Sequential adaptive introgression of the mitochondrial genome in Drosophila yakuba and Drosophila santomea

Interspecific hybridization provides the unique opportunity for species to tap into genetic variation present in a closely related species and potentially take advantage of beneficial alleles. It has become increasingly clear that when hybridization occurs, mitochondrial DNA (mtDNA) often crosses species boundaries, raising the possibility that it could serve as a recurrent target of natural selection and source of species' adaptations. Here we report the sequences of 46 complete mitochondrial genomes of Drosophila yakuba and Drosophila santomea, two sister species known to produce hybrids in nature (~3%). At least two independent events of mtDNA introgression are uncovered in this study, including an early invasion of the D. yakuba mitochondrial genome that fully replaced the D. santomea mtDNA native haplotypes and a more recent, ongoing event centred in the hybrid zone. Interestingly, this recent introgression event bears the signature of Darwinian natural selection, and the selective haplotype can be found at low frequency in Africa mainland populations of D. yakuba. We put forward the possibility that, because the effective population size of D. santomea is smaller than that of D. yakuba, the faster accumulation of mildly deleterious mutations associated with Muller's ratchet in the former species may have facilitated the replacement of the mutationally loaded mitochondrial genome of D. santomea by that of D. yakuba.     

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.     

Gene flow between Drosophila yakuba and Drosophila santomea in subunit V of cytochrome c oxidase: A potential case of cytonuclear cointrogression

Introgression is the effective exchange of genetic information between species through natural hybridization. Previous genetic analyses of the Drosophila yakuba—D. santomea hybrid zone showed that the mitochondrial genome of D. yakuba had introgressed into D. santomea and completely replaced its native form. Since mitochondrial proteins work intimately with nuclear‐encoded proteins in the oxidative phosphorylation (OXPHOS) pathway, we hypothesized that some nuclear genes in OXPHOS cointrogressed along with the mitochondrial genome. We analyzed nucleotide variation in the 12 nuclear genes that form cytochrome c oxidase (COX) in 33 Drosophila lines. COX is an OXPHOS enzyme composed of both nuclear‐ and mitochondrial‐encoded proteins and shows evidence of cytonuclear coadaptation in some species. Using maximum‐likelihood methods, we detected significant gene flow from D. yakuba to D. santomea for the entire COX complex. Interestingly, the signal of introgression is concentrated in the three nuclear genes composing subunit V, which shows population migration rates significantly greater than the background level of introgression in these species. The detection of introgression in three proteins that work together, interact directly with the mitochondrial‐encoded core, and are critical for early COX assembly suggests this could be a case of cytonuclear cointrogression.     

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.     

Discrepancy in divergence of the mitochondrial and nuclear genomes ofDrosophila teissieri andDrosophila yakuba

Summary Restriction sites were compared in the mitochondrial DNA (mtDNA) molecules from representatives of two closely related species of fruit flies: nine strains ofDrosophila teissieri and eight strains ofDrosophila yakuba. Nucleotide diversities amongD. teissieri strains and amongD. yakuba strains were 0.07% and 0.03%, respectively, and the nucleotide distance between the species was 0.22%. Also determined was the nucleotide sequence of a 2305-nucleotide pari (ntp) segment of the mtDNA molecule ofD. teissieri that contains the noncoding adenine+thymine (A+T)-rich region (1091 ntp) as well as the genes for the mitochondrial small-subunit rRNA, tRNA^f-met, tRNA^gln, and tRNA^ile, and portions of the ND2 and tRNA^val genes. This sequence differs from the corresponding segment of theD. yakuba mtDNA by base substitutions at 0.1% and 0.8% of the positions in the coding and noncoding regions, respectively. The higher divergence due to base substitutions in the A+T-rich region is accompanied by a greater number of insertions/deletions than in the coding regions. From alignment of theD. teissieri A+T-rich sequence with those ofD. yakuba andDrosophila virilis, it appears that the 40% of this sequence that lies adjacent to the tRNA^ile gene has been highly conserved. Divergence between the entireD. teissieri andD. yakuba mtDNA molecules, estimated from the sequences, was 0.3%; this value is close to the value (0.22%) obtained from the restriction analysis, but 10 times lower than the value estimated from published DNA hybridization results. From consideration of the relationships of mitochondrial nucleotide distance and allozyme genetic distance found among seven species of theDrosophila melanogaster subgroup, the mitochondrial nucleotide distance observed forD. teissieri andD. yakuba is anomalously low in relation to the nuclear genetic distance.     

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.     

