Gene Conversion Drives the Evolution of HINTW, an Ampliconic Gene on the Female-Specic Avian W Chromosome

Niclas Backstro m, Helene Ceplitis, Soa Berlin, and Hans Ellegren

Department of Evolutionary Biology, Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden The HINTW gene on the female-specic W chromosome of chicken and other birds is amplied and present in numerous copies. Moreover, as HINTW is distinctly different from its homolog on the Z chromosome (HINTZ), is a candidate gene in avian sex determination, and evolves rapidly under positive selection, it shows several common features to ampliconic and testis-specic genes on the mammalian Y chromosome. A phylogenetic analysis within galliform birds (chicken, turkey, quail, and pheasant) shows that individual HINTW copies within each species are more similar to each other than to gene copies of related species. Such convergent evolution is most easily explained by recurrent events of gene conversion, the rate of which we estimated at 106105 per site and generation. A signicantly higher GC content of HINTW than of other W-linked genes is consistent with biased gene conversion increasing the xation probability of mutations involving G and C nucleotides. Furthermore, and as a likely consequence, the neutral substitution rate is almost twice as high in HINTW as in other W-linked genes. The region on W encompassing the HINTW gene cluster is not covered in the initial assembly of the chicken genome, but analysis of raw sequence reads indicates that gene copy number is signicantly higher than a previous estimate of 40. While sexual selection is one of several factors that potentially affect the evolution of ampliconic, male-specic genes on the mammalian Y chromosome, data from HINTW provide evidence that gene amplication followed by gene conversion can evolve in female-specic chromosomes in the absence of sexual selection. The presence of multiple and highly similar copies of HINTW may be related to protein function, but, more generally, amplication and conversion offers a means to the avoidance of accumulation of deleterious mutations in nonrecombining chromosomes.

Introduction The female-specic avian W chromosome shares several characteristics with the mammalian Y chromosome; it is small, gene-poor, and rich in repetitive heterochromatic DNA (Ellegren 2000). The majority of genes so far found on the chicken W chromosome have a similar (gametologous) copy on the Z chromosome (e.g., CHD1W/CHD1Z and SPINW/SPINZ), a remnant of the ancestral homology of avian sex chromosomes prior to their differentiation. Given the high degree of amino acid sequence similarity between most Z- and W-linked gametologs, genes on the avian W chromosome rarely seem to have evolved female-specic function (Ellegren 2002). They can thus be seen as the avian equivalents to the class 1 genes of the nonrecombining (or male-specic) region of the human Y chromosome (e.g., SMCY and ZFY), i.e., genes with like-functioning X- and Y-linked gametologs encoding widely expressed housekeeping proteins (Lahn, Pearson, and Jegalian 2001). However, one W-linked gene in chicken, HINTW (formerly denoted Wpkci [Hori et al. 2000] and ASW [ONeill et al. 2000]; see discussion of nomenclature in Ceplitis and Ellegren 2004), is strikingly different from its Z chromosome gametolog, HINTZ (HINTW and HINTZ are orthologous to mammalian HINT). HINTW lacks a histidine triad normally found in HINT, including HINTZ, and other members of the HIT superfamily of proteins (Se raphin 1992) but contains a unique Leu- and Arg-rich region of unknown function (Hori et al. 2000; ONeill et al. 2000). As HINTW is expressed in the developing gonads of female embryos at the critical time of sexual differentiation, it has been suggested to play a role in avian sex determination
Key words: gene conversion, concerted evolution, multicopy gene, sex chromosomes, GC content, mutation rate, chicken. E-mail: hans.ellegren@ebc.uu.se.
Mol. Biol. Evol. 22(10):19921999. 2005 doi:10.1093/molbev/msi198 Advance Access publication June 22, 2005 The Author 2005. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oupjournals.org

