A rapidly evolving homeobox at the site of a hybrid sterility gene.

The homeodomain is a DNA binding motif that is usually conserved among diverse taxa. Rapidly evolving homeodomains are thus of interest because their divergence may be associated with speciation. The exact site of the Odysseus (Ods) locus of hybrid male sterility in Drosophila contains such a homeobox gene. In the past half million years, this homeodomain has experienced more amino acid substitutions than it did in the preceding 700 million years; during this period, it has also evolved faster than other parts of the protein or even the introns. Such rapid sequence divergence is driven by positive selection and may contribute to reproductive isolation.

The neutral theory in the genomic era.

A number of tests have been developed to detect positive selection at the molecular level. These tests are based on DNA polymorphism within and divergence between species. Applications of these tests have revealed a large collection of genes that have evolved under positive selection and some general insights into adaptive evolution. Recently, these tests have been applied on a genomic scale and have provided estimates of the frequency of adaptive substitutions and a critical test of the neutral theory.

Combined copy number and mutation analysis identifies oncogenic pathways associated with transformation of follicular lymphoma.

Follicular lymphoma (FL) is typically an indolent disease, but 30-40% of FL cases transform into an aggressive lymphoma (tFL) with a poor prognosis. To identify the genetic changes that drive this transformation, we sequenced the exomes of 12 cases with paired FL and tFL biopsies and identified 45 recurrently mutated genes in the FL-tFL data set and 39 in the tFL cases. We selected 496 genes of potential importance in transformation and sequenced them in 23 additional tFL cases. Integration of the mutation data with copy-number abnormality (CNA) data provided complementary information. We found recurrent mutations of miR-142, which has not been previously been reported to be mutated in FL/tFL. The genes most frequently mutated in tFL included KMT2D (MLL2), CREBBP, EZH2, BCL2 and MEF2B. Many recurrently mutated genes are involved in epigenetic regulation, the Janus-activated kinase-signal transducer and activator of transcription (STAT) or the nuclear factor-κB pathways, immune surveillance and cell cycle regulation or are TFs involved in B-cell development. Of particular interest are mutations and CNAs affecting S1P-activated pathways through S1PR1 or S1PR2, which likely regulate lymphoma cell migration and survival outside of follicles. Our custom gene enrichment panel provides high depth of coverage for the study of clonal evolution or divergence.

Inferences of species phylogeny in relation to segregation of ancient polymorphisms.

Standard formulas of gene frequency change under genetic drift are used to derive the probability of obtaining incorrect phylogenetic information for three species due to segregation of ancient polymorphisms. This probability depends upon the level of polymorphisms at the time of speciation and is generally quite high unless the two speciation events are far apart in time. If phylogenetic data from multiple loci are available, a likelihood ratio test can be used to reject the null hypothesis in favor of the best phylogeny. The appropriate null hypothesis is either a trichotomy or an alternative phylogeny, depending on the data set. The likelihood ratios required for accepting the best phylogeny are given. These ratios are obtained by exact enumeration when the number of loci is small (n less than 15) and by an asymptotic approach for larger n's. In general, more than five loci are needed to resolve the species phylogeny.

Virility Deficiency and the Sex-Ratio Trait in DROSOPHILA PSEUDOOBSCURA. I. Sperm Displacement and Sexual Selection.

The Sex-Ratio (SR) phenomenon is associated with the X chromosome of many species of Drosophila. Males carrying SR transmit predominantly sperm bearing the X chromosome. SR, therefore, has a very significant advantage in segregation. This paper provides an experimental analysis of the role of virility selection in preventing the fixation of SR. SR males are found to suffer substantial virility reduction when mated with nonvirgin females but not with virgin females. The reduced virility arises because they are weaker in sperm displacement and are discriminated against by nonvirgin females. The virility deficiency of SR males is even more severe at low temperatures.

Virility Deficiency and the Sex-Ratio Trait in DROSOPHILA PSEUDOOBSCURA. II. Multiple Mating and Overall Virility Selection.

Previous studies on fitness components of Drosophila have shown the over-whelming importance of virility selection. In this study, virility selection is further partitioned into two components-one with respect to virgin females and the other with respect to nonvirgin females. The relative importance of the two components to the overall virility selection depends on the remating tendency of females which is investigated here. A theoretical model is then proposed to estimate virility selection under the condition of frequent female remating. The model is tested experimentally. When this model is applied to the Sex-Ratio system of D. pseudoobscura, Sex-Ratio males are found to suffer substantial virility reduction. The significance of this finding to the Sex-Ratio problem is discussed.

