Population transcriptomics: insights from <Emphasis Type="Italic">Drosophila simulans,</Emphasis><Emphasis Type="Italic">Drosophila sechellia</Emphasis> and their hybrids

Sequence differentiation has been widely studied between populations and species, whereas interest in expression divergence is relatively recent. Using microarrays, we compared four geographically distinct populations of Drosophila simulans and a population of Drosophila sechellia, and interspecific hybrids. We observed few differences between populations, suggesting a slight population structure in D. simulans. This structure was observed in direct population comparisons, as well as in interspecific comparisons (hybrids vs. parents, D. sechellia vs. D. simulans). Expression variance is higher in the French and Zimbabwean populations than in the populations from the ancestral range of D. simulans (Kenya and Seychelles). This suggests a large scale phenomenon of decanalization following the invasion of a new environment. Comparing D. simulans and D. sechellia, we revealed 304 consistently differentially expressed genes, with striking overrepresentation of genes of the cytochrome P450 family, which could be related to their role in detoxification as well as in hormone regulation. We also revealed differences in genes involved in Juvenile hormone and Dopamine differentiation. We finally observed very few differentially expressed genes between hybrids and parental populations, with an overrepresentation of X-linked genes.     

The recent origin of the Sdic gene cluster in the melanogaster subgroup

Gene families are composed of closely related genes and are an important part of eukaryotic genomes. In the proximal region of the X chromosome of Drosophila melanogaster there is a cluster of four tandem Sdic genes, located between the gene Cdic and the gene AnnX. Sdic is a chimeric gene that encodes a novel protein with sperm-specific expression. It had been hypothesized that the Sdic gene cluster was formed after the split of D. melanogaster and D. simulans. To study the evolution of this cluster, the sequence of this region was studied in several Drosophilidae species. In all species analyzed, Sdic genes are absent and AnnX and Cdic are adjacent to each other. The results allowed the inference of the ancestral situation and the reconstruction of the evolution of the cluster, and confirm that the Sdic cluster was indeed formed in the lineage that gave rise to D. melanogaster, being one of the youngest gene clusters known. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Structural and genetic studies of the proliferation disrupter genes of Drosophila simulans and D. melanogaster

To examine whether structural and functional differences exist in the proliferation disrupter (prod) genes between Drosophila simulans and D. melanogaster, we analyzed and compared both genes. The exon–intron structure of the genes was found to be the same – three exons were interrupted by two introns, although a previous report suggested that only one intron existed in D. melanogaster. The prod genes of D. simulans and D. melanogaster both turn out to encode 346 amino acids, not 301 as previously reported for D. melanogaster. The numbers of nucleotide substitutions in the prod genes was 0.0747 ±  per synonymous site and 0.0116 ± 0.0039 per replacement site, both comparable to those previously known for homologous genes between D. simulans and D. melanogaster. Genetic analysis demonstrated that D. simulans PROD can compensate for a deficiency of D. melanogaster PROD in hybrids. The PRODs of D. simulans and D. melanogaster presumably share the same function and a conserved working mechanism. The prod gene showed no significant interaction with the lethality of the male hybrid between these species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Hybrid lethal systems in theDrosophila melanogaster species complex

Lethal phases of the hybrids betweenDrosophila melanogaster and its sibling species,D. simulans are classified into three types: (1) embryonic lethality in hybrids carryingD. simulans cytoplasm andD. melanogaster X chromosome, (2) larval lethality in hybrids not carryingD. simulans X, and (3) temperature-sensitive pupal lethality in hybrids carryingD. simulans X. The same lethal phases are also observed when either of the two other sibling species,D. mauritiana orD. sechellia, is employed for hybridization withD. melanogaster. Here, we describe genetic analyses of each hybrid lethality, and demonstrate that these three types of lethality are independent phenomena. We then propose two models to interpret the mechanisms of each hybrid lethality. The first model is a modification of the conventional X/autosome imbalance hypothesis assuming a lethal gene and a suppressor gene are involved in the larval lethality, while the second model is for embryonic lethality assuming an interaction between a maternal-effect lethal gene and a suppressor gene.     

