Genetic analysis of female mating recognition between Drosophila ananassae and Drosophila pallidosa: application of interspecific mosaic genome lines

Drosophila ananassae and Drosophila pallidosa are closely related species that can produce viable and fertile hybrids of both sexes, although strong sexual isolation exists between the two species. Females are thought to discriminate conspecific from heterospecific males based on their courtship songs. The genetic basis of female discrimination behavior was analyzed using isogenic females from interspecific mosaic genome lines that carry homozygous recombinant chromosomes. Multiple regression analysis indicated a highly significant effect of the left arm of chromosome 2 (2L) on the willingness of females to mate with D. ananassae males. Not only 2L but also the left arm of chromosome X (XL) and the right arm of chromosome 3 (3R) had significant effects on the females' willingness to mate with D. pallidosa males. All regions with strong effects on mate choice have chromosome arrangements characterized by species-specific inversions. Heterospecific combinations of 2L and 3R have previously been suggested to cause postzygotic reproductive isolation. Thus, genes involved in premating as well as postmating isolation are located in or near chromosomal inversions. This conclusion is consistent with the recently proposed hypothesis that "speciation genes" accumulate at a higher rate in non-recombining genome regions when species divergence occurs in the presence of gene flow.     

Rapid evolution of courtship song pattern in Drosophila willistoni sibling species

Courtship behaviours may provide a more reliable means of identifying reproductively isolated taxa than traits such as morphology or many genetic markers. Here we describe the courtship songs of the Drosophila willistoni sibling species group, which consists of several species and subspecies. We find that song pattern is species-specific, despite significant differences among strains within species. D. paulistorum has the most variable song pattern, which reflects this species' traditional subdivision into semispecies. All the other species could be unambiguously identified by song. The major differences among these species was in the interpulse interval, as has been found in other studies of fly song. However, the interpulse intervals of the species studied here were often multimodal. This was partly due to the presence of multiple song types within the courtship repertoire, but it also reflected changes in interpulse interval within a song type by some males. Unusually, some species had distinctively patterned variation in interpulse interval. Song must have evolved rapidly within the species complex, probably due to sexual selection.     

Behavioral Sequence Leading to Sexual Isolation Between <Emphasis Type="Italic">Drosophila ananassae</Emphasis> and <Emphasis Type="Italic">D. pallidosa</Emphasis>

Drosophila ananassae and D. pallidosa are closely related, sympatric species that lack postmating isolation. Sexual isolation has been considered important in maintaining them as independent species. To clarify the behavioral processes leading to sexual isolation, we analyzed behavioral sequences and examined the effect of courtship song on mating success and on behaviors of both sexes by surgically removing male wings (song generators), female aristae (song receivers), or female wings (means of fluttering). We found that heterospecific courtship songs evoked female wing fluttering, whereas conspecific courtship song did not. Furthermore, female wing fluttering made courting males discontinue courtship. These findings suggest that strong sexual isolation is achieved through the following behavioral sequence: heterospecific song→female wing fluttering→courtship discontinuation.     

The courtship song of African Drosophila melanogaster

For many years it was thought that Drosophila melanogaster was relatively panmictic, without differentiation in the Mate Recognition System. Recent studies have demonstrated that flies from Africa vary in pheromones and assortative mating. Strains from Zimbabwe show strong sexual isolation from others. We show that the interpulse interval (IPI) of courtship song, an important mating signal, is unusually short among African flies. Zimbabwean flies have the shortest IPI, but there is no correlation with assortative mating, suggesting little direct role in sexual isolation. Chromosome replacements show that the IPI difference is largely due to genes on chromosome III, with significant interactions involving other chromosomes. Several traits potentially influencing sexual isolation among the melanogaster group of Drosophila seem to be localized to this chromosome. A concentration of important genetic differences might mean that the interaction effects reflect secondary coadaptation of the genetic background to changes associated with chromosome III.     

