Meiotic, genomic and evolutionary properties of crossover distribution in Drosophila yakuba.

The number and location of crossovers across genomes are highly regulated during meiosis, yet the key components controlling them are fast evolving, hindering our understanding of the mechanistic causes and evolutionary consequences of changes in crossover rates. Drosophila melanogaster has been a model species to study meiosis for more than a century, with an available high-resolution crossover map that is, nonetheless, missing for closely related species, thus preventing evolutionary context. Here, we applied a novel and highly efficient approach to generate whole-genome high-resolution crossover maps in D. yakuba to tackle multiple questions that benefit from being addressed collectively within an appropriate phylogenetic framework, in our case the D. melanogaster species subgroup. The genotyping of more than 1,600 individual meiotic events allowed us to identify several key distinct properties relative to D. melanogaster. We show that D. yakuba, in addition to higher crossover rates than D. melanogaster, has a stronger centromere effect and crossover assurance than any Drosophila species analyzed to date. We also report the presence of an active crossover-associated meiotic drive mechanism for the X chromosome that results in the preferential inclusion in oocytes of chromatids with crossovers. Our evolutionary and genomic analyses suggest that the genome-wide landscape of crossover rates in D. yakuba has been fairly stable and captures a significant signal of the ancestral crossover landscape for the whole D. melanogaster subgroup, even informative for the D. melanogaster lineage. Contemporary crossover rates in D. melanogaster, on the other hand, do not recapitulate ancestral crossovers landscapes. As a result, the temporal stability of crossover landscapes observed in D. yakuba makes this species an ideal system for applying population genetic models of selection and linkage, given that these models assume temporal constancy in linkage effects. Our studies emphasize the importance of generating multiple high-resolution crossover rate maps within a coherent phylogenetic context to broaden our understanding of crossover control during meiosis and to improve studies on the evolutionary consequences of variable crossover rates across genomes and time.

Support for the Dominance Theory in Drosophila Transcriptomes.

Interactions among divergent elements of transcriptional networks from different species can lead to misexpression in hybrids through regulatory incompatibilities, some with the potential to generate sterility. While the possible contribution of faster-male evolution to this misexpression has been explored, the role of the hemizygous X chromosome (i.e., the dominance theory for transcriptomes) remains yet to be determined. Here, we study genome-wide patterns of gene expression in females and males of Drosophila yakuba, Drosophila santomea and their hybrids. We used attached-X stocks to specifically test the dominance theory, and we uncovered a significant contribution of recessive alleles on the X chromosome to hybrid misexpression. Our analyses also suggest a contribution of weakly deleterious regulatory mutations to gene expression divergence in genes with sex-biased expression, but only in the sex toward which the expression is biased (e.g, genes with female-biased expression when analyzed in females). In the opposite sex, we found stronger selective constraints on gene expression divergence. Although genes with a high degree of male-biased expression show a clear signal of faster-X evolution of gene expression, we also detected slower-X evolution in other gene classes (e.g., female-biased genes). This slower-X effect is mediated by significant decreases in cis- and trans-regulatory divergence. The distinct behavior of X-linked genes with a high degree of male-biased expression is consistent with these genes experiencing a higher incidence of positively selected regulatory mutations than their autosomal counterparts.

Faster-X evolution of gene expression is driven by recessive adaptive cis-regulatory variation in Drosophila.

The hemizygosity of the X (Z) chromosome fully exposes the fitness effects of mutations on that chromosome and has evolutionary consequences on the relative rates of evolution of X and autosomes. Specifically, several population genetics models predict increased rates of evolution in X-linked loci relative to autosomal loci. This prediction of faster-X evolution has been evaluated and confirmed for both protein coding sequences and gene expression. In the case of faster-X evolution for gene expression divergence, it is often assumed that variation in 5' noncoding sequences is associated with variation in transcript abundance between species but a formal, genomewide test of this hypothesis is still missing. Here, I use whole genome sequence data in Drosophila yakuba and D. santomea to evaluate this hypothesis and report positive correlations between sequence divergence at 5' noncoding sequences and gene expression divergence. I also examine polymorphism and divergence in 9,279 noncoding sequences located at the 5' end of annotated genes and detected multiple signals of positive selection. Notably, I used the traditional synonymous sites as neutral reference to test for adaptive evolution, but I also used bases 8-30 of introns <65 bp, which have been proposed to be a better neutral choice. X-linked genes with high degree of male-biased expression show the most extreme adaptive pattern at 5' noncoding regions, in agreement with faster-X evolution for gene expression divergence and a higher incidence of positively selected recessive mutations.