(Hori et al. 2000; ONeill et al. 2000; Pace and Brenner 2003). HINTW shows evidence of positive selection with highly elevated ratios of the rate of nonsynonymous to synonymous substitution (Ka/Ks) in the lineages leading to chicken and other galliform birds (Ceplitis and Ellegren 2004). In contrast, HINTZ is well-conserved with an extensive degree of amino acid sequence similarity to its orthologs in mammals (Hori et al. 2000). Another unique feature of HINTW is that it exists in multiple copies. Hori et al. (2000) estimated copy number in chicken to be 44 and found evidence of a large number of W-linked gene copies in a wide range of other bird species. Preliminary data indicate that members of the gene family are arranged in tandem and include functional genes as well as corrupted gene copies (Hori et al. 2000; ONeill et al. 2000). Unfortunately, the W chromosome is not well covered in the draft assembly of the chicken genome (ICGSC 2004), so no further information on the organization of the HINTW gene cluster exists. As a female-specic, multicopy gene that evolves rapidly under positive selection and is potentially involved in reproduction, HINTW makes an interesting comparison to several genes on the mammalian Y chromosome. Most testis-specic genes on the human Y chromosome are multicopy and/or lack a like-functioning X-linked homolog (class 2 genes according to Lahn, Pearson, and Jegalian 2001; see also Lahn and Page 1997; Makrinou et al. 2001; Skaletsky et al. 2003) similar to HINTW (note that Sry is multicopy in some mammals, Bullejos et al. 1997; Lundrigan and Tucker 1997). Also, many of these testisspecic genes evolve unusually rapidly (Wyckoff, Wang, and Wu 2000) and tend to consist of a mixture of active and silent copies (Manz et al. 1993; Elliott et al. 1997). However, while sexual selection is often invoked to explain the evolution of Y-linked genes involved in spermatogenesis (Lahn, Pearson, and Jegalian 2001), sexual selection is less obvious in affecting the evolution of female-specic genes in birds. To help understand how the common genomic

Gene Conversion on Avian W Chromosome 1993

features of avian HINTW and testis-specic genes on the mammalian Y chromosome may have evolved, we therefore set out to investigate the evolution of the HINTW gene family in galliform birds. Materials and Methods Polymerase Chain Reaction Amplication from Genomic DNA Whole blood or tissue samples were collected from morphologically sexed birds from four species (one female of each) of the order Galliformes: chicken (Gallus gallus, Ggal), turkey (Meleagris gallopavo, Mgal), quail (Coturnix coturnix, Ccot), and pheasant (Phasianus colchicus, Pcol). DNA preparation was done by incubating approximately 5 ll whole blood in 400 ll of 0.1 M NaCl, 10 mM Tris-HCl (pH 8.0), 5% sodium dodecyl sulfate and 10 ll of proteinase K (20 mg/ml) at 37C for approximately 12 h. DNA was puried with three rounds of phenol/chlorophorm treatment and nally, precipitated with ethanol, dried and dissolved in water. Exon 2, intron 2, and exon 3 of HINTW were polymerase chain reaction (PCR) amplied as a single product in 50 ll containing 100 ng DNA, 0.5 U Amplitaq Gold (PerkinElmer), 0.2 mM dNTP, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, and 0.2 lM primer PKCIW-F1 (TGC CTT GCG TTC CAT GAT A) and PKCIW-R1 (GTG AAA CCC ATT CGG TGG C) or PKCIW-R2 (TAT CCC CAT GCT CCA TCT TTA TT). For amplication of HINTZ, primers PKCIZ-F4b (TGC CTT GCG TTC CAT GAT ATC) and PKCIZ-R2b (CAA ATC TAT TTG CTA GTG ATT) were used. Amplication started with a 5-min denaturation step at 94C, followed by 40 cycles at 94C for 30 s, 52C (48C for HINTZ) for 30 s, and 72C for 45 s. A nal extension step at 72C for 10 min was added to complete the run. The absence of amplication products in at least one male of each species served as control to ensure W chromosomespecic amplication. Cloning and Sequencing PCR products were puried with QIAquick PCR purication kit (Qiagen) and ligated into pGEM-T Easy vector (Promega). Ligations were transformed into heatshock competent JM109 Escherichia coli cells (Promega) following the manufacturers protocol. Positive colonies were transferred to 50 ll cell lysis buffer (20 mM Tris-HCl pH 8.0, 1% (w/v) Triton X-100, and 0.5 mM EDTA), boiled for 2 min and centrifuged at 10,000 rpm for 5 min. Two microliter of the supernatant was used in PCR reactions to amplify the inserts with modied M13 primers (ACA GGA AAC AGC TAT GAC CAT GAT and CGA CGT TGT AAA ACG AGG CCA GT). The 50 ll PCR reaction contained 1.25 U Amplitaq Gold (PerkinElmer), 0.2 mM dNTP, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.0 mM MgCl2, and 0.2 lM of each primer. Amplication of the inserts was done starting with a 5 min denaturation step at 95C, followed by 30 cycles at 95C for 40 s, 50C for 60 s, and 72C for 60 s. A nal extension step at 72C for 7 min was added to complete the run. PCR products of the expected length were puried and sequenced in

