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.     

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.     

Evolution of Multiple Additive Loci Caused Divergence between Drosophila yakuba and D. santomea in Wing Rowing during Male Courtship

In Drosophila, male flies perform innate, stereotyped courtship behavior. This innate behavior evolves rapidly between fly species, and is likely to have contributed to reproductive isolation and species divergence. We currently understand little about the neurobiological and genetic mechanisms that contributed to the evolution of courtship behavior. Here we describe a novel behavioral difference between the two closely related species D. yakuba and D. santomea: the frequency of wing rowing during courtship. During courtship, D. santomea males repeatedly rotate their wing blades to face forward and then back (rowing), while D. yakuba males rarely row their wings. We found little intraspecific variation in the frequency of wing rowing for both species. We exploited multiplexed shotgun genotyping (MSG) to genotype two backcross populations with a single lane of Illumina sequencing. We performed quantitative trait locus (QTL) mapping using the ancestry information estimated by MSG and found that the species difference in wing rowing mapped to four or five genetically separable regions. We found no evidence that these loci display epistasis. The identified loci all act in the same direction and can account for most of the species difference.     

Genetic studies of two sister species in the Drosophila melanogaster subgroup, D. yakuba and D. santomea

We performed genetic analysis of hybrid sterility and of one morphological difference (sex-comb tooth number) on D. yakuba and D. santomea, the former species widespread in Africa and the latter endemic to the oceanic island of S?o Tomé, on which there is a hybrid zone. The sterility of hybrid males is due to at least three genes on the X chromosome and at least one on the Y, with the cytoplasm and large sections of the autosomes having no effect. F1 hybrid females carrying two X chromosomes from either species are perfectly fertile despite their genetic similarity to completely sterile F1 hybrid males. This implies that the appearance of Haldane's rule in this cross is at least partially due to the faster accumulation of genes causing male than female sterility. The larger effects of the X and Y chromosomes than of the autosomes, however, also suggest that the genes causing male sterility are recessive in hybrids. Some female sterility is also seen in interspecific crosses, but this does not occur between all strains. This is seen in pure-species females inseminated by heterospecific males (probably reflecting incompatibility between the sperm of one species and the female reproductive tract of the other) as well as in inseminated F1 and backcross females, probably reflecting genetically based incompatibilities in hybrids that affect the reproductive system. The latter 'innate' sterility appears to involve deleterious interactions between D. santomea chromosomes and D. yakuba cytoplasm. The difference in male sex-comb tooth number appears to involve fairly large effects of the X chromosome. We discuss the striking evolutionary parallels in the genetic basis of sterility, in the nature of sexual isolation, and in morphological differences between the D. santomea/D. yakuba divergence and two other speciation events in the D. melanogaster subgroup involving island colonization.     

TEMPERATURE-BASED EXTRINSIC REPRODUCTIVE ISOLATION IN TWO SPECIES OF DROSOPHILA

Drosophila santomea and D. yakuba are sister species that live on the African volcanic island of São Tomé, where they are ecologically isolated: D. yakuba inhabits low-altitude open and semiopen habitats while D. santomea lives in higher-elevation rain and mist forest. To determine whether this spatial isolation reflected differential preference for and tolerance of temperature, we estimated fitness components of both species at different temperatures as well as their behavioral preference for certain temperatures. At higher temperatures, especially 28°C, D. santomea was markedly inferior to D. yakuba in larval survival, egg hatchability, and longevity. Moreover, D. santomea females, unlike those of D. yakuba , become almost completely sterile after exposure to a temperature of 28°C, and conspecific males become semisterile. Drosophila santomea adults prefer temperatures 2–3°C lower than do adults of D. yakuba . Drosophila santomea , then, is poorly adapted to high temperature, partially explaining its restriction to cool, high habitats, which leads to extrinsic premating isolation and immigrant inviability. Rudimentary genetic analysis of the interspecific difference in egg hatchability and larval survival showed that these differences are due largely to cytoplasmic effects and to autosomal genes, with sex chromosomes playing little or no role.     

