Morphometric analysis of male genitalia in sibling species of Drosophila virilis Sturt

The structure of male genital organs in sibling species of the virilis group of Drosophila was examined using methods of multivariate statistics. The differences among these species were estimated using 33 indices and 2 angle parameters. The intraspecific and interspecific correlation structure of the examined characters and the order of their divergence were established. The key characters with respect to forming interspecific differences in the virilis species group were identified. Based on these results, the relative systematic positions of the sibling species are discussed as well as similarities and differences of the pattern of relationships among the species from that generally accepted for the virilis group.     

Courtship Behavior Analysis in Three Sibling Species of the Drosophila virilis Group

Entomological Review - Courtship behavior was studied in three sibling species of the Drosophila virilis group: D. virilis, D. lummei, and D. littoralis. The latter species was represented by two...     

Coevolution between Male and Female Genitalia in the Drosophila melanogaster Species Subgroup

In contrast to male genitalia that typically exhibit patterns of rapid and divergent evolution among internally fertilizing animals, female genitalia have been less well studied and are generally thought to evolve slowly among closely-related species. As a result, few cases of male-female genital coevolution have been documented. In Drosophila, female copulatory structures have been claimed to be mostly invariant compared to male structures. Here, we re-examined male and female genitalia in the nine species of the D. melanogaster subgroup. We describe several new species-specific female genital structures that appear to coevolve with male genital structures, and provide evidence that the coevolving structures contact each other during copulation. Several female structures might be defensive shields against apparently harmful male structures, such as cercal teeth, phallic hooks and spines. Evidence for male-female morphological coevolution in Drosophila has previously been shown at the post-copulatory level (e.g., sperm length and sperm storage organ size), and our results provide support for male-female coevolution at the copulatory level.     

Study of temperature-sensitive mutations in the virilis group of Drosophila. 4. Phenogenetics of a temperature-sensitive mutation affecting the development of imaginal discs in Drosophila virilis Sturt]

The phenogenetics of a new temperature-sensitive mutation diskless-ts (dl-ts, 2nd chromosome) has been studied in D. virilis. The rearing of larvae from the 1st instar at the temperature 31 degrees resulted in the complete or partial arrest of the development of imaginal discs and, consequently, the death of larvae prior to the pupation, at the prepupa stage. The change of temperature from 25 to 31 degrees during the second half of development in the 3rd instar affects the differentiation of imaginal discs. Some organs differentiate completely or partially (eyes, wings, legs) whereas the rest do not develop at all. The sensitive period embraces the whole larval development till the beginning of pupation.     

Ephemeral Association Between Gene CG5762 and Hybrid Male Sterility in Drosophila Sibling Species

Interspecies divergence in regulatory pathways may result in hybrid male sterility (HMS) when dominance and epistatic interactions between alleles that are functional within one genome are disrupted in hybrid genomes. The identification of genes contributing to HMS and other hybrid dysfunctions is essential for understanding the origin of new species (speciation). Previously, we identified a panel of male-specific loci misexpressed in sterile male hybrids of Drosophila simulans and D. mauritiana relative to parental species. In the current work, we attempt to dissect the genetic associations between HMS and one of the genes, CG5762, a Drosophila-unique locus characterized by rapid sequence divergence within the genus, presumably driven by positive natural selection. CG5762 is underexpressed in sterile backcross males compared with their fertile brothers. In CG5762 heterozygotes, the D. mauritiana allele is consistently overexpressed on both the D. simulans and D. mauritiana backcross genomic background, suggesting a cis-acting regulation factor. There is a significant association between heterozygosity and HMS in hybrid males from early but not later backcross generations. Microsatellite markers spanning CG5762 fail to associate with HMS. These observations lead to a conclusion that CG5762 is not a causative factor of HMS. Although genetic linkage between CG5762 and a neighboring causative introgression cannot be ruled out, it seems that the pattern is most consistent with CG5762 participating in epistatic interactions that are disrupted in flies with HMS.     