Comparative studies of allozyme loci in Drosophila simulans and Drosophila melanogaster. I. Three dipeptidase loci

Genetic variation at three dipeptidase loci (Dip-A, Dip-B, and Dip-C) in Drosophila simulans was analyzed by starch gel electrophoresis. Dip-A was found to be polymorphic in four populations, while Dip-B and Dip-C were found to be polymorphic in one. The numbers of different alleles found at each respective locus were: Dip-A, two; Dip-B, two; and Dip-C, three. Dip-A was genetically mapped at 57.9 on the second chromosome, and Dip-B and Dip-C at 80.9 and 87.9 on the third chromosome, respectively. Neither Dip-B nor Dip-C has been mapped in D. melanogaster because both loci are apparently monomorphic. Their map positions in D. simulans with respect to flanking markers whose homologous genes have been cytogenetically localized in D. melanogaster suggested that they might be mapped cytogenetically by using available deficiencies in D. melanogaster. Accordingly, by the construction of interspecific hybrids which carried deficiencies of melanogaster and an allele of simulans with a mobility different from that of the fixed melanogaster allele, Dip-B and Dip-C were localized between 87F12-14 and 88C1-3 and between 87B5-6 and 87B8-10, respectively, in the salivary gland chromosomes of D. melanogaster. The similarity between these two species is discussed on the basis of these findings.     

Male Sex Interspecies Divergence and Down Regulation of Expression of Spermatogenesis Genes in Drosophila Sterile Hybrids

Male sex genes have shown a pattern of rapid interspecies divergence at both the coding and gene expression level. A common outcome from crosses between closely-related species is hybrid male sterility. Phenotypic and genetic studies in Drosophila sterile hybrid males have shown that spermatogenesis arrest is postmeiotic with few exceptions, and that most misregulated genes are involved in late stages of spermatogenesis. Comparative studies of gene regulation in sterile hybrids and parental species have mainly used microarrays providing a whole genome representation of regulatory problems in sterile hybrids. Real-time PCR studies can reject or reveal differences not observed in microarray assays. Moreover, differences in gene expression between samples can be dependant on the source of RNA (e.g., whole body vs. tissue). Here we survey expression in D. simulans, D. mauritiana and both intra and interspecies hybrids using a real-time PCR approach for eight genes expressed at the four main stages of sperm development. We find that all genes show a trend toward under expression in the testes of sterile hybrids relative to parental species with only the two proliferation genes (bam and bgcn) and the two meiotic class genes (can and sa) showing significant down regulation. The observed pattern of down regulation for the genes tested can not fully explain hybrid male sterility. We discuss the down regulation of spermatogenesis genes in hybrids between closely-related species within the contest of rapid divergence experienced by the male genome, hybrid sterility and possible allometric changes due to subtle testes-specific developmental abnormalities.     

Rapid Male-Specific Regulatory Divergence and Down Regulation of Spermatogenesis Genes in Drosophila Species Hybrids

In most crosses between closely related species of Drosophila, the male hybrids are sterile and show postmeiotic abnormalities. A series of gene expression studies using genomic approaches have found significant down regulation of postmeiotic spermatogenesis genes in sterile male hybrids. These results have led some to suggest a direct relationship between down regulation in gene expression and hybrid sterility. An alternative explanation to a cause-and-effect relationship between misregulation of gene expression and male sterility is rapid divergence of male sex regulatory elements leading to incompatible interactions in an interspecies hybrid genome. To test the effect of regulatory divergence in spermatogenesis gene expression, we isolated 35 fertile D. simulans strains with D. mauritiana introgressions in either the X, second or third chromosome. We analyzed gene expression in these fertile hybrid strains for a subset of spermatogenesis genes previously reported as significantly under expressed in sterile hybrids relative to D. simulans. We found that fertile autosomal introgressions can cause levels of gene down regulation similar to that of sterile hybrids. We also found that X chromosome heterospecific introgressions cause significantly less gene down regulation than autosomal introgressions. Our results provide evidence that rapid male sex gene regulatory divergence can explain misexpression of spermatogenesis genes in hybrids.     

