Vege and Mar: two novel hAT MITE families from Drosophila willistoni

Two novel families of miniature inverted repeat transposable elements (MITEs), Vege and Mar, are described from Drosophila willistoni. Based on their structures, both element families are hypothesized to belong to the hAT superfamily of transposable elements. Both elements have perfect, inverted terminal repeats and 8-bp target site duplications and were found to have inserted within fixed copies of nonautonomous P elements. Vege is present in all studied D. willistoni populations and appears to have a relatively low copy number. Mar was identified in only a single D. willistoni population, and its copy number is presently unknown. Although MITEs occupy relatively large proportions of the genomes of a broad range of organisms, this may be their first unambiguous identification in any species of the genus Drosophila.     

Reproductive isolation caused by azoospermia in sterile male hybrids of Drosophila

Recently diverged populations in the early stages of speciation offer an opportunity to understand mechanisms of isolation and their relative contributions. Drosophila willistoni is a tropical species with broad distribution from Argentina to the southern United States, including the Caribbean islands. A postzygotic barrier between northern populations (North America, Central America, and the northern Caribbean islands) and southern populations (South American and the southern Caribbean islands) has been recently documented and used to propose the existence of two different subspecies. Here, we identify premating isolation between populations regardless of their subspecies status. We find no evidence of postmating prezygotic isolation and proceeded to characterize hybrid male sterility between the subspecies. Sterile male hybrids transfer an ejaculate that is devoid of sperm but causes elongation and expansion of the female uterus. In sterile male hybrids, bulging of the seminal vesicle appears to impede the movement of the sperm toward the sperm pump, where sperm normally mixes with accessory gland products. Our results highlight a unique form of hybrid male sterility in Drosophila that is driven by a mechanical impediment to transfer sperm rather than by an abnormality of the sperm itself. Interestingly, this form of sterility is reminiscent of a form of infertility (azoospermia) that is caused by lack of sperm in the semen due to blockages that impede the sperm from reaching the ejaculate.     

Identification of the sibling species of the Drosophila willistoni subgroup through the electrophoretical mobility of acid phosphatase-1

The Drosophila willistoni group consists of 23 species of which six are sibling species and belong to the D. willistoni subgroup: D. willistoni, Drosophila equinoxialis, Drosophila tropicalis, Drosophila insularis, Drosophila pavlovskiana and Drosophila paulistorum. These sibling species are abundant in the Neotropical region and can hardly be differentiated by the usual taxonomic traits. Four of them (D. willistoni, D. equinoxialis, D. tropicalis and D. paulistorum) cover extensive geographic distribution areas overlapping in places while two of them are endemic (D. insularis and D. pavlovskiana). In this study, we presented a method for the identification of five sibling species of the D. willistoni subgroup based on the allozyme variation of acid phosphatase‐1 (Acph‐1) in acrylamide gel electrophoresis. Our work showed that Acph‐1 allozyme differences can be used for species‐diagnostic characterization. This method was shown to be a more efficient tool for species identification than others because it is both quicker and produces reliable results.     

Rapid evolution of courtship song pattern in Drosophila willistoni sibling species

Courtship behaviours may provide a more reliable means of identifying reproductively isolated taxa than traits such as morphology or many genetic markers. Here we describe the courtship songs of the Drosophila willistoni sibling species group, which consists of several species and subspecies. We find that song pattern is species-specific, despite significant differences among strains within species. D. paulistorum has the most variable song pattern, which reflects this species' traditional subdivision into semispecies. All the other species could be unambiguously identified by song. The major differences among these species was in the interpulse interval, as has been found in other studies of fly song. However, the interpulse intervals of the species studied here were often multimodal. This was partly due to the presence of multiple song types within the courtship repertoire, but it also reflected changes in interpulse interval within a song type by some males. Unusually, some species had distinctively patterned variation in interpulse interval. Song must have evolved rapidly within the species complex, probably due to sexual selection.     

The genetics of reproductive isolation and the potential for gene exchange between Drosophila pseudoobscura and D. persimilis via backcross hybrid males

Hybrid male sterility, hybrid inviability, sexual isolation, and a hybrid male courtship dysfunction reproductively isolate Drosophila pseudoobscura and D. persimilis. Previous studies of the genetic bases of these isolating mechanisms have yielded only limited information about how much and what areas of the genome are susceptible to interspecies introgression. We have examined the genetic basis of these barriers to gene exchange in several thousand backcross hybrid male progeny of these species using 14 codominant molecular genetic markers spanning the five chromosomes of these species, focusing particularly on the autosomes. Hybrid male sterility, hybrid inviability, and the hybrid male courtship dysfunction were all associated with X-autosome interactions involving primarily the inverted regions on the left arm of the X-chromosome and the center of the second chromosome. Sexual isolation from D. pseudoobscura females was primarily associated with the left arm of the X-chromosome, although both the right arm and the center of the second chromosome also contributed to it. Sexual isolation from D. persimilis females was primarily associated with the second chromosome. The absence of isolating mechanisms being associated with many autosomal regions, including some large inverted regions that separate the strains, suggests that these phenotypes may not be caused by genes spread throughout the genome. We suggest that gene flow between these species via hybrid males may be possible at loci spread across much of the autosomes.     

