GENETIC ANALYSIS OF MATE DISCRIMINATION IN DROSOPHILA SIMULANS

Courtship is an elaborate behavior that conveys information about the identity of animal species and suitability of individual males as mates. In Drosophila, there is extensive evidence that females are capable of evaluating and comparing male courtships, and accepting or rejecting males as mates. These relatively simple responses minimize random sexual encounters involving subpar conspecific males and heterospecific males, and over generations can potentially select novel physical and behavioral traits. Despite its evolutionary and behavioral significance, little is still known about the genes involved in mating choice and how choices for novel males and females arise during evolution. Drosophila simulans and Drosophila sechellia are two recently diverged species of Drosophila in which females have a preference for conspecific males. Here we analyzed a total of 1748 F2 hybrid females between these two species and found a small number of dominant genes controlling the preference for D. simulans males. We also mapped two redundant X‐linked loci of mating choice, Macho‐XA and Macho‐XB, and show that neither one is required for female attractiveness. Together, our results reveal part of the genetic architecture that allows D. simulans females to recognize, mate, and successfully generate progenies with D. simulans males.     

THE REPRODUCTIVE RELATIONSHIPS OF DROSOPHILA SECHELLIA WITH D. MAURITIANA,D. SIMULANS,AND D. MELANOGASTER FROM THE AFROTROPICAL REGION

Hybridization tests among the four sibling species of the Drosophila melanogaster complex were made to determine the reproductive status of the recently discovered D. sechellia (which is endemic to a few islands and islets of the Seychelles archipelago) with regard to its three close relatives, D. mauritiana (endemic to Mauritius) and Afrotropical strains of the two cosmopolitan species D. melanogaster and D. simulans. Interstrain variation in the ability to hybridize with other species was also analyzed for D. melanogaster and D. simulans. D. mauritiana and D. simulans appear to be more weakly isolated from each other than either species is from D. sechellia. A striking unilateral mating success is observed in the cross of D. sechellia with D. simulans. The most extreme isolation is between D. melanogaster and its three siblings. Variation in the ability of strains to hybridize is observed in heterospecific crosses between D. simulans and either D. melanogaster or D. mauritiana.     

The role of species‐specific sensory cues in male responses to mating rivals in Drosophila melanogaster fruitflies

Complex sets of cues can be important in recognizing and responding to conspecific mating competitors and avoiding potentially costly heterospecific competitive interactions. Within Drosophila melanogaster, males can detect sensory inputs from conspecifics to assess the level of competition. They respond to rivals by significantly extending mating duration and gain significant fitness benefits from doing so. Here, we tested the idea that the multiple sensory cues used by D. melanogaster males to detect conspecifics also function to minimize “off‐target” responses to heterospecific males that they might encounter (Drosophila simulans, Drosophila yakuba, Drosophila pseudoobscura, or Drosophila virilis). Focal D. melanogaster males exposed to D. simulans or D. pseudoobscura subsequently increased mating duration, but to a lesser extent than following exposure to conspecific rivals. The magnitude of rivals’ responses expressed by D. melanogaster males did not align with genetic distance between species, and none of the sensory manipulations caused D. melanogaster to respond to males of all other species tested. However, when we removed or provided “false” sensory cues, D. melanogaster males became more likely to show increased mating duration responses to heterospecific males. We suggest that benefits of avoiding inaccurate assessment of the competitive environment may shape the evolution of recognition cues.     

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.     

INTRINSIC REPRODUCTIVE ISOLATION BETWEEN TWO SISTER SPECIES OF DROSOPHILA

Drosophila santomea and D. yakuba are sister species that live on the volcanic African island of São Tomé. Previous work has revealed several barriers to gene flow, including sexual isolation, hybrid sterility, and “extrinsic” ecological isolation based on differential adaptation to and preference for temperature. Here, we describe several new “intrinsic” barriers to gene flow—barriers that do not depend on the species’ ecology. These include reduced egg number, reduced egg hatchability, and faster depletion of sperm in interspecific compared to intraspecific matings. Further, hatching interval and egg‐to‐adult development time are significantly longer in interspecific than intraspecific crosses. If a female of either species is initially mated to a heterospecific male, she lays fewer and less‐fertile eggs than if she is first mated to a conspecific male, so that heterospecific matings permanently reduce female fertility. Finally, D. santomea females mated to D. yakuba males do not live as long as virgin or conspecifically mated females. The “poisoning” effects of heterospecific ejaculates may be byproducts of antagonistic sexual selection. Although these species diverged relatively recently, they are clearly separated by many isolating barriers that act both before and after mating.     