Genetics of incipient speciation in Drosophila mojavensis. I. Male courtship song, mating success, and genotype x environment interactions

Few studies have examined genotype by environment (GxE) effects on premating reproductive isolation and associated behaviors, even though such effects may be common when speciation is driven by adaptation to different environments. In this study, mating success and courtship song differences among diverging populations of Drosophila mojavensis were investigated in a two-environment quantitative trait locus (QTL) analysis. Baja California and mainland Mexico populations of D. mojavensis feed and breed on different host cacti, so these host plants were used to culture F2 males to examine host-specific QTL effects and GxE interactions influencing mating success and courtship songs. Linear selection gradient analysis showed that mainland females mated with males that produced songs with significantly shorter L(long)-IPIs, burst durations, and interburst intervals. Twenty-one microsatellite loci distributed across all five major chromosomes were used to localize effects of mating success, time to copulation, and courtship song components. Male courtship success was influenced by a single detected QTL, the main effect of cactus, and four GxE interactions, whereas time to copulation was influenced by three different QTLs on the fourth chromosome. Multiple-locus restricted maximum likelihood (REML) analysis of courtship song revealed consistent effects linked with the same fourth chromosome markers that influenced time to copulation, a number of GxE interactions, and few possible cases of epistasis. GxE interactions for mate choice and song can maintain genetic variation in populations, but alter outcomes of sexual selection and isolation, so signal evolution and reproductive isolation may be slowed in diverging populations. Understanding the genetics of incipient speciation in D. mojavensis clearly depends on cactus-specific expression of traits associated with courtship behavior and sexual isolation.     

Sexual isolation between Drosophila melanogaster females and D. simulans males: sexual maturation of males

Isofemale lines of D. simulans were examined to determine the age of sexual maturity of males with conspecific females, and for the frequency of hybridization with D. melanogaster females. Males started to mature sexually on the first day after eclosion but their ability to mate slowly increased during the following day. The estimates of both the age sexual maturation started and the switch from immature to mature males were strongly dependent on the female genotypes used in the tests. No clear differences in speed of maturation were apparent between male lines. In contrast, differences in frequency of hybridization with D. melanogaster females did occur. From the above results it is concluded that the differential hybridization success of male D. simulans lines is not related to the speed at which males mature sexually.

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Résumé Des lignées isofemelles de D. simulans ont été examinées pour déterminer l'âge de la maturité sexuelle des mâles avec des femelles conspécifiques et pour établir la fréquence de l'hybridation avec des femelles de D. melanogaster. Les mâles ont commencé à être sexuellement mûrs le premier jour après l'émergence, mais leur aptitude à la copulation a augmenté lentement pendant le jour suivant. Les estimations, tant de l'âge du début de la maturation sexuelle que de l'âge du passage de mâle immature à mâle sexuellement mûr dépendaient étroitement des génotypes des femelles utilisées dans les expériences. Il n'y avait pas de différences nettes entre les lignées de mâles. Par contre, des différences dans les fréquences d'hybridation avec les femelles de D. melanogaster ont été observées. De ces résultats, on peut conclure que les différences dans la réussite des hybridations des lignées de mâles de D. simulans n'étaient pas dues à la vitesse de maturation sexuelle des mâles.

     

Courtship song recognition in the Drosophila melanogaster complex: heterospecific songs make females receptive in D. melanogaster, but not in D. sechellia

Abstract. The courtship song emitted by male wing vibration has been regarded as one of the most important signals in sexual isolation in the species of the Drosophila melanogaster complex. Inter- and intraspecific crosses were observed using males whose wings were removed (mute) or females whose aristae were removed (deaf). Females of D. melanogaster, D. simulans , and D. mauritiana mated with heterospecific males in the song-present condition (cross between normal females and winged males) more often than in the no-song condition (cross between normal females and wingless males or between aristaless females and winged males) or they showed no preference between the two conditions. It is possible that in these females heterospecific courtship songs play a role as if they were conspecific. In contrast, the females of D. sechellia mated with D. melanogaster or D. simulans males in the no-song condition more often than in the song-present condition, suggesting that they reject males with heterospecific song. Female mate recognition depending on the courtship song in D. melanogaster, D. simulans , and D. mauritiana is considered to be relatively broader and that in D. sechellia narrower.     

