Ana Llopart

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.

GENETICS OF A DIFFERENCE IN PIGMENTATION BETWEEN DROSOPHILA YAKUBA AND DROSOPHILA SANTOMEA

Abstract Drosophila yakuba is a species widespread in Africa, whereas D. santomea, its newly discovered sister species, is endemic to the volcanic island of São Tomé in the Gulf of Guinea. Drosophila santomea probably formed after colonization of the island by its common ancestor with D. yakuba. The two species differ strikingly in pigmentation: D. santomea, unlike the other eight species in the D. melanogaster subgroup, almost completely lacks dark abdominal pigmentation. D. yakuba shows the sexually dimorphic pigmentation typical of the group: both sexes have melanic patterns on the abdomen, but males are much darker than females. A genetic analysis of this species difference using morphological markers shows that the X chromosome accounts for nearly 90% of the species difference in the area of abdomen that is pigmented and that at least three genes (one on each major chromosome) are involved in each sex. The order of chromosome effects on pigmentation area are the same in males and females, suggesting that loss of pigmentation in D. santomea may have involved the same genes in both sexes. Further genetic analysis of the interspecific difference between males in pigmentation area and intensity using molecular markers shows that at least five genes are responsible, with no single locus having an overwhelming effect on the trait. The species difference is thus oligogenic or polygenic. Different chromosomal regions from each of the two species influenced pigmentation in the same direction, suggesting that the species difference (at least in males) is due to natural or sexual selection and not genetic drift. Measurements of sexual isolation between the species in both light and dark conditions show no difference, suggesting that the pigmentation difference is not an important cue for interspecific mate discrimination. Using DNA sequence differences in nine noncoding regions, we estimate that D. santomea and D. yakuba diverged about 400,000 years ago, a time similar to the divergences between two other well-studied pair of species in the subgroup, both of which also involved island colonization.

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.

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 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).

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.

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.

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.

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.

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.