both directions using BigDye chemistry (Applied Biosystems) on an ABI 377 automated sequencer (PerkinElmer) and a DYEnamicTM ET Terminator Cycle Sequencing Kit (Amersham Biosciences) on a MegaBace 96-capillary instrument (Amersham Biosciences). All sequences observed in this study have been submitted to GenBank under the accession numbers AY713486AY713503. Sequence Analyses DNA sequences were edited in Sequence Navigator 1.0.1 (Applied Biosystems) and alignments made using ClustalW (Thompson, Higgins, and Gibson 1994, available from http://pbil.ibcp.fr). Intronic sequences were masked for microsatellite repeats using Sputnik (http://espressosoftware.com/pages/sputnik.jsp), and the rst and last 20 bp of introns were excluded as they may be subject to selective constraints (Chamary and Hurst 2004). A maximum likelihood (ML) consensus tree was constructed for pooled exon and intron sequences using MrBayes (Huelsenbeck and Ronquist 2001). Coding regions were analyzed with the codon model and intron sequences with the 4-by-4 model. Four chains were run simultaneously with Ngen 5 1,000,000, Samplefreq 5 100 and Burnin 5 500 resulting in a set of 9501 ML-trees from which the consensus tree was extracted. Sequence identity among gene copies within species was calculated in MEGA (Kumar, Tamura, and Nei 2004) using the TN93 model for noncoding sequence and the method of Nei and Gojobori (1986) with an estimated transition/transversion ratio of 2 for coding sequence. Divergence between species was estimated in the same way and the mean and standard error taken from all possible pairwise comparisons of copies between any two species. The lengths of three-species trees of chicken, turkey, and quail HINTW and HINTZ intron sequences were estimated in PAML (Yang 1997) using TN93. The procedure was repeated 1,000 times (rate variation among sites for each new alignment was estimated with a start value of a 5 0.5), in each case using a random gene copy from each species and then bootstrapping by site. The point estimate of each tree length was taken from the mean, and 95% condence intervals were obtained by excluding the 25 lowest and the 25 highest scores. The length of trees of the same species using introns from W chromosome and Z chromosome reference genes (CHD1W/CHD1Z, SPINW/SPINZ, and UBAP2W/UBAP2Z; unpublished data) was estimated in a similar way, although using a double bootstrapping approach (see Axelsson et al. 2004), resampling both by intron and by site. Similar analyses were also conducted without potential CpG sites by excluding all nucleotide pairs starting with C or T and ending with G or A, except sites xed for A and T in all copies, in any species. Estimates of GC content were obtained for the chicken W and Z chromosomes and the human Y chromosome genes listed in Supplementary Table 1. The male-to-female mutation rate ratio (am) was estimated by the formula of Miyata et al. (1987): am 5 3Z=W 1=2 where Z and W in this case are the total lengths of the threespecies trees constructed using Z chromosome and W

