Little effect of the tan locus on pigmentation in female hybrids between Drosophila santomea and D. melanogaster.

Previous work on Drosophila santomea suggested that its absence of abdominal pigmentation, compared to the other darkly pigmented species, is based on mutations in the cis-regulatory region of tan, inactivating the expression of that gene in the abdomen of D. santomea males and females. Our discovery that D. santomea males can produce viable hybrids when mated to D. melanogaster females enables us to use the armamentarium of genetic tools in the latter species to study the genetic basis of this interspecific difference in pigmentation. Hybridization tests using D. melanogaster deficiencies that include tan show no evidence that this locus is involved in the lighter pigmentation of D. santomea females; rather, the pigmentation difference appears to involve at least four other loci in the region. Earlier results implicating tan may have been based on a type of transgenic analysis that can give misleading results about the genes involved in an evolutionary change.

Inclusive fitness theory and eusociality.

Arising from M. A. Nowak, C. E. Tarnita & E. O. Wilson 466, 1057-1062 (2010); Nowak et al. reply. Nowak et al. argue that inclusive fitness theory has been of little value in explaining the natural world, and that it has led to negligible progress in explaining the evolution of eusociality. However, we believe that their arguments are based upon a misunderstanding of evolutionary theory and a misrepresentation of the empirical literature. We will focus our comments on three general issues.

Evolution of protein expression: new genes for a new diet.

A new study identifies gene duplication of a salivary enzyme as a recent adaptation to changes in diet among human populations, highlighting the diverse ways that gene regulation can evolve.

Rapid and Predictable Evolution of Admixed Populations Between Two Drosophila Species Pairs.

The consequences of hybridization are varied, ranging from the origin of new lineages, introgression of some genes between species, to the extinction of one of the hybridizing species. We generated replicate admixed populations between two pairs of sister species of Drosophila: D. simulans and D. mauritiana; and D. yakuba and D. santomea Each pair consisted of a continental species and an island endemic. The admixed populations were maintained by random mating in discrete generations for over 20 generations. We assessed morphological, behavioral, and fitness-related traits from each replicate population periodically, and sequenced genomic DNA from the populations at generation 20. For both pairs of species, species-specific traits and their genomes regressed to those of the continental species. A few alleles from the island species persisted, but they tended to be proportionally rare among all sites in the genome and were rarely fixed within the populations. This paucity of alleles from the island species was particularly pronounced on the X-chromosome. These results indicate that nearly all foreign genes were quickly eliminated after hybridization and that selection against the minor species genome might be similar across experimental replicates.

The genetic basis of sexual dimorphism in birds.

The genetic basis of sexual dimorphisms is an intriguing problem of evolutionary genetics because dimorphic traits are limited to one sex. Such traits can arise genetically in two ways. First, the alleles that cause dimorphisms could be limited in expression to only one sex at their first appearance. Alternatively, dimorphism alleles could initially be expressed in both sexes, but subsequently be repressed or promoted in only one sex by the evolution of modifier genes or regulatory elements. We investigated these alternatives by looking for the expression of sexually dimorphic traits in female hybrids between bird species whose males show different types of ornaments. If modifier alleles or regulatory elements involved in sex-limited traits are not completely dominant, the modification should break down in female hybrids, which might then show dimorphic traits resembling those seen in males. Of 13 interspecific hybridizations examined, we found not a single instance of the expression of male-limited ornaments in female hybrids. This suggests that male ornaments were sex limited from the outset or that those traits became sex limited through the evolution of dominant modifiers -- possibly cis-dominant regulatory elements. Observing hybrid phenotypes is a useful approach to studying the genetics and evolution of dimorphic traits.

The locus of evolution: evo devo and the genetics of adaptation.

