Z Chromosome Contains at Least Three Nonlinear Evolutionary Strata

The Chicken (Gallus gallus) Z Chromosome Contains at Least Three Nonlinear Evolutionary Strata

Kiwoong Nam and Hans Ellegren1 Department of Evolutionary Biology, Evolutionary Biology Centre, Uppsala University, SE-752 36 Uppsala, Sweden Manuscript received April 14, 2008 Accepted for publication August 19, 2008

ABSTRACT Birds have female heterogamety with Z and W sex chromosomes. These evolved from different autosomal precursor chromosomes than the mammalian X and Y. However, previous work has suggested that the pattern and process of sex chromosome evolution show many similarities across distantly related organisms. Here we show that stepwise restriction of recombination between the protosex chromosomes of birds has resulted in regions of the chicken Z chromosome showing discrete levels of divergence from W homologs (gametologs). The 12 genes analyzed fall into three levels of estimated divergence values, with the most recent divergence (dS ¼ 0.18–0.21) displayed by 6 genes in a region on the Z chromosome corresponding to the interval 1–11 Mb of the assembled genome sequence. Another 4 genes show intermediate divergence (dS ¼ 0.27–0.38) and are located in the interval 16–53 Mb. Two genes (at positions 42 and 50 Mb) with higher dS values are located proximal to the most distal of the 4 genes with intermediate divergence, suggesting an inversion event. The distribution of genes and their divergence indicate at least three evolutionary strata, with estimated times for cessation of recombination between Z and Wof 132–150 (stratum 1), 71–99 (stratum 2), and 47–57 (stratum 3) million years ago. An inversion event, or some other form of intrachromosomal rearrangement, subsequent to the formation of strata 1 and 2 has scrambled the gene order to give rise to the nonlinear arrangement of evolutionary strata currently seen on the chicken Z chromosome. These observations suggest that the progressive restriction of recombination is an integral feature of sex chromosome evolution and occurs also in systems of female heterogamety.

IFFERENTIATED sex chromosomes bear testi- Lahn and Page (1999) found a distinct divergence D mony of an ancestral autosomal state in the form pattern of human gametologs with respect to the of homologous sequences shared between the X and Y location of genes on the X (but not Y) chromosome. chromosomes. In the absence of recombination, such Specifically, genes located close to the pseudoautosomal homologous sequences will gradually diverge. Sequence region (PAR) on the terminal part of Xp had the lowest comparison of sex chromosome homologs, or game- X–Y divergence while genes on Xq had the highest. tologs (Garcıá-Moreno and Mindell 2000), can thus Between these regions, they found two segments on Xp shed light on both sex chromosome evolution and the with intermediate divergence, giving a total of four molecular evolutionary forces that differentially affect X ‘‘evolutionary strata.’’ Each stratum was characterized by and Y chromosomes (Hurst and Ellegren 1998; rather uniform dS estimates, with stratum 1 on Xq cor- Charlesworth and Charlesworth 2000; Li et al. responding to 240–320 million years of divergence and 2002; Marais and Galtier 2003; Charlesworth et al. stratum 4 at Xp of 20–30 million years of divergence. 2005). For example, for neutral sequences and assuming From an analysis of the finished sequence of the human a molecular clock, the observed divergence between X chromosome, a fifth stratum has subsequently also gametologs can be used to estimate the time when been suggested (Ross et al. 2005). These observations recombination was effectively suppressed between pro- suggest that sex chromosome differentiation occurred tosex chromosomes. While this type of analysis is in a stepwise fashion, with more or less simultaneous increasingly difficult for noncoding sequences as di- cessation of recombination in large blocks of the pro- vergence time increases, the rate of synonymous sub- tosex chromosomes (Charlesworth et al. 2005). stitution in coding sequences (dS) shared between sex In birds, females are the heterogametic sex with Z chromosomes provides a useful means for studies of how and W sex chromosomes; males are ZZ. Studies of sex and when sex chromosome differentiation was initiated. chromosome evolution in birds and other systems with female heterogamety are important because they offer independent replication of observations from X–Y 1Corresponding author: Department of Evolutionary Biology, Evolution- llegren ary Biology Centre, Uppsala University, Norbyva¨gen 18D, SE-752 36 species (E 2000). One such example is the dem- Uppsala, Sweden. E-mail: [email protected] onstration of evolutionary strata on the chicken (Gallus