Conspecific sperm precedence in sister species of Drosophila with overlapping ranges

Abstract Barriers to gene flow that act after mating but before fertilization are often overlooked in studies of reproductive isolation. Where species are sympatric, such "cryptic' isolating barriers may be important in maintaining species as distinct entities. Drosophilayakuba and its sister species D. santomea have overlapping ranges on the island of Sao Tome, off the coast of West Africa. Previous studies have shown that the two species are strongly sexually isolated. However, the degree of sexual isolation observed in the laboratory cannot explain the low frequency (–1%) of hybrids observed in nature. This study identifies two "cryptic" isolating barriers that may further reduce gene flow between D. yakuba andD. santomea where they are sympatric. First, noncompetitive gametic isolation has evolved between D. yakuba and D. santomea: heterospecific matings between the two species produce significantly fewer offspring than do conspecific matings. Second, conspecific sperm precedence (CSP) occurs when D. yakuba females mate with conspecific and heterospecific males. However, CSP is asymmetrical: D. santomea females do not show patterns of sperm usage consistent with CSP. Drosophila yakuba and D. santomea females also differ with respect to remating propensity after first mating with conspecific males. These results suggest that noncompetitive and competitive gametic isolating barriers may contribute to reproductive isolation between D. yakuba and D. santomea.     

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.     

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 S?o Tomé in the Atlantic Ocean, 280 km west of Gabon. On S?o 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).     

The evolution of gene regulation underlies a morphological difference between two Drosophila sister species

Understanding the mechanisms underlying the morphological divergence of species is one of the central goals of evolutionary biology. Here, we analyze the genetic and molecular bases of the divergence of body pigmentation patterns between Drosophila yakuba and its sister species Drosophila santomea. We found that loss of pigmentation in D. santomea involved the selective loss of expression of the tan and yellow pigmentation genes. We demonstrate that tan gene expression was eliminated through the mutational inactivation of one specific tan cis-regulatory element (CRE) whereas the Tan protein sequence remained unchanged. Surprisingly, we identify three independent loss-of-function alleles of the tan CRE in the young D. santomea lineage. We submit that there is sufficient empirical evidence to support the general prediction that functional evolutionary changes at pleiotropic loci will most often involve mutations in their discrete, modular cis-regulatory elements.     

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

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

Evolutionary novelties in islands: Drosophila santomea, a new melanogaster sister species from São Tomé

The finding of new melanogaster sister species may help us in understanding more about how the emergence of genetic novelties, particularly in insular habitats, can result in speciation. Here we report on the discovery of Drosophila santomea, which is the first melanogaster sibling found off West-equatorial Africa, on São Tomé, one of the Gulf of Guinea islands. Although the eight other melanogaster sister species are remarkably conservative in their morphology except for their terminalia, the new find has a morphological trait distinguishing it from all of these: a pure yellow body coloration of both sexes without the normal black abdominal banding. Evidence from the terminalia, polytene and mitotic chromosomes, period gene and allozymes are provided indicating that it is nonetheless the nearest relative of Drosophila yakuba with which it coexists on the island. The new find is a clear-cut taxon as shown by the production of sterile male hybrids, eventually with developmental defects, in both directions of cross with yakuba and by the existence of an altitudinal divide accompanied by a hybrid zone at mid-elevation on the island. Molecular and karyotypic data further support this conclusion. In contrast to the significant divergence of their nuclear DNAs, an intriguing similarity in their cytochrome b sequences was observed indicating a recent coalescence common to santomea, yakuba and also teissieri cytoplasms. These were shown to harbour the same Wolbachia endosymbiotic bacteria which could possibly be responsible for mitochondrial DNA hitchhiking across the species barrier.     

Extensive introgression of mitochondrial DNA relative to nuclear genes in the Drosophila yakuba species group