THE INFLUENCE OF ABDOMINAL PIGMENTATION ON DESICCATION AND ULTRAVIOLET RESISTANCE IN TWO SPECIES OF DROSOPHILA

Drosophila yakuba and D. santomea are sister species that differ in their levels of abdominal pigmentation; D. yakuba shows heavily pigmented posterior abdominal segments in both sexes, whereas D. santomea lacks dark pigment anywhere on its body. Using naturally collected lines, we demonstrate the existence of altitudinal variation in abdominal pigmentation in D. yakuba but not in D. santomea. We use the variation in pigmentation within D. yakuba and two body‐color mutants in D. yakuba to elucidate selective advantage of differences in pigmentation. Our results indicate that although differences in abdominal pigmentation have no effect on desiccation resistance, lighter pigmentation confers ultraviolet radiation resistance in this pair of species.     

An anomalous hybrid zone in Drosophila

Abstract 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 SaTo 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.     

The magnitude of behavioral isolation is affected by characteristics of the mating community

Gene exchange between species occurs in areas of secondary contact, where two species have the opportunity to hybridize. If heterospecific males are more common than conspecific males, females will experience more encounters with males of other species. These encounters might increase the likelihood of heterospecific matings, and lead to the production of hybrid progeny. I studied the mating behavior of two pairs of sibling species endemic to Africa: Drosophila yakuba/Drosophila santomea and Drosophila simulans/Drosophila sechellia. Drosophila yakuba and D. simulans are cosmopolitan species widely distributed in the African continent, while D. santomea and D. sechellia are island endemics. These pairs of species hybridize in nature and have the potential to exchange genes in natural conditions. I used these two pairs of Drosophila species, and constructed mating communities of different size and different heterospecific:conspecific composition. I found that both the total number of potential mates and the relative frequency of conspecific versus heterospecific males affect female mating decisions in the cosmopolitan species but not in the island endemics. These results suggest that the population characteristics, in which mating occurs, may affect the magnitude of premating isolation. Community composition might thus facilitate, or impair, gene flow between species.     

Lock‐and‐key structural isolation between sibling Drosophila species

Drosophila santomea Lachaise & Harry, which is endemic to the African island of São Tomé, and its sibling D. yakuba Burla comprise a new model system of speciation. They are morphologically distinguishable only by slight differences in the male genitalia and body coloration. As a previously undescribed difference, the aedeagus of D. yakuba bears a pair of stout spines (the ventral branches of the basal processes (VB)), instead of the paired humps found in D. santomea. Here, we show that this difference works as a lock‐and‐key isolating mechanism between the siblings. During conspecific copulation, D. yakuba females receive the spines in a pair of pocket‐shaped structures, which are protected by hardened plates, in the genitalia. The females of D. santomea, which lack such pockets, are wounded by the spines of the VB when mated with D. yakuba males. This genital mismatching resulted in leakage of the ejaculate, making 80% of the matings infertile and causing a prolonged struggle to separate pairs glued together by the ejaculate.     

INTRINSIC REPRODUCTIVE ISOLATION BETWEEN TWO SISTER SPECIES OF DROSOPHILA

Drosophila santomea and D. yakuba are sister species that live on the volcanic African island of São Tomé. Previous work has revealed several barriers to gene flow, including sexual isolation, hybrid sterility, and “extrinsic” ecological isolation based on differential adaptation to and preference for temperature. Here, we describe several new “intrinsic” barriers to gene flow—barriers that do not depend on the species’ ecology. These include reduced egg number, reduced egg hatchability, and faster depletion of sperm in interspecific compared to intraspecific matings. Further, hatching interval and egg‐to‐adult development time are significantly longer in interspecific than intraspecific crosses. If a female of either species is initially mated to a heterospecific male, she lays fewer and less‐fertile eggs than if she is first mated to a conspecific male, so that heterospecific matings permanently reduce female fertility. Finally, D. santomea females mated to D. yakuba males do not live as long as virgin or conspecifically mated females. The “poisoning” effects of heterospecific ejaculates may be byproducts of antagonistic sexual selection. Although these species diverged relatively recently, they are clearly separated by many isolating barriers that act both before and after mating.     

Embryonic lethality leads to hybrid male inviability in hybrids between Drosophila melanogaster and D. santomea

The study of the morphological defects unique to interspecific hybrids can reveal which developmental pathways have diverged between species. Drosophila melanogaster and D. santomea diverged more than 10 million years ago, and when crossed produce sterile adult females. Adult hybrid males are absent from all interspecific crosses. We aimed to determine the fate of these hybrid males. To do so, we tracked the development of hybrid females and males using classic genetic markers and techniques. We found that hybrid males die predominantly as embryos with severe segment‐specification defects while a large proportion of hybrid females embryos hatch and survive to adulthood. In particular, we show that most male embryos show a characteristic abdominal ablation phenotype, not observed in either parental species. This suggests that sex‐specific embryonic developmental defects eliminate hybrid males in this interspecific cross. The study of the developmental abnormalities that occur in hybrids can lead to the understanding of cryptic molecular divergence between species sharing a conserved body plan.     