Male courtship song and female preference variation between phylogeographically distinct populations of Drosophila montana

Understanding the variation within and between populations in important male mating traits and female preferences is crucial to theories concerning the origin of sexual isolation by coevolution or other processes. There have been surprisingly few studies on the extent of variation and covariation within and between populations, especially where the evolutionary relationships between populations are understood. Here we examine variation in female preferences and a sexually selected male song trait, the carrier frequency of the song, within and between populations from different phylogeographic clusters of Drosophila montana. Song is obligatory for successful mating in this species, and both playback and field studies implicate song carrier frequency as the most important parameter in male song. Carrier frequency varied among three recently collected populations from Oulanka (Finland), Vancouver (Canada), and Colorado (central United States), which represent the main phylogeographic groups in D. montana. Males from Colorado had the most distinct song frequency, which did not follow patterns of genetic differentiation. There was considerable variation in preference functions within, and some variation between, populations. Surprisingly, females from three lines from Colorado seem to have preferences disfavoring the extreme male trait found in this population. We discuss sources of selection on male song and female preference.     

Population and behaviour genetics of Drosophila ananassae

Drosophila ananassae is a cosmopolitan and domestic species. It occupies a nuique status among the Drosophila species due to certain peculiarities in its genetic behaviour. The most unusual feature of this species is spontaneous male recombination in appreciable frequency. The present review summarises the work done on population and behaviour genetics of D. ananassae from India. Population dynamics of three cosmopolitan inversions has been studied in Indian population of D. ananassae and it is evident from the results that there is a considerable degree of genetic divergence at the level of inversion polymorphism. In general, the populations from south India show more differentiation than those from the north. These three cosmopolitan inversions, which are coextensive with the species, exhibit heterosis. Interracial hybridization does not lead to beaakdown of heterosis, which suggests that evidence for coadaptation is lacking in geographic populations of D. ananassae. Heterosis appears to be simple luxuriance rather than populational heterosis (coadaptation). Unlinked inversions occur in random associations, indicating no interchromosomal interactions. However, two inversions of the third chromosome often show strong linkage disequilibrium in laboratory populations, which is due to epistatic gene interaction and suppression of crossing-over. Genetic variations for certain allozyme polymorphism and sternoleural bristle phenotypes in Indian populations of D. ananassae have also been observed.A number of investigations have also been carried out on certain aspects of behaviour genetics of Indian D. ananassae. There is evidence for sexual isolation within D. ananassae. Significant variations in mating propensity of several isofemale strains, inversion karyotypes, the diminishing effects of certain mutations on sexual activity of males and positive response to selection for high and low mating propensity provide evidence for genetic control of sexual behaviour in D. ananassae. Males contribute more to variation and thus are more subject to intra-sexual selection than females. Evidence for rare male mating advantage has also been presented. Geographic strains of D. ananassae show variation with respect to oviposition site preference. The results of studies on pupation site preference, which is an important component of larval behaviour, suggest that larval pupation behaviour in D. ananassae is under polygenic control with a substantial amount of additive genetic variation.     

Molecular and histological characterizations of the Om(2D) mutants in Drosophila ananassae.

A series of transposon-induced optic morphology (Om) mutants found in a hypermutable marker stock of Drosophila ananassae provides a useful system for analyzing the molecular mechanism of eye morphogenesis. In the present study, one of the 25 Om loci so far reported, Om(2D), has been subjected to histological and molecular analyses as a first step toward understanding the role of Om genes in eye morphogenesis. Histological abnormalities observed during eye morphogenesis of the mutant, i.e. cell death within the eye-antennal discs of third instar larvae, and loss of the lamina, disorganized ommatidia and atrophied optic lobes in adults, were all comparable to those reported with various eye morphology mutants of D. melanogaster. Approximately 25 kb of genomic DNA including the Om (2D) locus was cloned by tom tagging. Southern blot and cloning analyses of two alleles of the Om (2D) locus revealed that insertions of the tom element occurred at three sites within 359 bp; two tandemly arrayed toms sharing one long terminal repeat at the junction and an internally deleted tom were present 359 bp apart from each other in Om (2D) 63, while a single tom in reverse orientation was present within the 359 bp in Om (2D) 10a. Host DNA sequences at the three insertion sites were TATAT or AATAT, and ATAT was duplicated upon the tom insertion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences in male courtship song among the species of thequinaria group ofDrosophila

Courtship song is known to vary among several groups of closely related species ofDrosophila. The present study investigates differences in song among four members of thequinaria group of fungal-breedingDrosophila which are known to coexist in Britain and the near-continent. Results show that the species can be distinguished by several song parameters including interpulse interval.D. phalerata is very different from the other three species, while there is considerable overlap betweenD. kuntzei andD. limbata in several characters.D. transversa appears to be more similar to the latter species than it is toD. phalerata. The question of mate choice and the potential for reproductive character displacement are discussed.     