Genetics 180: 1131–1136 (October 2008) 1132 K. Nam and H. Ellegren gallus) Z chromosome (Ellegren and Carmichael Genome Sequencing Consortium 2004). This translates into 2001; Lawson Handley et al. 2004), suggesting that a mean lineage-specific rate of 2.6 3 109/site/year since the discontinuous recombination suppression over large split of the lineages leading to chicken and humans (divergence is twice the mean lineage-specific rates). It has been chromosomal segments is a general feature of sex suggested that chicken and turkey (Meleagris gallopavo) di- chromosome evolution, irrespective of male or female verged 30 MYA (Dimcheff et al. 2002). Axelsson et al. (2004) heterogamety. However, information on the evolution- observed 10% divergence between chicken and turkey auto- ary history of avian sex chromosomes is sparse, with only somal introns, which corresponds to lineage-specific rates of 3 9 five incomplete sequenced chicken genes analyzed by 1.8 10 . A preliminary analysis of orthologous autosomal single-copy coding sequences of chicken and turkey indicates Lawson Handley et al. (2004). Here, we present an 9 that dS is 0.08, giving a rate of 2 3 10 (data not shown). Due to extended analysis of how and when the avian Z and W male-biased mutation, the higher mutation rate in the male chromosomes diverged, on the basis of data from than in the female germline (Ellegren 2007), neutral Z and an additional seven gametologous gene pairs shared W chromosome sequences will evolve with different rates. The between these chromosomes. The new analysis re- Z chromosome is in the male germline two-thirds of the time, and thus, assuming a 1:1 sex ratio, its mutation rate is two-thirds veals at least three evolutionary strata on the chicken of the male mutation rate plus one-third of the female rate. Z chromosome that, in contrast to the situation on The W chromosome is only transmitted through the female the human X chromosome, are not linearly ordered germline and has therefore the female mutation rate. Previous with increasing divergence by increasing distance work in birds has indicated that the male mutation rate is two to artosch a¨rlid from the PAR. four times higher than the female rate (B -H et al. 2003; Axelsson et al. 2004; Berlin et al. 2006). In galliforms, the Z chromosome rate is slightly higher than the autosomal rate, whereas the W chromosome rate is about half the autosomal rate (Axelsson et al. 2004). If we assume, from MATERIALS AND METHODS the estimates mentioned above, an autosomal lineage-specific 9 Gametologous genes: W-linked chicken genes were ob- rate of 2.5 3 10 , the combined rate of Z–W divergence may be 9 9 tained from three sources. First, we extracted all genes 3.8 3 10 (2.6 1 1.2 3 10 ). This rate was used to estimate assigned to the W chromosome in the chicken genome divergence times between gametologs on the basis of estimates assembly version 2.1 (May 2006) (http://www.ensembl.org/ of dS. Gallus_gallus/index.html). Second, we used published information on two W-linked genes from Wahlberg et al. (2007). Third, we surveyed chicken microarray expression data (Ellegren et al. 2007) from male and female somatic and RESULTS reproductive tissue for genes of unknown chromosomal The most recent chicken genome assembly contains location consistently showing log2 fivefold higher expression level in females than in males, representing putative W eight genes on the W chromosome that have homolo- chromosomal genes. W linkage of these genes was subse- gous sequences on chromosome Z (ATP5A1, CHD1, quently confirmed by PCR amplification of male and female HINT, KCMF1, NIPBL, SMADS, SPIN, and UBAP2). Two DNA (see supplemental information). additional genes, ZFR and ZNF532, have recently been The sequences of all W-linked genes were BLASTed to the chicken genome sequence, which in all cases revealed a closely reported to map to the chicken W chromosome related paralog (gametolog) on the Z chromosome. Map (Wahlberg et al. 2007). Moreover, from chicken micro- location (physical position) of these genes on the Z was taken array data (Ellegren et al. 2007) we identified two from the genome assembly. genes, MIER3 and hnRNPK, which consistently show Estimates of divergence: The full coding sequences for strong female-biased expression across tissues, indica- chicken genes were downloaded through BIOMART (http:// www.biomart.org). After translation to protein sequences, tive of W-linkage, confirmed by PCR with W-specific gametologous gene pairs were aligned by ClustalW and then primers (supplemental Figure 1). A closely related gene checked by eye, and the corresponding DNA sequence align- copy on the Z chromosome was also identified for the ments were then used for further analysis. The synonymous four latter genes by BLAST searches (percentage amino (dS) and nonsynonymous (dN) divergence values were esti- acid identity of 95.6% for ZFR, 89.6% for ZNF532, 92.0% mated by the maximum likelihood method, using CODEML in the PAML package version 3.15 (Yang 1997), after removing for MIER3, and 99.3 for hnRNPK). In analogy with the poorly aligned sequences. Alignments are available upon re- nomenclature for gametologous gene pairs on the avian quest. Our divergence estimates differ from those of Lawson sex chromosomes (Ceplitis and Ellegren 2004), we Handley et al. (2004), because (although we used the same refer to these gene pairs as ZFRW/ZFRZ, ZNF532W/ sequences) we used a different alignment method (aligning ZNF532Z, MIER3W/MIER3Z, and hnRNPKW/hnRNPKZ. protein rather DNA sequences) and calculated divergence estimates differently (using maximum likelihood rather than The conservation between gametologs is generally the Nei and Gojobori method with Jukes–Cantor correction). high, with dN (the non-synonymous substitution rate) Estimating divergence times: The fossil record of birds is in the range of 0.003–0.068 (Table 1, and dN/dS of poor and hence there are few data points available for 0.01–0.32). The only exception is HINT for which the W- calibrating an avian molecular clock. Birds and mammals edges linked gametolog HINTW is known to have evolved diverged 320 million years ago (MYA) (H 2002). Sub- eplitis llegren stitution rates clearly vary among avian genes (Webster et al. rapidly under positive selection (C and E 2006); in a comparison of .7500 chicken–human orthologs, a 2004). Silent divergence (dS) ranges from 0.12 to 0.57 median dS of 1.66 was obtained (International Chicken (Table 1). Divergence estimates cluster in three dif- Evolutionary Strata on the Chicken Z 1133

