Homeobox genes encode transcription factors that function in various developmental processes and are usually evolutionarily conserved in their sequences. However, two X-chromosome-linked testis-expressed homeobox genes, one from rodents and the other from fruit flies, are known to evolve rapidly under positive Darwinian selection. Here we report yet another case, from primates. TGIFLX is an X-linked homeobox gene that originated by retroposition of the autosomal gene TGIF2, most likely in a common ancestor of rodents and primates. While TGIF2 is ubiquitously expressed, TGIFLX is exclusively expressed in adult testis. A comparison of the TGIFLX sequences among 16 anthropoid primates revealed a significantly higher rate of nonsynonymous nucleotide substitution (d_N) than synonymous substitution (d_S), strongly suggesting the action of positive selection. Although the high d_N/d_S ratio is most evident outside the homeobox, the homeobox has a d_N/d_S of 0.89 and includes two codons that are likely under selection. Furthermore, the rate of radical amino acid substitutions that alter amino acid charge is significantly greater than that of conservative substitutions, suggesting that the selection promotes diversity of the protein charge profile. More interestingly, an analysis of 64 orthologous homeobox genes from humans and mice shows substantially higher rates of amino acid substitution in X-linked testis-expressed genes than in other genes. These results suggest a general pattern of rapid evolution of mammalian X-linked testis-expressed homeobox genes. Although the physiological function of and the exact selective agent on TGIFLX and other rapidly evolving homeobox genes are unclear, the common expression pattern of these transcription factor genes led us to conjecture that the selection is related to one or more aspects of male reproduction and may contribute to speciation.

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HOMEOBOX genes are characterized by the presence of an 60-codon sequence motif known as the homeobox, which encodes a helix-turn-helix DNA-binding domain named the homeodomain (GEHRING et al. 1994a). Initially identified from fruit flies (MCGINNIS et al. 1984; SCOTT and WEINER 1984), homeobox-containing genes have now been found in fungi, plants, and animals and form a large gene superfamily (KAPPEN et al. 1993; BHARATHAN et al. 1997; KAPPEN 2000; BANERJEE-BASU and BAXEVANIS 2001). Homeobox genes function as transcription factors that regulate the expressions of their target genes in various developmental processes such as body-plan specification, pattern formation, and cell fate determination (GEHRING et al. 1994a). Because of their fundamental importance in development, homeobox genes are of substantial interest to evolutionary biologists as they may provide key information on the evolution of development (SHEPHERD et al. 1984; GARCIA-FERNANDEZ and HOLLAND 1994; ZHANG and NEI 1996; CARROLL et al. 2001). Earlier studies showed that homeobox genes, particularly the homeobox region, are conserved in evolution (MCGINNIS et al. 1984; GEHRING et al. 1994a), although two notable exceptions, Pem in rodents and OdsH in Drosophila, have been reported (SUTTON and WILKINSON 1997; TING et al. 1998). In both cases, high rates of amino acid substitution were found in the homeodomain and the action of positive selection was suggested. Interestingly, both genes are located on X chromosomes and are expressed in testis, although Pem is also expressed in female reproductive tissues. OdsH is in part responsible for the hybrid male sterility between Drosophila simulans and D. mauritiana (TING et al. 1998). These intriguing findings suggest that homeobox genes may also be involved in developmental processes that vary among closely related species. Because such developmental variations may lead to reproductive isolation and speciation (TING et al. 1998), it is of interest to identify new cases of rapidly evolving homeobox genes. Here we describe the identification of such a rapidly evolving homeobox gene, TGIFLX [TG-interacting factor (TGIF)-like X], from primates. TGIFLX is a member of TGIFs, a group of transcription factors of the three amino-acid loop extension (TALE) superclass of the homeodomain protein family (BERTOLINO et al. 1995; BLANCO-ARIAS et al. 2002). Earlier evolutionary analyses suggested that the X-chromosome-linked TGIFLX gene originated by retroposition of the autosomal TGIF2 gene, a member of TGIFs (BLANCO-ARIAS et al. 2002). In contrast to TGIF2, which is ubiquitously expressed, TGIFLX is specifically expressed in the germ cells of adult testis (BLANCO-ARIAS et al. 2002; LAI et al. 2002). In this report, we show that (1) the retroposition event predated the divergence of primates and rodents, (2) TGIFLX evolved rapidly in primates under positive selection, and (3) mammalian X-linked testis-expressed homeobox genes evolve rapidly in general.

