Population genetics and phylogenetics of DNA sequence variation at multiple loci within the Drosophila melanogaster species complex

Two regions of the genome, a 1-kbp portion of the zeste locus and a 1.1- kbp portion of the yolk protein 2 locus, were sequenced in six individuals from each of four species: Drosophila melanogaster, D. simulans, D. mauritiana, and D. sechellia. The species and strains were the same as those of a previous study of a 1.9-kbp region of the period locus. No evidence was found for recent balancing or directional selection or for the accumulation of selected differences between species. Yolk protein 2 has a high level of amino acid replacement variation and a low level of synonymous variation, while zeste has the opposite pattern. This contrast is consistent with information on gene function and patterns of codon bias. Polymorphism levels are consistent with a ranking of effective population sizes, from low to high, in the following order: D. sechellia, D. melanogaster, D.mauritiana, and D. simulans. The apparent species relationships are very similar to those suggested by the period locus study. In particular, D. simulans appears to be a large population that is still segregating variation that arose before the separation of D. mauritiana and D. sechellia. It is estimated that the separation of ancestral D. melanogaster from the other species occurred 2.5-3.4 Mya. The separations of D. sechellia and D. mauritiana from ancestral D. simulans appear to have occurred 0.58- 0.86 Mya, with D. mauritiana having diverged from ancestral D. simulans 0.1 Myr more recently than D. sechellia.      

The population genetics of the origin and divergence of the Drosophila simulans complex species

The origins and divergence of Drosophila simulans and close relatives D. mauritiana and D. sechellia were examined using the patterns of DNA sequence variation found within and between species at 14 different genes. D. sechellia consistently revealed low levels of polymorphism, and genes from D. sechellia have accumulated mutations at a rate that is approximately 50% higher than the same genes from D. simulans. At synonymous sites, D. sechellia has experienced a significant excess of unpreferred codon substitutions. Together these observations suggest that D. sechellia has had a reduced effective population size for some time, and that it is accumulating slightly deleterious mutations as a result. D. simulans and D. mauritiana are both highly polymorphic and the two species share many polymorphisms, probably since the time of common ancestry. A simple isolation speciation model, with zero gene flow following incipient species separation, was fitted to both the simulans/mauritiana divergence and the simulans/sechellia divergence. In both cases the model fit the data quite well, and the analyses revealed little evidence of gene flow between the species. The exception is one gene copy at one locus in D. sechellia, which closely resembled other D. simulans sequences. The overall picture is of two allopatric speciation events that occurred quite near one another in time.     

Patterns of Microsatellite Variability in the Drosophila Melanogaster Complex

Forty-seven microsatellite loci were amplified in Drosophila melanogaster, Drosophila simulans, Drosophila mauritiana and Drosophila sechellia. The two cosmopolitan species D. melanogaster and D. simulans were found to be the most variable ones, followed by D. mauritiana and D. sechellia. A model based clustering algorithm was applied to the population samples of D. melanogaster, D. simulans and D. sechellia. No evidence for population substructure was detected within species--most likely due to insufficient power. A Markov chain Monte Carlo method developed for demographic inference based on microsatellites provided unambiguous evidence for population contraction in D. melanogaster, D. simulans and D. sechellia, despite that the D. melanogaster and D. simulans population samples were of non-African origin and represented recently expanded populations.     

Evidence for Complex Genic Interactions between Conspecific Chromosomes Underlying Hybrid Female Sterility in the Drosophila Simulans Clade

F(1) hybrid females between the sibling species Drosophila simulans, Drosophila mauritiana and Drosophila sechellia are completely fertile. However, we have found that female sterility can be observed in F(2) backcross females who are homozygous for D. simulans X chromosomes and homozygous for autosomal regions from either D. mauritiana or D. sechellia. Our results indicate that neither D. mauritiana autosome (2 or 3) can cause complete female sterility in a D. simulans background. The simultaneous presence of homozygous regions from both the second and third chromosomes of D. mauritiana, however, causes nearly complete female sterility which cannot be accounted for by their individual effects. The two autosomes of D. sechellia may show a similar pattern. From the same crosses, we also obtained evidence against a role for cytoplasmic or maternal effects in causing hybrid male sterility between these species. Taken with the results presented elsewhere, these observations suggest that epistatic interactions between conspecific genes in a hybrid background may be the prevalent mode of hybrid sterility between recently diverged species.     