1994 Backstro m et al.

chromosome sequences. Bootstrapping was done by randomly pairing bootstrapped Z and W length estimates 1,000 times. The rate of gene conversion was estimated according to the formula of Ohta (1982): c 1 5 3a 1 6v *=K = 3a 1 6v * where c^ 1 is the average sequence identity among gene copies, K is the nite number of allelic states per site (actually four), and v* is Kv/(K1), v being the mutation rate per site and generation. a denotes a gene conversion parameter equal to 2k/(n1) where k is the rate of gene conversion per site and generation and n is the number of gene copies. A partial likelihood sliding window approach was used to test whether some regions of HINTW showed deviating phylogeny, using the program PLATO with default settings (Grassly and Holmes 1997). The trace archive of chicken genome sequences was searched for single reads corresponding to HINTW (the complete gene sequence identied by Hori et al. [2000] and represented in clone Wpkci7) using BLAST at http:// genome.wustl.edu/projects/chicken/chicken_analysis/. Reads covering the whole of particular exons or at least half the length of either of the two introns were extracted and pairwise sequence identities were estimated as above for exons and introns separately. Results Phylogenetic Analysis Reveals Species-Specic Clustering of HINTW Gene Copies As HINTW is multicopy it is not straightforward to obtain the complete sequence of individual copies by PCR using overlapping amplicons. However, most of exon 2 (87 of 106 bp), the entire intron 2 (about 850 bp), and exon 3 (159 bp) could be readily amplied as a single amplicon. Following amplication and sequencing, 34 different gene copies were identied in chicken, pheasant, quail, and turkey. At least four sequences (one each in chicken and pheasant and two in turkey) represented nonfunctional copies as indicated by frameshift or nonsense mutations. With strong posterior probability support, phylogenetic analysis revealed species-specic clustering of all gene copies (g. 1). In other words, all gene sequences were consistently more similar to other copies of the same species than to any gene family member in other species. This suggests a monophyletic origin of copies within lineages, either with ancestral amplication of HINTW followed by homogenization of gene copies (concerted evolution) within each lineage or with independent amplication of HINTW after lineage splitting. A sliding window analysis using PLATO failed to identify any anomalous region, indicating that the whole gene has been subject to similar evolutionary forces. Intronic sequence divergence among gene copies was much lower in quail (0.004 6 0.002) than in chicken (0.031 6 0.005), pheasant (0.039 6 0.006), and turkey (0.052 6 0.007). The same trend was evident for synonymous sites in coding sequence, although based on limited amount of data only (quail, 0.013 6 0.01; chicken, 0.026 6 0.013; turkey, 0.047 6 0.019; pheasant, 0.084 6 0.022).
100
^

Mgal1 Mgal2 Mgal3 Pcol3 Pcol4 Pcol1 Pcol2 Ggal4 100 Ggal1 100 Ggal2 Ggal3 Wpkci7 Wpkci8 Ccot3 Ccot1 Ccot2 Ccot4

100

FIG. 1.Maximum likelihood tree showing the phylogeny of different copies of HINTW from chicken (Ggal), quail (Ccot), turkey (Mgal), and pheasant (Pcol). Numbers on species branches indicate posterior probability values (%). Wpkci7 and Wpkci8 are from Hori et al. (2000) and ASW from ONeill et al. (2000), all other sequences are from this study.