Influence of antibiotic treatment and Wolbachia curing on sexual isolation among Drosophila melanogaster cage populations

Speciation depends on the establishment of reproductive isolation between populations of the same species. Whether assortative mating evolves as a by-product of adaptation is a major question relevant to the origin of species by reproductive isolation. The long-term selection populations used here were originally established 30 years ago from a single cage population (originating from a maternal one) and subsequently subjected to divergent selection for tolerance of toxins in food (heavy metals versus ethanol) to investigate this question. Those populations now differ in sexual isolation and Wolbachia infection status. Wolbachia are common and widespread bacteria infecting arthropods and nematodes. Attention has recently focused on their potential role in insect speciation, due to post-mating sperm-egg incompatibilities induced by the bacteria. In this paper we examine the potential effect of Wolbachia on the level of sexual isolation. By antibiotic curing, we show that removal of Wolbachia decreases levels of mate discrimination (sexual isolation index) between populations by about 50%. Backcrossing experiments confirm that this effect is due to infection status rather than to genetic changes in the populations resulting from antibiotic treatment. Antibiotic treatment has no effect on mate discrimination level between uninfected populations. Our findings suggest that the presence of Wolbachia (or another undetected bacterial associate) act as an additive factor contributing to the level of pre-mating isolation between these Drosophila melanogaster populations. Given the ubiquity of bacterial associates of insects, such effects could be relevant to some speciation events.     

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.     

The search for causal traits of speciation: Divergent female mate preferences target male courtship song,not pheromones,in Drosophila athabasca species complex

Understanding speciation requires the identification of traits that cause reproductive isolation. This remains a major challenge since it is difficult to determine which of the many divergent traits actually caused speciation. To overcome this difficulty, we studied the sexual cue traits and behaviors associated with rapid speciation between EA and WN sympatric behavioral races of Drosophila athabasca that diverged only 16,000–20,000 years ago. First, we found that sexual isolation was essentially complete and driven primarily by divergent female mating preferences. To determine the target of female mate choice, we found that, unlike cuticular hydrocarbons (CHCs), male courtship song is highly divergent between EA and WN in both allopatry and sympatry and is not affected by latitudinal variation. We then used pheromone rub‐off experiments to show no effect of CHCs on divergent female mate choice. In contrast, both male song differences and male mating success in hybrids exhibited a large X‐effect and playback song experiments confirmed that male courtship song is indeed the target of sexual isolation. These results show that a single secondary sexual trait is a major driver of speciation and suggest that we may be overestimating the number of traits involved in speciation when we study older taxa.     

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.     

Nucleotide and repeat length variation at the nonA gene of the Drosophila virilis group species and its effects on male courtship song

Genes found to affect male courtship song characters in Drosophila melanogaster are good candidates when tracing genes responsible for species-specific songs in other Drosophila species. It has previously been shown that Thr–Gly repeat length variation at the period gene affects song traits in D. melanogaster, which gives the repetitive regions a special interest. In this work, we have characterised the patterns of nucleotide variation for gene regions containing two Gly and one Gln–Ala repeat in another D. melanogaster song gene, no-on-transient A, in D. virilis group species. The levels of nucleotide variability in D. virilis nonA were similar to those found for other genes of the species, and the gene sequences showed no signs of deviation from neutrality. The Gly 2 repeat preceding the central domain of the gene exhibited length variation, which did not, however, correlate with song variation either within D. virilis or between the species of D. virilis group. The Gly 3 repeat located on the other side of the central domain showed amino acid divergence parallel to the consensus phylogeny of the D. virilis group species. The species of the virilis subgroup having Asn after the first three glycines in this repeat have simple songs with no species-specificity, while the species of the montana subgroup having two Gly or Asn–Ser in this site have unique courtship songs. Amino acid differences between the species in this repeat may, however, reflect species phylogeny rather than have an effect on song divergence per se.     

