Genetics of a Pheromonal Difference Affecting Sexual Isolation between Drosophila Mauritiana and D. Sechellia

Females of the sibling species Drosophila sechellia and D. mauritiana differ in their cuticular hydrocarbons: the predominant compound in D. sechellia is 7,11-heptacosadiene (7,11-HD), while that in D. mauritiana is 7-tricosene (7-T). We investigate the genetic basis of this difference and its involvement in reproductive isolation between the species. Behavioral studies involving hydrocarbon transfer suggest that these compounds play a large role in the sexual isolation between D. mauritiana males and D. sechellia females, while sexual isolation in the reciprocal hybridization results more from differences in female behavior than hydrocarbons. This interspecific difference in hydrocarbon profile is due to evolutionary change at a minimum of six loci, all on the third chromosome. The localization of evolutionary change to the third chromosome has been seen in every other genetic analysis of female hydrocarbon differences in the D. melanogaster group. We suggest that the high 7,11-HD phenotype seen in two species evolved twice independently from ancestors having the high 7-T phenotype, and that the recurrent third-chromosome effects are evolutionary convergences that may be due to a concentration of ``hydrocarbon genes' on that chromosome.     

Genetics of Differences in Pheromonal Hydrocarbons between Drosophila Melanogaster and D. Simulans

Females of Drosophila melanogaster and its sibling species D. simulans have very different cuticular hydrocarbons, with the former bearing predominantly 7,11-heptacosadiene and the latter 7-tricosene. This difference contributes to reproductive isolation between the species. Genetic analysis shows that this difference maps to only the third chromosome, with the other three chromosomes having no apparent effect. The D. simulans alleles on the left arm of chromosome 3 are largely recessive, allowing us to search for the relevant regions using D. melanogaster deficiencies. At least four nonoverlapping regions of this arm have large effects on the hydrocarbon profile, implying that several genes on this arm are responsible for the species difference. Because the right arm of chromosome 3 also affects the hydrocarbon profile, a minimum of five genes appear to be involved. The large effect of the thrid chromosome on hydrocarbons has also been reported in the hybridization between D. simulans and its closer relative D. sechellia, implying either an evolutionary convergence or the retention in D. sechellia of an ancestral sexual dimorphism.     

The genetics of mating recognition between Drosophila simulans and D. sechellia

During courtship, visual and chemical signals are often exchanged between the sexes. The proper exchange of such signals ensures intraspecific recognition. We have examined the genetic basis of interspecific differences in male mating behaviour and pheromone concentration between Drosophila simulans and D. sechellia by using Drosophila simulans/D. sechellia introgression lines. Our results show a majority of quantitative trait loci (QTLs) explaining variation in both male mating behaviour and pheromone concentration to be located on the third chromosome. One QTL found on the third chromosome explains variation in time needed to start courtship and copulation as well as time spent courting. The position of such QTL (approximately 84A-88B) with effects on courtship and copulation aspects of mating includes the candidate sex determination gene doublesex (84E5-6) and Voila (86E1-2), a gene that affects male courtship in D. melanogaster. One additional third chromosome QTL explained variation in 7-tricosene pheromone concentrations among males. The interval mapping position of this QTL (approximately 68E-76E) did not overlap with the position detected for differences in mating behaviour and the intervals did not include candidate genes previously identified as having an effect on D. melanogaster cuticular hydrocarbon production. We did not detect any directionality of the effect of Drosophila sechellia allele introgressions in male mating recognition.     

The Effects of Interspecific Y Chromosome Replacements on Hybrid Sterility within the Drosophila Simulans Clade

We attempted to introgress Y chromosomes between three sibling species of Drosophila: D. simulans, D. sechellia and D. mauritiana. Four D. sechellia Y chromosomes were introgressed into D. simulans without loss of fertility whereas the four reciprocal introgressions (D. simulans Y introgressed into D. sechellia) all result in sterility. Both reciprocal Y introgressions of D. simulans and D. mauritiana (four of each) also result in sterility. Compared with D. simulans males, the males with the D. sechellia Y chromosome in D. simulans background had lower productivity but only after multiple matings with virgin females. These males also were inferior compared with pure species males in sperm displacement and/or remating ability. The two different Y genotype males, however, were comparable in viability, longevity and mating success in female choice tests. We also use our results to estimate the effective number of autosomal loci interacting with X-linked genes to produce hybrid male sterility.     

