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.     

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.     

Conflicting intersexual mate choices maintain interspecific sexual interactions

Reproductive interference, interspecific sexual interactions that affect reproductive success, is found in various taxa and has been considered as a fundamental source of reproductive character displacement (RCD). Once RCD has occurred, persistent interspecific sexual interactions between species pairs are expected to diminish. However, reproductive interference has been reported from some species pairs that sympatrically coexist. Thus, the question arises, can reproductive interference persist even after RCD? We modeled the evolutionary dynamics of signal traits and mate recognition that determine whether interspecific sexual interactions occur. Our models incorporate male decision making based on the recognition of signal traits, whereas most previous models incorporate only female decision making in mate selection. Our models predict the following: (1) even when male decision making is incorporated, males remain promiscuous; (2) nevertheless, the frequency of interspecific mating is maintained at a low level after trait divergence; (3) the rarity of interspecific mating is due to strict female mate recognition and the consequent refusal of interspecific courtship by females; and (4) the frequency of interspecific mating becomes higher as the cost to females of refusing interspecific courtship increases. These predictions are consistent with empirical observations that males of some species engage in infrequent heterospecific mating. Thus, our models predict that reproductive interference can persist even after RCD occurred.     

Female mate recognition and sexual isolation depending on courtship song in Drosophila sechellia and its siblings

In Drosophila sechellia, females accept males that sing heterospecific songs less than those that do not sing, whereas in D. melanogaster and D. simulans, females accept males that sing heterospecific song more than those that do not sing. Here we studied the sexual isolation of D. sechellia and its siblings using interspecific hybrids to reveal the mechanisms underlying female mate recognition. The females of hybrids mated more with winged males of the parent species than with wingless ones, suggesting that the discrimination against heterospecific songs by D. sechellia females is recessive. Female preference for courtship songs seems to be inherited additively or semidominantly. In addition, we examined female receptivity without the stimuli of courtship songs by comparing the mating frequencies between the crosses using wingless males and found that it is also inherited additively or semidominantly.     

Genetics of Drosophila simulans male mating discrimination in crosses with D. melanogaster

The genetic bases of sexual isolation between Drosophila melanogaster and D. simulans have been mainly studied in females, and there is little information about the role of the males in interspecific mating discrimination. Using D. simulans synthetic lines with compound chromosomes from a population of the Seychelles Islands (high frequency of interspecific mating) and a multimarker strain (low frequency), we show that D. simulans males play an important role in discriminating D. melanogaster females. The genetics of male discrimination fits well with the inheritance mode of a single locus, dominant for sexual isolation, located in chromosome II near the net mutation (2L-0.0). The heterospecific mating success of the male was not related to his sexual vigor. The specific load of male cuticular hydrocarbons was counted as a possible source of discrimination used by the D. melanogaster female.     

The courtship behaviour of the spotted dusky salamander, Desmognathus fuscus conanti (Amphibia: Caudata: Plethodontidae)

The courtship behaviour of the North American southern dusky salamander, Desmognathus fuscus conanti (Plethodontidae), is described for the first time. The male is the most active participant, as is typical for caudate amphibians, and much of his behaviour can be understood in terms of orientation to and stimulation (or persuasion) of the female. The latter is largely accomplished by the transfer of courtship pheromones from the male's mental (chin) gland. The orientation and persuasion phases of courtship appear necessary if sperm transfer by means of a spermatophore deposited upon the substratum is to be successful. The courtship of D. fuscus conanti is qualitatively identical to that of D. santeetlah , with which it is sexually compatible in the laboratory and with which it may hybridize in nature. However, a broader comparison of species in the genus Desmognathus reveals that the greatest interspecific differences occur during the orientation and persuasion phases of courtship. A complete understanding of the evolution of courtship behaviour in desmognathine salamanders awaits availability of ethograms for all other taxa and a robust, independent phytogeny for the entire subfamily.     