Sequence Analysis of Active Mariner Elements in Natural Populations of Drosophila Simulans

Active and inactive mariner elements from natural and laboratory populations of Drosophila simulans were isolated and sequenced in order to assess their nucleotide variability and to compare them with previously isolated mariner elements from the sibling species Drosophila mauritiana and Drosophila sechellia. The active elements of D. simulans are very similar among themselves (average 99.7% nucleotide identity), suggesting that the level of mariner expression in different natural populations is largely determined by position effects, dosage effects and perhaps other factors. Furthermore, the D. simulans elements exhibit nucleotide identities of 98% or greater when compared with mariner elements from the sibling species. Parsimony analysis of mariner elements places active elements from the three species into separate groups and suggests that D. simulans is the species from which mariner elements in D. mauritiana and D. sechellia are most likely derived. This result strongly suggests that the ancestral form of mariner among these species was an active element. The two inactive mariner elements sequenced from D. simulans are very similar to the inactive peach element from D. mauritiana. The similarity may result from introgression between D. simulans and D. mauritiana or from selective constraints imposed by regulatory effects of inactive elements.     

Molecular population genetics of male accessory gland proteins in the Drosophila simulans complex

Accessory gland proteins are a major component of Drosophila seminal fluid. These proteins have a variety of functions and may be subject to sexual selection and/or antagonistic evolution between the sexes. Most population genetic data from these proteins are from D. melanogaster and D. simulans. Here, we extend the population genetic analysis of Acp genes to the other simulans complex species, D. mauritiana and D. sechellia. We sequenced population samples of seven Acp's from D. mauritiana, D. sechellia, and D. simulans. We investigated the population genetics of these genes on individual simulans complex lineages and compared Acp polymorphism and divergence to polymorphism and divergence from a set of non-Acp loci in the same species. Polymorphism and divergence data from the simulans complex revealed little evidence for adaptive protein evolution at individual loci. However, we observed a dramatically inflated index of dispersion for amino acid substitutions in the simulans complex at Acp genes, but not at non-Acp genes. This pattern of episodic bursts of protein evolution in Acp's provides the strongest evidence to date that the population genetic mechanisms driving Acp divergence are different from the mechanisms driving evolution at most Drosophila genes.     

Variation in fiber number of a male-specific muscle between Drosophila species: a genetic and developmental analysis

We characterize a newly discovered morphological difference between species of the Drosophila melanogaster subgroup. The muscle of Lawrence (MOL) contains about four to five fibers in D. melanogaster and Drosophila simulans and six to seven fibers in Drosophila mauritiana and Drosophila sechellia. The same number of nuclei per fiber is present in these species but their total number of MOL nuclei differs. This suggests that the number of muscle precursor cells has changed during evolution. Our comparison of MOL development indicates that the species difference appears during metamorphosis. We mapped the quantitative trait loci responsible for the change in muscle fiber number between D. sechellia and D. simulans to two genomic regions on chromosome 2. Our data eliminate the possibility of evolving mutations in the fruitless gene and suggest that a change in the twist might be partly responsible for this evolutionary change.     

The Genetic Basis of Haldane''s Rule and the Nature of Asymmetric Hybrid Male Sterility among Drosophila Simulans, Drosophila Mauritiana and Drosophila Sechellia

Haldane's rule (i.e., the preferential hybrid sterility and inviability of heterogametic sex) has been known for 70 years, but its genetic basis, which is crucial to the understanding of the process of species formation, remains unclear. In the present study, we have investigated the genetic basis of hybrid male sterility using Drosophila simulans, Drosophila mauritiana and Drosophila sechellia. An introgression of D. sechellia Y chromosome into a fairly homogenous background of D. simulans did not show any effect of the introgressed Y on male sterility. The substitution of D. simulans Y chromosome into D. sechellia, and both reciprocal Y chromosome substitutions between D. simulans and D. mauritiana were unsuccessful. Introgressions of cytoplasm between D. simulans and D. mauritiana (or D. sechellia) also did not have any effect on hybrid male sterility. These results rule out the X-Y interaction hypothesis as a general explanation of Haldane's rule in this species group and indicate an involvement of an X-autosome interaction. Models of symmetrical and asymmetrical X-autosome interaction have been developed which explain the Y chromosome substitution results and suggest that evolution of interactions between different genetic elements in the early stages of speciation is more likely to be of an asymmetrical nature. The model of asymmetrical X-autosome interaction also predicts that different sets of interacting genes may be involved in different pairs of related species and can account for the observation that hybrid male sterility in many partially isolated species is often nonreciprocal or unidirectional.     