The Genetic Basis of Haldane''s Rule and the Nature of Asymmetric Hybrid Male Sterility among Drosophila Simulans, Drosophila Mauritiana and Drosophila Sechellia

Haldane's rule (i.e., the preferential hybrid sterility and inviability of heterogametic sex) has been known for 70 years, but its genetic basis, which is crucial to the understanding of the process of species formation, remains unclear. In the present study, we have investigated the genetic basis of hybrid male sterility using Drosophila simulans, Drosophila mauritiana and Drosophila sechellia. An introgression of D. sechellia Y chromosome into a fairly homogenous background of D. simulans did not show any effect of the introgressed Y on male sterility. The substitution of D. simulans Y chromosome into D. sechellia, and both reciprocal Y chromosome substitutions between D. simulans and D. mauritiana were unsuccessful. Introgressions of cytoplasm between D. simulans and D. mauritiana (or D. sechellia) also did not have any effect on hybrid male sterility. These results rule out the X-Y interaction hypothesis as a general explanation of Haldane's rule in this species group and indicate an involvement of an X-autosome interaction. Models of symmetrical and asymmetrical X-autosome interaction have been developed which explain the Y chromosome substitution results and suggest that evolution of interactions between different genetic elements in the early stages of speciation is more likely to be of an asymmetrical nature. The model of asymmetrical X-autosome interaction also predicts that different sets of interacting genes may be involved in different pairs of related species and can account for the observation that hybrid male sterility in many partially isolated species is often nonreciprocal or unidirectional.     

The genetic basis of behavioral isolation between Drosophila mauritiana and D. sechellia

Understanding how species form is a fundamental question in evolutionary biology. Identifying the genetic bases of barriers that prevent gene flow between species provides insight into how speciation occurs. Here, I analyze a poorly understood reproductive isolating barrier, prezygotic reproductive isolation. I perform a genetic analysis of prezygotic isolation between two closely related species of Drosophila, D. mauritiana and D. sechellia. I first confirm the existence of strong behavioral isolation between D. mauritiana females and D. sechellia males. Next, I examine the genetic basis of behavioral isolation by (1) scanning an existing set of introgression lines for chromosomal regions that have a large effect on isolation; and (2) mapping quantitative trait loci (QTL) that underlie behavioral isolation via backcross analysis. In particular, I map QTL that determine whether a hybrid backcross female and a D. sechellia male will mate. I identify a single significant QTL, on the X chromosome, suggesting that few major-effect loci contribute to behavioral isolation between these species. In further work, I refine the map position of the QTL to a small region of the X chromosome.     

Quantitative trait loci for cuticular hydrocarbons associated with sexual isolation between Drosophila simulans and D. sechellia

The identification of genes with large effects on sexual isolation and speciation is an important link between classic evolutionary genetics and molecular biology. Few genes that affect sexual isolation and speciation have been identified, perhaps because many traits influencing sexual isolation are complex behaviors. Cuticular hydrocarbons (CHs) of species of the Drosophila melanogaster group play a large role in sexual isolation by functioning as contact pheromones influencing mate recognition. Some of the genes that play key roles in determining species-specific CHs have been identified. We have performed separate quantitative trait locus (QTL) analyses of 7-tricosene (7-T) and 7,11-heptacosadiene (7,11-HD), the two major female CHs differing between D. simulans and D. sechellia. We find that approximately 40% of the phenotypic variance in each CH is associated with two to four chromosomal regions. A region on the right arm of chromosome 3 contains QTL that affect both traits, but other QTL are in distinct chromosomal regions. Epistatic interactions were detected between two pairs of QTL for 7,11-HD such that if either were homozygous for the D. simulans allele, the fly was similar to D. simulans in phenotype, with a low level of 7,11-HD. We discuss the location of these regions with regard to candidate genes for CH production, including those for desaturases.     

