Department of Biology II, Section of Evolutionary Biology, University of Munich (LMU), Munich, Germany
Correspondence: E-mail: parsch{at}zi.biologie.uni-muenchen.de.
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Abstract |
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Key Words: codon bias comparative genomics gene expression sexual selection
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Introduction |
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Studies of interspecific hybridization have also suggested the rapid evolution of male reproductive characters. Haldane (1922) noted a common pattern regarding the viability and fertility of species hybrids. His observation, known as Haldane's Rule, was that when hybrid offspring of only one sex are either inviable or infertile, it is most often the heterogametic sex. In many taxa, such as mammals and Drosophila, the males are heterogametic (XY), and, thus, hybrid male offspring are more prone to be inviable or sterile. Two major hypotheses have been proposed to explain Haldane's rule. The first hypothesis, known as the "dominance" hypothesis posits that hybrid incompatibilities are often recessive and, thus, are only observed in the sex with hemizygous sex chromosomes (Turelli and Orr 1995). The second hypothesis is known as "faster male evolution" and posits that genes involved in male reproduction evolve faster than genes involved in female reproduction or genes with nonreproductive function (Wu and Davis 1993). This hypothesis can only completely explain Haldane's rule for taxa in which the males are heterogametic and is expected to apply primarily to hybrid sterility, not inviability. However, the two hypotheses are not mutually exclusive, and it is likely that both faster male evolution and dominance play a role in hybrid breakdown (Presgraves and Orr 1998). Faster male evolution may also explain the overwhelming preponderance of male sterility factors relative to inviability factors that have been identified from Drosophila hybridizations (Wu and Davis 1993; True, Weir, and Laurie 1996; Tao et al. 2003).
Recent studies have indicated that sex-related genes show increased rates of evolution in their protein/DNA sequences, and it has been suggested that sexual selection affects the evolution of a broad range of genes with reproductive functions (see reviews by Civetta and Singh [1999], Singh and Kulathinal [2000], and Swanson and Vacquier [2002]). In Drosophila, the rapid evolution of reproductive proteins is suggested by the relatively large interspecific differences in migration pattern observed for these proteins on two-dimensional electrophoresis gels (Coulthart and Singh 1988; Civetta and Singh 1995) and by the elevated rate of amino acid substitution between D. melanogaster and D. simulans observed for a large number of male-specific accessory gland proteins (Swanson et al. 2001). In addition, a number of male-specific genes showing evidence for rapid evolution caused by positive selection have been identified, including Acp26Aa (Tsaur and Wu 1997; Aguadé 1998; Tsaur, Ting, and Wu 1998), OdsH (Ting et al. 1998), Sdic (Nurminsky et al. 1998; Nurminsky et al. 2001), Dntf-2r (Betrán and Long 2003), and jan-ocn (Parsch et al. 2001; Parsch, Meiklejohn, and Hartl 2001). Recently, observations of faster male evolution have been extended to the level of gene expression (Meiklejohn et al. 2003; Ranz et al. 2003). These studies used competitive cDNA microarray hybridizations to demonstrate that genes with male-biased expression show greater expression differences both within and between species than do either female-biased or unbiased genes.
In this article, we use data from published cDNA microarray experiments to identify Drosophila genes showing either male bias or female bias in their expression. We then investigate the rates of evolution of these genes (using the ratio of nonsynonymous/synonymous substitution rates, dN/dS) among Drosophila species and compare them with a collection of control genes that show no sex bias in their expression. Our results indicate that male-biased genes have a significantly higher rate of evolution than both female-biased and unbiased genes. A similar pattern is observed for the evolution of highly sex-biased genes between D. melanogaster and D. pseudoobscura, with male-biased genes showing significantly greater divergence between these two species than do either female-biased or unbiased genes. Polymorphism and divergence data suggest that these differences are caused by increased positive selection on male-biased genes.
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Materials and Methods |
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Comparative sequence data for 81 orthologous genes from D. melanogaster, D. erecta, D. pseudoobscura, D. willistoni, and D. virilis (Bergman et al. 2002) were downloaded from the Berkeley Drosophila Genome Project Web site (http://www.fruitfly.org/comparative/index.html). These data come from sequenced fosmid clones (40 kb each) corresponding to the D. melanogaster genomic regions containing the apterous, even-skipped, fushi tarazu, twist, and Rhodopsin 1, 2, 3, and 4 genes. Of the 81 genes, 50 could be classified as male biased, female biased, or unbiased based on the microarray data (table 1). For these genes, values of dN, dS, and dN/dS were calculated for all pairwise comparisons of species using PAML (codeml runmode2). For genes with sequences available from three or more species, dN/dS was also calculated for each gene using all available sequences and assuming a constant dN/dS over all branches of the phylogenetic tree (codeml runmode 0, model 0). In addition, we applied the "free-ratio" model (Yang 1998) that allows dN/dS to vary over all branches of the tree (codeml runmode 0, model 1) and compared the likelihood ratio of the two models using a
2 test with the degrees of freedom equal to the difference in parameter number (i.e., the number of branches in the tree minus 1). For these analyses, the phylogenetic relationship of Drosophila species given in Bergman et al. (2002) was used.