The Effects of Interspecific Y Chromosome Replacements on Hybrid Sterility within the Drosophila Simulans Clade

We attempted to introgress Y chromosomes between three sibling species of Drosophila: D. simulans, D. sechellia and D. mauritiana. Four D. sechellia Y chromosomes were introgressed into D. simulans without loss of fertility whereas the four reciprocal introgressions (D. simulans Y introgressed into D. sechellia) all result in sterility. Both reciprocal Y introgressions of D. simulans and D. mauritiana (four of each) also result in sterility. Compared with D. simulans males, the males with the D. sechellia Y chromosome in D. simulans background had lower productivity but only after multiple matings with virgin females. These males also were inferior compared with pure species males in sperm displacement and/or remating ability. The two different Y genotype males, however, were comparable in viability, longevity and mating success in female choice tests. We also use our results to estimate the effective number of autosomal loci interacting with X-linked genes to produce hybrid male sterility.     

Wolbachia Infections and the Expression of Cytoplasmic Incompatibility in Drosophila Sechellia and D. Mauritiana

Various stocks of Drosophila mauritiana and D. sechellia were found to be infected with Wolbachia, a Rickettsia-like bacterium that is known to cause cytoplasmic incompatibility and other reproductive abnormalities in arthropods. Testing for the expression of cytoplasmic incompatibility in these two species showed partial incompatibility in D. sechellia but no expression of incompatibility in D. mauritiana. To determine whether absence of cytoplasmic incompatibility in D. mauritiana was due to either the bacterial or host genome, we transferred bacteria from D. mauritiana into an uninfected strain of D. simulans, a host species known to express high levels of incompatibility with endogenous Wolbachia. We also performed the reciprocal transfer of the natural D. simulans Riverside infection into a tetracycline-treated stock of D. mauritiana. In each case, the ability to express incompatibility was unaltered by the different host genetic background. These experiments indicate that in D. simulans and D. mauritiana expression of the cytoplasmic incompatibility phenotype is determined by the bacterial strain and that D. mauritiana harbors a neutral strain of Wolbachia.     

Genetics of Reproductive Isolation in the Drosophila Simulans Clade: Complex Epistasis Underlying Hybrid Male Sterility

We have analyzed the sterility associated with introgressions of the distal one-fourth of the X chromosome from either Drosophila mauritiana or Drosophila sechellia into the genome of Drosophila simulans using a series of visible and DNA markers. Because in Drosophila hybrids, male sterility is usually complete and is often tightly linked with each of several markers used in crosses, a simple genetic basis has generally been assumed. In our low resolution mapping experiment, we were not able to reject the null hypothesis that a single gene, introgressed from either D. mauritiana or D. sechellia, is the cause of male sterility. High resolution mapping, however, reveals a much more complex picture. At least three distinct factors from D. mauritiana, or two from D. sechellia, were identified that need to be jointly present to confer full sterility. Each individual factor by itself is relatively ineffective in causing sterility, or even a partial spermatogenic defect. Moreover, there appear to be more sterility factors on comparable introgressions from D. mauritiana than from D. sechellia. On the basis of these observations, we propose a model which suggests that multilocus weak allele interactions are a very common cause of reproductive incompatibility between closely related species. We also present theoretical argument and empirical evidence against extrapolating the results of within-species analysis to interpret the genetic basis of species differences. The implications of this model on the theories of evolution of species differences and the attempt to understand the mechanisms of hybrid sterility/inviability at the molecular level are discussed.     