Genetics of sexual isolation in females of the Drosophila simulans species complex.

Genetic analysis of hybrids between Drosophila simulans and D. sechellia shows that sexual isolation in females is caused by at least two genes, one on each major autosome, while the X chromosome has no effect. These results are similar to those of a previous study of hybrids between D. simulans and another sibling species, D. mauritiana. In this latter hybridization, each arm of the second chromosome carries genes causing sexual isolation in females, implying a total divergence of at least three loci. The genetic similarity between the D. simulans/D. mauritiana and D. simulans/D. sechellia hybridizations probably results from independent evolution and not phylogenetic artifacts, because the dominance relationships and behavioural interactions differ between the two hybridizations. The lack of an X-chromosome effect on sexual isolation contrasts with genetic studies of post-zygotic reproductive isolation, which invariably show strong effects of this chromosome.     

Chromosome localization of the lambda20p1.4 clone of the Drosophila melanogaster nuclear lamina DNA in the melanogaster species subgroup of the genus Drosophila (Sophophora)

Chromosome localization of sequences homologous to lambda 20p1.4 of the Drosophila melanogaster nuclear lamina DNA (nlDNA) was established by in situ hybridization in species of the melanogaster subgroup. DNA of the lambda 20p1.4 clone was shown to be located in the chromocenter in all the species examined. Laboratory strains of D. simulans, D. mauritiana, and D. sechellia exhibited interspecific differences in localization of lambda 20p1.4 nlDNA on chromosome arms. In eight natural populations, intraspecific polymorphism of lambda 20p1.4 nlDNA chromosome localization was shown to be present in D. simulans but absent in D. melanogaster. The possible participation of transposable elements in nlDNA relocation is discussed.     

Nup96-dependent hybrid lethality occurs in a subset of species from the simulans clade of Drosophila

The cross of Drosophila melanogaster females to D. simulans males typically produces lethal F(1) hybrid males. F(1) male lethality is suppressed when the D. simulans Lhr(1) hybrid rescue strain is used. Viability of these F(1) males carrying Lhr(1) is in turn substantially reduced when the hybrids are heterozygous for some mutant alleles of the D. melanogaster Nup96 gene. I show here that similar patterns of Nup96-dependent lethality occur when other hybrid rescue mutations are used to create F(1) males, demonstrating that Nup96 does not reduce hybrid viability by suppressing the Lhr(1) rescue effect. The penetrance of this Nup96-dependent lethality does not correlate with the penetrance of the F(1) hybrid rescue, arguing that these two phenomena reflect genetically independent processes. D. simulans, together with two additional sister species, forms a clade that speciated after the divergence of their common ancestor from D. melanogaster. I report here that Nup96(-) reduces F(1) viability in D. melanogaster hybrids with one of these sister species, D. sechellia, but not with the other, D. mauritiana. These results suggest that Nup96-dependent lethality evolved after the speciation of D. melanogaster from the common ancestor of the simulans clade and is caused by an interaction among Nup96, unknown gene(s) on the D. melanogaster X chromosome, and unknown autosomal gene(s), at least some of which have diverged in D. simulans and D. sechellia but not in D. mauritiana. The genetic properties of Nup96 are also discussed relative to other hybrid lethal genes.     