DNA amplification and sequencing:

The TGIFLX coding region does not contain introns. The coding region was amplified from genomic DNAs of two Old World (OW) monkeys (green monkey Cercopithecus aethiops and douc langur Pygathrix nemaeus) and five New World (NW) monkeys (marmoset Callithrix jacchus, tamarin Saguinus oedipus, owl monkey Aotus trivirgatus, squirrel monkey Saimiri sciureus, and woolly monkey Lagothrix lagotricha), using polymerase chain reaction (PCR). For green monkey and douc langur, primers 2XL (5'-TTTGAATATGGAGGCCGCTG) and 2XR (5'-CATCATCAATCATGGATTAG) were used; for tamarin, woolly monkey, and marmoset, primers 2XL and XIA1 (5'-GGATTAGACTCTTGCTTCTTCT) were used; for owl monkey and squirrel monkey, primers X2 (5'-ATATGGAGGCCGCTGCAgAAGAC) and X3 (5'-GGCTCTTGCTTCTTCTCTAGC) were used. PCRs were performed with MasterTaq under conditions recommended by the manufacturer (Eppendorf, Hamburg, Germany). The products were then purified and sequenced from both directions, using the dideoxy chain termination method with an automated sequencer.

Analysis of TGIFLX gene sequences:

The DNA sequences of the TGIFLX coding region from five hominoids (humans and apes) and four OW monkeys (BLANCO-ARIAS et al. 2002) were obtained from GenBank. The accession numbers are: human (Homo sapiens), AJ427749; chimpanzee (Pan troglodytes), AJ345073; gorilla (Gorilla gorilla), AJ345074; orangutan (Pongo pygmaeus), AJ345075; gibbon (Hylobates lar), AJ345076; talapoin (Miopithecus talapoin), AJ345077; rhesus monkey (Macaca mulatta), AJ345078; crab-eating macaque (M. fascicularis), AJ345079; and baboon (Papio hamadryas), AJ345080. These publicly available sequences are analyzed together with those determined in this study. Seven amino acids at the N terminus and 10 amino acids at the C terminus of the sequences are encoded by the primer sequences and were not included in data analysis. A total of 16 TGIFLX protein sequences were aligned using the software DAMBE (XIA and XIE 2001) followed by manual adjustments. The DNA sequence alignment was then made following the protein alignment. The MEGA2 program (KUMAR et al. 2001) was used for phylogenetic analysis. The number of synonymous nucleotide substitutions per synonymous site (d_S) and that of nonsynonymous substitutions per nonsynonymous site (d_N) were computed using the modified Nei-Gojobori method (NEI and GOJOBORI 1986; ZHANG et al. 1998), with an estimated transition/transversion ratio of 1.6. On the basis of the phylogeny of the 16 primates, we inferred ancestral TGIFLX sequences at all interior nodes of this tree, using the distance-based Bayesian method (ZHANG and NEI 1997). The numbers of synonymous (s) and nonsynonymous (n) substitutions on each branch of the tree were then counted. Radical and conservative nonsynonymous substitutions with regard to amino acid charge and polarity were computed following ZHANG (2000). Positive selection at individual codons was tested using the likelihood-based (YANG et al. 2000) and parsimony-based (SUZUKI and GOJOBORI 1999) methods.

Analysis of other homeobox genes of human and mouse:

We searched for homeobox genes from the human genome resources (http://www.ncbi.nlm.nih.gov/genome/guide/human/) and then found their mouse orthologs using the UniGene tool (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=unigene). We downloaded the human and mouse protein sequences, aligned them using DAMBE, and computed protein p-distances (proportional differences; NEI and KUMAR 2000) between human and mouse orthologs. The information on gene location and expression pattern was found using human genome resources and the LocusLink tool (http://www.ncbi.nlm.nih.gov/LocusLink/).