Linking intra‐ and interspecific assortative mating: Consequences for asymmetric sexual isolation

Assortative mating is of interest because of its role in speciation and the maintenance of species boundaries. However, we know little about how within‐species assortment is related to interspecific sexual isolation. Most previous studies of assortative mating have focused on a single trait in males and females, rather than utilizing multivariate trait information. Here, we investigate how intraspecific assortative mating relates to sexual isolation in two sympatric and congeneric damselfly species (genus Calopteryx). We connect intraspecific assortment to interspecific sexual isolation by combining field observations, mate preference experiments, and enforced copulation experiments. Using canonical correlation analysis, we demonstrate multivariate intraspecific assortment for body size and body shape. Males of the smaller species mate more frequently with heterospecific females than males of the larger species, which showed less attraction to small heterospecific females. Field experiments suggest that sexual isolation asymmetry is caused by male preferences for large heterospecific females, rather than by mechanical isolation due to interspecific size differences or female preferences for large males. Male preferences for large females and male–male competition for high quality females can therefore counteract sexual isolation. This sexual isolation asymmetry indicates that sexual selection currently opposes a species boundary.     

Mating asymmetry and the direction of evolution in the Hawaiian cricket genus Laupala

Based on studies from native Hawaiian Drosophila, a model was proposed to explain sexual isolation and mating asymmetry, from which one could potentially infer the 'direction of evolution'. We examined sexual isolation between allopatric cricket species of the genus Laupala, another endemic Hawaiian insect with an elaborate mating system, to begin to explore the nature of sexual isolation and mating asymmetry in closely related Hawaiian organisms. We studied sexual isolation and mating asymmetry in two contrasts. First, an inter-island comparison, including L. makaio from the older island of Maui and L. paranigra from the younger island of Hawaii, and second, an intra-island (Hawaii) comparison, including L. nigra from the older volcano of Mauna Kea and L. paranigra with a primary distribution on the younger volcanoes of Mauna Loa and Kilauea. We used a 'no-choice' experimental design, pairing individual males and females in homospecific or heterospecific combinations. Several behavioural aspects of courtship (proportion of male singing, latency to male singing, production of spermatophores and courtship initiation speed) were quantified as well as the success or failure of matings. We demonstrate asymmetry in sexual isolation between reciprocal combinations of L. makaio and L. paranigra. This result is examined in light of the differences in courtship behaviour manifest in the experiments with these two species. We did not find evidence of asymmetry in sexual isolation between L. nigra and L. paranigra, although differences in courtship initiation speed were evident between reciprocal combinations of these two species. In addition to the geological argument that species on older islands and older volcanoes give rise to species on younger islands and younger volcanoes, we discuss phylogenetic evidence consistent with these biogeographic hypotheses of relationships among the focal taxa. The patterns of asymmetrical sexual isolation and mating asymmetry are consistent with those found in the native Hawaiian Drosophila.     

Courtship signals and mate choice of the flies of inbred Drosophila montana strains

We studied genetic variation in fly mating signals and mate choice in crosses within and between inbred strains of Drosophila montana. Male songs and the cuticular hydrocarbons of both sexes as well as some of the flies’ behavioural traits differed significantly between strains. This did not, however, cause sexual isolation between strains. In fact, courtship was shorter if the female was courted by a male of a foreign strain than when courted by their own male. Heterosis was found for courtship duration and the carrier frequency of male song. Diallel analysis of male song revealed additive genetic variation in four out of the five traits studied. Two traits showed dominance variation and one of these, carrier frequency, expressed unidirectional dominance with alleles for higher carrier frequency being dominant. Direction of dominance in carrier frequency was the same as the direction of sexual selection exercised by D. montana females on this trait, which suggests that sexual selection could be a driving force in the evolution of song towards a higher carrier frequency.     