A High Neutral Substitution Rate and GC-Content in HINTW For three of the species (chicken, quail, and turkey) analyzed in this study we have recently estimated the intronic substitution rate of other W-linked genes (unpublished data). The total length of a phylogenetic tree with these three species is 0.100 (95% CI 0.0880.113) for 7786 bp of W-linked CHD1W, SPINW, and UBAP2W introns, while it is almost twice as high, 0.185 (0.1460.229), for HINTW intron 2 (P , 0.001, by bootstrapping). The GC content of the two HINTW introns in chicken (55.3%) is signicantly higher than that of introns of other W-linked genes (40.0%). A similar difference is seen for GC3 (58.2% vs. 37.1%). High GC may cause elevated substitution rates, e.g., by a higher incidence of hypermutable CpG sites (Zhao and Boerwinkle 2002). However, when removing all CpG sites from the analysis, the total length of the HINTW tree is still signicantly (P 5 0.016) longer (0.120, 0.0890.150) than that of other W-linked introns (0.081, 0.0720.093), although the difference is not as pronounced as with including CpG sites. The intronic GC content of HINTZ, the Z-linked gametolog of HINTW, is 40.5% in chicken, which is similar to that of other Z-linked gametologs (36.3%). This indicates that the GC content of HINTW has increased following

Gene Conversion on Avian W Chromosome 1995

900 800

1800 1600

# pairwise comparisons

# pairwise comparisons

700 600 500 400 300 200 100 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34

1400 1200 1000 800 600 400 200 0 0 2 4 6 8 10 12 14 16 18 20 22 24

Pairwise divergence (%)

Pairwise divergence (%)

FIG. 2.Pairwise sequence divergence among unique HINTW exon variants (n 5 132) identied from chicken genome sequencing.

FIG. 3.Pairwise sequence divergence among unique HINTW intron variants (n 5 188) identied from chicken genome sequencing.

differentiation of sex chromosomes, in contrast to the situation for most other W-linked genes in which GC has decreased (data not shown). Moreover, the intronic substitution rate of HINTZ (0.205, 0.1640.286; total tree length for the same three-species comparison as indicated above) is similar to that of other Z-linked genes (0.194, 0.1690.214). Copy Number and Overall Degree of Sequence Identity Among HINTW Gene Copies in Chicken The region on the chicken W chromosome encompassing HINTW is not covered in the initial assembly of the chicken genome, a combined consequence of the repetitive nature of the gene, the fact that W was sequenced to three times coverage only and the shortage of anchor markers on W (ICGSC 2004). Moreover, the high similarity among gene copies means that it is difcult to construct contigs with some condence. We extracted single reads corresponding to HINTW from the chicken genome project and determined the number of different sequence variants for each exon and intron separately (no read would cover the entire gene). Clearly, any sequence error would imply that copy number is overestimated so we tallied the number of variants using a minimum requirement of 1, 2, or 3 bp differences between copies (Supplementary Table 2). This analysis indicates that HINTW gene copy number in chicken is higher than the previous estimate of 40 obtained from quantitative hybridization experiments (Hori et al. 2000). For instance, for exon 1 only we found 13 functional variants and an additional 28 variants suggested to represent pseudogenes using a stringent criteria of 2-bp differences between any two copies. Similar levels of sequence variation in the other two exons as well as in introns suggest that the total number of gene copies is signicantly higher than 40. Note that the genome sequence was obtained from one female bird so that all data from the W chromosome come from a single haplotype. Mean pairwise sequence divergence between variants was 0.077 6 0.0005 for exonic sequence and 0.044 6 0.0003 for introns. As these estimates are based on distinct variants only, the possible existence of identical copies, something which we cannot address, would imply lower averages. On the other hand, the estimates may potentially