Comparison of Highly Sex-Biased Genes Between Drosophila Genomes
To investigate the evolutionary rates of genes showing the strongest sex bias in expression, we selected the 50 genes with the highest and lowest male/female (or testes/ovaries) ratios from both the Parisi et al. (2003) and Ranz et al. (2003) data sets. As a control, we selected the 50 genes showing a male/female ratio closest to 1 from each data set. Only genes corresponding to predicted transcripts in the D. melanogaster genome release 3.0 (Celniker et al. 2002) were included. Of the 100 genes selected for each expression class, 93 male-biased, 92 female-biased, and 99 unbiased genes remained after removing redundancies (table 1). The paucity of overlapping genes between the two data sets was primarily the result of differences in array composition; that is, genes present in one data set but absent in the other (50% of the genes). For another 47% of the genes, the difference was only in the level of sex bias, that is, the gene was in the top 50 of its expression class in one data set but not the other. For 3% of the genes, there was a conflict such that a gene was sex biased in one data set but unbiased in the other. There were no cases of conflicting male/female sex-bias classification among these genes. The coding sequences of these genes from D. melanogaster were used for a BlastN version 2.0 (Altschul et al. 1999) search of the D. pseudoobscura genome sequence using the Baylor College of Medicine Drosophila Genome Project Web site (http://www.hgsc.bcm.tmc.edu/projects/Drosophila/). Because the coding sequences of many of the genes could not be aligned between the two species, we report divergence as either Blast e-values or Blast scores. For score calculation, the default values of 5 and 2 were used for the gap creation and gap extension penalties, respectively. Levels of codon bias were calculated as described above using the full-length coding sequences from D. melanogaster.
Comparison of Polymorphism and Divergence
Because the largest number of DNA sequence polymorphism surveys have been conducted in D. melanogaster, we chose this species to investigate levels of polymorphism in sex-biased genes. We began with a database of 101 protein-encoding genes extracted from the literature and GenBank (provided by D. Presgraves) for which multiple (at least six) D. melanogaster alleles had been sequenced and at least one D. simulans allele was available for divergence analysis. A total of 55 genes could be classified as male biased, female biased, or unbiased based on the microarray expression data (table 1). A list of the individual genes and their associated references is provided as Supplementary Material online. One gene (Dntf-2r) was classified as unbiased based on the Parisi et al. (2003) data set, although it had been shown to be testis-specific by RT-PCR (Betrán and Long 2003). In this case, we classified the gene as male biased. The conflicting microarray result may be caused by cross-hybridization between Dntf-2r and its close paralog Dntf-2, which is expressed in both sexes (Betrán and Long 2003). DnaSP version 4 (Rozas et al. 2003) was used to calculate levels of polymorphism and divergence and to perform the MK test (McDonald and Kreitman 1991). All available D. melanogaster sequences were used for polymorphism calculations, and a single D. simulans allele was used for divergence.
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Results and Discussion |
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Comparison of Orthologous Genomic Regions Among Drosophila Species
To assess the impact of comparative sequence data on genome annotation, Bergman et al. (2002) sequenced fosmid clones (40 kb each) corresponding to the D. melanogaster genomic regions containing the apterous, even-skipped, fushi tarazu, twist, and Rhodopsin 1, 2, 3, and 4 genes in four diverse Drosophila species: D. erecta, D. pseudoobscura, D. willistoni, and D. virilis. These autosomal regions contain 81 known or predicted genes in D. melanogaster. However, orthologous sequences from all of these genes were not obtained from every species because of either incomplete overlap of fosmid clones or genomic rearrangements between species. Based on microarray data, we were able to classify 50 of the genes as male biased, female biased, or unbiased in their expression and compare evolutionary rates among the three classes (fig. 2A). Using all available sequences and assuming a constant dN/dS over all branches of the phylogenetic tree, we calculated average dN/dS values of 0.11, 0.02, and 0.07 for male-biased, female-biased, and unbiased genes, respectively. The difference between male-biased and female-biased genes was significant (Mann-Whitney test, P = 0.04), although comparisons among other classes were not significant (P > 0.05) because of the limited sample size. For 30 genes (eight male biased, four female biased, and 18 unbiased) for which sequences from three or more species were available, we applied a "free-ratio" model (Yang 1998) that allowed dN/dS to vary over all branches of the phylogenetic tree. This model did not provide a significantly better fit to the data for any of the female-biased genes, but it did provide a significantly better fit for four (22%) of the unbiased genes and five (63%) of the male-biased genes. This indicates significant evolutionary rate heterogeneity among lineages, particularly for male-biased genes. If dN/dS is calculated as an average over all branches (scaled by their length), then the average dN/dS values for male-biased, female-biased, and unbiased genes are 0.14, 0.02, and 0.10, respectively. The difference between male-biased and female-biased genes is significant (Mann-Whitney test, P = 0.03), although other comparisons are not significant.