Retroposition predated the human-mouse separation:

To determine when the retroposition that generated TGIFLX occurred in evolution, we conducted a BLAST search in the GenBank for homologous sequences to TGIFLX and its mother gene TGIF2. We identified a homeobox gene Tex1 (also known as Tgifx1-pending) in the mouse that is mapped to a region of the X chromosome that is syntenic with human Xq21.3, where TGIFLX is located. Tex1 is also specifically expressed in the germ cells of mouse testis (LAI et al. 2002). These facts suggest that mouse Tex1 is orthologous to human TGIFLX. Furthermore, we obtained the gene sequences of human and mouse TGIF2 from GenBank and conducted a phylogenetic analysis of these sequences. The human and mouse TGIF sequences are used as outgroups. The gene tree shows high bootstrap support for the retroposition that gave birth to TGIFLX occurring in a common ancestor of primates and rodents (Figure 1) .

View larger version (8K): In this window In a new window Download PPT slide   FIGURE 1.— Phylogenetic tree of TGIFLX, TGIF2, and TGIF genes. The tree is reconstructed with the neighbor-joining method with the protein Poisson distances. Bootstrap percentages from 1000 replications are shown on tree branches. Branch lengths show the numbers of amino acid substitutions per site. TGIF genes are used as outgroups.

 
Although retroposition usually generates pseudogenes, a number of retroposition-mediated functional genes have been identified (LONG 2001). TGIFLX is apparently a functional gene as its open reading frame has been maintained throughout mammalian evolution. Retroposition is a mutation-prone process due to a high error rate in retrotranscription. Also, newly duplicated genes often have elevated rates of evolution due to relaxation of functional constraints and/or positive selection (ZHANG 2003). Thus, one may expect to see a burst of substitutions in the TGIFLX branch immediately following the retroposition. Interestingly, Figure 1 shows that TGIFLX evolves more rapidly than TGIF2 not only in this branch, but also throughout its evolutionary history. We found that the number of amino acid substitutions per site (Poisson distance) between the orthologous human and mouse TGIFLX genes is 0.814 ± 0.080, and the corresponding number for TGIF2 is 0.031 ± 0.013, their difference being statistically significant (P < 0.001). Of 1880 orthologous human and rodent genes analyzed by MAKALOWSKI and BOGUSKI (1998), only 6 have substitution rates greater than that of TGIFLX, suggesting that it is evolving at an exceptionally high rate. To further characterize the substitution rate of TGIFLX, we conducted a detailed evolutionary study of this gene in primates.

Positive selection on primate TGIFLX:

The TGIFLX coding sequences from five hominoids and four OW monkeys were reported by BLANCO-ARIAS et al. (2002). We here determined the orthologous sequences in two additional OW monkeys and five NW monkeys. Thus, a total of 16 primate sequences are analyzed here. The alignment of these 16 protein sequences shows that they are highly variable (Figure 2) . The nonhomeodomain regions show the highest variability, although 25 of the 63 amino acid positions in the homeodomain are also variable among the 16 primates. Hydrophobic amino acids are usually conserved in homeodomains; in the present case 22 of the 29 hydrophobic sites are completely conserved among the primate sequences, and in the rest 7 also involve only hydrophobic amino acid changes. In the third helix of the homeodomain, four amino acids (W51, F52, N54, and R56; positions in the homeodomain) are known to be conserved (BANERJEE-BASU and BAXEVANIS 2001), which is also the case here. Position 53 is usually occupied by a polar amino acid in homeodomains, but was found to have a small, nonpolar amino acid in a previous analysis of TALE homeodomains (BURGLIN 1997). In our sequences, position 53 is variable with either polar or nonpolar amino acids. To examine whether the high sequence variability is a result of positive selection, we computed the synonymous (d_S) and nonsynonymous (d_N) distances between each pair of the sequences. For the entire coding region, higher d_N than d_S is observed in 93 of 120 pairwise comparisons (Figure 3A) , suggesting the possible action of positive selection. This pattern is more apparent when only the nonhomeodomain regions are analyzed, as 98 of the comparisons show d_N > d_S (Figure 3B). For the homeodomain, however, only 39 of the comparisons show d_N > d_S (Figure 3C). These results indicate that the substitution rate and pattern may be different between amino acid positions inside and outside the homeodomain.