Widespread Positive Selection Drives Differentiation of Centromeric Proteins in the Drosophila melanogaster subgroup.

Rapid evolution of centromeric satellite repeats is thought to cause compensatory amino acid evolution in interacting centromere-associated kinetochore proteins. Cid, a protein that mediates kinetochore/centromere interactions, displays particularly high amino acid turnover. Rapid evolution of both Cid and centromeric satellite repeats led us to hypothesize that the apparent compensatory evolution may extend to interacting partners in the Condensin I complex (i.e., SMC2, SMC4, Cap-H, Cap-D2, and Cap-G) and HP1s. Missense mutations in these proteins often result in improper centromere formation and aberrant chromosome segregation, thus selection for maintained function and coevolution among proteins of the complex is likely strong. Here, we report evidence of rapid evolution and recurrent positive selection in seven centromere-associated proteins in species of the Drosophila melanogaster subgroup, and further postulate that positive selection on these proteins could be a result of centromere drive and compensatory changes, with kinetochore proteins competing for optimal spindle attachment.

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

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

Parallel faster-X evolution of gene expression and protein sequences in Drosophila: beyond differences in expression properties and protein interactions.

Population genetics models predict that the X (or Z) chromosome will evolve at faster rates than the autosomes in XY (or ZW) systems. Studies of molecular evolution using large datasets in multiple species have provided evidence supporting this faster-X effect in protein-coding sequences and, more recently, in transcriptomes. However, X-linked and autosomal genes differ significantly in important properties besides hemizygosity in males, including gene expression levels, tissue specificity in gene expression, and the number of interactions in which they are involved (i.e., protein-protein or DNA-protein interactions). Most important, these properties are known to correlate with rates of evolution, which raises the question of whether differences between the X chromosome and autosomes in gene properties, rather than hemizygosity, are sufficient to explain faster-X evolution. Here I investigate this possibility using whole genome sequences and transcriptomes of Drosophila yakuba and D. santomea and show that this is not the case. Additional factors are needed to account for faster-X evolution of both gene expression and protein-coding sequences beyond differences in gene properties, likely a higher incidence of positive selection in combination with the accumulation of weakly deleterious mutations.

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

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

Introgression in the Drosophila subobscura--D. Madeirensis sister species: evidence of gene flow in nuclear genes despite mitochondrial differentiation.

Species hybridization, and thus the potential for gene flow, was once viewed as reproductive mistake. However, recent analysis based on large datasets and newly developed models suggest that gene exchange is not as rare as originally suspected. To investigate the history and speciation of the closely related species Drosophila subobscura, D. madeirensis, and D. guanche, we obtained polymorphism and divergence data for 26 regions throughout the genome, including the Y chromosome and mitochondrial DNA. We found that the D. subobscura X/autosome ratio of silent nucleotide diversity is significantly smaller than the 0.75 expected under neutrality. This pattern, if held genomewide, may reflect a faster accumulation of beneficial mutations on the X chromosome than on autosomes. We also detected evidence of gene flow in autosomal regions, while sex chromosomes remain distinct. This is consistent with the large X effect on hybrid male sterility seen in this system and the presence of two X chromosome inversions fixed between species. Overall, our data conform to chromosomal speciation models in which rearrangements are proposed to serve as gene flow barriers. Contrary to other observations in Drosophila, the mitochondrial genome appears resilient to gene flow in the presence of nuclear exchange.

The rapid evolution of X-linked male-biased gene expression and the large-X effect in Drosophila yakuba, D. santomea, and their hybrids.