Reproductive interference by male Drosophila subobscura on female D. persimilis: A laboratory experiment

While females often reject courtship attempts by heterospecific males, reproductive interference by harassment from such males can nonetheless occur, potentially reducing female fitness. Such effects may be profound following a range expansion, when males from a new species may suddenly encounter (and perhaps even become abundant relative to) females of related native species. Drosophila subobscura recently invaded North America and may impact native species through reproductive interference and other processes. We test for the potential for reproductive interference by D. subobscura males on D. persimilis females in the laboratory. D. subobscura males aggressively copulated with D. persimilis females, including many females that exhibit rejection behaviors. Despite females attempting to dismount the males, the heterospecific copulations are on average longer than conspecific copulations, and females exhibit some reluctance to remate with conspecific males following this harassment. Females confined with both conspecific and heterospecific males produce fewer adult progeny than those with either conspecific males only or with conspecific males and distantly related D. simulans males that do not engage in female harassment. Overall, our results illustrate how reproductive interference by an invasive species can have negative effects on resident natural populations.     

Drosophila melanogaster males respond differently at the behavioural and genome‐wide levels to Drosophila melanogaster and Drosophila simulans females

Drosophila melanogaster are found in sympatry with Drosophila simulans, and matings between the species produce nonfertile hybrid offspring at low frequency. Evolutionary theory predicts that females choose mates, so males should alter their behaviour in response to female cues. We show that D. melanogaster males quickly decrease courtship towards D. simulans females. Courtship levels are reduced within 5 min of exposure to a heterospecific female, and overall courtship is significantly lower than courtship towards conspecific females. To understand changes at the molecular level during mate choice, we performed microarray analysis on D. melanogaster males that courted heterospecific D. simulans females and found nine genes have altered expression compared with controls. In contrast, males that court conspecific females alter expression of at least 35 loci. The changes elicited by conspecific courtship likely modulate nervous system function to reinforce positive conspecific signals and dampen the response to heterospecific signals.     

Sexual isolation between two sibling species with overlapping ranges: Drosophila santomea and Drosophila yakuba

Abstract.— .Drosophila yakuba is widespread in Africa, whereas D. santomea, its newly discovered sister species, is endemic to the volcanic island of São Tomé in the Gulf of Guinea. Drosophila santomea probably formed after colonization of the island by a D. yakuba‐like ancestor. The species presently have overlapping ranges on the mountain Pico do São Tome, with some hybridization occurring in this region. Sexual isolation between the species is uniformly high regardless of the source of the populations, and, as in many pairs of Drosophila species, is asymmetrical, so that hybridizations occur much more readily in one direction than the other. Despite the fact that these species meet many of the conditions required for the evolution of reinforcement (the elevation of sexual isolation by natural selection to avoid maladaptive interspecific hybridization), there is no evidence that sexual isolation between the species is highest in the zone of overlap. Sexual isolation is due to evolutionary changes in both female preference for heterospecific males and in the vigor with which males court heterospecific females. Heterospecific matings are also slower to take place than are homospecific matings, constituting another possible form of reproductive isolation. Genetic studies show that, when tested with females of either species, male hybrids having a D. santomea X chromosome mate much less frequently with females of either species than do males having a D. yakuba X chromosome, suggesting that the interaction between the D. santomea X chromosome and the D. yakuba genome causes behavioral sterility. Hybrid F₁ females mate readily with males of either species, so that sexual isolation in this sex is completely recessive, a phenomenon seen in other Drosophila species. There has also been significant evolutionary change in the duration of copulation between these species; this difference involves genetic changes in both sexes, with at least two genes responsible in males and at least one in females.     

