Department of Evolutionary Biology, Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden
Correspondence: E-mail: hans.ellegren{at}ebc.uu.se.
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Abstract |
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Key Words: W chromosome adaptive evolution positive selection sex chromosomes birds
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Introduction |
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For birds, it is unclear whether genes on the W chromosome are necessary for female development (a dominant role, cf. the mammalian Y chromosome) or whether the number of Z chromosomes regulates sex differentiation (a dosage effect, cf. Drosophila) (Clinton 1998; Clinton and Haines 1999; Ellegren 2001). Only a few genes have been identified on the avian W chromosome, and most of them have very similar homologs (gametologs) on Z, providing no indication of evolution of female-specific functions (Griffiths and Korn 1997; Carmichael et al. 2000; Ellegren 2000; Fridolfsson and Ellegren 2000; Itho et al. 2001). However, the amino acid sequence of one W-linked gene, HINTW, is distinctly different from that of its Z-linked gametolog, HINTZ (Hori et al. 2000; O'Neill et al. 2000; Pace and Brenner 2003; see Notes added in proofs concerning the nomenclature of these genes). The gene encoding a histidine triad nucleotide binding protein (HINT) is likely to have been present as a single-copy gene also on the ancestral protosex chromosomes of birds. After the differentiation of the avian sex chromosomes into Z and W, initiated some 100 to 170 MYA (dating based on the degree of divergence between gametologous gene pairs on Z and W [Lawson-Handley et al. 2004]), HINT diverged into two independently evolving copies, HINTW and HINTZ (Hori et al. 2000) (see fig. 1). Avian HINTZ is highly conserved; for instance, the chicken HINTZ amino acid sequence shows an overall 87% identity to human HINT and even higher similarity in functional domains (Hori et al. 2000). In contrast, and despite more recent common ancestry, chicken HINTZ shows only 65% amino acid identity to its W-linked gametolog HINTW (Hori et al. 2000). Moreover, the latter lacks a histidine triad (HIT; HisHis
His
where
is a hydrophobic amino acid) found in HINT and other members of the HIT superfamily of proteins (Séraphin 1992; Brenner et al. 1999; Brenner 2002) but contains a unique Leu-rich and Arg-rich region of unknown function (Hori et al. 2000; O'Neill et al. 2000).
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Materials and Methods |
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Phylogenetic Analysis
Maximum parsimony was used to construct phylogenetic trees in PAUP* version 4.0b10 (Swofford 2000). A tree of cDNA sequences of HINTZ and HINTW from both chicken and quail and of HINT from human was constructed using a 399-bp alignment with HINT from fugu as an outgroup (fig. 1). Levels of support were obtained from 1,000 bootstrap replicates. For subsequent molecular evolutionary analyses, an unrooted tree (without fugu) was also constructed (fig. 2). Alignments of cDNA sequences were done using ClustalW (available from http://pbil.ibcp.fr [Thompson, Higgins, and Gibson 1994]) and when necessary edited by eye.
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Ka/Ks ratios were also estimated from pairwise comparisons between sequences (table 1). We used PAML to calculate the likelihood values under a model in which Ka/Ks was fixed at 1 (L0) and under a model in which the Ka/Ks ratio were estimated from the data (L1). The likelihood ratio test statistics (LRT = 2[ln L0 ln L1]) was then compared with the 2 distribution with one degree of freedom to test for significant deviations from neutrality (table 1). Because frameshift mutations occur in HINT genes, the whole gene sequence could not be used in every alignment. Specifically, the reading frame of HINTW differs from other HINT genes in regions marked 1 and 3 in figure 3. For the region marked 2 in figure 3, the reading frame of avian HINTW and HINTZ differs from human HINT. These regions were thus excluded from the corresponding alignments and analyses.
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Results |
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An analysis of Ka/Ks along individual branches (fig. 2a and table 2) revealed high Ka/Ks ratios (1.31 to 1.96) for HINTW branches, indicating positive selection and adaptive molecular evolution towards new function in a female-specific gene. In contrast, HINTZ has been subject to strong purifying selection during avian evolution (Ka/Ks = 0 to 0.13).