The X chromosome has a large effect on hybrid dysfunction, particularly on hybrid male sterility. Although the evidence for this so-called large-X effect is clear, its molecular causes are not yet fully understood. One possibility is that, under certain conditions, evolution proceeds faster in X-linked than in autosomal loci (i.e., faster-X effect) due to both natural selection and their hemizygosity in males, an effect that is expected to be greatest in genes with male-biased expression. Here, I study genome-wide variation in transcript abundance between Drosophila yakuba and D. santomea, within these species and in their hybrid males to evaluate both the faster-X and large-X effects at the level of expression. I find that in X-linked male-biased genes (MBGs) expression evolves faster than in their autosomal counterparts, an effect that is accompanied by a unique reduction in expression polymorphism. This suggests that Darwinian selection is driving expression differences between species, likely enhanced by the hemizygosity of the X chromosome in males. Despite the recent split of the two sister species under study, abundant changes in both cis- and trans-regulatory elements underlie expression divergence in the majority of the genes analyzed, with significant differences in allelic ratios of transcript abundance between the two reciprocal F(1) hybrid males. Cis-trans coevolution at molecular level, evolved shortly after populations become isolated, may therefore contribute to explain the breakdown of the regulation of gene expression in hybrid males. Additionally, the X chromosome plays a large role in this hybrid male misexpression, which affects not only MBG but also, to a lesser degree, nonsex-biased genes. Interestingly, hybrid male misexpression is concentrated mostly in autosomal genes, likely facilitated by the rapid evolution of sex-linked trans-acting factors. I suggest that the faster evolution of X-linked MBGs, at both protein and expression levels, contributes to explain the large effect of the X chromosome on hybrid male sterility, likely mediating widespread autosomal misexpression through the preferential recognition of cis-regulatory elements by conspecific trans-acting factors (i.e., cis-trans conspecific recognition).

Recurrent events of positive selection in independent Drosophila lineages at the spermatogenesis gene roughex.

Our understanding of the role of positive selection in the evolution of genes with male-biased expression can be hindered by two observations. First, male-biased genes tend to be overrepresented among lineage-specific genes. Second, novel genes are prone to experience bursts of adaptive evolution shortly after their formation. A thorough study of the forces acting on male-biased genes therefore would benefit from phylogenywide analyses that could distinguish evolutionary trends associated with gene formation and later events, while at the same time tackling the interesting question of whether adaptive evolution is indeed idiosyncratic. Here we investigate the roughex (rux) gene, a dose-dependent regulator of Drosophila spermatogenesis with a C-terminal domain responsible for nuclear localization that shows a distinct amino acid sequence in the melanogaster subgroup. We collected polymorphism and divergence data in eight populations of six Drosophila species, for a total of 99 rux sequences, to study rates and patterns of evolution at this male-biased gene. Our results from two phylogeny-based methods (PAML and HyPhy) as well as from population genetics analyses (McDonald-Kreitman-based tests) indicate that amino acid replacements have contributed disproportionately to divergence, consistent with adaptive evolution at the Rux protein. Analyses based on extant variation show also the signature of recent selective sweeps in several of the populations surveyed. Most important, we detect the significant and consistent signature of positive selection in several independent Drosophila lineages, which evidences recurrent and concurrent events of adaptive evolution after rux formation.

A new test for selection applied to codon usage in Drosophila simulans and D. mauritiana.

In many organisms, synonymous codon usage is biased by a history of natural selection. However, codon bias, itself, does not indicate that selection is ongoing; it may be a vestige of past selection. Simple statistical tests have been devised to infer ongoing selection on codon usage by comparing the derived state frequency spectra at polymorphic sites segregating either derived preferred codons or derived unpreferred codons; if selection is effective, the frequency of derived states should be higher in the former. We propose a new test that uses the inferred degree of preference, essentially calculating the correlation of derived state frequency and the difference in preference between the derived and the ancestral states; the correlation should be positive if selection is effective. When implementing the test, derived and ancestral states can be assigned by parsimony or on the basis of relative probability. In either case, statistical significance is estimated by a simple permutation test. We explored the statistical power of the test by sampling polymorphism data from 14 loci in 16 strains of D. simulans, finding that the test retains 80% power even when quite a few of the data are discarded. The power of the test likely reflects better use of multiple features of the data, combining population frequencies of polymorphic variants and quantitative estimates of codon preferences. We also applied this novel test to 14 newly sequenced loci in five strains of D. mauritiana, showing for the first time ongoing selection on codon usage in this species.

The genetic basis of prezygotic reproductive isolation between Drosophila santomea and D. yakuba due to mating preference.