Genetics of a Difference in Male Cuticular Hydrocarbons between Two Sibling Species, Drosophila Simulans and D. Sechellia

In seven of the eight species of the Drosophila melanogaster group, the predominant cuticular hydrocarbon of males is 7-tricosene, but in the island endemic species D. sechellia it is 6-tricosene. The phylogeny of the group implies that the novel hydrocarbon profile of D. sechellia is a derived character. Genetic analysis of hybrids between D. sechellia and its close relative D. simulans show that each of the five major chromosome arms carries at least one gene affecting the ratio of the two tricosene isomers, with the right arm of the third chromosome having the largest effect. The species difference in this character is therefore polygenic with the effects of the different chromosome arms generally additive, although there is some epistasis among third-chromosome genes. Observations of courtship by males who have been coated with foreign hydrocarbons suggest that a male's hydrocarbon profile may slightly affect the degree of sexual isolation in one of the reciprocal hybridizations between these species, but that this role is small compared to that played by hydrocarbon differences between females.     

Rapid Divergent Evolution of Male Genitalia Among Populations of Drosophila buzzatii

Increasing evidence from multiple animal systems suggests that genital evolution and diversification are driven by rapid and strong evolutionary forces. Particularly, the morphology of male genital structures is considered to be among the fastest evolving traits in animal groups with internal fertilization. In this study, we investigated patterns of male genital variation within and between natural populations of the cactophilic fly Drosophila buzzatii in its original geographic distribution range in the Neotropics. We detected significant morphological differences among populations and distinguished five differentiated groups. Moreover, among population differentiation in genital morphology was associated with the degree of geographic isolation among populations and clearly contrasted with the general homogeneity detected for the putatively neutral mitochondrial gene COI. Integrating our present data with previous molecular population genetic surveys, our results suggest that male genital morphology has rapidly diverged after the recent demographic expansion that D. buzzatii has undergone in the arid zones of South America. Because the “lock and key” hypothesis failed to explain the present pattern, we explored alternative explanations for the observed pattern of genital diversification including drift-facilitated sexual selection.     

Transposable Element Dynamics in Two Sibling Species: Drosophila Melanogaster and Drosophila Simulans

Transposable elements (TEs) in the two sibling species, Drosophila melanogaster and D. simulans, differ considerably in amount and dynamics, with D. simulans having a smaller amount of TEs than D. melanogaster. Several hypotheses have been proposed to explain these differences, based on the evolutionary history of the two species, and claim differences either in the effective size of the population or in genome characteristics. Recent data suggest, however, that the higher amount of TEs in D. melanogaster could be associated with the worldwide invasion of D. melanogaster a long time ago while D. simulans is still under the process of such geographical spread. Stresses due to new environmental conditions and crosses between migrating populations could explain the mobilization of TEs while the flies colonize. Colonization and TE mobilization may be strong evolutionary forces that have shaped and are still shaping the eukaryote genomes.     

Mitochondrial DNA Polymorphism in Natural Populations of the Drosophila virilis Species Group

Restriction fragment length polymorphism (RFLP) analysis has been used to evaluate mitochondrial DNA (mtDNA) variation in 12 sibling species forming the Drosophila virilis species group. The variation thresholds corresponding to the interspecific and interstrain levels have been determined. The results indicate that interspecific hybridization has significantly contributed to the evolutionary history of the virilis species group.     

Genetics of Hybrid Male Sterility between Drosophila Sibling Species: A Complex Web of Epistasis Is Revealed in Interspecific Studies

To study the genetic differences responsible for the sterility of their male hybrids, we introgressed small segments of an X chromosome from Drosophila simulans into a pure Drosophila mauritiana genetic background, then assessed the fertility of males carrying heterospecific introgressions of varying size. Although this analysis examined less than 20% of the X chromosome (roughly 5% of the euchromatic portion of the D. simulans genome), and the segments were introgressed in only one direction, a minimum of four factors that contribute to hybrid male sterility were revealed. At least two of the factors exhibited strong epistasis: males carrying either factor alone were consistently fertile, whereas males carrying both factors together were always sterile. Distinct spermatogenic phenotypes were observed for sterile introgressions of different lengths, and it appeared that an interaction between introgressed segments also influenced the stage of spermatogenic defect. Males with one category of introgression often produced large quantities of motile sperm and were observed copulating, but never inseminated females. Evidently these two species have diverged at a large number of loci which have varied effects on hybrid male fertility. By extrapolation, we estimate that there are at least 40 such loci on the X chromosome alone. Because these species exhibit little DNA-sequence divergence at arbitrarily chosen loci, it seems unlikely that the extensive functional divergence observed could be due mainly to random genetic drift. Significant epistasis between conspecific genes appears to be a common component of hybrid sterility between recently diverged species of Drosophila. The linkage relationships of interacting factors could shed light on the role played by epistatic selection in the dynamics of the allele substitutions responsible for reproductive barriers between species.     