A Combined Classical Genetic and High Resolution Two-Dimensional Electrophoretic Approach to the Assessment of the Number of Genes Affecting Hybrid Male Sterility in Drosophila Simulans and Drosophila Sechellia

We have attempted to estimate the number of genes involved in postzygotic reproductive isolation between two closely related species, Drosophila simulans and Drosophila sechellia, by a novel approach that involves the use of high resolution two-dimensional gel electrophoresis (2DE) to examine testis proteins in parents, hybrids and fertile and sterile backcross progenies. The important results that have emerged from this study are as follows: (1) about 8% of about 1000 proteins examined showed divergence (presence/absence) between the two species; (2) by tracing individual proteins in parental, hybrid and backcross males, we were able to associate the divergent proteins with different chromosomes and found that most divergent proteins are associated with autosomes and very few with X chromosome, Y chromosome and cytoplasm; (3) when proteins showing both quantitative and qualitative differences between the two species were examined in F(1) hybrid males, most (97.4%) proteins were expressed at levels between the two parents and no sign of large scale changes in spot density was observed. All the proteins observed in the two parental species were present in F(1) hybrid males except two species-specific proteins that may be encoded (or regulated) by sex chromosomes; (4) when different fertile and sterile backcross male testes were compared, a few D. sechellia-specific proteins were identified to be consistently associated with male sterility. These results along with the observation that a large proportion (23.6%) of first generation backcross males were fertile show that hybrid male sterility between D. simulans and D. sechellia involves a relatively small number of genes. Role of large scale genetic changes due to general genome incompatibility is not supported. The results also suggest that the large effect of X chromosome on hybrid male sterility is not due to higher divergence of X chromosome than autosomes.     

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 egg production in Drosophila sechellia

Drosophila sechellia, an island endemic that specializes on a single host plant, has a lower rate of egg production than its generalist sister species D. melanogaster, D. simulans, and D. mauritiana. Earlier work showed that part of this difference in egg production was due to a reduction in the number of ovarioles in D. sechellia relative to its sister species. Here, I extend this earlier work by genetically analyzing the difference in egg production between D. sechellia and D. simulans. In all, 10 genetic markers were used in several interspecific backcrosses to identify chromosome regions that affected the rate of egg production. While previously mapped factors affecting ovariole number appear to impact the rate of egg production, new, non-ovariole factors were also identified. Overall, the difference in egg production between D. sechellia and D. simulans appears to be a polygenic trait. The relationship between these factors and genes involved the adaptation of D. sechellia to its host plant is not yet clear. The data are consistent with the hypothesis that decline in egg production is, in part, a negative pleiotropic effect of genetic changes required for host specialization in D. sechellia, although finer-scale genetic analysis of both traits is needed to truly test this hypothesis.     

Mate choice in Drosophila melanogaster and D. sechellia: criteria and their variation depending on courtship song

Courtship song is one of the most important male signals in Drosophila mating. A female D. melanogaster copulates more readily when given a conspecific type of courtship song. Female D. melanogaster accepted winged D. sechellia more than wingless ones in a no choice test. Copulation frequencies varied between strains and were significantly correlated with male mating propensity of D. sechellia. Females from three of five strains of D. sechellia accepted winged D. melanogaster less than wingless ones in no choice tests, suggesting that D. melanogaster songs reduce mating in D. sechellia females. Multiple choice tests showed that males prefer conspecifics. In female choice tests, D. melanogaster females copulated with winged heterospecific D. sechellia males more than with wingless conspecific D. melanogaster males in the confined condition, suggesting that song differences between D. melanogaster and D. sechellia affect D. melanogaster females less than the absence of courtship song. Copyright 2000 The Association for the Study of Animal Behaviour.     

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.     

Discord between the Phylogenies Inferred from Molecular versus Functional Data: Uneven Rates of Functional Evolution or Low Levels of Gene Flow?