Sexual isolating mechanisms that act before fertilization are often considered the most important genetic barriers leading to speciation in animals. While progress has been made toward understanding the genetic basis of the postzygotic isolating mechanisms of hybrid sterility and inviability, little is known about the genetic basis of prezygotic sexual isolation. Here, we map quantitative trait loci (QTL) contributing to prezygotic reproductive isolation between the sibling species Drosophila santomea and D. yakuba. We mapped at least three QTL affecting discrimination of D. santomea females against D. yakuba males: one X-linked and one autosomal QTL affected the likelihood of copulation, and a second X chromosome QTL affected copulation latency. Three autosomal QTL also affected mating success of D. yakuba males with D. santomea. No epistasis was detected between QTL affecting sexual isolation. The QTL do not overlap between males and females and are not disproportionately concentrated on the X chromosome. There was some overlap in map locations of QTL affecting sexual isolation between D. santomea and D. yakuba with QTL affecting sexual isolation between D. simulans and D. mauritiana and with QTL affecting differences in pigmentation between D. santomea and D. yakuba. Future high-resolution mapping and, ultimately, positional cloning, will reveal whether these traits do indeed have a common genetic basis.

The genetic basis of postzygotic reproductive isolation between Drosophila santomea and D. yakuba due to hybrid male sterility.

A major unresolved challenge of evolutionary biology is to determine the nature of the allelic variants of "speciation genes": those alleles whose interaction produces inviable or infertile interspecific hybrids but does not reduce fitness in pure species. Here we map quantitative trait loci (QTL) affecting fertility of male hybrids between D. yakuba and its recently discovered sibling species, D. santomea. We mapped three to four X chromosome QTL and two autosomal QTL with large effects on the reduced fertility of D. yakuba and D. santomea backcross males. We observed epistasis between the X-linked QTL and also between the X and autosomal QTL. The X chromosome had a disproportionately large effect on hybrid sterility in both reciprocal backcross hybrids. However, the genetics of hybrid sterility differ between D. yakuba and D. santomea backcross males, both in terms of the magnitude of main effects and in the epistatic interactions. The QTL affecting hybrid fertility did not colocalize with QTL affecting sexual isolation in this species pair, but did colocalize with QTL affecting the marked difference in pigmentation between D. yakuba and D. santomea. These results provide the basis for future high-resolution mapping and ultimately, molecular cloning, of the interacting genes that contribute to hybrid sterility.

Quantitative trait loci affecting the difference in pigmentation between Drosophila yakuba and D. santomea.

Using quantitative trait locus (QTL) mapping, we studied the genetic basis of the difference in pigmentation between two sister species of Drosophila: Drosophila yakuba, which, like other members of the D. melanogaster subgroup, shows heavy black pigmentation on the abdomen of males and females, and D. santomea, an endemic to the African island of São Tomé, which has virtually no pigmentation. Here we mapped four QTL with large effects on this interspecific difference in pigmentation: two on the X chromosome and one each on the second and third chromosomes. The same four QTL were detected in male hybrids in the backcrosses to both D. santomea and D. yakuba and in the female D. yakuba backcross hybrids. All four QTL exhibited strong epistatic interactions in male backcross hybrids, but only one pair of QTL interacted in females from the backcross to D. yabuka. All QTL from each species affected pigmentation in the same direction, consistent with adaptive evolution driven by directional natural selection. The regions delimited by the QTL included many positional candidate loci in the pigmentation pathway, including genes affecting catecholamine biosynthesis, melanization of the cuticle, and many additional pleiotropic effects.

Multilocus analysis of introgression between two sympatric sister species of Drosophila: Drosophila yakuba and D. santomea.

Drosophila yakuba is widely distributed in sub-Saharan Africa, while D. santomea is endemic to the volcanic island of São Tomé in the Atlantic Ocean, 280 km west of Gabon. On São Tomé, D. yakuba is found mainly in open lowland forests, and D. santomea is restricted to the wet misty forests at higher elevations. At intermediate elevations, the species form a hybrid zone where hybrids occur at a frequency of approximately 1%. To determine the extent of gene flow between these species we studied polymorphism and divergence patterns in 29 regions distributed throughout the genome, including mtDNA and three genes on the Y chromosome. This multilocus approach, together with the comparison to the two allopatric species D. mauritiana and D. sechellia, allowed us to distinguish between forces that should affect all genes and forces that should act on some genes (e.g., introgression). Our results show that D. yakuba mtDNA has replaced that of D. santomea and that there is also significant introgression for two nuclear genes, yellow and salr. The majority of genes, however, has remained distinct. These two species therefore do not form a "hybrid swarm" in which much of the genome shows substantial introgression while disruptive selection maintains distinctness for only a few traits (e.g., pigmentation and male genitalia).

