Susannah Elwyn

GENETICS OF A DIFFERENCE IN PIGMENTATION BETWEEN DROSOPHILA YAKUBA AND DROSOPHILA SANTOMEA

Abstract Drosophila yakuba is a species 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 its common ancestor with D. yakuba. The two species differ strikingly in pigmentation: D. santomea, unlike the other eight species in the D. melanogaster subgroup, almost completely lacks dark abdominal pigmentation. D. yakuba shows the sexually dimorphic pigmentation typical of the group: both sexes have melanic patterns on the abdomen, but males are much darker than females. A genetic analysis of this species difference using morphological markers shows that the X chromosome accounts for nearly 90% of the species difference in the area of abdomen that is pigmented and that at least three genes (one on each major chromosome) are involved in each sex. The order of chromosome effects on pigmentation area are the same in males and females, suggesting that loss of pigmentation in D. santomea may have involved the same genes in both sexes. Further genetic analysis of the interspecific difference between males in pigmentation area and intensity using molecular markers shows that at least five genes are responsible, with no single locus having an overwhelming effect on the trait. The species difference is thus oligogenic or polygenic. Different chromosomal regions from each of the two species influenced pigmentation in the same direction, suggesting that the species difference (at least in males) is due to natural or sexual selection and not genetic drift. Measurements of sexual isolation between the species in both light and dark conditions show no difference, suggesting that the pigmentation difference is not an important cue for interspecific mate discrimination. Using DNA sequence differences in nine noncoding regions, we estimate that D. santomea and D. yakuba diverged about 400,000 years ago, a time similar to the divergences between two other well-studied pair of species in the subgroup, both of which also involved island colonization.

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 Tomé, 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.

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.

The Genetic Basis of Postzygotic Reproductive Isolation Between Drosophila santomea and D. yakuba Due to Hybrid Male Sterility

A major unresolved challenge of evolutionary biology is to determine the nature of the allelic variants of “speciation genes”: those alleles whose interaction produces inviable or infertile interspecific hybrids but does not reduce fitness in pure species. Here we map quantitative trait loci (QTL) affecting fertility of male hybrids between D. yakuba and its recently discovered sibling species, D. santomea. We mapped three to four X chromosome QTL and two autosomal QTL with large effects on the reduced fertility of D. yakuba and D. santomea backcross males. We observed epistasis between the X-linked QTL and also between the X and autosomal QTL. The X chromosome had a disproportionately large effect on hybrid sterility in both reciprocal backcross hybrids. However, the genetics of hybrid sterility differ between D. yakuba and D. santomea backcross males, both in terms of the magnitude of main effects and in the epistatic interactions. The QTL affecting hybrid fertility did not colocalize with QTL affecting sexual isolation in this species pair, but did colocalize with QTL affecting the marked difference in pigmentation between D. yakuba and D. santomea. These results provide the basis for future high-resolution mapping and ultimately, molecular cloning, of the interacting genes that contribute to hybrid sterility.

DOES THE DESATURASE-2 LOCUS IN DROSOPHILA MELANOGASTER CAUSE ADAPTATION AND SEXUAL ISOLATION?

Abstract The desaturase-2 (desat2) locus of Drosophila melanogaster has two alleles whose frequencies vary geographically: one (the “Z” allele) is found primarily in east Africa and the Caribbean, and the other (the “M” allele) occurs in other parts of the world. It has been suggested that these alleles not only cause sexual isolation between races, but that their distribution reflects differential adaptation to climate: Z alleles are supposedly adapted to tropical conditions and M alleles to temperate ones. This has thus been viewed as a case of reproductive isolation evolving as a pleiotropic byproduct of adaptation. Here we reinvestigate this presumed climatic adaptation, using transgenic lines differing in the nature of their desat2 alleles. We were unable to replicate earlier results showing that carriers of M alleles are uniformly more cold resistant and less starvation resistant than carriers of Z alleles. It is thus doubtful whether the distribution of these alleles reflects natural selection involving climate. Mating studies of transgenic lines show some evidence for sexual isolation due to desat2. However, work on other, wild-type lines, as well as observations on the nature of sexual isolation, suggest that this conclusion—and thus the relationship between this locus and mating discrimination between races of D. melanogaster—may also be doubtful.