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Although our data indicate, albeit not with statistical support, that the avian HINTW gene has been subject to positive selection, it is possible that the gene also contains conserved amino acid positions, subject to purifying selection, that reduce the overall Ka/Ks ratio (Endo, Ikeo, and Gojobori 1996; Nielsen and Yang 1998; Siltberg and Liberles 2002). As a matter of fact, an analysis of the amino acid identity between chicken HINTW and HINTZ has revealed two regions with strong sequence similarity, including an -helix and a C-terminal loop (Hori et al. 2000) (fig. 3). Three-dimensional analysis suggests that these regions are crucial for dimer formation of HINT proteins (Lima et al. 1996), and their conservation in HINTW could indicate that this protein is similarly involved in dimerization. We therefore constructed a phylogenetic tree based on an alignment without the regions encoding the
-helix and the C-terminal loop, and repeated Ka/Ks analyses (fig. 2b). Estimates of Ka/Ks ratios were now very high along the HINTW branches (1.51 to
). Note that a very high Ka/Ks ratio (48.98) was found in the internal branch leading to galliform HINTW sequences, indicating that positive selection in HINTW is not only a recent phenomenon during avian evolution. A pairwise comparison of chicken and quail HINTW without the sequence encoding the
-helix and C-terminal loop gives Ka/Ks = 3.74 (table 1), in this case providing statistical support for deviation from neutrality (LRT = 4.23, df = 1, P = 0.04). We therefore conclude that while parts of avian HINTW are likely to have been constrained by negative selection, other parts show evidence of adaptive molecular evolution.
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Discussion |
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Positive selection is not uncommon among genes involved in male reproduction (Yang and Bielawski 2000; Wyckoff, Wang, and Wu 2000; Tsaur, Ting, and Wu 1998; Lee, Ohta, and Vacquier 1995). Sexual selection arising from sperm competition can be invoked as an important driving force in such cases, but this is not applicable in the case of the female-specific HINTW gene. However, a few mammalian genes related to female reproduction have also shown evidence of adaptive evolution (Swanson et al. 2001; Swanson, Nielsen, and Yang 2003; Civetta 2003). These include autosomally encoded genes with a sex-specific pattern of expression. However, avian HINTW is the first gene restricted to the female genome documented with adaptive evolution.
The avian W chromosome shows intriguingly low levels of nucleotide diversity (Berlin and Ellegren 2001; Montell, Fridolfsson, and Ellegren 2001). For instance, a study of 3.4-kb intronic W chromosome sequence of seven different bird species failed to identify a single segregating site (Montell, Fridolfsson, and Ellegren 2001). This limited genetic diversity is unaccounted for (Ellegren 2003), but the demonstration of positive selection in HINTW now offers a clue. As W is a single segregating unit (with the exception of the pseudoautosomal region), positive selection on any W-linked gene will introduce selective sweeps, reducing genetic diversity at all loci on the chromosome. Strong directional selection on HINTW may represent a causal link between selection and levels of polymorphism on the avian W chromosome.
It is of interest to note that HINTW is the only gene found in multiple copies on a female-specific W chromosome (Hori et al. 2000; O'Neill et al. 2000). This resembles the situation for several testis-specific genes that are amplified on the mammalian Y chromosome (Lahn, Pearson, and Jegalian 2001). Amplification of gene copy number might be a way of counteracting the decay of genes on the sex-limited chromosome (Vogel and Schmidtke 1998; Dechend et al. 2000; Lahn, Pearson, and Jegalian 2001), especially in cases of adaptively evolving genes, but could also be driven by sexual selection. In any case, and in summary, avian HINTW displays several characteristics that make it represent a potential female parallel to the organization and evolution of Y chromosome genes involved in male reproduction and development.
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Note Added in Proofs |
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Subsequent to the acceptance of this paper it was brought to our attention that the genes originally described as PKCI-1 and PKCI-2 in mammals actually do not encode for protein kinase C inhibitors (Charles Brenner, Dartmouth Medical School, Lebanon). Based on the characteristic histidine triad motif in this group of proteins, the name histidine triad nucleotide-binding protein has been suggested (Brenner et al. 1997), with gene symbol HINT. Although not yet consequently used, HINT has recently been introduced as a gene symbol for the Z-linked copy in birds (e.g., http://www.thearkdb.org/).
Accepting HINT and to follow the abovementioned nomenclature for gametologous genes shared between avian Z and W sex chromosomes, we suggest that the ASW|Wpkci|PKCIW gene should be referred to as HINTW, which is thus used in this paper. It follows that the Z copy should be referred to as HINTZ. This nomenclature is not specific for birds, but is also applied for gametologous genes shared between mammalian X and Y (e.g., ZFX|ZFY, SMCX|SMCY, DBX|DBY, etc.) and plant X and Y (SlX1|SlY1, SlX4|SlY4). One possible drawback is that HINTW differs from HINTZ in that it lacks the part of the gene encoding the histidine triad motif although other parts of the gene are conserved. For this reason HINTW does not code for a histidine triad nucleotide-binding protein, and has thus lost its nucleotide binding ability (Pace and Brenner 2003), so it is better seen as coding for a HINT-related protein. One may potentially argue that this difference would resemble the situation for the gametologous gene pair SOX3|SRY of mammals, which constitutes the only exception to the standard nomenclature on X and Y in terms of gene symbols. However, SOX3 and SRY are clearly more differentiated than HINTZ and HINTW and only show conservation in a limited region (the HMG box) so, overall, it would seem reasonable to follow standard nomenclature for HINTZ| HINTW.
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Acknowledgements |
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Footnotes |
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