Female Remating in Drosophila ananassae: Evidence for the Effect of Density on Female Remating Frequency

Drosophila ananassae, a cosmopolitan and domestic species, is largely circumtropical in distribution and belongs to the ananassae species complex of the ananassae subgroup of the melanogaster species group. In the present study, experiments were conducted to investigate the effect of density on female remating frequency by employing different wild-type and mutant strains of D. ananassae. Two experimental designs, i.e., 2-h daily observation and continuous confinement, were used. The results show that there is significant dependence of remating frequency on density in all strains tested under both experimental designs except in a wild-type strain (Bhutan), which shows no dependence of remating frequency on density under 2-h daily observation design. This finding provides evidence that density may increase the frequency of female remating in D. ananassae.     

Courtship behavior and sexual isolation between Drosophila auraria and D. triauraria in darkness and light

Sexual isolation between two species of the Drosophila auraria complex, D. auraria and D. triauraria is different in darkness and light. In darkness there is complete isolation while in light it is only partial. The sensory bases of these differences were investigated by behavioral studies in darkness and light. In darkness there was no normal courtship sequence but males of both species displayed attempted copulation to homospecific and heterospecific females, as well as homospecific males. After a few hours together, homospecific copulations were observed after females displayed a characteristic posture: spreading the wings. Such an acceptance posture, also observed in other Drosophila species, was probably released by the wing vibration of a homospecific male. In light, visual signals alone are able to elicit male's orientation and following, whether flies are housed together or physically separated. Intense homosexual courtships were observed. All these data suggest that males have a low discrimination ability and females play the main role in sexual isolation.     

Two distinct genomic regions, harbouring the period and fruitless genes, affect male courtship song in Drosophila montana

Acoustic signals often have a significant role in pair formation and in species recognition. Determining the genetic basis of signal divergence will help to understand signal evolution by sexual selection and its role in the speciation process. An earlier study investigated quantitative trait locus for male courtship song carrier frequency (FRE) in Drosophila montana using microsatellite markers. We refined this study by adding to the linkage map markers for 10 candidate genes known to affect song production in Drosophila melanogaster. We also extended the analyses to additional song characters (pulse train length (PTL), pulse number (PN), interpulse interval, pulse length (PL) and cycle number (CN)). Our results indicate that loci in two different regions of the genome control distinct features of the courtship song. Pulse train traits (PTL and PN) mapped to the X chromosome, showing significant linkage with the period gene. In contrast, characters related to song pulse properties (PL, CN and carrier FRE) mapped to the region of chromosome 2 near the candidate gene fruitless, identifying these genes as suitable loci for further investigations. In previous studies, the pulse train traits have been found to vary substantially between Drosophila species, and so are potential species recognition signals, while the pulse traits may be more important in intra-specific mate choice.     

Sexual Isolation Between Drosophila Melanogaster, D. Simulans and D. Mauritiana: Sex and Species Specific Discrimination

The sexual isolation among the related species Drosophila melanogaster, D. simulans and D. mauritiana is asymmetrical. While D. mauritiana males mate well with both D. melanogaster and D. simulans females, females of D. mauritiana discriminate strongly against males of these two species. Similarly, D. simulans males mate with D. melanogaster females but the reciprocal cross is difficult. Interspecific crosses between several populations of the three species were performed to determine if (i) males and females of the same species share a common sexual isolation genetic system, and (ii) males (or females) use the same genetic system to discriminate against females (or males) of the other two species. Results indicate that although differences in male and female isolation depend on the populations tested, the isolation behaviour between a pair of species is highly correlated despite the variations. However, the rank order of the isolation level along the populations was not correlated in both sexes, which suggests that different genes act in male and female sexual isolation. Neither for males nor for females, the isolation behaviour of one species was paralleled in the other two species, which indicates that the genetic systems involved in this trait are species-pair specific. The implications of these results are discussed. This revised version was published online in August 2006 with corrections to the Cover Date.