Hybridization occurs between Drosophila simulans and D. sechellia in the Seychelles archipelago

Drosophila simulans and D. sechellia are sister species that serve as a model to study the evolution of reproductive isolation. While D. simulans is a human commensal that has spread all over the world, D. sechellia is restricted to the Seychelles archipelago and is found to breed exclusively on the toxic fruit of Morinda citrifolia. We surveyed the relative frequency of males from these two species in a variety of substrates found on five islands of the Seychelles archipelago. We sampled different fruits and found that putative D. simulans can be found in a variety of substrates, including, surprisingly, M. citrifolia. Putative D. sechellia was found preferentially on M. citrifolia fruits, but a small proportion was found in other substrates. Our survey also shows the existence of putative hybrid males in areas where D. simulans is present in Seychelles. The results from this field survey support the hypothesis of current interbreeding between these species in the central islands of Seychelles and open the possibility for fine measurements of admixture between these two Drosophila species to be made.     

VARIATIONS IN CUTICULAR HYDROCARBONS AMONG THE EIGHT SPECIES OF THE DROSOPHILA MELANOGASTER SUBGROUP

In addition to protecting against desiccation, some of the hydrocarbons of the waxy cuticle have previously been shown to be mating pheromones in Drosophila melanogaster and D. simulans. Therefore, cuticular hydrocarbons were compared among the eight species in the D. melanogaster subgroup. For the two cosmopolitan species and several geographic strains that were studied, all males are quite similar with very abundant monoenes. The major compound in most cases is 7-tricosene. Only three exceptions were found: D. sechellia, and the Afrotropical strains of D. melanogaster and D. simulans. A significant sexual dimorphism exists in three species: D. melanogaster, D. erecta, and D. sechellia. Greater variation was observed in females than in males. D. erecta is singular in the production of long-chain molecules (31–33 carbons). Only three species (D. melanogaster, D. erecta, and D. sechellia) produce diene in significant amounts. Such products, especially 7,11-heptacosadiene, are known to act as aphrodisiacs for D. melanogaster males. In the five other species, females show only quantitative differences from males, generally with 7-tricosene as the most abundant compound. This compound is an aphrodisiac for D. simulans males. Some species such as D. yakuba, D. teissieri, D. orena, D. mauritiana, and the Seychelles strain of D. simulans are almost identical in the chemical composition of cuticular hydrocarbons. In contrast, important variations are observed between geographic populations of D. melanogaster and D. simulans.     

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.     

Microsatellite variation suggests a recent fine-scale population structure of <Emphasis Type="Italic">Drosophila sechellia</Emphasis>, a species endemic of the Seychelles archipelago

Drosophila sechellia is closely related to the cosmopolitan and widespread model species, D. simulans. This species, endemic to the Seychelles archipelago, is specialized on the fruits of Morinda citrifolia, and harbours the lowest overall genetic diversity compared to other species of Drosophila. This low diversity is associated with a small population size. In addition, no obvious population structure has been evidenced so far across islands of the Seychelles archipelago. Here, a microsatellite panel of 17 loci in ten populations from nine islands of the Seychelles was used to assess the effect of the D. sechellia’s fragmented distribution on the fine-scale population genetic structure, the migration pattern, as well as on the demography of the species. Contrary to previous results, also based on microsatellites, no evidence for population contraction in D. sechellia was found. The results confirm previous studies based on gene sequence polymorphism that showed a long-term stable population size for this species. Interestingly, a pattern of Isolation By Distance which had not been described yet in D. sechellia was found, with evidence of first-generation migrants between some neighbouring islands. Bayesian structuring algorithm results were consistent with a split of D. sechellia into two main groups of populations: Silhouette/Mahé versus all the other islands. Thus, microsatellites suggest that variability in D. sechellia is most likely explained by local genetic exchanges between neighbouring islands that have recently resulted in slight differentiation of the two largest island populations from all the others.     