be biased by sequencing errors increasing the number of only slightly different gene copies. However, the frequency distributions of pairwise differences do not show a strong excess of singleton variants (gs. 2 and 3). We conclude that the observed mean divergences from genomic reads agree well with what we found in the amplication and sequencing of individual copies of HINTW described above. Discussion Gene Conversion Drives the Evolution of HINTW The multicopy HINTW gene on the female-specic W chromosome of chicken and other galliform birds shows a number of distinctive evolutionary features: (1) gene copies show a very high degree of sequence identity, (2) the phylogenetic relationships among gene copies reveal concerted evolution within lineages, (3) GC content in HINTW is considerably higher than in other genes on the W chromosomes, (4) the neutral substitution rate of HINTW is almost twice as high as in other W-linked genes. These observations may be taken to suggest that gene conversion drives the evolution of HINTW gene family. In the following paragraphs we examine the evidence. Species-specic clustering of gene copies, without reticulations within the network, is most easily explained by sequence homogenization arising from gene conversion, i.e., nonreciprocal recombination where one allele replaces the other allele at the same locus, or an allele at another locus. According to this scenario, gene amplication predated the split of the four galliform lineages studied, after which gene conversion has led to concerted evolution among paralogous copies (cf. Fig. 1b in Teshima and Innan 2004). As the female-specic region of the avian W chromosome is haploid and not involved in sexual recombination, gene conversion may occur between paralogous copies on the same chromatide or between sister chromatids (ectopic gene conversion). Ectopic gene conversion may be facilitated by the close physical linkage of members of a gene family (Jackson and Fink 1981; Galtier 2003), as is the case for the tandemly repeated HINTW gene copies. Given that we found a high degree of sequence identity among copies over the whole gene, our data indicate that gene conversion affects coding as well as intronic sequence.

1996 Backstro m et al.

In theory, the observed pattern of species-specic clustering would also be consistent with independent amplication of gene copies after lineage splitting. However, multiple copies of HINTW seems to be a ubiquitous feature of neognath birds (Hori et al. 2000). If the high degree of sequence similarity among gene copies seen in the species studied herein is representative for other avian orders, then gene amplication would for some reason have occurred independently numerous times within a relatively short time interval across phylogenetically divergent avian lineages. We nd this unlikely and do not consider this possibility further. A conventional molecular clock cannot be applied to amplied sequences undergoing concerted evolution. However, we can use the observed mean intraspecic divergence among members of the HINTW gene family to roughly estimate the rate of intrachromosomal gene conversion according to the formula of Ohta (1982). We assume n 5 40 and v 3.5 3 109, the latter obtained by noting that chicken-turkey intronic divergence for HINTW is 0.098 (table 1), that chicken-turkey have an estimated divergence time of 3040 Myr (Dimcheff, Drovetski, and Mindell 2002; van Tuinen and Dyke 2004), and that galliform birds have an average generation time of 2 years. This suggests that the rate of gene conversion per site and generation in HINTW is approximately 34 106 for chicken, turkey, and pheasant, and 3 105 for quail. If gene copy number is higher, as our analyses suggest, the rate would be somewhat higher. We note that the estimated gene conversion rate is several orders of magnitude higher than the rate of point mutation. Evidence for concerted evolution of repeated sequences on the human Y chromosome has recently been presented. A large proportion of human Y constitutes palindromic repeats within which several testis-specic multicopy genes are located (Skaletsky et al. 2003). These ampliconic genes also exist in multiple copies in great apes, and because pairs of palindromic arms show up to 99.97% sequence identity in humans, concerted evolution arising from gene conversion has been interpreted (Rozen et al. 2003). Gene conversion has also been implicated between endogenous retroviral sequences (HERV elements) on the human Y chromosome (Bosch et al. 2004). These observations resemble what we found in this study, and indicate that gene conversion in multicopy genes on the hemizygote sex chromosome (Y in mammals, W in birds) may occur irrespective of whether there is male or female heterogamety. Moreover, this indicates that sexual selection is not a necessary component in the evolution of gene conversion on sex chromosomes. Sexual selection has been demonstrated among ornamented females of some bird species (reviewed in Amundsen 2000). However, showy female colors are generally absent in galliform birds, and other than contributing sperm, males invest little in offspring. While sexual selection resulting from sperm competition among males is indeed intense in e.g., chicken (Birkhead et al. 1999, Pizzari and Birkhead 2000) and has resulted in extravagant male ornaments in several galliforms, there is little to suggest that sexual selection among females is prevalent in the species studied herein.