An important tenet of evolutionary developmental biology ("evo devo") is that adaptive mutations affecting morphology are more likely to occur in the cis-regulatory regions than in the protein-coding regions of genes. This argument rests on two claims: (1) the modular nature of cis-regulatory elements largely frees them from deleterious pleiotropic effects, and (2) a growing body of empirical evidence appears to support the predominant role of gene regulatory change in adaptation, especially morphological adaptation. Here we discuss and critique these assertions. We first show that there is no theoretical or empirical basis for the evo devo contention that adaptations involving morphology evolve by genetic mechanisms different from those involving physiology and other traits. In addition, some forms of protein evolution can avoid the negative consequences of pleiotropy, most notably via gene duplication. In light of evo devo claims, we then examine the substantial data on the genetic basis of adaptation from both genome-wide surveys and single-locus studies. Genomic studies lend little support to the cis-regulatory theory: many of these have detected adaptation in protein-coding regions, including transcription factors, whereas few have examined regulatory regions. Turning to single-locus studies, we note that the most widely cited examples of adaptive cis-regulatory mutations focus on trait loss rather than gain, and none have yet pinpointed an evolved regulatory site. In contrast, there are many studies that have both identified structural mutations and functionally verified their contribution to adaptation and speciation. Neither the theoretical arguments nor the data from nature, then, support the claim for a predominance of cis-regulatory mutations in evolution. Although this claim may be true, it is at best premature. Adaptation and speciation probably proceed through a combination of cis-regulatory and structural mutations, with a substantial contribution of the latter.

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.

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?

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.

Multilocus analysis of introgression between two sympatric sister species of Drosophila: Drosophila yakuba and D. santomea.

Drosophila yakuba is widely distributed in sub-Saharan Africa, while D. santomea is endemic to the volcanic island of São Tomé in the Atlantic Ocean, 280 km west of Gabon. On São Tomé, D. yakuba is found mainly in open lowland forests, and D. santomea is restricted to the wet misty forests at higher elevations. At intermediate elevations, the species form a hybrid zone where hybrids occur at a frequency of approximately 1%. To determine the extent of gene flow between these species we studied polymorphism and divergence patterns in 29 regions distributed throughout the genome, including mtDNA and three genes on the Y chromosome. This multilocus approach, together with the comparison to the two allopatric species D. mauritiana and D. sechellia, allowed us to distinguish between forces that should affect all genes and forces that should act on some genes (e.g., introgression). Our results show that D. yakuba mtDNA has replaced that of D. santomea and that there is also significant introgression for two nuclear genes, yellow and salr. The majority of genes, however, has remained distinct. These two species therefore do not form a "hybrid swarm" in which much of the genome shows substantial introgression while disruptive selection maintains distinctness for only a few traits (e.g., pigmentation and male genitalia).

Quantitative trait loci affecting the difference in pigmentation between Drosophila yakuba and D. santomea.

Using quantitative trait locus (QTL) mapping, we studied the genetic basis of the difference in pigmentation between two sister species of Drosophila: Drosophila yakuba, which, like other members of the D. melanogaster subgroup, shows heavy black pigmentation on the abdomen of males and females, and D. santomea, an endemic to the African island of São Tomé, which has virtually no pigmentation. Here we mapped four QTL with large effects on this interspecific difference in pigmentation: two on the X chromosome and one each on the second and third chromosomes. The same four QTL were detected in male hybrids in the backcrosses to both D. santomea and D. yakuba and in the female D. yakuba backcross hybrids. All four QTL exhibited strong epistatic interactions in male backcross hybrids, but only one pair of QTL interacted in females from the backcross to D. yabuka. All QTL from each species affected pigmentation in the same direction, consistent with adaptive evolution driven by directional natural selection. The regions delimited by the QTL included many positional candidate loci in the pigmentation pathway, including genes affecting catecholamine biosynthesis, melanization of the cuticle, and many additional pleiotropic effects.

Hubby and Lewontin on Protein Variation in Natural Populations: When Molecular Genetics Came to the Rescue of Population Genetics.