Male mate choice via cuticular hydrocarbon pheromones drives reproductive isolation between Drosophila species

Mate discrimination is a key mechanism restricting gene flow between species. While studied extensively with respect to female mate choice, mechanisms of male mate choice between species are far less studied. Thus, we have little knowledge of the relative frequency, importance, or overall contribution of male mate discrimination to reproductive isolation. In the present study, we estimated the relative contributions of male and female choice to reproductive isolation between Drosophila simulans and D. sechellia, and show that male mate discrimination accounts for the majority of the current isolation between these species. We further demonstrate that males discriminate based on female cuticular hydrocarbon pheromones, and collect evidence supporting the hypothesis that male mate discrimination may alleviate the costs associated with heterospecific courtship and mating. Our findings highlight the potentially significant contribution of male mate choice to the formation of reproductive isolating barriers, and thus the speciation process.     

Adult female hamsters require long and sustained exposures to heterospecific males to avoid interspecific mating

Interspecific mating normally decreases female fitness. In many species, females avoid heterospecific males innately or by imprinting on their parents. Alternatively, adult females could learn to discriminate against heterospecific males after exposure to such males. For example, Syrian hamster (Mesocricetus auratus) females learn to discriminate between conspecific males and Turkish hamster (M. brandti) males during adulthood by exposure to males of both species. Adult females not previously exposed to Turkish hamster males will mate similarly with conspecific and heterospecific males. However, in a previous study we showed that exposure to a heterospecific male and a conspecific male for 8 days led to mating avoidance and aggression towards the heterospecific male. Here we conducted two experiments to investigate how much exposure to the heterospecific male was required for females to avoid mating with the heterospecific male (Experiment 1) and how long that avoidance lasted in the absence of continuous exposure to heterospecific stimuli (Experiment 2). Fast and durable learning would indicate the evolution of an efficient avoidance response. In Experiment 1, females were exposed to a heterospecific male for 1, 4 h, 4 or 8 days and then paired with that male. We found more avoidance of interspecific mating after 4 or 8 days of exposure than after 1 or 4 h of exposure. In Experiment 2, females were exposed to a heterospecific male for 8 days and then paired with that male either 10 min later or 8 days later. We found that after an 8-day delay females were highly sexually receptive to the heterospecific male. Additionally, a comparison between the current experiments and a previous study indicates that female Syrian hamsters do not require concurrent exposure to a conspecific male and a heterospecific male to learn to avoid interspecific mating; exposure to a heterospecific male is sufficient.     

Reproductive interference between Aedes albopictus and Aedes flavopictus at a place of their origin

Aedes (Stegomyia) albopictus and Aedes (Stegomyia) flavopictus are related species that have overlapping distributions from southern to central Japan. To understand how they interact, we studied reproductive interference between them, particularly focusing on the body size difference between the mating pair. Here, we examined the effects of conspecific, heterospecific and double mating (i.e. heterospecific mating followed by conspecific mating) on copulation duration, egg production and hatchability of eggs using mosquitoes that varied in body size. Females mated only with heterospecific males produced few viable eggs, indicating that post‐mating isolation is almost complete. When mated with heterospecific males before conspecific mating, the production of viable eggs was lower than when mated only with conspecific males, revealing the occurrence of reproductive interference. The degree of reproductive interference was larger in Ae. flavopictus than in Ae. albopictus when the female size was small but did not differ between them when the female size was large. Aedes albopictus females appear to be able to distinguish Ae. flavopictus males from conspecific males and larger females are more successful in the rejection of heterospecific males. On the other hand, Ae. flavopictus were not able to discriminate between conspecific and heterospecific males.     

Mating Behavior Differences and the Cost of Mating in <Emphasis Type="Italic">Allonemobius fasciatus</Emphasis> and <Emphasis Type="Italic">A. socius</Emphasis>

Close range prezygotic barriers are assumed to be present between sister taxa who have overlapping distributions. Here we report the results of studies designed to test the existence of prezygotic barriers between two closely related species, A. fasciatus and A. socius. We finely dissected the courtship and mating rituals and performed Monte Carlo analysis on lengths of time and number of occurrences of particular events in the courtship mating sequence. These detailed investigations of the courtship and mating behavior of conspecific and heterospecific pairs demonstrate that behavioral isolation is non-existent. We also measure the adult lifespan and number of progeny produced from singly and multiply mated males and females in conspecific and heterospecific trials. We found that cost of a heterospecific mating is asymmetric between the sexes with males paying a higher cost.     