Divergence between Drosophila santomea and allopatric or sympatric populations of D. yakuba using paralogous amylase genes and migration scenarios along the Cameroon volcanic line

We have used two paralogous genes (Amyrel and Amy) of the amylase multigene family to reconstruct the phylogeny of the nine Drosophila melanogaster subgroup sister species, including D. santomea, the newly discovered endemic from São Tomé island. The evolutionary divergence of these genes is of special interest as it is suspected to result from physiological evolution via gene duplication. This paper describes the relationship between the geographical origin of the various strains and the patterns of mating and phylogeny, focusing on the evolution of D. santomea and its relationship to other species and their niches. The Amyrel and Amy data indicate that, contrary to expectations, the sympatric insular D. yakuba population is less closely related to D. santomea than allopatric mainland ones, suggesting that the extant insular D. yakuba population on São Tomé results from a recent secondary colonization. Data for sympatric and allopatric D. yakuba suggest that D. santomea arose from a mainland D. yakuba parental stock when montane habitats of the Cameroon volcanic line extended to lower altitudes during colder and less humid periods. Despite their different modes of evolution and different functions, the Amyrel and Amy genes provide remarkably consistent topologies and hence reflect the same history, that of the species.     

Reinforcement of Gametic Isolation in Drosophila

Reinforcement, a process by which natural selection increases reproductive isolation between populations, has been suggested to be an important force in the formation of new species. However, all existing cases of reinforcement involve an increase in mate discrimination between species. Here, I report the first case of reinforcement of postmating prezygotic isolation (i.e., barriers that act after mating but before fertilization) in animals. On the slopes of the African island of São Tomé, Drosophila yakuba and its endemic sister species D. santomea hybridize within a well-demarcated hybrid zone. I find that D. yakuba females from within this zone, but not from outside it, show an increase in gametic isolation from males of D. santomea, an apparent result of natural selection acting to reduce maladaptive hybridization between species. To determine whether such a barrier could evolve under laboratory conditions, I exposed D. yakuba lines derived from allopatric populations to experimental sympatry with D. santomea, and found that both behavioral and gametic isolation become stronger after only four generations. Reinforcement thus appears to be the best explanation for the heightened gametic isolation seen in sympatry. This appears to be the first example in animals in which natural selection has promoted the evolution of stronger interspecific genetic barriers that act after mating but before fertilization. This suggests that many other genetic barriers between species have been increased by natural selection but have been overlooked because they are difficult to study.     

SPERM PRODUCTION AND STERILITY IN HYBRIDS BETWEEN TWO SUBSPECIES OF DROSOPHILA PSEUDOOBSCURA

Subspecies of Drosophila pseudoobscura, one occurring in the United States and the other in Bogota, Columbia, exhibit Haldane's Rule in one direction of the cross. Additionally, D. pseudoobscura produces two sperm types: short, sterile sperm and long, fertile, sperm. Here I examine the relationship between the production of short and long sperm and hybrid sterility. Fertile and sterile hybrid males produce a greater proportion of short sperm compared to parental males with sterile hybrids producing mainly short, immotile sperm. Sperm transfer and storage patterns were similar between fertile hybrid and parental strains; and unexpectedly, short, immotile sperm from sterile hybrids were stored. These findings raise the question of whether different genetic mechanisms disrupt both sperm heteromorphic production and sperm motility and whether this indicates that females exert some control over sperm storage.     

Development time and pupation behavior in theDrosophila melanogaster subgroup (Diptera: Drosophilidae)

This study is an in-depth analysis of intersexual, intraspecific, and interspecific variability in larvopupal developmental time, pupation site preference, and larval and pupal survival of a number of isofemale lines of the speciesDrosophila mauritiana, D. melanogaster, D. sechellia, D. simulans, D. teissieri, andD. yakuba. There was no significant sex differences in pupation height, but females eclosed significantly earlier than males in all species. In addition, the suggestion of a strong negative correlation between larval developmental time and pupation height could not be confirmed in this study. The hypothesis that differences in pupation height provide a basis for niche partitioning between closely related species with overlapping distributions was tested by three planned orthogonal contrast analyses of variance. First, the two speciesD. teissieri andD. yakuba, with largely overlapping distribution, were significantly different in pupation height. Second, the two allopatric, nonoverlapping island speciesD. mauritiana andD. sechellia did not significantly differ in pupation height. However, the absence of a significant difference in the final contrast between the two cosmopolitan speciesD. melanogaster andD. simulans, which are often found together, makes us cautious to accept the hypothesis.     