View larger version (34K): In this window In a new window Download PPT slide   FIGURE 2.— Alignment of TGIFLX sequences of 16 primates. A dot indicates identity to the human sequence and a dash indicates a gap. The first 7 and last 10 amino acid positions are primer encoded in various sequences and are not used in subsequent sequence analysis. The four positively selected sites with posterior probabilities >90% (see text) are in boldface type.

 

View larger version (13K): In this window In a new window Download PPT slide   FIGURE 3.— Pairwise comparisons of dS and dN among 16 primate TGIFLX sequences for (A) the entire sequence, (B) nonhomeodomain regions, and (C) the homeodomain.

 
To test the hypothesis of positive selection more rigorously, we used a phylogeny-based approach (ZHANG et al. 1997). The phylogentic relationships of the 16 primates are assumed to follow the tree in Figure 4 . This phylogeny is relatively well established, especially for the major divisions (GOODMAN et al. 1998; PAGE and GOODMAN 2001; SINGER et al. 2003; STEIPER and RUVOLO 2003), and use of alternative trees does not affect our main conclusion. On the basis of this tree, we inferred the ancestral TGIFLX gene sequences at all interior nodes of the tree and counted the numbers of synonymous (s) and nonsynonymous (n) substitutions on each tree branch (Figure 4). We found that the sums of n and s for all branches are 195.5 and 58.5, respectively, for the nonhomeodomain regions. The potential numbers of nonsynonymous (N) and synonymous (S) sites are 322 and 128, respectively. Thus n/s = 3.34 is significantly greater than N/S = 2.51 (P = 0.031, binomial test). The binomial test used here is more conservative than Fisher's exact test used in ZHANG et al. (1997) and is more appropriate here because of multiple substitutions that may have occurred at individual sites (ZHANG and ROSENBERG 2002). Fisher's exact test would have given a P value of 0.002 here. We also analyzed n/s in hominoids, OW monkeys, and NW monkeys separately, but did not find significant differences (Figure 4). The average number of synonymous substitutions per site is 0.155 between hominoids and New World monkeys and 0.0819 between hominoids and Old World monkeys. These values are virtually identical to the corresponding numbers obtained from multiple intron and noncoding sequences of primate genomes (0.149 and 0.079, respectively; LI 1997, pp. 221–224), suggesting that the synonymous substitution rate in TGIFLX is normal. Thus, our results strongly suggest that positive selection is responsible for the rapid evolution at nonsynonymous sites of the nonhomeodomain regions.

View larger version (29K): In this window In a new window Download PPT slide   FIGURE 4.— Numbers of synonymous (s) and nonsynonymous (n) substitutions in the evolution of primate TGIFLX genes. Shown above each branch is n/s for the nonhomeodomain regions and below each branch is n/s for the homeodomain. N and S are the potential numbers of nonsynonymous and synonymous sites, respectively (see text).

 
For the homeodomain, we found that n/s (2.42) is slightly lower than N/S (2.73) and that the null hypothesis of n/s = N/S cannot be rejected. This may suggest that the homeodomain is under no functional constraints. It may also suggest that some sites in the homeodomain are under positive selection while other sites are under purifying selection, giving an overall pattern of similar average substitution rates at synonymous and nonsynonymous sites (see below). When we examine the substitution patterns of hominoids, OW monkeys, and NW monkeys separately, we find that the n/s ratio is higher among hominoids and OW monkeys (23.5/4.5 = 5.22) than among NW monkeys (25/12 = 2.08; Figure 4). However, this difference is not significant (P = 0.132). The n/s ratio is not significantly different from N/S for hominoids and OW monkeys (P = 0.150).