Comparative analysis of transposable elements in the melanogaster subgroup sequenced genomes

Transposable elements (TEs) are indwelling components of genomes, and their dynamics have been a driving force in genome evolution. Although we now have more information concerning their amounts and characteristics in various organisms, we still have little data from overall comparisons of their sequences in very closely-related species. While the Drosophila melanogaster genome has been extensively studied, we have only limited knowledge regarding the precise TE sequences in the genomes of the related species Drosophila simulans, Drosophila sechellia and Drosophila yakuba. In this study we analyzed the number and structure of TE copies in the sequenced genomes of these four species. Our findings show that, unexpectedly, the number of TE insertions in D. simulans is greater than that in D. melanogaster, but that most of the copies in D. simulans are degraded and in small fragments, as in D. sechellia and D. yakuba. This suggests that all three species were invaded by numerous TEs a long time ago, but have since regulated their activity, as the present TE copies are degraded, with very few full-length elements. In contrast, in D. melanogaster, a recent activation of TEs has resulted in a large number of almost-identical TE copies. We have detected variants of some TEs in D. simulans and D. sechellia, that are almost identical to the reference TE sequences in D. melanogaster, suggesting that D. melanogaster has recently been invaded by active TE variants from the other species. Our results indicate that the three species D. simulans, D. sechellia, and D. yakuba seem to be at a different stage of their TE life cycle when compared to D. melanogaster. Moreover, we show that D. melanogaster has been invaded by active TE variants for several TE families likely to come from D. simulans or the ancestor of D. simulans and D. sechellia. The numerous horizontal transfer events implied to explain these results could indicate introgression events between these species.     

Rapid sequence turnover at an intergenic locus in Drosophila

Closely related species of Drosophila tend to have similar genome sizes. The strong imbalance in favor of small deletions relative to insertions implies that the unconstrained DNA in Drosophila is unlikely to be passively inherited from even closely related ancestors, and yet most DNA in Drosophila genomes is intergenic and potentially unconstrained. In an attempt to investigate the maintenance of this intergenic DNA, we studied the evolution of an intergenic locus on the fourth chromosome of the Drosophila melanogaster genome. This 1.2-kb locus is marked by two distinct, large insertion events: a nuclear transposition of a mitochondrial sequence and a transposition of a nonautonomous DNA transposon DNAREP1_DM. Because we could trace the evolutionary histories of these sequences, we were able to reconstruct the length evolution of this region in some detail. We sequenced this locus in all four species of the D. melanogaster species complex: D. melanogaster, D. simulans, D. sechellia, and D. mauritiana. Although this locus is similar in size in these four species, less than 10% of the sequence from the most recent common ancestor remains in D. melanogaster and all of its sister species. This region appears to have increased in size through several distinct insertions in the ancestor of the D. melanogaster species complex and has been shrinking since the split of these lineages. In addition, we found no evidence suggesting that the size of this locus has been maintained over evolutionary time; these results are consistent with the model of a dynamic equilibrium between persistent DNA loss through small deletions and more sporadic DNA gain through less frequent but longer insertions. The apparent stability of genome size in Drosophila may belie very rapid sequence turnover at intergenic loci.     

A Reanalysis of Protein Polymorphism in Drosophila Melanogaster, D. Simulans, D. Sechellia and D. Mauritiana: Effects of Population Size and Selection