Copulatory courtship behavior and sine song as a mate recognition cue in Drosophila lini and its sibling species

Most Drosophila species sing species-specific pulse songs during their "precopulatory courtship." Three sibling species of the Drosophila montium species subgroup performed "copulatory courtship": males generated courtship songs by vibrating either wing only after mounting and during copulation. In these three species, strong sexual isolation was detected between D. ohnishii and D. lini and between D. ohnishii and D. ogumai, but not between D. lini and D. ogumai. Female showed strong repelling behavior when they were mounted by a heterospecific male in the species combinations including D. ohnishii, resulting in failure of the copulation attempt of the male. Acoustic analyses of courtship songs revealed that the pulse song was irregular, without any species-specific parameters, but that the frequency of the sine song was different among the three species in accordance with the modes of sexual isolation between them; it was significantly lower in D. ohnishii (mean ± SE = 193.0 ± 1.7 Hz) but higher in D. lini (253.4 ± 2.7 Hz) and D. ogumai (246.7 ± 5.3 Hz). We suggest that this difference in the sine song frequency is a sexual signal in the Specific Mate Recognition System (SMRS) among these three Drosophila species.     

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.     

Drosophila bipectinata species complex

The Drosophila bipectinata species complex belongs to the ananassae subgroup of the melanogaster species group (Genus Drosophila, Subgenus Sophophora). The members of the complex are: D. bipectinata, D. parabipectinata, D. malerkotliana, and D. pseudoananassae. Of the four species, D. bipectinata is most widely distributed. Females are indistinguishable, but males are distinguishable by their sex-comb teeth number and pattern and by abdominal colouration. Chromosomal inversions have been detected in these species. In natural populations of D. bipectinata the frequency of inversions and the level of inversion heterozygosity were found to be very low but in laboratory stocks inversions persisted for more than 20 generations due to heterotic buffering. On an average 9.3 fixed interspecific inversions separate each species pair. Non-random association between linked inversions indicated epistatic interaction in natural populations of D. bipectinata. Certain spontaneous mutations were detected and mapped for the first time in D. bipectinata. Low frequency of spontaneous male recombination has also been reported in D. bipectinata. Sexual isolation study in the complex indicated strong preference for homogamic mating. The results also indicated incomplete sexual isolation among different members of this complex. The isolation estimate among six different geographic populations of D. bipectinata ranged from 0.54 - 0.92 representing positive assortative mating which is an evidence for incipient sexual isolation. Incipient sexual isolation was also found within D. malerkotliana and D. parabipectinata . Chromosomal, hybridization and allozyme studies revealed close phylogenetic relationship among the four species of the bipectinata complex. Mitochondrial DNA study revealed net nucleotide difference (delta) between these species to be very small (0.0002 +/- 0.0008) reflecting closeness. Evidence for genetic control of sexual activity and existence of sexual selection in D. bipectinata has been shown on the basis of mating propensity tests carried out on geographic strains, their hybrids and diallel crosses. Significant variation was found among the strains tested with respect to courtship time, duration of copulation and fertility. A positive correlation between duration of copulation and fertility in D. bipectinata was found. Evidence for rare-male mating advantage was also found in D. bipectinata. A positive response to selection for high and low mating activity provided evidence for polygenic control of this phenomenon in D. bipectinata. Bilateral outgrowths on thorax, a unique phenotype, reported for the first time in D. bipectinata has been shown to affect mate recognition ability. Results of the study on pupation site preference (larval behaviour) and oviposition site preference (non-sexual behaviour) have also been included.     

Divergence in Multiple Courtship Song Traits between Drosophila santomea and D. yakuba

The courtship song of Drosophila is useful for species recognition and sexual selection. A new species of the melanogaster group of Drosophila , D. santomea , has recently been described from the island of São Tomé in the Gulf of Guinea. We describe the courtship song of D. santomea and compare it with that of its sibling species D. yakuba . Both species have a relatively unusual song pattern for melanogaster-group species, in that they have two types of pulse song but no sine song. There are large differences in the inter-pulse interval of both types of song, but no major differences in pulse shape or intrapulse frequency between the species. The song of D. yakuba is similar in lines from the African mainland (allopatric to D. santomea ) and from São Tomé (sympatric). We test if song pattern might influence sexual isolation by examining the mating success of wingless males with homo- and hetero-specific females. We show that song pattern contributes to sexual stimulation, but the differences in song patterns alone are unlikely to explain patterns of sexual isolation such as the asymmetrical isolation seen between species.     