According to measures of molecular divergence, the three species of the Drosophila simulans clade are closely related to and essentially equidistant from each other. We introgressed 10% of the D. sechellia X chromosome into a pure D. simulans genetic background and found that males carrying this introgressed region were consistently fertile; in contrast, males carrying the same segment from D. mauritiana are sterile and suffer from incompatibilities at a minimum of four loci. Together with other recent results, these data suggest that D. simulans and D. sechellia are much more closely related to each other than either is to D. mauritiana. How can we reconcile the phylogeny inferred from the density of hybrid sterility genes with that inferred from molecular divergence? If the molecular phylogeny is correct, the discrepancy might be explained by uneven rates of functional evolution, resulting in the uneven accumulation of substitutions with corresponding negative effects in hybrids. If the functional phylogeny is correct, then low levels of gene flow across nascent species boundaries, particularly for loci not tightly linked to a hybrid sterility gene, may have erased the original pattern of lineage splitting. We propose tests that will allow us to discriminate between these hypotheses.     

Wolbachia segregation dynamics and levels of cytoplasmic incompatibility in Drosophila sechellia

In Drosophila sechellia, the endocellular bacterium Wolbachia induces cytoplasmic incompatibility (CI): in crosses involving infected males, a partial or complete embryonic mortality occurs unless the female bears the same Wolbachia. D. sechellia is known to harbour two Wolbachia variants, namely wSh and wSn, closely related to wHa and wNo, respectively, two strains infecting the populations of D. simulans from the Seychelles archipelago and New Caledonia. Strikingly, the two species show similar infection patterns: in D. sechellia, wSh can be present on its own or in double infection with wSn, but individuals carrying wSn only do not occur; in D. simulans, wHa can be present on its own or in double infection with wNo, but individuals carrying wNo only do not occur, or occur at very low frequency. Previous experiments on D. simulans showed that lines singly infected by wNo can be obtained by segregation, and stably maintained. Here we investigate this issue in D. sechellia through an 18 generation experiment, and show that wSn and wSh singly infected lines can arise by segregation. Using singly infected lines obtained in this experiment, we estimate the CI intensities of wSh and wSn in D. sechellia, and compare these to the CI intensities of the same Wolbachia injected into D. simulans. Our results do not suggest any consistent effect of the host species on the CI induced by wSh. On the contrary, it seems that wSn expression is repressed by host factors in D. sechellia.     

Variation in fiber number of a male-specific muscle between Drosophila species: a genetic and developmental analysis

We characterize a newly discovered morphological difference between species of the Drosophila melanogaster subgroup. The muscle of Lawrence (MOL) contains about four to five fibers in D. melanogaster and Drosophila simulans and six to seven fibers in Drosophila mauritiana and Drosophila sechellia. The same number of nuclei per fiber is present in these species but their total number of MOL nuclei differs. This suggests that the number of muscle precursor cells has changed during evolution. Our comparison of MOL development indicates that the species difference appears during metamorphosis. We mapped the quantitative trait loci responsible for the change in muscle fiber number between D. sechellia and D. simulans to two genomic regions on chromosome 2. Our data eliminate the possibility of evolving mutations in the fruitless gene and suggest that a change in the twist might be partly responsible for this evolutionary change.     

The population genetics of the origin and divergence of the Drosophila simulans complex species

The origins and divergence of Drosophila simulans and close relatives D. mauritiana and D. sechellia were examined using the patterns of DNA sequence variation found within and between species at 14 different genes. D. sechellia consistently revealed low levels of polymorphism, and genes from D. sechellia have accumulated mutations at a rate that is approximately 50% higher than the same genes from D. simulans. At synonymous sites, D. sechellia has experienced a significant excess of unpreferred codon substitutions. Together these observations suggest that D. sechellia has had a reduced effective population size for some time, and that it is accumulating slightly deleterious mutations as a result. D. simulans and D. mauritiana are both highly polymorphic and the two species share many polymorphisms, probably since the time of common ancestry. A simple isolation speciation model, with zero gene flow following incipient species separation, was fitted to both the simulans/mauritiana divergence and the simulans/sechellia divergence. In both cases the model fit the data quite well, and the analyses revealed little evidence of gene flow between the species. The exception is one gene copy at one locus in D. sechellia, which closely resembled other D. simulans sequences. The overall picture is of two allopatric speciation events that occurred quite near one another in time.