Statistical methods for identifying individual codons that are under positive selection have been developed in recent years (SUZUKI and GOJOBORI 1999; YANG et al. 2000). We first applied the likelihood method (YANG et al. 2000) to the TGIFLX data and compared the likelihoods under models 7 and 8. Here model 7 assumes that the d_N/d_S ratio for individual sites follows a ß-distribution between 0 and 1, while model 8 adds an extra class of sites to model 7. We found that model 8 fits the data significantly better than model 7 (² = 15.2, d.f. = 2, P < 0.001), with an additional class of sites of d_N/d_S = 2.42. Four codons were identified to be under positive selection with posterior probabilities >90%, and they are marked on the sequences shown in Figure 2. Similar results were obtained when models 1 and 2 were compared (see YANG et al. 2000 for details of the model description). Because the likelihood method has been shown to generate false-positive results occasionally (SUZUKI and NEI 2002), we examined the evidence for selection at the four codons by a more conservative parsimony-based method (SUZUKI and GOJOBORI 1999). None of the four codons show significant results of positive selection when they are tested individually (P = 0.19–0.59). When they are tested together, however, significant evidence for positive selection is found (average d_N/d_S = 5.10, P = 0.021), suggesting that one or more of the four codons are under positive selection. It is interesting to note that two of the four codons are located within the homeodomain while the other two are adjacent to the 3' end of the homeodomain, suggesting that the homeodomain may indeed be under positive selection (Figure 2). The two residues within the homeodomain are not among the completely conserved residues of all homeodomains, indicating that substitutions at these sites are unlikely to disrupt the basic structure and function of homeodomains. Furthermore, crystal structures of homeodomains show that the first of the two residues is involved in DNA-protein binding and that it contributes significantly to the functional specificity of homeodomains (GEHRING et al. 1994b). The second of the two residues belongs to helix I of the homeodomain, and it may also be involved in DNA-protein binding, although a more specific molecular function has yet to be defined.

Selection promotes the diversity of charge profile:

To investigate what types of nonsynonymous substitutions are favored by selection, we counted the numbers of conservative and radical nonsynonymous substitutions on each branch of the tree in Figure 4. Conservative nonsynonymous substitutions are those that do not alter the charge of the encoded amino acids and radical substitutions are those that alter the charge of the amino acids. We found a total number of r = 91.5 radical substitutions and c = 104 conservative substitutions in the tree for the nonhomeodomain regions. The potential numbers of radical and conservative sites are R = 128 and C = 195, respectively. The radical substitution rate (r/R = 0.715) is significantly greater than the conservative substitution rate (c/C = 0.533) at P = 0.027 (binomial test). This is in sharp contrast to the situation in most mammalian genes where the radical substitution rate is below the conservative rate (ZHANG 2000). This result suggests that selection may favor alternations of amino acid charge in TGIFLX evolution. We also tested the hypothesis that selection may favor an alternation of amino acid polarity, but obtained no supporting evidence. For the homeodomain, there is no evidence for selection promoting the diversity of either amino acid polarity or charge.

In the above, we compared the number of radical substitutions per radical site (r/R) with the number of conservative substitutions per conservative site (c/C). This comparison provides information on differential selections at radical vs. conservative sites, as long as the four parameters (r, c, R, and C) are correctly estimated (SMITH 2003). In contrast, comparisons between r and c can be misleading, because the potential numbers of radical (R) and conservative (C) sites in a gene sequence are usually different and they are affected by many factors unrelated to selection (DAGAN et al. 2002).

Rapid evolution of mammalian X-linked testis-expressed homeobox genes:

As mentioned, two other homeobox genes, Pem and OdsH, were reported to evolve rapidly (SUTTON and WILKINSON 1997; TING et al. 1998). The d_N/d_S ratio of Pem ranges from 0.65 to 1.56 for the homeodomain between Mus musculus and several related rodents (SUTTON and WILKINSON 1997). We reanalyzed the OdsH homeodomain sequences from D. simulans and D. mauritiana (TING et al. 1998) and obtained a d_N/d_S ratio of 1.55. Interestingly, TGIFLX, Pem, and OdsH are all located in X chromosomes and are all testis expressed. This observation prompted us to wonder whether it is a general pattern for X-linked testis-expressed homeobox genes to evolve rapidly. To test this hypothesis, we searched for orthologous homeobox genes from the human and mouse genome sequences. Our search was not exhaustive, but random. Of the 64 genes found, 4 are X-linked and testis expressed, 3 are X-linked and non-testis expressed, 13 are autosomal and testis expressed, and 44 are autosomal and non-testis expressed. Note that there appear to be only 7 X-linked homeobox genes, as a further exhaustive search did not find additional genes. Here "testis expression" simply means that the gene is expressed in testis, regardless of its expression in other tissues. We aligned the sequences and computed the amino acid p-distance for each orthologous pair. As shown in Table 1 and Figure 5A , when the entire protein is considered, autosomal homeobox genes (regardless of the expression pattern) and X-chromosomal non-testis-expressed homeobox genes have similar amino acid p-distances on average, which are an order of magnitude lower than those of X-linked testis-expressed homeobox genes, and their difference is statistically significant (P < 0.0001, permutation test). The same pattern is observed when only the homeodomain or nonhomeodomain regions are considered (Table 1; Figure 5, B and C). These results suggest that it is a general pattern for mammalian X-linked testis-expressed homeobox genes to evolve rapidly. In addition to TGIFLX, the other X-linked testis-expressed homeobox genes are ESX1L, OTEX, and PEPP-2. While the mouse ortholog of human ESX1L is clearly defined by a phylogenetic analysis (data not shown) and chromosomal locations, the orthologs of human OTEX and PEPP-2 are not uniquely defined, probably because of independent gene duplications in rodents and primates after their separation (WAYNE et al. 2002). From the mouse genome sequence, we identified a total of 15 homologs of the human OTEX and PEPP-2 genes and conducted a phylogenetic analysis of these genes. The phylogeny is not well resolved and has low bootstrap supports (see supplementary Figure 1 at http://www.genetics.org/supplemental/). To be conservative, we computed protein p-distances for the human OTEX with each of the 15 mouse genes and presented the smallest distance in Table 1. We also did the same for the human PEPP-2 gene. Considering possible nonindependent comparisons involved, we also repeated all the statistical tests when only one of the OTEX and PEPP-2 genes was used. We found that the statistical results remain unchanged.

View this table: In this window In a new window   TABLE 1 Protein p-distances between orthologous human and mouse homeobox genes

 

View larger version (21K): In this window In a new window Download PPT slide   FIGURE 5.— Distribution of the evolutionary rate of 64 mammalian homeobox genes. The evolutionary rate is measured by protein p-distance between the human and mouse orthologous genes for (A) the entire sequence, (B) the homeodomain, and (C) nonhomeodomain regions. Solid bars, X-linked testis-expressed genes; shaded bars, X-linked non-testis expressed; hatched bars, autosomal testis expressed; open bars, autosomal non-testis expressed.

 

In this report, we provide evidence that TGIFLX evolves rapidly under positive selection in primates and that the selection favors diversity in charge profile. Although positive selection acts mainly in the nonhomeodomain regions of the protein, it may also operate at a few sites in the homeodomain. The homeodomain is used in binding DNA sequences in transcription regulation, while the nonhomeodomain regions in TGIFLX might be used in protein-protein interaction as in the case of TGIF and TGIF2 (BERTOLINO et al. 1995; MELHUISH and WOTTON 2000; MELHUISH et al. 2001). Rapid evolution at these sites thus may alter the DNA- and protein-binding properties of TGIFLX. In mouse, the TGIFLX ortholog Tex1 is exclusively expressed in the germ cells at the spermatid stage (LAI et al. 2002) and apparently escapes the inactivation that most X-linked genes are supposed to experience in spermatogenesis (LIFSCHYTZ and LINDSLEY 1972). Although the physiological function of TGIFLX is unknown, the restricted temporal and spatial expression pattern suggests a role of this gene in spermatogenesis and the detected positive selection on TGIFLX may be related to spermatogenesis as well.