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Comparison of Highly Sex-Biased Genes Between D. melanogaster and D. pseudoobscura
To investigate the evolutionary rates of genes showing the most extreme levels of sex-biased expression, we extracted the 50 genes with the highest and lowest male/female expression ratios from both the Parisi et al. (2003) and Ranz et al. (2003) data sets. As a control, the 50 genes showing male/female ratios closest to 1 were extracted from each data set. After removing redundancies, the final list contained 93 male-biased, 92 female-biased, and 99 unbiased genes (table 1). The coding sequences from these genes were used for a Blast search of the recently completed D. pseudoobscura genome. The three sex-bias classes showed significant differences in the number of genes with Blast matches over a wide range of e-value cutoffs (fig. 3). In all cases, male-biased genes showed the least conservation between the two species. For example, using a conservative e-value cutoff of 109, 46% of the male-biased genes did not have a significant Blast match. The corresponding numbers for female-biased and unbiased genes were 20% and 4%, respectively. Female-biased genes were less conserved than were unbiased genes for all cutoff values (fig. 3), but even in the most extreme case (e-value of cutoff of 106), the difference between female-biased and unbiased genes was not significant (2 = 1.51, P = 0.22).
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Polymorphism and Divergence in Sex-Biased Genes
The most plausible explanation for the observed evolutionary rate differences among male-biased, female-biased, and unbiased genes is variation in the strength and/or type of natural selection acting on genes of the three expression classes. One possibility is that male-biased genes are under relaxed selective constraints relative to genes of the other two classes and, thus, accumulate a larger fraction of neutral amino acid replacements between species. Alternatively, male-biased genes could be subject to increased positive selection because of male-male or male-female interactions/conflicts and, thus, accumulate more adaptive amino acid substitutions between species. To distinguish between these two possibilities, we examined DNA sequence polymorphism in 55 D. melanogaster protein-encoding genes that could be classified as male-biased, female-biased, or unbiased in their expression based on the microarray data (table 1). The divergence of these genes between D. melanogaster and D. simulans showed the same pattern observed for the other comparative genomic data sets, with male-biased genes showing the greatest divergence and female-biased genes showing the least (table 4). If the increased dN/dS ratio observed for male-biased genes were the result of relaxed selective constraints, then one would expect male-biased genes to show a corresponding increase in their ratio of nonsynoymous/synonymous polymorphism (N/
S) relative to female-biased and unbiased genes. However, the opposite pattern was observed. Male-biased genes had lower average
N/
S than both female-biased and unbiased genes (table 4). Thus, the polymorphism data do not support a general reduction of selective constraint on male-biased genes.
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The above results should be interpreted cautiously for several reasons. First, they are based on published surveys of DNA polymorphism that used different sample sizes and population sampling schemes, including African and non-African samples. Thus, the results may be affected by demographic factors, such as bottlenecks or population subdivision. Second, polymorphism is known to be much more sensitive to chromosomal environment (e.g., local recombination rate) than is divergence (Begun and Aquadro 1992), and because of the limited available data, we were unable to partition genes based on chromosomal location. By considering only the ratio of nonsynonymous to synonymous polymorphism (and not the separate values), we could partially control for the above two factors. However, it is possible that these factors also influence the N/
S ratio. Finally, there is likely an ascertainment bias in the polymorphism data present in the literature. Some of the genes may have been surveyed with an a priori expectation of positive (or balancing) selection based on functional or divergence data. In addition, there may be a publication bias towards genes that depart from neutrality rather than those that fit the neutral model. These limitations can be addressed in future studies that use common population samples and select genes based only on expression class without a priori expectations of selection.
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Conclusions |
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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References |
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