TABLE 1 Data on gametologous genes shared between the chicken Z and W chromosomes

Position Gene Divergence on Z length time Gene pair Ensembl ID (Z/W) (kb) (bp) dS dN (MYA) ZNF532Z/ZNF532W ENSGALG00000002852/ ... 14003 747 2,974 0.210 0.065 55 SMAD2Z/SMAD2W a ENSGALG00000014697/ ... 10056, 1,290 1,326 0.213 0.005 56 14184 ATP5A1Z/ATP5A1W ENSGALG00000014644/ ... 1756 1,938 1,192 0.202 0.033 53 UBAP2Z/UBAP2W ENSGALG00000013809/ ... 5785 6,876 3,166 0.215 0.068 57 ZFRZ/ZFRW ENSGALG00000003235/ ... 14545 9,104 3,069 0.180 0.024 47 NIPBLZ/NIPBLW a ENSGALG00000003605/ ... 13312, 10,720 5,320 0.198 0.033 52 14715, 22678, 21294 MIER3Z/MIER3W ENSGALG00000014721/ ... 14584 16,837 604 0.378 0.061 99 hnRNPKZ/hnRNPKW ENSGALG00000012591/ ... 14366 39,553 1,281 0.281 0.004 74 SPINZ/SPINW ENSGALG00000014916/ ... 14641 42,572 444 0.269 0.003 71 HINTZ/HINTW ENSGALG00000000428/ ... 22685 44,169 327b 0.569 0.317 150 CHD1Z/CHD1W ENSGALG00000014642/ ... 15278 50,156 3,567 0.502 0.050 132 KCMF1Z/KCMF1W ENSGALG00000015391/ ... 14441 52,658 1,144 0.301 0.040 79 Position is the location on the Z chromosome (in kilobases) in the chicken May 2006 genome assembly. ID, identiﬁcation. a The complete coding sequence of these W-linked genes is not assembled in the chicken genome and are therefore represented by different Ensembl identities. b Due to a high rate of adaptive evolution in HINTW (Ceplitis and Ellegren 2004), including insertions and deletions, the complete coding sequences of HINTW and HINTZ could not be aligned.