Chromosomal replication in Drosophila virilis

About half of the diploid genome of D. virilis is -heterochromatic (Heitz, 1934) and contains the satellite sequences found in isopycnic CsCl density gradients (Gall et al, 1971; Steinemann, 1976). The thymidine incorporation behavior of this material in the course of S phase was monitored by autoradiography. Labelled interphase nuclei show three types of labelling patterns, label exclusively confined to either eu- or -heterochromatin, and simultaneous labelling of both fractions. Using the fraction of labelled mitotic index method, the duration of the DNA-synthetic period, t_s = 11.9 ± 4.3 h and G₂ period, t_{G2 + 1/2M} = 6.9 ± 3.8 h, were determined. On the assumption that the investigated brain cells belong to an exponentially growing cell population, the cell cycle is 22.9 h long and the G₁ period lasts t_G1=4.1 h. The a-heterochromatin begins to replicate later than euchromatin and continues alone after a phase of common replication of both fractions. Noteworthy is the asynchronous termination in the proximal -heterochromatic segments of different chromosomes. Within the S phase, the first 1 h of DNA replication is exclusively confined to euchromatin, followed by 8 h of replication in both eu- and -heterochromatin and terminated by 3 h of exclusive -heterochromatin replication. Thus euchromatin has a doubling time of about 9 h and -heterochromatin of about 11 h. The -heterochromatin of D. virilis is late and slow replicating.     

Chromosomal replication in Drosophila virilis

DNA fiber autoradiography was used to determine parameters underlying the DNA replication of the eukaryotic chromosome in Drosophila diploid brain cells in organ culture. The average rate of fork movement, estimated from 4 different labelling intervals, is 0.35 μm/min at 25 ° C. Of the tandem arrays 93% show patterns which are compatible with bidirectional replication, 7% show unidirectional replication. The unidirectional mode of replication is interpreted as being a consequence of the experimental schedule (using hot-cold pulse labelling) combined with the occurrence of termination signals. — Some autoradiograms showed the expected two grain tracks of different densities; others showed only a high density track. The latter were most prominent in arrays of short replicons (<10 μm) which correlate with replicating satellite sequences. — The majority of replicons fall into size classes < 100 μm. The frequency distribution is skewed towards larger replicon sizes; it spans 2–238 μm, has a mean of ˉx = 35.6 μm and a median of = 21.0 μm. If the distribution is corrected for supposed satellite replicons, the median increases to = 31.0 μm. — In experiments using warmhot pulse labelling, arrays were scored which must have been a consequence of fixed termination signals. Furthermore, grain tracks diverging from weak labelled centers often have different lengths, indicating that these replicons contain two diverging replicating sections of unequal length. Presented to Professor Dr. Wolfgang Beermann on the occasion of his 60th birthday with my best wishes     

X chromosome DNA variation in Drosophila virilis.

Comparisons of polymorphism patterns between distantly related species are essential in order to determine their generality. However, most work on the genus Drosophila has been done only with species of the subgenus Sophophora. In the present work, we have sequenced one intron and surrounding coding sequences of 6 X-linked genes (chorion protein s36, elav, fused, runt, suppressor of sable and zeste) from 21 strains of wild-type Drosophila virilis (subgenus Drosophila). From these data, we have estimated the average level of DNA polymorphism, inferred the effective population size and population structure of this species, and compared the results with those obtained for other Drosophila species. There is no reduction in variation at two loci close to the centromeric heterochromatin, in contrast to Drosophila melanogaster.     