Adaptive gene expression divergence inferred from population genomics

Detailed studies of individual genes have shown that gene expression divergence often results from adaptive evolution of regulatory sequence. Genome-wide analyses, however, have yet to unite patterns of gene expression with polymorphism and divergence to infer population genetic mechanisms underlying expression evolution. Here, we combined genomic expression data—analyzed in a phylogenetic context—with whole genome light-shotgun sequence data from six Drosophila simulans lines and reference sequences from D. melanogaster and D. yakuba. These data allowed us to use molecular population genetics to test for neutral versus adaptive gene expression divergence on a genomic scale. We identified recent and recurrent adaptive evolution along the D. simulans lineage by contrasting sequence polymorphism within D. simulans to divergence from D. melanogaster and D. yakuba. Genes that evolved higher levels of expression in D. simulans have experienced adaptive evolution of the associated 3′ flanking and amino acid sequence. Concomitantly, these genes are also decelerating in their rates of protein evolution, which is in agreement with the finding that highly expressed genes evolve slowly. Interestingly, adaptive evolution in 5′ cis-regulatory regions did not correspond strongly with expression evolution. Our results provide a genomic view of the intimate link between selection acting on a phenotype and associated genic evolution.     

Conservation and change in structural and 5′ flanking sequences of esterase 6 in siblingDrosophila species

Esterase 6 (Est-6/EST6) is the major β-carboxylesterase inD. melanogaster and its siblingsD. simulans andD. mauritiana. It is expressed in several tissues but its major site of expression is the sperm ejaculatory duct of the adult male. Although EST6 activity affects reproductive fitness, there are high levels of electrophoretic and activity polymorphism, at least withinD. melanogaster andD. simulans. Here we present the nucleotide sequences of anEst-6 allele and its flanking regions from each ofD. simulans andD. mauritiana and compare them with the publishedD. melanogaster sequences. As might be expected, replacement sites are significantly less divergent than exon silent sites in all comparisons, suggesting that selection is acting to maintain EST6 structure and function among the three species. Nevertheless, the ratio of the levels of replacement to silent site divergence is still much higher forEst-6 than for seven of ten other genes (including both isozyme-coding loci) for which comparable data have been published for these species. This is consistent with the high levels of EST6 electrophoretic polymorphism withinD. melanogaster andD. simulans and implies that selective constraints against amino acid change are relatively weak for EST6. By contrast, comparisons involving promotor sequences show that the level of divergence in the first 350bp 5′ of the gene is significantly lower than those for four of the six other loci for which comparable data have been published for these species. In particular, there are two perfectly conserved stretches (−1 to −158bp and −219 to −334bp) each over 100bp long included in this 350bp region. Thus the data suggest a relatively low level of selective constraint on the amino acid sequence of EST6 but a relatively high level of constraint on sequences affecting aspects of its expression.     

Correlated Evolution of Synonymous and Nonsynonymous Sites in <Emphasis Type="Italic">Drosophila</Emphasis>

Recent work has shown that Drosophila melanogaster genes with fast-evolving nonsynonymous sites have lower codon usage bias. This pattern has been attributed to interference between positive selection at nonsynonymous sites and weak selection on codon usage. Here we have looked for this correlation in a much larger and less biased dataset, comprising 630 gene pairs from D. melanogaster and D. yakuba. We confirmed that there is a negative correlation between the rate of nonsynonymous substitutions (d_N) and codon bias in D. melanogaster. We then tested the interference hypothesis and other alternative explanations, including one involving gene expression. We found that d_N indeed correlates with the level of gene expression. Given that gene expression is a strong determinant of codon bias, the relationship between d_N and codon bias might be a by-product of gene expression. However, our tests show that none of the hypotheses we consider seem to explain the data fully.This article contains online supplementary material.Reviewing Editor: Dr. John Huelsenbeck     

Molecular evolution of sex-biased genes in the Drosophila ananassae subgroup

Background  

Genes with sex-biased expression often show rapid molecular evolution between species. Previous population genetic and comparative genomic studies of Drosophila melanogaster and D. simulans revealed that male-biased genes have especially high rates of adaptive evolution. To test if this is also the case for other lineages within the melanogaster group, we investigated gene expression in D. ananassae, a species that occurs in structured populations in tropical and subtropical regions. We used custom-made microarrays and published microarray data to characterize the sex-biased expression of 129 D. ananassae genes whose D. melanogaster orthologs had been classified previously as male-biased, female-biased, or unbiased in their expression and had been studied extensively at the population-genetic level. For 43 of these genes we surveyed DNA sequence polymorphism in a natural population of D. ananassae and determined divergence to the sister species D. atripex and D. phaeopleura.     