Table 1 Mean Pairwise Divergence Estimates for HINTW Exonic (Below Diagonal) and Intronic (Above Diagonal) Sequences in Chicken, Turkey, Quail, and Pheasant
Species Chicken Turkey Quail Pheasant 0.1010 (0.0192) 0.1081 (0.0209) 0.1054 (0.0189) Chicken Turkey 0.0987 (0.0095) 0.1307 (0.0223) 0.0990 (0.0172) Quail 0.1298 (0.0133) 0.1263 (0.0127) 0.1566 (0.0255) Pheasant 0.1456 (0.0129) 0.1205 (0.0113) 0.1774 (0.0161)

NOTE.Standard errors are given in parentheses.

Gene Conversion Affects GC Content and Substitution Rates in HINTW The higher GC content of HINTW than of other W-linked genes as well as of HINTZ adds to the observations from mammalian genomes that biased gene conversion (BGC) is a potent force to alter the GC content of segregating sites (Galtier et al. 2001; Duret et al. 2002; Galtier 2003; Webster, Smith, and Ellegren 2003; Kudla, Helwak, and Lipinskii 2004). BGC refers to the nonrandom xation of mismatches toward G and C and is expected to have a particularly strong effect when recombination-associated processes occur at high rates. BGC has been suggested to explain the higher GC content of microthan of macrochromosomes in chicken (ICGSC 2004, Axelsson et al. 2005); the recombination rate of the former is about twice as high as that of the latter (ICGSC 2004). The W chromosome has the lowest GC content of all chromosomes in the chicken genome (ICGSC 2004) which is consistent with most of the W representing haploid sequence not involved in recombination. However, we suggest that the markedly higher GC content of HINTW than of other W-linked genes, and of its gametolog on the Z chromosome HINTZ, is a direct consequence of frequent events of gene conversion among gene copies resulting in preferential xation of AT / GC mutations. The lower GC content of single-copy than of multicopy genes on the human Y chromosome gives further support to the idea that gene conversion affects GC on the hemizygote sex chromosome. In class 1 single-copy human Y chromosome genes, the mean GC content is 38.4% for introns and 38.8% for GC3, while for ampliconic class 2 genes it is 42.8% for introns and 52.2% for GC3. Gene conversion has also been implicated to affect the GC content of repeated autosomal sequences, like the histone gene family in which GC increases with increasing number of gene copies (Galtier 2003). The high neutral substitution rate of HINTW may be a consequence of an increase in GC from gene conversion, although the case is complicated by the complex interactions between different genomic features. Apart from the higher incidence of mutable CpG sites in GC-rich sequences, there are several observations of positive correlation between GC and substitution rate in mammalian genomes (Bielawski, Dunn, and Yang 2000; Hurst and Williams 2000; Smith, Webster, and Ellegren 2002). Base composition may directly alter regional substitution rates if