ferent groups, corresponding to dS ¼ 0.18–0.22 (six the lineage leading to the chicken seems to have erased genes), 0.28–0.36 (four genes), and 0.50–0.52 (two the orderly pattern to give a nonperfect correlation genes) (Figure 1). These genes’ dS values differ signifi- between gametologous divergence and position on the cantly. In post hoc tests, the group located in the Z region Z chromosome. Critical to this interpretation is the from 1–11 Mb (stratum 3) have significantly lower distal location and relatively moderate divergence of divergence than the group located more distally (stra- KCMF1Z/KCMF1W. One potential alternative possibil- tum 2) (t ¼ 5.09, P ¼ 0.0009, two-tailed). The third ity would be that this gene pair has undergone gene group (stratum 1), despite being located among the conversion that homogenized the sequences of other- latter genes, have a significantly higher dS and thus wise independently evolving Z and W chromosomes being the earliest region to have lost recombination (t ¼ copies.Geneconversionbetweendifferentiatedsexchro- 5.42, P ¼ 0.0056). We therefore conclude that the mosome gametologs seems rare but has been reported different regions stopped recombining at three differ- for several mammals (Pecon Slattery et al. 2000; Ross ent times. et al. 2005). Subsequent to the formation of the two oldest clusters GC content and recombination rate tend to be or strata (strata 1 and 2), a chromosomal inversion in positively correlated in vertebrate genomes (Galtier 2003; Meunier and Duret 2004), including in birds (Webster et al. 2006), due to biased gene conversion. For sex chromosomes, this leads to the predictions of the highest GC for genes that recombine in both sexes (the pseudoautosomal region), intermediate GC for genes that recombine in just one sex (X linked/Z linked), and lowest GC for nonrecombining genes (Y linked/W linked), predictions that are supported by empirical data (Iwase et al. 2003; Backstro¨m et al. 2005). A comparison of GC content in gametologous genes can be used to reveal possible cases of gene conversion (Marias and Galtier 2003). Figure 2 shows the GC content of all gametologs on the chicken Z and W chromosomes. All Z-linked gene sequences, except discussion Figure 1.—Distribution of gametologous genes on the HINTZ (see ), have higher GC3 than their chicken Z chromosome and the corresponding Z–W diver- W-linked gametologs. This suggests that interchromo- gence (dS) with 95% confidence intervals. somal gene conversion has not played an important role 1134 K. Nam and H. Ellegren

Figure 2.—GC content at third codon positions (GC3) of gametologous gene pairs on the chicken Z (open bars) and W (solid bars) chromosomes. Gene pairs are given in the order in which they occur on the Z chromosome.