The Genetic Basis of Prezygotic Reproductive Isolation Between Drosophila santomea and D. yakuba Due to Mating Preference

Sexual isolating mechanisms that act before fertilization are often considered the most important genetic barriers leading to speciation in animals. While progress has been made toward understanding the genetic basis of the postzygotic isolating mechanisms of hybrid sterility and inviability, little is known about the genetic basis of prezygotic sexual isolation. Here, we map quantitative trait loci (QTL) contributing to prezygotic reproductive isolation between the sibling species Drosophila santomea and D. yakuba. We mapped at least three QTL affecting discrimination of D. santomea females against D. yakuba males: one X-linked and one autosomal QTL affected the likelihood of copulation, and a second X chromosome QTL affected copulation latency. Three autosomal QTL also affected mating success of D. yakuba males with D. santomea. No epistasis was detected between QTL affecting sexual isolation. The QTL do not overlap between males and females and are not disproportionately concentrated on the X chromosome. There was some overlap in map locations of QTL affecting sexual isolation between D. santomea and D. yakuba with QTL affecting sexual isolation between D. simulans and D. mauritiana and with QTL affecting differences in pigmentation between D. santomea and D. yakuba. Future high-resolution mapping and, ultimately, positional cloning, will reveal whether these traits do indeed have a common genetic basis.

DESATURASE-2, ENVIRONMENTAL ADAPTATION, AND SEXUAL ISOLATION IN DROSOPHILA MELANOGASTER

In a previous paper (Coyne and Elwyn 2006), we repeated environmental stress experiments in Drosophila melanogaster that were originally conducted by Greenberg et al. (2003). In their study, Greenberg et al. used targeted gene replacement to construct lines having different alleles of the desaturase-2 (desat2) locus—a gene involved in synthesis of cuticular hydrocarbons. They found that different alleles had different effects on flies’ responses to cold and starvation stress: carriers of the African and Caribbean allele (ds2Z) consistently had less cold resistance and greater starvation resistance than carriers of the Cosmopolitan allele (ds2M). Greenberg et al. (2003) proposed that these differences are adaptive because one might expect African flies to encounter higher temperatures and less available food. Because there was earlier evidence for sexual isolation between carriers of different desat2 alleles (African females homozygous for ds2Z discriminate against non-African males homozygous for ds2M; Fang et al. 2002), possibly because different cuticular hydrocarbons act as mating cues, Greenberg et al. (2003) interpreted the sexual isolation between African and Cosmopolitan races as a byproduct of adaptation to different environments. Using the same lines described by Greenberg et al. (2003), but with a larger sample of transgenic constructs and of tested individuals, Coyne and Elwyn (2006) failed to find consistent ‘‘adaptive’’ differences between ds2Z and ds2M alleles in their tolerance to cold and starvation stress. Rather, the association was more or less random, with differences between the alleles sometimes nonsignificant and sometimes significant in directions opposite to those reported by Greenberg et al. (2003). We attributed our observations of ‘‘nonadaptive’’ differences in stress tolerance between different alleles, as well as the differences among replicate constructs of the same desat2 allele, to differences in the genetic backgrounds of transgenic constructs—differences that arose during their synthesis and that could affect stress tolerance independently of the desat2 genotype. We have no explanation for the discrepancy between the results of Coyne and Elwyn (2006) and those of Greenberg et al. (2003). Coyne and Elwyn also (2006) also reported mating experiments between carriers of various genetically engineered desat2 alleles and found that, although there was some evidence that desat2 may be involved in sexual isolation, this evidence was weak and was contradicted by other observations. After learning of Coyne and Elwyn’s new results, Green-