The 1966 GENETICS papers by John Hubby and Richard Lewontin were a landmark in the study of genome-wide levels of variability. They used the technique of gel electrophoresis of enzymes and proteins to study variation in natural populations of Drosophila pseudoobscura, at a set of loci that had been chosen purely for technical convenience, without prior knowledge of their levels of variability. Together with the independent study of human populations by Harry Harris, this seminal study provided the first relatively unbiased picture of the extent of genetic variability in protein sequences within populations, revealing that many genes had surprisingly high levels of diversity. These papers stimulated a large research program that found similarly high electrophoretic variability in many different species and led to statistical tools for interpreting the data in terms of population genetics processes such as genetic drift, balancing and purifying selection, and the effects of selection on linked variants. The current use of whole-genome sequences in studies of variation is the direct descendant of this pioneering work.

Impact of experimental design on Drosophila sexual isolation studies: direct effects and comparison to field hybridization data.

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 São 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.

An anomalous hybrid zone in Drosophila.

Despite the genetic tractability of many of Drosophila species, the genus has few examples of the "classic" type of hybrid zone, in which the ranges of two species overlap with a gradual transition from one species to another through an area where hybrids are produced. Here we describe a classic hybrid zone in Drosophila that involves two sister species, Drosophila yakuba and D. santomea, on the island of São Tomé. Our transect of this zone has yielded several surprising and anomalous findings. First, we detected the presence of an additional hybrid zone largely outside the range of both parental species. This phenomenon is, to our knowledge, unique among animals. Second, the genetic analysis using diagnostic molecular markers of the flies collected in this anomalous hybrid zone indicates that nearly all hybrid males are F1s that carry the D. santomea X chromosome. This F1 genotype is much more difficult to produce in the laboratory compared to the genotype from the reciprocal cross, showing that sexual isolation as seen in the laboratory is insufficient to explain the genotypes of hybrids found in the wild. Third, there is a puzzling absence of hybrid females. We suggest several tentative explanations for the anomalies associated with this hybrid zone, but for the present they remain a mystery.

Quantitative trait loci for sexual isolation between Drosophila simulans and D. mauritiana.

Sexual isolating mechanisms that act before fertilization are often considered the most important genetic barriers leading to speciation in animals. While recent 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 simulans and D. mauritiana. We mapped at least seven QTL affecting discrimination of D. mauritiana females against D. simulans males, three QTL affecting D. simulans male traits against which D. mauritiana females discriminate, and six QTL affecting D. mauritiana male traits against which D. simulans females discriminate. QTL affecting sexual isolation act additively, are largely different in males and females, and are not disproportionately concentrated on the X chromosome: The QTL of greatest effect are located on chromosome 3. Unlike the genetic components of postzygotic isolation, the loci for prezygotic isolation do not interact epistatically. The observation of a few QTL with moderate to large effects will facilitate positional cloning of genes underlying sexual isolation.

Mathematical consequences of the genealogical species concept.

A genealogical species is defined as a basal group of organisms whose members are all more closely related to each other than they are to any organisms outside the group ("exclusivity"), and which contains no exclusive group within it. In practice, a pair of species is so defined when phylogenies of alleles from a sample of loci shows them to be reciprocally monophyletic at all or some specified fraction of the loci. We investigate the length of time it takes to attain this status when an ancestral population divides into two descendant populations of equal size with no gene exchange, and when genetic drift and mutation are the only evolutionary forces operating. The number of loci used has a substantial effect on the probability of observing reciprocal monophyly at different times after population separation, with very long times needed to observe complete reciprocal monophyly for a large number of loci. In contrast, the number of alleles sampled per locus has a relatively small effect on the probability of reciprocal monophyly. Because a single mitochondrial or chloroplast locus becomes reciprocally monophyletic much faster than does a single nuclear locus, it is not advisable to use mitochondrial and chloroplast DNA to recognize genealogical species for long periods after population divergence. Using a weaker criterion of assigning genealogical species status when more than 50% of sampled nuclear loci show reciprocal monophyly, genealogical species status depends much less on the number of sampled loci, and is attained at roughly 4-7 N generations after populations are isolated, where N is the historically effective population size of each descendant. If genealogical species status is defined as more than 95% of sampled nuclear loci showing reciprocal monophyly, this status is attained after roughly 9-12 N generations.

Genetics of a difference in pigmentation between Drosophila yakuba and Drosophila santomea.

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.