Drosophila Interspecific Hybrids Phenocopy piRNA-Pathway Mutants

The Piwi-interacting RNA (piRNA) pathway defends the germline of animals from the deleterious activity of selfish transposable elements (TEs) through small-RNA mediated silencing. Adaptation to novel invasive TEs is proposed to occur by incorporating their sequences into the piRNA pool that females produce and deposit into their eggs, which then propagates immunity against specific TEs to future generations. In support of this model, the F1 offspring of crosses between strains of the same Drosophila species sometimes suffer from germline derepression of paternally inherited TE families, caused by a failure of the maternal strain to produce the piRNAs necessary for their regulation. However, many protein components of the Drosophila piRNA pathway exhibit signatures of positive selection, suggesting that they also contribute to the evolution of host genome defense. Here we investigate piRNA pathway function and TE regulation in the F1 hybrids of interspecific crosses between D. melanogaster and D. simulans and compare them with intraspecific control crosses of D. melanogaster. We confirm previous reports showing that intraspecific crosses are characterized by derepression of paternally inherited TE families that are rare or absent from the maternal genome and piRNA pool, consistent with the role of maternally deposited piRNAs in shaping TE silencing. In contrast to the intraspecific cross, we discover that interspecific hybrids are characterized by widespread derepression of both maternally and paternally inherited TE families. Furthermore, the pattern of derepression of TE families in interspecific hybrids cannot be attributed to their paucity or absence from the piRNA pool of the maternal species. Rather, we demonstrate that interspecific hybrids closely resemble piRNA effector-protein mutants in both TE misregulation and aberrant piRNA production. We suggest that TE derepression in interspecific hybrids largely reflects adaptive divergence of piRNA pathway genes rather than species-specific differences in TE-derived piRNAs.     

EVOLUTIONARY GENETICS OF TWO SIBLING SPECIES,DROSOPHILA SIMULANS AND D. SECHELLIA

Drosophila simulans and D. sechellia are sibling species, the former cosmopolitan and the latter restricted to the Seychelles Islands. We used classical genetic analysis to measure the numbers and effects of genes responsible for reproductive isolation and morphological differences in male genitalia between these species. At least five loci are responsible for male sterility in hybrids, with the strongest effects produced by at least two genes on the X chromosome. At least three (and probably four) loci are responsible for the interspecific difference in the size of the posterior process of the male genital arch. These genetic results, as well as the pattern of morphological divergence between the species, show several parallels with the divergence between D. simulans and its other island relative, D. mauritiana. We also present the DNA sequence of a 4.5 kilobase region containing the alcohol dehydrogenase (Adh) locus of D. sechellia, and combine this with previous data to reconstruct the phylogenies of the three species and their more distant relative D. melanogaster. Both D. mauritiana and D. sechellia are very closely related to D. simulans. Although most phylogenies show the two island species to be independent offshoots of the D. simulans lineage (with D. sechellia the more recent), the branch points are too close to make this conclusion unambiguous. The genetic and evolutionary parallels between the simulans/mauritiana and the simulans/sechellia divergences may therefore represent either a striking evolutionary convergence or a close common ancestry of the island species. A comparison of Adh alleles within species shows that the divergence among them may be almost as large as among alleles from different species. We conclude that many of the nucleotide differences among these species actually represent polymorphisms within common ancestors. It may be difficult to build accurate phylogenies using only a single DNA sequence from each species.     