Studies of gene flow between recently diverged species can illuminate the role of natural selection in the formation of new species. Drosophila santomea and D. yakuba are recently diverged, partially reproductively isolated species that continue to hybridize in the wild, and appear to be reproductively isolated from the more distantly related species D. teissieri. We examine patterns of nucleotide polymorphism and divergence in these three species at multiple X-linked, Y-linked, and mitochondrial markers. All three species harbor drastically reduced variability on the Y chromosome relative to the X, as expected for a nonrecombining chromosome subject to variation-reducing selection. The three species are generally well differentiated at the nuclear markers, with little evidence for recent introgression for either the X- or Y-linked genes. Based on the nuclear genes, we estimate that D. santomea and D. yakuba diverged about one-half million years ago and split from D. teissieri about one million years ago. In contrast to the pattern at nuclear loci, all three species share a very similar mtDNA haplotype. We show that the mtDNA must have recently introgressed across species boundaries in the D. yakuba subgroup and that its fixation was driven by either selection on the mitochondria itself or other cytoplasmic factors. These results demonstrate that different regions of the genome can have distinct evolutionary dynamics in the context of species formation. Although natural selection is usually thought of as accentuating divergence between species, our results imply that it can also act as a homogenizing force.     

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.     

An intron loss of Dfak gene in species of the Drosophila melanogaster subgroup and phylogenetic analysis

Drosophila focal adhesion kinase (Dfak) gene is a single-copy nuclear gene. Previous study revealed that Drosophila melanogaster and Drosophila simulans had lost an intron precisely within the tyrosine kinase (TyK) domain of this gene. However, this did not happen in several other Drosophila species, including Drosophila elegans, Drosophila ficusphila, Drosophila biarmipes, Drosophila jambulina, Drosophila prostipennis, Drosophila takahashii, and Drosophila pseudoobscura. In the current study, homologous sequences of Drosophila sechellia, Drosophila mauritiana, Drosophila yakuba, Drosophila teissieri, Drosophila santomea, and Drosophila erecta were amplified by polymerase chain reaction, and further sequencing analysis indicated that these species were missing a TyK domain intron, indicating they were closely related. The relationship of the D. melanogaster species group was reconstructed using TyK domain nucleotide sequences. The resulting phylogenetic tree revealed that these 8 species were the most related species in the melanogaster group. These results strongly support previously proposed classifications based on morphological and molecular data.     

Genetic basis of sex-specific color pattern variation in Drosophila malerkotliana

Pigmentation is a rapidly evolving trait that can play important roles in mimicry, sexual selection, thermoregulation, and other adaptive processes in many groups of animals. In Drosophila, pigmentation can differ dramatically among closely related taxa, presenting a good opportunity to dissect the genetic changes underlying species divergence. In this report, we investigate the genetic basis of color pattern variation between two allopatric subspecies of Drosophila malerkotliana, a widespread member of the ananassae species subgroup. In D. malerkotliana malerkotliana, the last three abdominal segments are darkly pigmented in males but not in females, while in D. malerkotliana pallens both sexes lack dark pigmentation. Composite interval mapping in F(2) hybrid progeny shows that this difference is largely controlled by three quantitative trait loci (QTL) located on the 2L chromosome arm, which is homologous to the 3R of D. melanogaster (Muller element E). Using highly recombinant introgression strains produced by repeated backcrossing and phenotypic selection, we show that these QTL do not correspond to any of the candidate genes known to be involved in pigment patterning and synthesis in Drosophila. These results, in combination with similar analyses in other Drosophila species, indicate that different genetic and molecular changes are responsible for the evolution of similar phenotypic traits in different lineages. This feature makes Drosophila color patterns a powerful model for investigating how the genetic basis of trait evolution is influenced by the intrinsic organization of regulatory pathways controlling the development of these traits.     

Natural Wolbachia infections in the Drosophila yakuba species complex do not induce cytoplasmic incompatibility but fully rescue the wRi modification

In this study, we report data about the presence of Wolbachia in Drosophila yakuba, D. teissieri, and D. santomea. Wolbachia strains were characterized using their wsp gene sequence and cytoplasmic incompatibility assays. All three species were found infected with Wolbachia bacteria closely related to the wAu strain, found so far in D. simulans natural populations, and were unable to induce cytoplasmic incompatibility. We injected wRi, a CI-inducing strain naturally infecting D. simulans, into the three species and the established transinfected lines exhibited high levels of CI, suggesting that absence of CI expression is a property of the Wolbachia strain naturally present or that CI is specifically repressed by the host. We also tested the relationship between the natural infection and wRi and found that it fully rescues the wRi modification. This result was unexpected, considering the significant evolutionary divergence between the two Wolbachia strains.