Hitchhiking under positive Darwinian selection.

Positive selection can be inferred from its effect on linked neutral variation. In the restrictive case when there is no recombination, all linked variation is removed. If recombination is present but rare, both deterministic and stochastic models of positive selection show that linked variation hitchhikes to either low or high frequencies. While the frequency distribution of variation can be influenced by a number of evolutionary processes, an excess of derived variants at high frequency is a unique pattern produced by hitchhiking (derived refers to the nonancestral state as determined from an outgroup). We adopt a statistic, H, to measure an excess of high compared to intermediate frequency variants. Only a few high-frequency variants are needed to detect hitchhiking since not many are expected under neutrality. This is of particular utility in regions of low recombination where there is not much variation and in regions of normal or high recombination, where the hitchhiking effect can be limited to a small (<1 kb) region. Application of the H test to published surveys of Drosophila variation reveals an excess of high frequency variants that are likely to have been influenced by positive selection.

Genetics of hybrid male sterility between drosophila sibling species: a complex web of epistasis is revealed in interspecific studies.

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

An empirical test of the meiotic drive models of hybrid sterility: sex-ratio data from hybrids between Drosophila simulans and Drosophila sechellia.

Recently, there has been much discussion regarding the hypothesis that divergence of meiotic drive systems in isolated populations can generate the patterns of reproductive isolation observed in animal hybridizations. One prediction from this hypothesis is that the sex ratio of hybrids with heterospecific sex chromosomes should greatly deviate from the Mendelian expectation of 50% female. From sex-ratio data in our Drosophila hybridization studies, we find no such deviation: the sex ratio of offspring of males with introgressed heterospecific Y chromosomes with various autosomal backgrounds does not differ from that of the pure species. We also discuss other aspects of the current meiotic drive models.

Further characterization of the Odysseus locus of hybrid sterility in Drosophila: one gene is not enough.

Previously we mapped by genetical and molecular means a gene that contributes to hybrid-male sterility between Drosophila mauritiana and D. simulans to the cytological interval of 16D. In this report, we refine the mapping of this gene, Odysseus (Ods) and show that it can be delineated to a region the size of an average gene. We further demonstrate that, while Ods appears to be a discrete element, it requires other nearby gene(s) to be cointrogressed to confer full hybrid sterility effect. This observation is in agreement with the view that reproductive isolation between closely related species of Drosophila is usually caused by several genes of weak effect from the same species that interact strongly among themselves as well as with the foreign genetic background.

The genetics of reproductive isolation in the Drosophila simulans clade: X vs. autosomal effects and male vs. female effects.

A strong effect of homozygous autosomal regions on reproductive isolation was found for crosses between the species in the Drosophila simulans clade. Second chromosome regions were introgressed from D. mauritiana and D. sechellia into D. simulans and tested for their homozygous effects on hybrid male and hybrid female sterility and inviability. Most introgressions are fertile as heterozygotes, yet produce sterile male offspring when made homozygous. The density of homozygous autosomal factors contributing to hybrid male sterility is comparable to the density of X chromosome factors for this level of resolution. Female sterility was also revealed, yet the disparity between male and female levels of sterility was great, with male sterility being up to 23 times greater than female sterility. Complete hybrid inviability was also associated with some regions of the second chromosome, yet there were no strong sex differences. In conclusion, we find no evidence to support a strong X chromosome bias in the evolution of hybrid sterility or inviability but do find a very strong sex bias in the evolution of hybrid sterility. In light of these findings, we reevaluate the current models proposed to explain the genetic pattern of reproductive isolation.

The broom of the sorcerer's apprentice: the fine structure of a chromosomal region causing reproductive isolation between two sibling species of Drosophila.

How many genes contribute to reproductive isolation between closely related species? We determined the number of genes located in the 9D-12B region of the Drosophila mauritiana X chromosome that cause hybrid male sterility in a D. simulans background. Previous low resolution studies suggested that a single hybrid sterility factor was associated with this region. In this study, by taking advantage of a cluster of visible and DNA markers, we identified three D. mauritiana factors in this region and then subjected one of them to detailed analysis. This factor again turned out to be comprised of three factors; one of which, mapped to within 200 kb, may in fact be two factors. The title refers to this exercise of splitting sterile introgressions into ever smaller ones, each of which retains partial or full sterility effects. In a region representing a mere 3% of the Drosophila genome, no fewer than six loci of hybrid sterility were identified between two sibling species that have not shown clear divergence at the molecular level. These results suggest that levels of genetic divergence between closely related species may be quite high for functionally important traits even when the opposite is true for randomly chosen loci.