Comparison of synonymous and nonsynonymous variation/substitution within and between species at individual genes has become a widely used general approach to detect the effect of selection versus drift. The sibling species group comprised of two cosmopolitan (Drosophila melanogaster and Drosophila simulans) and two island (Drosophila mauritiana and Drosophila sechellia) species has become a model system for such studies. In the present study we reanalyzed the pattern of protein variation in these species, and the results were compared against the patterns of nucleotide variation obtained from the literature, mostly available for melanogaster and simulans. We have mainly focused on the contrasting patterns of variation between the cosmopolitan pair. The results can be summarized as follows: (1) As expected the island species D. mauritiana and D. sechellia showed much less variation than the cosmopolitan species D. melanogaster and D. simulans. (2) The chromosome 2 showed significantly less variation than chromosome 3 and X in all four species which may indicate effects of past selective sweeps. (3) In contrast to its overall low variation, D. mauritiana showed highest variation for X-linked loci which may indicate introgression from its sibling, D. simulans. (4) An average population of D. simulans was as heterozygous as that of D. melanogaster (14.4% v.s. 13.9%) but the difference was large and significant when considering only polymorphic loci (37.2% v.s. 26.1%). (5) The species-wise pooled populations of these two species showed similar results (all loci = 18.3% v.s. 20.0%, polymorphic loci = 47.2% v.s. 37.6%). (6) An average population of D. simulans had more low-frequency alleles than D. melanogaster, and the D. simulans alleles were found widely distributed in all populations whereas the D. melanogaster alleles were limited to local populations. As a results of this, pooled populations of D. melanogaster showed more polymorphic loci than those of D. simulans (48.0% v.s. 32.0%) but the difference was reduced when the comparison was made on the basis of an average population (29.1% v.s. 21.4%). (7) While the allele frequency distributions within populations were nonsignificant in both D. melanogaster and D. simulans, melanogaster had fewer than simulans, but more than expected from the neutral theory, low frequency alleles. (8) Diallelic loci with the second allele with a frequency less than 20% had similar frequencies in all four species but those with the second allele with a frequency higher than 20% were limited to only melanogaster the latter group of loci have clinal (latitudinal) patterns of variation indicative of balancing selection. (9) The comparison of D. simulans/D. melanogaster protein variation gave a ratio of 1.04 for all loci and 1.42 for polymorphic loci, against a ratio of approximately 2-fold difference for silent nucleotide sites. This suggests that the species ratios of protein and silent nucleotide polymorphism are too close to call for selective difference between silent and allozyme variation in D. simulans. In conclusion, the contrasting levels of allozyme polymorphism, distribution of rare alleles, number of diallelic loci and the patterns of geographic differentiation between the two species suggest the role of natural selection in D. melanogaster, and of possibly ancient population structure and recent worldwide migration in D. simulans. Population size differences alone are insufficient as an explanation for the patterns of variation between these two species.     

Genetics of egg production in Drosophila sechellia

Drosophila sechellia, an island endemic that specializes on a single host plant, has a lower rate of egg production than its generalist sister species D. melanogaster, D. simulans, and D. mauritiana. Earlier work showed that part of this difference in egg production was due to a reduction in the number of ovarioles in D. sechellia relative to its sister species. Here, I extend this earlier work by genetically analyzing the difference in egg production between D. sechellia and D. simulans. In all, 10 genetic markers were used in several interspecific backcrosses to identify chromosome regions that affected the rate of egg production. While previously mapped factors affecting ovariole number appear to impact the rate of egg production, new, non-ovariole factors were also identified. Overall, the difference in egg production between D. sechellia and D. simulans appears to be a polygenic trait. The relationship between these factors and genes involved the adaptation of D. sechellia to its host plant is not yet clear. The data are consistent with the hypothesis that decline in egg production is, in part, a negative pleiotropic effect of genetic changes required for host specialization in D. sechellia, although finer-scale genetic analysis of both traits is needed to truly test this hypothesis.     

Genetics of sexual isolation in females of the Drosophila simulans species complex.

Genetic analysis of hybrids between Drosophila simulans and D. sechellia shows that sexual isolation in females is caused by at least two genes, one on each major autosome, while the X chromosome has no effect. These results are similar to those of a previous study of hybrids between D. simulans and another sibling species, D. mauritiana. In this latter hybridization, each arm of the second chromosome carries genes causing sexual isolation in females, implying a total divergence of at least three loci. The genetic similarity between the D. simulans/D. mauritiana and D. simulans/D. sechellia hybridizations probably results from independent evolution and not phylogenetic artifacts, because the dominance relationships and behavioural interactions differ between the two hybridizations. The lack of an X-chromosome effect on sexual isolation contrasts with genetic studies of post-zygotic reproductive isolation, which invariably show strong effects of this chromosome.     

Horizontal transfer of hobo transposable elements within the Drosophila melanogaster species complex: evidence from DNA sequencing.

The hobo family of transposable elements, one of three transposable-element families that cause hybrid dysgenesis in Drosophila melanogaster, appears to be present in all members of the D. melanogaster species complex: D. melanogaster, D. simulans, D. mauritiana, and D. sechellia. Some hobo-hybridizing sequences are also found in the other members of the melanogaster subgroup and in many members of the related montium subgroup. Surveys of older isofemale lines of D. melanogaster suggest that complete hobo elements were absent prior to 50 years ago and that hobo has recently been introduced into the species by horizontal transfer. To test the horizontal transfer hypothesis, the 2.6-kb XhoI fragments of hobo elements from D. melanogaster, D. simulans, and D. mauritiana were cloned and sequenced. The DNA sequences reveal an extremely low level of divergence and support the conclusion that the active hobo element has been horizontally transferred into or among these species in the recent past.     