in the evolution of avian gametologs since they ceased to et al. 2005). Two possible, but not mutually exclusive, recombine. The high GC of HINTW is in accordance with mechanisms for such segmental steps of sex chromo- the fact that while HINTZ is a single-copy gene, HINTW some divergence are inversions on Y (W) and recombi- has been amplified on the avian W chromosome to be- nation restriction without inversions. Neither of these come a multicopy gene (in the chicken there are 40 alternatives can currently be tested, as the poor assembly copies, Hori et al. 2000) and undergoes frequent intra- of the repeat-rich chicken W chromosome precludes chromosomal gene conversion on the W (Backstro¨m gene-order analysis, and no antagonistic effects of W et al. 2005). HINTW became amplified after the original genes are known. When a W chromosome physical map HINT gene ceased to recombine, hence all HINTW becomes available, comparisons with the Z gene order copies are orthologs to HINTZ. should reveal any inversions that have occurred. Divergence estimates for gametologous genes in the We cannot exclude the possibility that additional strata three different evolutionary strata on the chicken Z exist in those regions where we lack data from gametol- chromosome can be used to estimate when recombina- ogous genes (17–39 and 53–75 Mb, respectively). More- tion between homologs ceased. As diverging sex chro- over, it is theoretically possible that the heterogeneity in mosomes are differentially affected by male and female divergence estimates seen within each inferred stratum mutation rates, we assumed a molecular clock that took reflects the existence of additional strata within these into account sex-specific mutation rates for the Z and regions. However, the stochastic variation in divergence the W chromosome lineage (3.8 3 109/site/year; see estimates from finite sequences coupled with mutation materials and methods). This gives estimates of rate variation on a local scale, as previously demonstrated divergence times for stratum 1 of 132–150 MYA; for for the avian sex chromosomes (Berlin et al. 2006), seem stratum 2, 71–99 MYA; and for stratum 3, 47–57 MYA, to be more likely explanations to such heterogeneity. In respectively (Table 1). any case, the interpretation of three evolutionary strata on the chicken Z chromosome should be seen as a minimum number. Moreover, although divergence esti- DISCUSSION mates cluster in three different intervals, we cannot The evolutionary history of genes shared between the formally distinguish between a process of discrete events chicken Z and W chromosomes is consistent with a of cessation of recombination and a process of more process of sex chromosome evolution including arrest of gradual recombination restriction. recombination at different times. Moreover, the con- In mammals, the evolutionary strata identified on the sistent observation of a Z-linked gametolog for genes on human X chromosome are also seen on the mouse X the chicken W chromosome suggests that the protein- chromosome (Sandstedt and Tucker 2004). However, coding content of avian W largely derives from a de- these strata are not ordered linearly along the mouse X, generating protosex chromosome, without the addition similar to what we observe for the chicken Z. This has of autosomal sequences, through processes that include been explained by X chromosome rearrangements in translocation or transposition. The consecutive expan- the rodent lineage subsequent to the split from pri- sions of the nonrecombining region on the avian Z mates, breaking up the strict correlation between di- chromosome mirrors the situation in both mammals vergence of X–Y gametologs and physical position on the (Lahn and Page 1999; Sandstedt and Tucker 2004; X(Sandstedt and Tucker 2004). A similar explanation Ross et al. 2005) and dioecious plants of the genus Silene is also reasonable for birds. While the body of evidence (Filatov 2005; Nicolas et al. 2005; Zluvova et al. 2005; suggests that the gene content of the avian Z chromo- Bergero et al. 2007), two systems with male heterogam- some has remained conserved during the evolution of ety. The observation of this pattern in highly divergent extant birds (Shetty et al.1999;Backstro¨m et al. 2006; phylogenetic lineages of male as well as female hetero- Itoh et al. 2006), comparative mapping indicates that gamety suggests that progressive restriction of recombi- intrachromosomal rearrangements have occurredseveral nation generating evolutionary strata is an inherent times during avian Z chromosome evolution (Shibusawa theme of sex chromosome evolution (Charlesworth et al. 2004a,b; Backstro¨met al. 2006; Griffin et al. 2007), Evolutionary Strata on the Chicken Z 1135 including within Galliformes (chicken and its allies). genes in the nonrecombining region of W will decrease The scrambled location of gametologous genes on the with the time since recombination stopped. The density chicken Z chromosome, with respect to Z–W divergence, of remaining gametologs shared between Z and W is consistent with this and suggests at least one inversion should then decrease with the age of the evolutionary event that distinguishes the contemporary gene order in strata, as we observe. A similar pattern is seen on the the chicken from the ancestral organization of the chro- human X chromosome (Lahn and Page 1999). mosome. One possibility is that a fragment including The observed pattern of avian sex chromosome HINTZ, CHD1Z,andKCMF1Z was inverted after the organization is similar to that seen in male heteroga- evolution of strata 1 and 2. However, there are several metic animals and plants. Interestingly, the three most alternative scenarios that cannot be excluded. well-studied systems with respect to the formation of sex The estimated times of origin of evolutionary strata chromosomal strata represent three different temporal on the chicken Z chromosome are of relevance for scales of sex chromosome evolution. Mammalian sex temporal aspects of avian evolution. Highly differenti- chromosome evolution was initiated 320–240 MYA ated sex chromosomes are characteristic of birds from (stratum 1; but see Wallis et al. 2007, who place the the dominant clade of contemporary bird lineages, the start at 210–180 MYA, and Potrzebowski et al. 2007) Neognathae (everything but ratites and tinamous, and was followed by the formation of a second stratum which belong to Paleognathae). In Paleognathae, the 170–130 MYA. In contrast, in Silene sex chromosomes Z and W chromosomes have largely remained recom- evolved ,10 MYA. The avian sex chromosomes are bining, including HINT, CHD1, and other genes that do intermediate, with Z and W starting to diverge 150 not recombine in the chicken (Nishida-Umehara et al. MYA. The three systems thus offer possibilities for 2007). As we estimate the age of stratum 1 to 132–150 comparative studies of sex chromosome evolution on MYA, the Neognathae and Paleognathae in this view different time scales. should have diverged earlier than this. The estimated Niclas Backstro¨m, Judith Mank, Deborah Charlesworth, and two divergence time between Neognathae and Paleogna- anonymous reviewers are acknowledged for helpful comments. thae is, however, slightly later (120 MYA) (Van Tuinen Financial support was obtained from the Swedish Research Council. and Hedges 2001), but the discrepancy is small and is probably due to the uncertainty of rate of the molecular clock. LITERATURE CITED Our previous work showed that genes from stratum 1 Axelsson, E., N. G. 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