Rapid evolution of a coadapted gene complex: evidence from the Segregation Distorter (SD) system of meiotic drive in Drosophila melanogaster.

Segregation Distorter (SD) is a system of meiotic drive found in natural populations of Drosophila melanogaster. Males heterozygous for an SD second chromosome and a normal homologue (SD+) produce predominantly SD-bearing sperm. The coadapted gene complex responsible for this transmission advantage spans the second chromosome centromere, consisting of three major and several minor interacting loci. To investigate the evolutionary history of this system, we surveyed levels of polymorphism and divergence at six genes that together encompass this pericentromeric region and span seven map units. Interestingly, there was no discernible divergence between SD and SD+ chromosomes for any of these molecular markers. Furthermore, SD chromosomes harbored much less polymorphism than did SD+ chromosomes. The results suggest that the SD-system evolved recently, swept to appreciable frequencies worldwide, and carried with it the entire second chromosome centromeric region (roughly 10% of the genome). Despite its well-documented genetic complexity, this coadapted system appears to have evolved on a time scale that is much shorter than can be gauged using nucleotide substitution data. Finally, the large genomic region hitchhiking with SD indicates that a multilocus, epistatically selected system could affect the levels of DNA polymorphism observed in regions of reduced recombination.

Evidence for Extensive Genetic Differentiation between the Sex-Ratio and the Standard Arrangement of DROSOPHILA PSEUDOOBSCURA and D. PERSIMILIS and Identification of Hybrid Sterility Factors.

This study deals with sex-ratio genes tightly linked within the Sex-Ratio inversion. By taking advantage of the fact that the Sex-Ratio chromosome of Drosophila persimilis [SR(B)] is homosequential to the Standard chromosome of D. pseudoobscura [ST(A)], we carried out two reciprocal introgression experiments. Individual segments of SR(B) or ST(A) were introgressed into the genome of D. pseudoobscura or D. persimilis, respectively. Males possessing a hybrid SR(B)-ST(A) X chromosome and a genetic background derived from either of the two species were tested for fertility and sex-ratio expression.-It was found that, in terms of the meiotic drive genes, the Sex-Ratio chromosome differs extensively from the Standard chromosome. Because recombinations of these genes result in a complete loss of sex-ratio expression, this finding lends strong support to the hypothesis of gene coadaptation. Coadaptation, in this context, is the advantage of being transmitted preferentially. In light of this finding, the evolution of the sex-ratio system in these two sibling species is discussed.-Introgression experiments also yielded information about hybrid sterility. With reciprocal introgression, sterility interactions were found to be "asymmetric." The asymmetry is fully expected from the viewpoint of evolution of postmating reproductive isolation.

Modeling linkage disequilibrium between a polymorphic marker locus and a locus affecting complex dichotomous traits in natural populations.

Linkage disequilibrium is an important topic in evolutionary and population genetics. An issue yet to be settled is the theory required to extend the linkage disequilibrium analysis to complex traits. In this study, we present theoretical analysis and methods for detecting or estimating linkage disequilibrium (LD) between a polymorphic marker locus and any one of the loci affecting a complex dichotomous trait on the basis of samples randomly or selectively collected from natural populations. Statistical properties of these methods were investigated and their powers were compared analytically or by use of Monte Carlo simulations. The results show that the disequilibrium may be detected with a power of 80% by using phenotypic records and marker genotype when both the trait and marker variants are common (30%) and the LD is relatively high (40-100% of the theoretical maximum). The maximum-likelihood approach provides accurate estimates of the model parameters as well as detection of linkage disequilibrium. The likelihood method is preferred for its higher power and reliability in parameter estimation. The approaches developed in this article are also compared to those for analyzing a continuously distributed quantitative trait. It is shown that a larger sample size is required for the dichotomous trait model to obtain the same level of power in detecting linkage disequilibrium as the continuous trait analysis. Potential use of these estimates in mapping the trait locus is also discussed.

Models of evolution of reproductive isolation.