Evolution of mating isolation between populations of Drosophila ananassae

Prezygotic mating isolation has been a major interest of evolutionary biologists during the past several decades because it is likely to represent one of the first stages in the transition from populations to species. Mate discrimination is one of the most commonly measured forms of prezygotic isolation and appears to be relatively common among closely related species. In some cases, it has been used as a measure to distinguish populations from subspecies, races, and sister species, yet the influences of various evolutionary mechanisms that may generate mate discrimination are largely unknown. In this study, we measured the level and pattern of mate discrimination among 18 populations of a cosmopolitan drosophilid species, Drosophila ananassae , from throughout its geographical range and its sister species, Drosophila pallidosa, which has a restricted geographical distribution in the South Pacific Islands. In addition, we measured genetic differentiation between all 18 populations using mitochondrial DNA polymorphism data. Mate discrimination varies considerably throughout the species range, being higher among populations outside the ancestral Indonesian range, and highest in the South Pacific. Our results suggest that colonization and genetic differentiation may have an influence on the evolutionary origin of mate discrimination. Our phylogeographical approach clarifies the ancestral relationships of several populations from the South Pacific that show particularly strong mate discrimination and suggests that they may be in the early stages of speciation. Furthermore, both the genetic and behavioral results cast doubt on the status of D. pallidosa as a good species.     

Genetics of hybrid male sterility among strains and species in the Drosophila pseudoobscura species group

Taxa in the early stages of speciation may bear intraspecific allelic variation at loci conferring barrier traits in hybrids such as hybrid sterility. Additionally, hybridization may spread alleles that confer barrier traits to other taxa. Historically, few studies examine within- and between-species variation at loci conferring reproductive isolation. Here, we test for allelic variation within Drosophila persimilis and within the Bogota subspecies of D. pseudoobscura at regions previously shown to contribute to hybrid male sterility. We also test whether D. persimilis and the USA subspecies of D. pseudoobscura share an allele conferring hybrid sterility in a D. pseudoobscura bogotana genetic background. All loci conferred similar hybrid sterility effects across all strains studied, although we detected some statistically significant quantitative effect variation among D. persimilis alleles of some hybrid incompatibility QTLs. We also detected allelism between D. persimilis and D. pseudoobscura USA at a second chromosome hybrid sterility QTL. We hypothesize that either the QTL is ancestral in D. persimilis and D. pseudoobscura USA and lost in D. pseudoobscura bogotana, or gene flow transferred the QTL from D. persimilis to D. pseudoobscura USA. We discuss our findings in the context of population features that may contribute to variation in hybrid incompatibilities.     

Body size and mating success inDrosophila willistoni are uncorrelated under laboratory conditions

Mating activity and wing length were investigated in the F₁ progeny ofDrosophila willistoni females collected in the field to examine any possible relationship between body size and mating success. The flies were observed in a mating chamber under laboratory conditions. No significant differences in wing length were observed between copulating and noncopulating flies, and there was no significant correlation between wing length and copulation latency for both males and females. These results therefore suggest that the commonly accepted view that large body size is positively correlated with mating success inDrosophila does not always hold true. The results support the view that the extent of environmentally induced variation in body size may be an important factor in determining whether an association between body size and mating success is observed inDrosophila species.     

Reproductive isolation among allopatric Drosophila montana populations

An outstanding goal in speciation research is to trace the mode and tempo of the evolution of barriers to gene flow. Such research benefits from studying incipient speciation, in which speciation between populations has not yet occurred, but where multiple potential mechanisms of reproductive isolation (RI: i.e., premating, postmating‐prezygotic (PMPZ), and postzygotic barriers) may act. We used such a system to investigate these barriers among allopatric populations of Drosophila montana. In all heteropopulation crosses we found premating (sexual) isolation, which was either symmetric or asymmetric depending on the population pair compared. Postmating isolation was particularly strong in crosses involving males from one of the study populations, and while sperm were successfully transferred, stored, and motile, we experimentally demonstrated that the majority of eggs produced were unfertilized. Thus, we identified the nature of a PMPZ incompatibility. There was no evidence of intrinsic postzygotic effects. Measures of absolute and relative strengths of pre‐ and postmating barriers showed that populations differed in the mode and magnitude of RI barriers. Our results indicate that incipient RI among populations can be driven by different contributions of both premating and PMPZ barriers occurring between different population pairs and without the evolution of postzygotic barriers.     