Sexual isolation between two newt species, Triturus vulgaris and T. montandoni (Amphibia, Urodela, Salamandridae)

In the present study we investigated sexual isolation between Triturus vulgaris and 77 montandoni in mating experiments run under semi-natural conditions. The two newt species offer a suitable model for studying evolution of reproductive isolation and mating preferences because they arc genetically the most similar species within the genus and readily hybridize in nature. Separate experiments were conducted in which groups of virgin females were placed together (in artificial pools) with groups of homospecific, heterospecific or both types of males. The estimates of reproductive isolation and mating propensity were based on the numbers of females producing hybrids and/or non-hybrid progeny. The levels of reproductive isolation, isolation asymmetry (IA) and propensity asymmetry (PA) were significant only for experiments in which females were given a choice between conspecific and heterospecific males. This implies that mating experiments with no interspecific choice may reduce discrimination and affect patterns of IA and PA. Asymmetry in reproductive isolation was also significant when the analysis was confined to just inseminated females. Differences in habitat preferences and condition of females possibly contributed to the relatively high values of PA.     

Courtship Roles of Male and Female European Earwigs, Forficula auricularia L. (Dermaptera: Forficulidae), and Sexual Use of Forceps

Detailed observations of the courtship and mating of the European earwig Forficula auricularia revealed a complex of sexual behaviors for both males and females. A sequential analysis of the transition frequencies between male preceding-following behaviors showed that courtship is intricate and nonstereotyped. The significance of the male forceps was demonstrated by their use in early courtship with displays and later use as a tactile stimulus for the female. A study of males from which the forceps had been removed showed no mating by altered males. Male forcep length was bimodally distributed and positively allometric, while female forcep length was normally distributed. Males with longer forceps did not have a mating advantage. Receptive females were behaviorally active during courtship. The possible evolutionary development of the sexual dimorphism of the earwig is discussed.     

Behavioural mechanisms of sexual isolation involving multiple modalities and their inheritance

Speciation research dissects the genetics and evolution of reproductive barriers between parental species. Hybrids are the “gatekeepers” of gene flow, so it is also important to understand the behavioural mechanisms and genetics of any potential isolation from their parental species. We tested the role of multiple behavioural barriers in reproductive isolation among closely related field crickets and their hybrids (Teleogryllus oceanicus and Teleogryllus commodus). These species hybridize in the laboratory, but the behaviour of hybrids is unusual and there is little evidence for gene flow in the wild. We found that heterospecific pairs exhibited reduced rates of courtship behaviour due to discrimination by both sexes, and that this behavioural isolation was symmetrical. However, hybrids were not sexually selected against and exhibited high rates of courtship behaviour even though hybrid females are sterile. Using reciprocal hybrid crosses, we characterized patterns of interspecific divergence and inheritance in key sexual traits that might underlie the mating patterns we found: calling song, courtship song and cuticular hydrocarbons (CHCs). Song traits exhibited both sex linkage and transgressive segregation, whereas CHCs exhibited only the latter. Calculations of the strength of isolation exerted by these sexual traits suggest that close‐range signals are as important as long‐distance signals in contributing to interspecific sexual isolation. The surprisingly weak mating barriers observed between hybrids and parental species highlight the need to examine reproductive isolating mechanisms and their genetic bases across different potential stages of introgressive hybridization.     

SEX DIFFERENCES IN MATE RECOGNITION AND CONSPECIFIC PREFERENCE IN SPECIES WITH MUTUAL MATE CHOICE

Sexual isolation is often assumed to arise because choosy females recognize and reject heterospecific males as mates. Yet in taxa in which both males and females are choosy, males might also recognize and reject heterospecific females. Here, we asked about the relative contribution of the sexes to the strong sexual isolation found in limnetic–benthic species pairs of threespine sticklebacks, which show mutual mate choice. We asked whether males and females of the two species recognize conspecifics and also prefer to mate with them. We found evidence for mate recognition by both sexes but only females prefer conspecifics. The nature of male courtship depended on which species of female they were courting, indicating that males recognized conspecific females and differentiated them from heterospecifics. However, males courted both species of females with equal vigor and changed courtship in a manner that would increase the chance of mating with heterospecifics. Females both recognized conspecifics and strongly preferred them. They responded very little to heterospecific male courtship and almost never mated with them. Therefore, males are likely to undermine sexual isolation, but females uphold it. Despite mutual mate choice and mate recognition in both sexes, females are primarily responsible for sexual isolation in these taxa.     