Structural evolution of the Drosophila 5S ribosomal genes

We compare the 5S gene structure from nine Drosophila species. New sequence data (5S genes of D. melanogaster, D. mauritiana, D. sechellia, D. yakuba, D. erecta, D. orena, and D. takahashii) and already-published data (5S genes of D. melanogaster, D. simulans, and D. teissieri) are used in these comparisons. We show that four regions within the Drosophila 5S genes display distinct rates of evolution: the coding region (120 bp), the 5-flanking region (54–55 bp), the 3-flanking region (21–22 bp), and the internal spacer (149–206 bp). Intra- and interspecific heterogeneity is due mainly to insertions and deletions of 6–17-bp oligomers. These small rearrangements could be generated by fork slippages during replication and could produce rapid sequence divergence in a limited number of steps. Correspondence to: M. Wegnez     

Regulatory Evolution of a Duplicated Heterodimer Across Species and Tissues of Allopolyploid Clawed Frogs (<Emphasis Type="Italic">Xenopus</Emphasis>)

Changes in gene expression contribute to reproductive isolation of species, adaptation, and development and may impact the genetic fate of duplicated genes. African clawed frogs (genus Xenopus) offer a useful model for examining regulatory evolution, particularly after gene duplication, because species in this genus are polyploid. Additionally, these species can produce viable hybrids, and expression divergence between coexpressed species-specific alleles in hybrids can be attributed exclusively to cis-acting mechanisms. Here we have explored expression divergence of a duplicated heterodimer composed of the recombination activating genes 1 and 2 (RAG1 and RAG2). Previous work identified a phylogenetically biased pattern of pseudogenization of RAG1 wherein one duplicate—RAG1β—was more likely to become a pseudogene than the other one—RAG1α. In this study we show that ancestral expression divergence between these duplicates could account for this. Using comparative data we demonstrate that regulatory divergence between species and between duplicated genes varies significantly across tissue types. These results have implications for understanding of variables that influence pseudogenization of duplicated genes generated by polyploidization, and for interpretation of the relative contributions of cis versus trans mechanisms to expression divergence at the cellular level. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A Genomic Comparison of Faster-Sex, Faster-X, and Faster-Male Evolution Between Drosophila melanogaster and Drosophila pseudoobscura

A genomic comparison of Drosophila melanogaster and Drosophila pseudoobscura provides a unique opportunity to investigate factors involved in sequence divergence. The chromosomal arrangements of these species include an autosomal segment in D. melanogaster which is homologous to part of the X chromosome in D. pseudoobscura. Using orthologues to calculate rates of nonsynonymous (d_N) substitutions, we found genes on the X chromosome to be significantly more diverged than those on the autosomes, but it is not true for segment 3L-XR which is autosomal in D. melanogaster (3L) and X-linked in D. pseudoobscura (XR). We also found that the median d_N values for genes having reproductive functions in either the male, the female, or both sexes are higher than those for sequences without reproductive function and even higher for sequences involved in male-specific function. These estimates of divergence for male sex-related sequences are most likely underestimates, as the very rapidly evolving reproductive genes would tend to lose homology sooner and thus not be included in the comparison of orthologues. We also noticed a high proportion of male reproductive genes among the othologous genes with the highest rates of d_N. Reproductive genes with and without an orthologue in D. pseudoobscura were compared among D. melanogaster, D. simulans, and D. yakuba and it was found that there were in fact higher rates of divergence in the group without a D. pseudoobscura orthologue. These results, from widely separated taxa, bolster the thesis that sexual system genes experience accelerated rates of change in comparison to nonsexual genes in evolution and speciation. [Reviewing Editor: Dr. Willie J. Swanson]     

High genetic differentiation between an African and a non-African strain of <Emphasis Type="Italic">Drosophila simulans</Emphasis> revealed by segregation distortion and reduced crossover frequency

Drosophila simulans strains originating from Madagascar and nearby islands in the Indian Ocean often differ from those elsewhere in the number of sex comb teeth and the degree of morphological anomaly in hybrids with D. melanogaster. Here, we report a strong segregation distortion in the F1 intercross between two D. simulans strains originating from Madagascar and the US, possibly at both the gametic and zygotic levels. Strong bias against alleles of the Madagascar strain was observed for all ten marker loci distributed over the entire second chromosome in the F1 intercross, but only a few showed a weak distortion in the isogenic backgrounds of either strains. Significant deviations of genotype frequencies from Hardy–Weinberg proportions were consistently observed for the second chromosome. By contrast, the X and third chromosomes did not show any strong segregation distortion. Crossover frequency on the second chromosome was uniformly reduced in isogenic backgrounds whereas the map lengths in the F1 intercross were comparable to or larger than that of the standard D. melanogaster map. We discuss these findings in relation to previous studies on other traits and interspecific differences between D. mauritiana, which is endemic to Mauritius Island, and D. simulans.     