Differences in Crossover Frequency and Distribution among Three Sibling Species of Drosophila

Comparisons of the genetic and cytogenetic maps of three sibling species of Drosophila reveal marked differences in the frequency and cumulative distribution of crossovers during meiosis. The maps for two of these species, Drosophila melanogaster and D. simulans, have previously been described, while this report presents new map data for D. mauritiana, obtained using a set of P element markers. A genetic map covering nearly the entire genome was constructed by estimating the recombination fraction for each pair of adjacent inserts. The P-based genetic map of mauritiana is ~1.8 times longer than the standard melanogaster map. It appears that mauritiana has higher recombination along the entire length of each chromosome, but the difference is greatest in centromere-proximal regions of the autosomes. The mauritiana autosomes show little or no centromeric recombinational suppression, a characteristic that is prominent in melanogaster. D. simulans appears to be intermediate both in terms of total map length and intensity of the autosomal centromeric effect. These interspecific differences in recombination have important evolutionary implications for DNA sequence organization and variability. In particular, mauritiana is expected to differ from melanogaster in patterns and amounts of sequence variation and transposon insertions.     

Simple Y-Autosomal Incompatibilities Cause Hybrid Male Sterility in Reciprocal Crosses Between Drosophila virilis and D. americana

Postzygotic reproductive isolation evolves when hybrid incompatibilities accumulate between diverging populations. Here, I examine the genetic basis of hybrid male sterility between two species of Drosophila, Drosophila virilis and D. americana. From these analyses, I reach several conclusions. First, neither species carries any autosomal dominant hybrid male sterility alleles: reciprocal F₁ hybrid males are perfectly fertile. Second, later generation (backcross and F₂) hybrid male sterility between D. virilis and D. americana is not polygenic. In fact, I identified only three genetically independent incompatibilities that cause hybrid male sterility. Remarkably, each of these incompatibilities involves the Y chromosome. In one direction of the cross, the D. americana Y is incompatible with recessive D. virilis alleles at loci on chromosomes 2 and 5. In the other direction, the D. virilis Y chromosome causes hybrid male sterility in combination with recessive D. americana alleles at a single QTL on chromosome 5. Finally, in contrast with findings from other Drosophila species pairs, the X chromosome has only a modest effect on hybrid male sterility between D. virilis and D. americana.SPECIATION occurs when populations evolve one or more barriers to interbreeding (Dobzhansky 1937; Mayr 1963). One such barrier is intrinsic postzygotic isolation, which typically evolves when diverging populations accumulate different alleles at two or more loci that are incompatible when brought together in hybrid genomes; negative epistasis between these alleles renders hybrids inviable or sterile (Bateson 1909; Dobzhansky 1937; Muller 1942). Classical and recent studies in diverse animal taxa have provided support for two evolutionary patterns that often characterize the genetics of postzygotic isolation (Coyne and Orr 1989a). The first, Haldane''s rule, observes that when there is F₁ hybrid inviability or sterility that affects only one sex, it is almost always the heterogametic sex (Haldane 1922). Over the years, many researchers have tried to account for this pattern, but only two ideas are now thought to provide a general explanation: the “dominance theory,” which posits that incompatibility alleles are generally recessive in hybrids, and the “faster-male theory,” which posits that genes causing hybrid male sterility diverge more rapidly than those causing hybrid female sterility (Muller 1942; Wu and Davis 1993; Turelli and Orr 1995; reviewed in Coyne and Orr 2004). In some cases, however, additional factors might contribute to Haldane''s rule, including meiotic drive, a faster-evolving X chromosome, dosage compensation, and Y chromosome incompatibilities (reviewed in Laurie 1997; Turelli and Orr 2000; Coyne and Orr 2004).The second broad pattern affecting the evolution of postzygotic isolation is the disproportionately large effect of the X chromosome on heterogametic F₁ hybrid sterility (Coyne 1992). This “large X effect” has been documented in genetic analyses of backcross hybrid sterility (e.g., Dobzhansky 1936; Grula and Taylor 1980; Orr 1987; Masly and Presgraves 2007) and inferred from patterns of introgression across natural hybrid zones (e.g., Machado