Mathematical models are presented for the evolution of postmating and premating reproductive isolation. In the case of postmating isolation it is assumed that hybrid sterility or inviability is caused by incompatibility of alleles at one or two loci, and evolution of reproductive isolation occurs by random fixation of different incompatibility alleles in different populations. Mutations are assumed to occur following either the stepwise mutation model or the infinite-allele model. Computer simulations by using Itô's stochastic differential equations have shown that in the model used the reproductive isolation mechanism evolves faster in small populations than in large populations when the mutation rate remains the same. In populations of a given size it evolves faster when the number of loci involved is large than when this is small. In general, however, evolution of isolation mechanisms is a very slow process, and it would take thousands to millions of generations if the mutation rate is of the order of 10(-5) per generation. Since gene substitution occurs as a stochastic process, the time required for the establishment of reproductive isolation has a large variance. Although the average time of evolution of isolation mechanisms is very long, substitution of incompatibility genes in a population occurs rather quickly once it starts. The intrapopulational fertility or viability is always very high. In the model of premating isolation it is assumed that mating preference or compatibility is determined by male- and female-limited characters, each of which is controlled by a single locus with multiple alleles, and mating occurs only when the male and female characters are compatible with each other. Computer simulations have shown that the dynamics of evolution of premating isolation mechanism is very similar to that of postmating isolation mechanism, and the mean and variance of the time required for establishment of premating isolation are very large. Theoretical predictions obtained from the present study about the speed of evolution of reproductive isolation are consistent with empirical data available from vertebrate organisms.

Characterization of two Segregation distorter revertants: evidence that the tandem duplication is necessary for Sd activity in Drosophila melanogaster.

Segregation Distorter (SD) is a naturally occurring system of meiotic drive in Drosophila melanogaster. Males heterozygous for an SD second chromosome and a normal homolog (SD+) transmit predominantly SD-bearing sperm. To accomplish this, the Segregation distorter (Sd) locus induces the dysfunction of those spermatids that receive the SD+ chromosome. Recently, P. A. Powers and B. Ganetzky isolated overlapping DNA clones spanning the region of DNA known to contain the Sd gene and identified a 5-kb tandem duplication that is present on all SD chromosomes examined, but is apparently absent from wild-type chromosomes. Here we report a molecular analysis of two spontaneous revertants from an Australian SD chromosome (SD-Arm28). Both of these revertants have lost the 5-kb tandem duplication along with the ability to distort transmission; the critical observation, however, is that they retain the DNA haplotype in the flanking regions (both proximally and distally) that is characteristic of the original SD-Arm28. We propose unequal sister chromatid exchange between the tandem repeats as the only plausible explanation for loss of a repeat while retaining flanking markers. This provides direct evidence that the tandem duplication is indeed necessary for the Sd phenotype. Further, we examined testes-specific levels of both RNA and protein for the nearby Topoisomerase 2 gene. Neither revealed a consistent difference between SD and SD+ strains. We also measured testes-specific levels of RNA using the tandem duplication itself as probe. Our results suggest that there is strong up-regulation of one or several 2.0-2.3-kb transcripts from the duplicated region in the testes of an SD strain.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for complex genic interactions between conspecific chromosomes underlying hybrid female sterility in the Drosophila simulans clade.

F1 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 F2 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.

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.

Population genetics of tandem repeats in centromeric heterochromatin: unequal crossing over and chromosomal divergence at the Responder locus of Drosophila melanogaster.

The Responder (Rsp) locus in Drosophila melanogaster is the target locus of segregation distortion and is known to be comprised of a tandem array of 120-bp repetitive sequences. In this study, we first determined the large scale molecular structure of the Rsp locus, which extends over a region of 600 kb on the standard sensitive (cn bw) chromosome. Within the region, small Rsp repeat arrays are interspersed with non-Rsp sequences and account for 10-20% of the total sequences. We isolated and sequenced 32 Rsp clones from three different chromosomes. The main results are: (1) Rsp repeats isolated from the same chromosome are not more similar than those from different chromosomes. This implies either that there are more homologous exchanges at the Rsp locus than expected or, alternatively, that the second chromosomes of D. melanogaster have diverged from one another more recently at the centromeric heterochromatin than at the nearby euchromatin. (2) The repeats usually have a dimeric structure with an average difference of 16% between the left and right halves. The differences allow us to easily identify the products of unequal exchanges. Despite the large differences between the two halves, exchanges have occurred frequently and the majority of them fall within a 29-bp interval of identity between the two halves. Our data thus support the suggestion that recombination depends on short stretches of complete identity rather than long stretches of general homology. (3) Frequent unequal crossover events obscure the phylogenetic relationships between repeats; therefore, different parts of any single repeat could often have different phylogenetic histories. The high rate of unequal crossing over may also help explain the evolutionary dynamics of the Rsp locus.