of speciation. Much research has focused on female choice, yet the role of male competition in ecological speciation has been understudied. Here, we test how mating habitats impact sexual selection and speciation through male competition. Using limnetic and benthic species of threespine stickleback fish, we find that different mating habitats select differently on male traits through male competition. In mixed habitat with both vegetated and open areas, selection favours two trait combinations of male body size and nuptial colour: large with little colour and small with lots of colour. This matches what we see in reproductively isolated stickleback species, suggesting male competition could promote trait divergence and reproductive isolation. In contrast, when only open habitat exists, selection favours one trait combination, large with lots of colour, which would hinder trait divergence and reproductive isolation. Other behavioural mechanisms in male competition that might promote divergence, such as avoiding aggression with heterospecifics, are insufficient to maintain separate species. This work highlights the importance of mating habitats in male competition for both sexual selection and speciation.     

Sexual conflict and speciation.

We review the significance of two forms of sexual conflict (different evolutionary interests of the two sexes) for genetic differentiation of populations and the evolution of reproductive isolation. Conflicting selection on the alleles at a single locus can occur in males and females if the sexes have different optima for a trait, and there are pleiotropic genetic correlations between the sexes for it. There will then be selection for sex limitation and hence sexual dimorphism. This sex limitation could break down in hybrids and reduce their fitness. Pleiotropic genetic correlations between the sexes could also affect the likelihood of mating in interpopulation encounters. Conflict can also occur between (sex-limited) loci that determine behaviour in males and those that determine behaviour in females. Reproductive isolation may occur by rapid coevolution of male trait and female mating preference. This would tend to generate assortative mating on secondary contact, hence promoting speciation. Sexual conflict resulting from sensory exploitation, polyspermy and the cost of mating could result in high levels of interpopulation mating. If females evolve resistance to make pre- and postmating manipulation, males from one population could be more successful with females from the other, because females would have evolved resistance to their own (but not to the allopatric) males. Between-locus sexual conflict could also occur as a result of conflict between males and females of different populations over the production of unfit hybrids. We develop models which show that females are in general selected to resist such matings and males to persist, and this could have a bearing on both the initial level of interpopulation matings and the likelihood that reinforcement will occur. In effect, selection on males usually acts to promote gene flow and to restrict premating isolation, whereas selection on females usually acts in the reverse direction. We review theoretical models relevant to resolution of this conflict. The winning role depends on a balance between the ''value of winning'' and ''power'' (relating to contest or armament costs): the winning role is likely to correlate with high value of winning and low costs. Sperm-ovum (or sperm-female tract) conflicts (and their plant parallels) are likely to obey the same principles. Males may typically have higher values of winning, but it is difficult to quantify ''power'', and females may often be able to resist mating more cheaply than males can force it. We tentatively predict that sexual conflict will typically result in a higher rate of speciation in ''female-win'' clades, that females will be responsible for premating isolation through reinforcement, and that ''female-win'' populations will be less genetically diverse.     

Genetics of sexual isolation based on courtship song between two sympatric species: Drosophila ananassae and D. pallidosa

Sexual isolation has been considered one of the primary causes of speciation and its genetic study has the potential to reveal the genetics of speciation. In Drosophila, the importance of courtship songs in sexual isolation between closely related species has been well investigated, but studies analysing the genetic basis of the difference in the courtship songs associated with sexual isolation are less well documented. Drosophila ananassae and Drosophila pallidosa are useful for studies of sexual isolation, because of their sympatric distribution and absence of postmating isolation. Courtship songs are known to play a crucial role in sexual isolation between these two species, and the female discrimination behaviour against the courting male has been revealed to be controlled by a very narrow region on the second chromosome. In this study we investigated the genetic basis controlling the song differences associated with their sexual isolation, using intact and wingless males with chromosomes substituted between species. The results obtained from F₁ hybrid males between these species indicate the dominance of the song characters favoured by D. pallidosa females. In addition, the results obtained from backcross F₂ males indicate that chromosome 2 had a major effect on the control of the song characters associated with sexual isolation.     

Mate choice, sexual imprinting, and speciation: a test of a one-allele isolating mechanism in sympatric sticklebacks

One-allele isolating mechanisms should make the evolution of reproductive isolation between potentially hybridizing taxa easier than two-allele mechanisms, but the generality of one-allele mechanisms in nature has yet to be established. A potentially important one-allele mechanism is sexual imprinting, where the mate preferences of individuals are based on the phenotype of their parents. Here I test the possibility that sexual imprinting promotes reproductive isolation using sympatric species of threespine sticklebacks (Gasterosteus aculeatus). Sympatric species of sticklebacks consist of large benthic species and small limnetic species that are reproductively isolated and adapted to feeding in different environments. I fostered families of F1 hybrids between the species to males of both species. Preferences of these fostered females for males of either type revealed little or no effect of sexual imprinting on assortative mating. However, F1 females showed preferences for males that were similar to themselves in length, suggesting that size-assortative mating may be more important than sexual imprinting for promoting reproductive isolation between species pairs of threespine sticklebacks.