et al. 2002; Saetre et al. 2003; Payseur et al. 2004). However, in only one case has the cause of the large X effect been unambiguously determined: incompatibilities causing hybrid male sterility between Drosophila mauritiana and D. sechellia occur at a higher density on the X than on the autosomes (Masly and Presgraves 2007). Testing the generality of this pattern will require additional high-resolution genetic analyses in diverse taxa (Presgraves 2008). But whatever its causes, there is now general consensus that the X chromosome often plays a special role in the evolution of postzygotic isolation (Coyne and Orr 2004).The contribution of the Y chromosome to animal speciation is less clear. Y chromosomes have far fewer genes than the X or autosomes, and most of these genes are male specific (Lahn and Page 1997; Carvalho et al. 2009). In Drosophila species, the Y chromosome is typically required for male fertility, but not for viability (Voelker and Kojima 1971). How often, then, does the Y chromosome play a role in reproductive isolation? In crosses between Drosophila species, hybrid male sterility is frequently caused by incompatibilities between the X and Y chromosomes (Schafer 1978; Heikkinen and Lumme 1998; Mishra and Singh 2007) or between the Y and heterospecific autosomal alleles (Patterson and Stone 1952; Vigneault and Zouros 1986; Lamnissou et al. 1996). In crosses between D. yakuba and D. santomea, the Y chromosome causes F₁ hybrid male sterility, and accordingly, shows no evidence for recent introgression across a species hybrid zone (Coyne et al. 2004; Llopart et al. 2005). In mammals, reduced introgression of Y-linked loci (relative to autosomal loci) has been shown across natural hybrid zones of mice (Tucker et al. 1992) and rabbits (Geraldes et al. 2008), suggesting that the Y chromosome contributes to reproductive barriers.Here I examine the genetic basis of hybrid male sterility between two species of Drosophila, D. virilis and D. americana. These species show considerable genetic divergence (K_s ∼0.11, Morales-Hojas et al. 2008) and are currently allopatric: D. virilis is a human commensal worldwide with natural populations in Asia, and D. americana is found in riparian habitats throughout much of North America (Throckmorton 1982; McAllister 2002). Nearly 70 years ago, Patterson et al. (1942) showed that incompatibilities between the D. americana Y chromosome and the second and fifth chromosomes from D. virilis cause hybrid male sterility, a result that was confirmed in a more recent study (Lamnissou et al. 1996). Another study suggested that the X chromosome might play the predominant role in causing hybrid male sterility between D. virilis and D. americana (Orr and Coyne 1989). But because previous genetic analyses had to rely on only a few visible markers to map hybrid male sterility, they lacked the resolution to examine the genomic distribution of incompatibility loci.Using the D. virilis genome sequence, I have developed a dense set of molecular markers to investigate the genetic architecture of hybrid male sterility between D. virilis and D. americana. In this study, I perform a comprehensive set of crosses to address several key questions: What is the effect of the X chromosome on hybrid male sterility between D. virilis and D. americana? What is the effect of the Y chromosome? Approximately how many loci contribute to hybrid male sterility between these Drosophila species? Perhaps surprisingly, the answers to these questions differ dramatically from what has been found for other Drosophila species, including the well-studied D. melanogaster group.     

DNA-protein interactions in the Drosophila virilis mitochondrial chromosome.

The location of proteins on the mitochondrial DNA (mtDNA) of Drosophila virilis was investigated by Me3 psoralen photoreaction of mitochondria isolated from embryos. After photoreaction the mtDNA was purified and the pattern of DNA cross-linking was determined by electron microscopy of the DNA under totally denaturing conditions. The transcribed regions of the mtDNA molecule contained some uncross-linked regions, but such regions were infrequent and randomly distributed. In contrast, the A + T-rich region around the origin of replication of the mtDNA was usually protected from psoralen cross-linking. The data were best fit by two protected sites, each approximately 400 base pairs, compared to the four 400 base pair sites observed in the equivalent region of D. melanogaster mtDNA [Potter et al. (1980) Proc. Nat. Acad. Sci. USA 77, 4118-4122]. Thus this region of the mtDNA appears to be involved in a DNA-protein structure that is highly conserved even though the DNA sequence has diverged rapidly relative to protein-coding sequences.