A molecular phylogeny for the Drosophila melanogaster subgroup and the problem of polymorphism data

Drosophila melanogaster belongs to a closely related group of eight species collectively known as the melanogaster subgroup; all are native to sub-Saharan Africa and islands off the east coast of Africa. The phylogenetic relationships of most species in this subgroup have been well documented; however, the three most closely related species, D. simulans, D. sechellia, and D. mauritiana, have remained problematic from a phylogenetic standpoint as no data set has unambiguously resolved them. We present new DNA sequence data on the nullo and Serendipity-alpha genes and combine them with all available nuclear DNA sequence data; the total data encompass 12 genes and the ITS of rDNA. A methodological problem arose because nine of the genes had information on intraspecific polymorphisms in at least one species. We explored the effect of inclusion/exclusion of polymorphic sites and found that it had very little effect on phylogenetic inferences, due largely to the fact that 82% of polymorphisms are autapomorphies (unique to one species). We have also reanalyzed our previous DNA-DNA hybridization data with a bootstrap procedure. The combined sequence data set and the DNA-DNA hybridization data strongly support the sister status of the two island species, D. sechellia and D. mauritiana. This at least partially resolves what had been a paradox of parallel evolution in these two species.      

Courtship song recognition in the Drosophila melanogaster complex: heterospecific songs make females receptive in D. melanogaster, but not in D. sechellia

Abstract. The courtship song emitted by male wing vibration has been regarded as one of the most important signals in sexual isolation in the species of the Drosophila melanogaster complex. Inter- and intraspecific crosses were observed using males whose wings were removed (mute) or females whose aristae were removed (deaf). Females of D. melanogaster, D. simulans , and D. mauritiana mated with heterospecific males in the song-present condition (cross between normal females and winged males) more often than in the no-song condition (cross between normal females and wingless males or between aristaless females and winged males) or they showed no preference between the two conditions. It is possible that in these females heterospecific courtship songs play a role as if they were conspecific. In contrast, the females of D. sechellia mated with D. melanogaster or D. simulans males in the no-song condition more often than in the song-present condition, suggesting that they reject males with heterospecific song. Female mate recognition depending on the courtship song in D. melanogaster, D. simulans , and D. mauritiana is considered to be relatively broader and that in D. sechellia narrower.     

Incompatibility between X chromosome factor and pericentric heterochromatic region causes lethality in hybrids between Drosophila melanogaster and its sibling species

The Dobzhansky-Muller model posits that postzygotic reproductive isolation results from the evolution of incompatible epistatic interactions between species: alleles that function in the genetic background of one species can cause sterility or lethality in the genetic background of another species. Progress in identifying and characterizing factors involved in postzygotic isolation in Drosophila has remained slow, mainly because Drosophila melanogaster, with all of its genetic tools, forms dead or sterile hybrids when crossed to its sister species, D. simulans, D. sechellia, and D. mauritiana. To circumvent this problem, we used chromosome deletions and duplications from D. melanogaster to map two hybrid incompatibility loci in F(1) hybrids with its sister species. We mapped a recessive factor to the pericentromeric heterochromatin of the X chromosome in D. simulans and D. mauritiana, which we call heterochromatin hybrid lethal (hhl), which causes lethality in F(1) hybrid females with D. melanogaster. As F(1) hybrid males hemizygous for a D. mauritiana (or D. simulans) X chromosome are viable, the lethality of deficiency hybrid females implies that a dominant incompatible partner locus exists on the D. melanogaster X. Using small segments of the D. melanogaster X chromosome duplicated onto the Y chromosome, we mapped a dominant factor that causes hybrid lethality to a small 24-gene region of the D. melanogaster X. We provide evidence suggesting that it interacts with hhl(mau). The location of hhl is consistent with the emerging theme that hybrid incompatibilities in Drosophila involve heterochromatic regions and factors that interact with the heterochromatin.