Interactions of the Males and Females of Three Sympatric Drosophila virilis-Group Species, D. montana, D. littoralis, and D. lummei, (Diptera: Drosophilidae) in Intra- and Interspecific Courtships in the Wild and in the Laboratory

Four species of the Drosophila virilis group, D. montana, D. littoralis, D. lummei, and D. ezoana, occur sympatrically in several locations in northern Europe. Courtship interactions between the flies of the three first-mentioned species were observed at malt baits in Kemi, northern Finland, to find out how the flies of different species recognize conspecific individuals and how interspecific courtships differ from intraspecific ones in the wild. Intraspecific courtships (including females of different reproductive stages) and interspecific courtships were also videotaped and analyzed in laboratory. In the wild the males courted both conspecific and allospecific females, even though the species varied in how much the males were attracted to females of different species. Interspecific courtships usually broke off when the male touched the female or when the male and/or the female vibrated his/her wings, producing acoustic cues. In the laboratory males courted conspecific females irrespective of the reproductive stage of the female, even though the courtships directed toward immature and fertilized females usually included only orienting and touching (no licking and singing). D. littoralis, and very rarely D. montana and D. lummei, males courted also allospecific females. In the few interspecific courtships between these three species, where the male proceeded to singing, females responded to male singing by vibrating their wings. This ended the courtship. It is suggested that both the chemical cues affecting female attractivity and the acoustic signals of males and females, which are produced by wing vibration, function in maintaining sexual isolation between these three species.     

Search for species-specific mating signal in courtship songs of sympatric sibling species,Drosophila ananassae and D. pallidosa

Sexual isolation is one of the most important mechanisms that may lead to speciation. Drosophila ananassae and D. pallidosa are useful for the study of sexual isolation, because of their sympatric distribution and no postmating isolation between them. Courtship songs are considered to play a crucial role in sexual isolation between D. ananassae and D. pallidosa. We recorded and analyzed male courtship songs of D. ananassae and D. pallidosa for eight and four geographical strains, respectively. Courtship behaviors of the two species were consistent with those previously described, however, male's middle leg shaking, which had not before been described, was observed in both species. Males sing by wing vibration only during courtship. Their song oscillograms were distinct between species, but those of conspecific strains were very similar, in spite of their different geographical derivation. We found species-specificity in burst length, pulse length, cycle number in a pulse, and frequency spectra of bursts; these results suggest that these song parameters may play a role in mate recognition that enforces their sexual isolation. The specific values of interpulse interval, cycle number in a pulse and intrapulse frequency were involved with the determination of specificity in frequency spectra of bursts. We discussed the possibility that the specific frequency spectra of bursts are recognized by females as the species-specific signal rather than each parameter individually.     

Evidence for no sexual isolation between Drosophila albomicans and D. nasuta

Sexual isolation, the reduced tendency to mate, is one of the reproductive barriers that prevent gene flow between different species. Various species‐specific signals during courtship contribute to sexual isolation between species. Drosophila albomicans and D. nasuta are closely related species of the nasuta subgroup within the Drosophila immigrans group and are distributed in allopatry. We analyzed mating behavior and courtship as well as cuticular hydrocarbon profiles within and between species. Here, we report that these two species randomly mated with each other. We did not observe any sexual isolation between species or between strains within species by multiple‐choice tests. Significant difference in the courtship index was detected between these two species, but males and females of both species showed no discrimination against heterospecific partners. Significant quantitative variations in cuticular hydrocarbons between these two species were also found, but the cuticular hydrocarbons appear to play a negligible role in both courtship and sexual isolation between these two species. In contrast to the evident postzygotic isolation, the lack of sexual isolation between these two species suggests that the evolution of premating isolation may lag behind that of the intergenomic incompatibility, which might be driven by intragenomic conflicts.