Molecular Phylogeny of the <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> Species Subgroup

Although molecular and phenotypic evolution have been studied extensively in Drosophila melanogaster and its close relatives, phylogenetic relationships within the D. melanogaster species subgroup remain unresolved. In particular, recent molecular studies have not converged on the branching orders of the D. yakuba–D. teissieri and D. erecta–D. orena species pairs relative to the D. melanogaster–D. simulans–D. mauritiana–D. sechellia species complex. Here, we reconstruct the phylogeny of the melanogaster species subgroup using DNA sequence data from four nuclear genes. We have employed vectorette PCR to obtain sequence data for orthologous regions of the Alcohol dehydrogenase (Adh), Alcohol dehydrogenase related (Adhr), Glucose dehydrogenase (Gld), and rosy (ry) genes (totaling 7164 bp) from six melanogaster subgroup species (D. melanogaster, D. simulans, D. teissieri, D. yakuba, D. erecta, and D. orena) and three species from subgroups outside the melanogaster species subgroup [D. eugracilis (eugracilis subgroup), D. mimetica (suzukii subgroup), and D. lutescens (takahashii subgroup)]. Relationships within the D. simulans complex are not addressed. Phylogenetic analyses employing maximum parsimony, neighbor-joining, and maximum likelihood methods strongly support a D. yakuba–D. teissieri and D. erecta–D. orena clade within the melanogaster species subgroup. D. eugracilis is grouped closer to the melanogaster subgroup than a D. mimetica–D. lutescens clade. This tree topology is supported by reconstructions employing simple (single parameter) and more complex (nonreversible) substitution models. Present address (Ryan M. David): University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284, USA     

Gene expression analysis of the ovary of hybrid females of <Emphasis Type="Italic">Xenopus laevis</Emphasis> and <Emphasis Type="Italic">X. muelleri</Emphasis>

Background  

Interspecific hybrids of frogs of the genus Xenopus result in sterile hybrid males and fertile hybrid females. Previous work has demonstrated a dramatic asymmetrical pattern of misexpression in hybrid males compared to the two parental species with relatively few genes misexpressed in comparisons of hybrids and the maternal species (X. laevis) and dramatically more genes misexpressed in hybrids compared to the paternal species (X. muelleri). In this work, we examine the gene expression pattern in hybrid females of X. laevis × X. muelleri to determine if this asymmetrical pattern of expression also occurs in hybrid females.     

A morphological trait involved in reproductive isolation between Drosophila sister species is sensitive to temperature

Male genitalia are usually extremely divergent between closely related species, but relatively constant within one species. Here we examine the effect of temperature on the shape of the ventral branches, a male genital structure involved in reproductive isolation, in the sister species Drosophila santomea and Drosophila yakuba. We designed a semi‐automatic measurement machine learning pipeline that can reliably identify curvatures and landmarks based on manually digitized contours of the ventral branches. With this method, we observed that temperature does not affect ventral branches in D. yakuba but that in D. santomea ventral branches tend to morph into a D. yakuba‐like shape at lower temperature. We found that male genitalia structures involved in reproductive isolation can be relatively variable within one species and can resemble the shape of closely related species’ genitalia through plasticity to temperature. Our results suggest that reproductive isolation mechanisms can be dependent on the environmental context.     

Radiation effects on Drosophila suzukii (Diptera: Drosophilidae) reproductive behaviour

Female remating is a widespread behaviour, reported in several insect species. This behaviour can affect the efficiency of sterile insect technique (SIT); however, little is known about the postcopulatory behaviour of some pest species considered as candidates to be controlled by this technique, such as Drosophila suzukii (Matsumura, 1931) (Diptera: Drosophilidae). In this study, we investigated the effects of male and female sterilization on mating and remating behaviour of D. suzukii. First, we tested the occurrence of multiple mating in different combinations between sterile and fertile males and females. Then, we tested the effects of male and female sterility on female propensity to mate and remate. We found an overall low remating rate by D. suzukii females. Male sterility did not influence mating and remating likelihood; however, copula duration of sterile males was shorter compared to fertile males. On the other hand, sterile females were less likely to mate. Our findings encourage further research regarding the use of SIT to control D. suzukii.