Our analysis of homeobox genes of humans and mice revealed a general pattern of rapid evolution of X-linked, testis-expressed homeobox genes, although the number of such genes is relatively small. It is interesting to note that among autosomal homeobox genes, testis-expressed genes and non-testis-expressed genes show similar rates of amino acid substitution (Figure 5). Thus, testis expression alone does not explain high rates of protein evolution. Among non-testis-expressed homeobox genes, there is also no significant difference in substitution rate between autosomal genes and X-linked genes, suggesting that chromosomal location alone also does not explain the difference in amino acid substitution rate. We noted in collecting the expression pattern data that 3 of the 4 X-linked testis-expressed genes (TGIFLX, OTEX, and PEPP-2), but only 1 (NKX3.1) of the 13 autosomal testis-expressed genes, have exclusive or highly selective expressions in testis. This difference suggests that the majority of the autosomal testis-expressed genes may be under greater functional constraints due to their multifaceted roles in many tissues and developmental processes and thus evolve more slowly. Indeed, NKX3.1, which is expressed only in testis, has the highest substitution rate among the 13 autosomal testis-expressed genes (Table 1). On the contrary, most of the X-linked testis-expressed homeobox genes are expressed exclusively or highly in testis and may thus be specifically involved in male reproduction. Many authors showed that genes involved in male reproduction evolve rapidly under positive selection (e.g., LEE et al. 1995; SWANSON and VACQUIER 1995; METZ and PALUMBI 1996; TSAUR and WU 1997; ROONEY and ZHANG 1999; WYCKOFF et al. 2000; SWANSON and VACQUIER 2002; PODLAHA and ZHANG 2003). In particular, TORGERSON and SINGH (2003) recently showed that mammalian X-linked sperm proteins evolve faster than autosomal ones. Our finding of rapid evolution of mammalian X-linked testis-expressed homeobox genes is thus consistent with these previous observations.

WANG et al. (2001) reported that the mammalian X chromosome harbors disproportionately more spermatogonia-expressed genes than autosomes. Spermatogonia are the mitotic germ cells of the testis from which sperm arise by spermatogenesis. Spermatogonia-expressed genes are probably involved in male reproduction. In our random sample of 64 homeobox genes, 57% of the 7 X-linked genes and 23% of the 57 autosomal genes are testis expressed. Thus, even for homeobox genes, the X chromosome appears to harbor a higher proportion of testis-expressed genes than autosomes (P = 0.074). If only those genes that are exclusively (or highly selectively) expressed in testis are considered, the X chromosome harbors an even higher percentage of such genes (3/7 = 43%) than autosomes (1/57 = 2%), and their difference is significant (P = 0.003). Sex-chromosome meiotic drive and/or sexual antagonism have been invoked as possible explanations for a higher proportion of X-linked genes to function in male reproduction, and these hypotheses have been discussed extensively in WANG et al. (2001).

It has also been proposed that X-linked genes evolve more rapidly than autosomal genes (CHARLESWORTH et al. 1987). This is particularly so when the X-linked genes are expressed only in males, because all newly arising advantageous alleles, dominant or recessive, are exposed to positive Darwinian selection. In contrast, recessive advantageous alleles at autosomal loci are effectively neutral when the allele frequencies are very low. This might explain the effectiveness of positive selection on X-linked testis-expressed genes.

The X chromosome has been shown to be of special importance in hybrid sterility between closely related species (reviewed in COYNE 1992). The importance of homeobox genes in hybrid sterility, however, is not well recognized, probably because most homeobox genes are evolutionarily conserved. It was thus a surprise to identify the rapidly evolving OdsH, an X-linked testis-expressed homeobox gene that is in part responsible for the hybrid male sterility between D. simulans and D. mauritiana (TING et al. 1998). This study showed that it is a general pattern for mammalian X-linked testis-expressed homeobox genes to evolve rapidly. This suggests the intriguing possibility that it is a rule rather than an exception that homeobox genes such as OdsH play important roles in reproductive isolation. In the future, it will be of great interest to work out the developmental pathways in which these homeobox genes function and the biological significance of their rapid pace of evolution.

We thank Lizhi Gao, Ondrej Podlaha, David Webb, and two anonymous reviewers for valuable comments. This work was supported by a startup fund from the University of Michigan and a National Institutes of Health grant (GM67030) to J.Z.

Sequence data from this article have been deposited in the EMBL/GenBank Data Libraries under accession nos. AY449635, AY449636, AY449637, AY449638, AY449639, AY449640, AY449641.

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