An anomalous hybrid zone in Drosophila.

Despite the genetic tractability of many of Drosophila species, the genus has few examples of the "classic" type of hybrid zone, in which the ranges of two species overlap with a gradual transition from one species to another through an area where hybrids are produced. Here we describe a classic hybrid zone in Drosophila that involves two sister species, Drosophila yakuba and D. santomea, on the island of São Tomé. Our transect of this zone has yielded several surprising and anomalous findings. First, we detected the presence of an additional hybrid zone largely outside the range of both parental species. This phenomenon is, to our knowledge, unique among animals. Second, the genetic analysis using diagnostic molecular markers of the flies collected in this anomalous hybrid zone indicates that nearly all hybrid males are F1s that carry the D. santomea X chromosome. This F1 genotype is much more difficult to produce in the laboratory compared to the genotype from the reciprocal cross, showing that sexual isolation as seen in the laboratory is insufficient to explain the genotypes of hybrids found in the wild. Third, there is a puzzling absence of hybrid females. We suggest several tentative explanations for the anomalies associated with this hybrid zone, but for the present they remain a mystery.

Genetic studies of two sister species in the Drosophila melanogaster subgroup, D. yakuba and D. santomea.

We performed genetic analysis of hybrid sterility and of one morphological difference (sex-comb tooth number) on D. yakuba and D. santomea, the former species widespread in Africa and the latter endemic to the oceanic island of São Tomé, on which there is a hybrid zone. The sterility of hybrid males is due to at least three genes on the X chromosome and at least one on the Y, with the cytoplasm and large sections of the autosomes having no effect. F1 hybrid females carrying two X chromosomes from either species are perfectly fertile despite their genetic similarity to completely sterile F1 hybrid males. This implies that the appearance of Haldane's rule in this cross is at least partially due to the faster accumulation of genes causing male than female sterility. The larger effects of the X and Y chromosomes than of the autosomes, however, also suggest that the genes causing male sterility are recessive in hybrids. Some female sterility is also seen in interspecific crosses, but this does not occur between all strains. This is seen in pure-species females inseminated by heterospecific males (probably reflecting incompatibility between the sperm of one species and the female reproductive tract of the other) as well as in inseminated F1 and backcross females, probably reflecting genetically based incompatibilities in hybrids that affect the reproductive system. The latter 'innate' sterility appears to involve deleterious interactions between D. santomea chromosomes and D. yakuba cytoplasm. The difference in male sex-comb tooth number appears to involve fairly large effects of the X chromosome. We discuss the striking evolutionary parallels in the genetic basis of sterility, in the nature of sexual isolation, and in morphological differences between the D. santomea/D. yakuba divergence and two other speciation events in the D. melanogaster subgroup involving island colonization.

Genetics of a difference in pigmentation between Drosophila yakuba and Drosophila santomea.

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.

Intron presence-absence polymorphism in Drosophila driven by positive Darwinian selection.

Comparisons of intron-exon structures between homologous genes in different eukaryotic species have revealed substantial variation in the number of introns. These observations imply that, in each case, an intron presence-absence polymorphism must have existed in the past. Such a polymorphism, created by a recent intron-loss mutation, is reported here in a eukaryotic organism. This gene structure, detected in the jingwei (jgw) gene, segregates at high frequency (77%) in natural populations of Drosophila teissieri and is associated with a marked change in mRNA levels. Furthermore, the intron loss does not result from a mRNA-mediated mechanism as is usually proposed, but from a partial deletion at the DNA level that also results in the addition of four new amino acids to the JGW protein. Population genetic analyses of the pattern of nucleotide variation surrounding the intron polymorphism indicate the action of positive Darwinian selection on the intron-absent variant. Forward simulations suggest that the intensity of this selection is weak to moderate, roughly equal to the selection intensity on most replacement mutations in Drosophila.