Gene Conversion on Avian W Chromosome 1997

global rates of AT / GC and GC / AT mutations differ, although the effect is largely dependent on the equilibrium GC content a sequence is evolving toward (Piganeau et al. 2002). Recent studies indicate that GC-rich regions are decaying in mammalian genomes (Duret et al. 2002; Smith, Webster, and Ellegren 2002; Arndt, Petrov, and Hwa 2003; Webster, Smith, and Ellegren 2003) which should result in higher mutation rates in regions of high GC. However, because it is not yet known whether avian base composition is at equilibrium the expected effect of GC content on mutation rate in birds is unclear. One practical consequence of the unusually high neutral substitution rate of HINTW is that it evolves at about the same neutral rate as its gametolog on the Z chromosome HINTZ. This is in contrast to other gametologous gene pairs on the Z and W sex chromosomes, as male-biased mutation usually implies that W is exposed to fewer mutations than other regions of the avian genome (Ellegren and Fridolfsson 1997). An estimate of the male mutation bias (am) using HINTW/HINTZ intron sequences according to Miyata et al. (1987) gives am 5 1.22 (0.722.00), while we obtain am 5 2.41 (1.932.87) for other gametologous gene pairs (P 5 0.014). Axelsson et al. (2004) used 43 kb of autosomal and sex chromosome sequence in the chicken-turkey comparison and estimated am at 2.47 (2.272.68). HINTW/ HINTZ may thus not be particularly useful for deriving representative estimates of am in birds. Why Have Multiple Copies of HINTW Evolved? Classical models suggest that gene duplication allows the evolution of new gene function (Ohno 1970). Moreover, it is often assumed that the long-term survival of duplicated genes requires their functional diversication (Hughes 1994), either through acquisition of novel function by one or both of the paralogs (neofunctionalization) or partitioning of the original function among the duplicates (subfunctionalization). However, concerted evolution of HINTW gene copies suggests that amplication of HINTW is not associated with functional diversication. Instead, gene amplification could represent a means for increasing the amount of HINTW gene product. In mammals, amplication of testis-specic genes on the Y chromosome may be driven by sexual selection, assuming that increased amount of testis proteins offer a selective advantage to males in sperm competition. Although this is not applicable to genes on the female-specic W chromosome of birds nor of other organisms with female heterogamety, other scenarios are possible. HINTW is a candidate gene to be involved in avian sex determination, either by triggering the cascade of gene expression toward the differentiation of ovary or by blocking the differentiation of testis (Hori et al. 2000; ONeill et al. 2000). HINT is a dimer and while HINTW has evolved quite characteristic features compared to HINTZ, the amino acid residues that form the dimer binding site have remained constant in HINTW. It has been suggested that HINTW could act as a dominant negative by forming a heterodimer with HINTZ in female birds and thereby interfere with the function of the HINTZ homodimer, leading to feminization (Hori et al. 2000; Pace and Brenner 2003; Parks et al. 2004). It could be speculated that multiplicity of highly similar

HINTW proteins provides a means to minimize HINTZ homodimerization, i.e., to ensure that the ratio of HINTZ: HINTW to HINTZ:HINTZ dimers is sufciently high to block testis differentiation. However, the observation of concerted evolution in the HINTW gene family offers an alternative, although not mutually exclusive, explanation to the ampliconic nature of HINTW. Genes in haploid chromosomes are subject to a number of degenerative forces as the absence of recombination leads to accumulation of deleterious mutation and pervasive decay (reviewed in B. Charlesworth and D. Charlesworth 2000). However, the presence of multiple copies can buffer against harmful mutation, increasing the likelihood that at least some members of the gene family remain t (Lahn, Pearson, and Jegalian 2001). Moreover, gene conversion will allow both positive and negative selection to operate on chromosomes with respectively high and low number of functionable copies. The high rate of gene conversion found indicate that this process may be frequent enough to counteract Mullers ratchet. Amplication/conversion should be particularly benecial to genes that have specialized in function subsequent to sex chromosome differentiation. Consistent with this, gene amplication and conversion in HINTW coincides with this being the only gene on the avian W chromosome so far found to have evolved distinct female-specic function. Similarly, gene amplication and conversion is only seen in the malespecic genes of the human Y chromosome. Supplementary Material Supplementary Tables 1 and 2 are available at Molecular Biology and Evolution online (http://www.mbe. oxfordjournals.org/). Acknowledgments We thank Mikael Brandstro m for help with data analysis, Hideki Innan for advice on gene conversion, and Go ran Arnqvist, Anna Qvarnstro m, and Staffan Ulfstrand for useful discussion. Financial support was obtained from the Swedish Research Council. H.E. is a Royal Academy of Sciences Research Fellow supported by the Knut and Alice Wallenberg Foundation. Literature Cited
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Scott Edwards, Associate Editor

Accepted June 1, 2005