Identification of One Intron Loss and Phylogenetic Evolution of Dfak Gene in the Drosophila melanogaster Species Group

Intron loss and its evolutionary significance have been noted in Drosophila. The current study provides another example of intron loss within a single-copy Dfak gene in Drosophila. By using polymerase chain reaction (PCR), we amplified about 1.3 kb fragment spanning intron 5–10, located in the position of Tyr kinase (TyK) domain of Dfak gene from Drosophila melanogaster species group, and observed size difference among the amplified DNA fragments from different species. Further sequencing analysis revealed that D. melanogaster and D. simulans deleted an about 60 bp of DNA fragment relative to other 7 Drosophila species, such as D. elegans, D. ficusphila, D. biarmipes, D. takahashii, D. jambulina, D. prostipennis and D. pseudoobscura, and the deleted fragment located precisely in the position of one intron. The data suggested that intron loss might have occurred in the Dfak gene evolutionary process of D. melanogaster and D. simulans of Drosophila melanogaster species group. In addition, the constructed phylogenetic tree based on the Dfak TyK domains clearly revealed the evolutionary relationships between subgroups of Drosophila melanogaster species group, and the intron loss identified from D. melanogaster and D. simulans provides a unique diagnostic tool for taxonomic classification of the melanogaster subgroup from other group of genus Drosophila.     

Clonal analysis in hybrids between Drosophila melanogaster and Drosophila simulans

We have analysed the viability of cellular clones induced by mitotic recombination in Drosophila melanogaster/D. simulans hybrid females during larval growth. These clones contain a portion of either melanogaster or simulans genomes in homozygosity. Analysis has been carried out for the X and the second chromosomes, as well as for the 3L chromosome arm. Clones were not found in certain structures, and in others they appeared in a very low frequency. Only in abdominal tergites was a significant number of clones observed, although their frequency was lower than in melanogaster abdomens. The bigger the portion of the genome that is homozygous, the less viable is the recombinant melano-gaster/simulans hybrid clone. The few clones that appeared may represent cases in which mitotic recombination took place in distal chromosome intervals, so that the clones contained a small portion of either melanogaster or simulans chromosomes in homozygosity. Moreover, Lhr, a gene of D. simulans that suppresses the lethality of male and female melanogaster/simulans hybrids, does not suppress the lethality of the recombinant melanogaster/simulans clones. Thus, it appears that there is not just a single gene, but at least one per tested chromosome arm (and maybe more) that cause hybrid lethality. Therefore, the two species, D. melanogaster and D. simulans, have diverged to such a degree that the absence of part of the genome of one species cannot be substituted by the corresponding part of the genome of the other, probably due to the formation of co-adapted gene complexes in both species following their divergent evolution after speciation. The disruption of those coadapted gene complexes would cause the lethality of the recombinant hybrid clones.     

A New Retroposed Gene in <Emphasis Type="Italic">Drosophila</Emphasis> Heterochromatin Detected by Microarray-Based Comparative Genomic Hybridization

A genomic pattern of new gene origination is often dependent on a genomic method that can efficiently identify a statistically adequate number of recently originated genes. The heterochromatic regions have often been viewed as genomic deserts with low coding potential and thus a low flux of new genes. However, increasing reports revealed unexpected roles of heterochromatic regions in the evolution of genes and genomes. We identified recently retroposed genes that originated in heterochromatic regions in Drosophila, by developing microarray-based comparative genomic hybridization (CGH) with multiple species. This new gene family, named Ifc-2h, originated in the common ancestor of the clade of D. simulans, D. mauritiana, and D. sechellia. The sequence features and phylogenetic distribution indicated that Ifc-2h resulted from the retroposition from its parental gene, Infertile crescent (Ifc), and integrated into heterochromatic region of common ancester of the three sibling species 2 million years ago. Expression analysis revealed that Ifc-2h had developed a new expression pattern by recruiting a putative regulatory element from its target sequence. The distribution of indel variation in Ifc-2h of D. simulans and D. mauritiana revealed a significant sequence constraint, suggesting that the Ifc-2h gene may be functional. These analyses cast fresh insight into the evolution of heterochromatin and the origin of its coding regions. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Martin Kreitman]