High divergence of reproductive tract proteins and their association with postzygotic reproductive isolation in Drosophila melanogaster and Drosophila virilis group species

The possible association between gonadal protein divergence and postzygotic reproductive isolation was investigated among species of the Drosophila melanogaster and D. virilis groups. Protein divergence was scored by high-resolution two-dimensional electrophoresis (2DE). Close to 500 protein spots from gonadal tissues (testis and ovary) and nongonadal tissues (malpighian tubules and brain) were analyzed and protein divergence was calculated based on presence vs absence. Both testis and ovary proteins showed higher divergence than nongonadal proteins, and also a highly significant positive correlation with postzygotic reproductive isolation but a weaker correlation with prezygotic reproductive isolation. Particularly, a positive and significant correlation was found between proteins expressed in the testis and postzygotic reproductive isolation among closely related species such as the within-phylad species in the D. virilis group. The high levels of male-reproductive-tract protein divergence between species might be associated with F₁ hybrid male sterility among closely related species. If so, a lower level of ovary protein divergence should be expected on the basis that F₁ female hybrids are fully fertile. However, this is not necessarily true if relatively few genes are responsible for the reproductive isolation observed between closely related species, as recent studies seem to suggest. We suggest that the faster rate of evolution of gonadal proteins in comparison to nongonadal proteins and the association of that rate with postzygotic reproductive isolation may be the result of episodic and/or sexual selection on male and female molecular traits. Correspondence to: A. Civetta     

A test of the chromosomal rearrangement model of speciation in Drosophila pseudoobscura

Recent studies suggest that chromosomal rearrangements play a significant role in speciation by preventing recombination and maintaining species persistence despite interspecies gene flow. Factors conferring adaptation or reproductive isolation are maintained in rearranged regions in the face of hybridization, while such factors are eliminated from collinear regions. As a direct test of this rearrangement model, we evaluated the genetic basis of hybrid male sterility in a sympatric species pair, Drosophila pseudoobscura pseudoobscura and D. persimilis, and an allopatric species pair, D. pseudoobscura bogotana and D. persimilis. Our results are consistent with the proposed model: virtually all of the sterility factors in the former pair are associated with three inverted regions, whereas sterility factors are present in the collinear regions in the latter pair. These findings indicate recombination and selection may have eliminated sterility factors outside the inverted regions between D. p. pseudoobscura and D. persimilis, suggesting chromosomal rearrangements may facilitate species persistence despite hybridization.     

Reproductive isolation and the period gene of Drosophila

The identification of genes of large effect on ecologically important traits is an important aim of molecular ecology. The period gene of Drosophila is a candidate for a gene with a large influence on premating isolation between Drosophila species, as it determines species specific aspects of courtship behaviour. Strains of D. melanogaster are available which have been genetically transformed with the period gene of either D. melanogaster or D. simulans. Here we show that D. melanogaster females do not discriminate between two such strains. This suggests that period may only make a small contribution to total premating isolation between these species. We discuss the use of genetically transformed strains in assessing the influence of single genes on complex traits.     

Influence of antibiotic treatment and Wolbachia curing on sexual isolation among Drosophila melanogaster cage populations

Speciation depends on the establishment of reproductive isolation between populations of the same species. Whether assortative mating evolves as a by-product of adaptation is a major question relevant to the origin of species by reproductive isolation. The long-term selection populations used here were originally established 30 years ago from a single cage population (originating from a maternal one) and subsequently subjected to divergent selection for tolerance of toxins in food (heavy metals versus ethanol) to investigate this question. Those populations now differ in sexual isolation and Wolbachia infection status. Wolbachia are common and widespread bacteria infecting arthropods and nematodes. Attention has recently focused on their potential role in insect speciation, due to post-mating sperm-egg incompatibilities induced by the bacteria. In this paper we examine the potential effect of Wolbachia on the level of sexual isolation. By antibiotic curing, we show that removal of Wolbachia decreases levels of mate discrimination (sexual isolation index) between populations by about 50%. Backcrossing experiments confirm that this effect is due to infection status rather than to genetic changes in the populations resulting from antibiotic treatment. Antibiotic treatment has no effect on mate discrimination level between uninfected populations. Our findings suggest that the presence of Wolbachia (or another undetected bacterial associate) act as an additive factor contributing to the level of pre-mating isolation between these Drosophila melanogaster populations. Given the ubiquity of bacterial associates of insects, such effects could be relevant to some speciation events.     

Cryptic postzygotic isolation in an eruptive species of bark beetle (Dendroctonus ponderosae)

Studies of postzygotic isolation often involve well-differentiated taxa that show a consistent level of incompatibility, thereby limiting our understanding of the initial stages and development of reproductive barriers. Dendroctonus ponderosae provides an informative system because recent evidence suggests that distant populations produce hybrids with reproductive incompatibilities. Dendroctonus ponderosae shows an isolation-by-distance gene flow pattern allowing us to characterize the evolution of postzygotic isolation (e.g., hybrid inviability, hybrid sterility) by crossing populations along a continuum of geographic/genetic divergence. We found little evidence of hybrid inviability among these crosses. However, crosses between geographically distant populations produced sterile males (consistent with Haldane's rule). This effect was not consistent with the fixation of mutations in an isolation-by-distance pattern, but instead is spatially localized. These reproductive barriers are uncorrelated with a reduction in gene flow suggesting their recent development. Crosses between geographically proximal populations bounding the transition from compatibility to hybrid male sterility showed evidence of unidirectional reduction in hybrid male fecundity. Our study describes significant postzygotic isolation occurring across a narrow and molecularly cryptic geographic zone between the states of Oregon and Idaho. This study provides a view of the early stages of postzygotic isolation in a geographically widespread species.     

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.