Reinforcement of Gametic Isolation in Drosophila

Reinforcement, a process by which natural selection increases reproductive isolation between populations, has been suggested to be an important force in the formation of new species. However, all existing cases of reinforcement involve an increase in mate discrimination between species. Here, I report the first case of reinforcement of postmating prezygotic isolation (i.e., barriers that act after mating but before fertilization) in animals. On the slopes of the African island of São Tomé, Drosophila yakuba and its endemic sister species D. santomea hybridize within a well-demarcated hybrid zone. I find that D. yakuba females from within this zone, but not from outside it, show an increase in gametic isolation from males of D. santomea, an apparent result of natural selection acting to reduce maladaptive hybridization between species. To determine whether such a barrier could evolve under laboratory conditions, I exposed D. yakuba lines derived from allopatric populations to experimental sympatry with D. santomea, and found that both behavioral and gametic isolation become stronger after only four generations. Reinforcement thus appears to be the best explanation for the heightened gametic isolation seen in sympatry. This appears to be the first example in animals in which natural selection has promoted the evolution of stronger interspecific genetic barriers that act after mating but before fertilization. This suggests that many other genetic barriers between species have been increased by natural selection but have been overlooked because they are difficult to study.     

The evolution of reproductive isolation in the Drosophila yakuba complex of species

In the Drosophila melanogaster subgroup, the yakuba species complex, D. yakuba, D. santomea and D. teissieri have identical mitochondrial genomes in spite of nuclear differentiation. The first two species can be readily hybridized in the laboratory and produce fertile females and sterile males. They also form hybrids in natural conditions. Nonetheless, the third species, D. teissieri, was thought to be unable to produce hybrids with either D. yakuba or D. santomea. This in turn posed the conundrum of why the three species shared a single mitochondrial genome. In this report, we show that D. teissieri can indeed hybridize with both D. yakuba and D. santomea. The resulting female hybrids from both crosses are fertile, whereas the hybrid males are sterile. We also characterize six isolating mechanisms that might be involved in keeping the three species apart. Our results open the possibility of studying the history of introgression in the yakuba species complex and dissecting the genetic basis of interspecific differences between these three species by genetic mapping.     

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.     

Predation Risk Reduces a Female Preference for Heterospecific Males in the Green Swordtail

The presence of a predator can result in the alteration, loss or reversal of a mating preference. Under predation risk, females often change their initial preference for conspicuous males, favouring less flashy males to reduce the risk of being detected by predators. Previous studies on predator‐induced plasticity in mate preferences have given females a choice between more and less conspicuous conspecific males. However, in species that naturally hybridize, it is also possible that females might choose an inconspicuous heterospecific male over a conspicuous conspecific male under predation risk. Our study addresses this question using the green swordtail (Xiphophorus helleri) and the southern platyfish (Xiphophorus maculatus), which are sympatric in the wild. We hypothesized that X. helleri females would prefer the sworded conspecific males in the absence of a predator but favour the less conspicuous, swordless, heterospecific males in the presence of a predator. Contrary to our expectation, females associated more with the heterospecific male than the conspecific male in the control (no predator) treatment, and they were non‐choosy in the predator treatment. This might reflect that females were attracted to the novel male phenotype when there was no risk of predation but became more neophobic after predator exposure. Regardless of the underlying mechanism, our results suggest that predation pressure may affect female preferences for conspecific versus heterospecific males. We also found striking within‐population, between‐individual variation in behavioural plasticity: females differed in the strength and direction of their preferences, as well as in the extent to which they altered their preferences in response to changes in perceived predation risk. Such variation in female preferences for heterospecific males could potentially lead to temporal and spatial variation in hybridization rates in the wild.