Download The Chicken (Gallus gallus) Z Chromosome Contains at Least Three

Copyright Ó 2008 by the Genetics Society of America
DOI: 10.1534/genetics.108.090324
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 W of 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.
D
IFFERENTIATED sex chromosomes bear testimony of an ancestral autosomal state in the form
of homologous sequences shared between the X and Y
chromosomes. In the absence of recombination, such
homologous sequences will gradually diverge. Sequence
comparison of sex chromosome homologs, or gametologs (Garcı́a-Moreno and Mindell 2000), can thus
shed light on both sex chromosome evolution and the
molecular evolutionary forces that differentially affect X
and Y chromosomes (Hurst and Ellegren 1998;
Charlesworth and Charlesworth 2000; Li et al.
2002; Marais and Galtier 2003; Charlesworth et al.
2005). For example, for neutral sequences and assuming
a molecular clock, the observed divergence between
gametologs can be used to estimate the time when
recombination was effectively suppressed between protosex chromosomes. While this type of analysis is
increasingly difficult for noncoding sequences as divergence time increases, the rate of synonymous substitution in coding sequences (dS) shared between sex
chromosomes provides a useful means for studies of how
and when sex chromosome differentiation was initiated.
1
Corresponding author: Department of Evolutionary Biology, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18D, SE-752 36
Uppsala, Sweden. E-mail: [email protected]
Genetics 180: 1131–1136 (October 2008)
Lahn and Page (1999) found a distinct divergence
pattern of human gametologs with respect to the
location of genes on the X (but not Y) chromosome.
Specifically, genes located close to the pseudoautosomal
region (PAR) on the terminal part of Xp had the lowest
X–Y divergence while genes on Xq had the highest.
Between these regions, they found two segments on Xp
with intermediate divergence, giving a total of four
‘‘evolutionary strata.’’ Each stratum was characterized by
rather uniform dS estimates, with stratum 1 on Xq corresponding to 240–320 million years of divergence and
stratum 4 at Xp of 20–30 million years of divergence.
From an analysis of the finished sequence of the human
X chromosome, a fifth stratum has subsequently also
been suggested (Ross et al. 2005). These observations
suggest that sex chromosome differentiation occurred
in a stepwise fashion, with more or less simultaneous
cessation of recombination in large blocks of the protosex chromosomes (Charlesworth et al. 2005).
In birds, females are the heterogametic sex with Z
and W sex chromosomes; males are ZZ. Studies of sex
chromosome evolution in birds and other systems with
female heterogamety are important because they offer independent replication of observations from X–Y
species (Ellegren 2000). One such example is the demonstration of evolutionary strata on the chicken (Gallus
1132
K. Nam and H. Ellegren
gallus) Z chromosome (Ellegren and Carmichael
2001; Lawson Handley et al. 2004), suggesting that
discontinuous recombination suppression over large
chromosomal segments is a general feature of sex
chromosome evolution, irrespective of male or female
heterogamety. However, information on the evolutionary history of avian sex chromosomes is sparse, with only
five incomplete sequenced chicken genes analyzed by
Lawson Handley et al. (2004). Here, we present an
extended analysis of how and when the avian Z and W
chromosomes diverged, on the basis of data from
an additional seven gametologous gene pairs shared
between these chromosomes. The new analysis reveals at least three evolutionary strata on the chicken
Z chromosome that, in contrast to the situation on
the human X chromosome, are not linearly ordered
with increasing divergence by increasing distance
from the PAR.
MATERIALS AND METHODS
Gametologous genes: W-linked chicken genes were obtained from three sources. First, we extracted all genes
assigned to the W chromosome in the chicken genome
assembly version 2.1 (May 2006) (http://www.ensembl.org/
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
reproductive tissue for genes of unknown chromosomal
location consistently showing log2 fivefold higher expression
level in females than in males, representing putative W
chromosomal genes. W linkage of these genes was subsequently confirmed by PCR amplification of male and female
DNA (see supplemental information).
The sequences of all W-linked genes were BLASTed to the
chicken genome sequence, which in all cases revealed a closely
related paralog (gametolog) on the Z chromosome. Map
location (physical position) of these genes on the Z was taken
from the genome assembly.
Estimates of divergence: The full coding sequences for
chicken genes were downloaded through BIOMART (http://
www.biomart.org). After translation to protein sequences,
gametologous gene pairs were aligned by ClustalW and then
checked by eye, and the corresponding DNA sequence alignments were then used for further analysis. The synonymous
(dS) and nonsynonymous (dN) divergence values were estimated by the maximum likelihood method, using CODEML in
the PAML package version 3.15 (Yang 1997), after removing
poorly aligned sequences. Alignments are available upon request. Our divergence estimates differ from those of Lawson
Handley et al. (2004), because (although we used the same
sequences) we used a different alignment method (aligning
protein rather DNA sequences) and calculated divergence
estimates differently (using maximum likelihood rather than
the Nei and Gojobori method with Jukes–Cantor correction).
Estimating divergence times: The fossil record of birds is
poor and hence there are few data points available for
calibrating an avian molecular clock. Birds and mammals
diverged 320 million years ago (MYA) (Hedges 2002). Substitution rates clearly vary among avian genes (Webster et al.
2006); in a comparison of .7500 chicken–human orthologs, a
median dS of 1.66 was obtained (International Chicken
Genome Sequencing Consortium 2004). This translates into
a mean lineage-specific rate of 2.6 3 109/site/year since the
split of the lineages leading to chicken and humans (divergence is twice the mean lineage-specific rates). It has been
suggested that chicken and turkey (Meleagris gallopavo) diverged 30 MYA (Dimcheff et al. 2002). Axelsson et al. (2004)
observed 10% divergence between chicken and turkey autosomal introns, which corresponds to lineage-specific rates of
1.8 3 109. A preliminary analysis of orthologous autosomal
single-copy coding sequences of chicken and turkey indicates
that dS is 0.08, giving a rate of 2 3 109 (data not shown). Due to
male-biased mutation, the higher mutation rate in the male
than in the female germline (Ellegren 2007), neutral Z and
W chromosome sequences will evolve with different rates. The
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
of the male mutation rate plus one-third of the female rate.
The W chromosome is only transmitted through the female
germline and has therefore the female mutation rate. Previous
work in birds has indicated that the male mutation rate is two to
four times higher than the female rate (Bartosch-Härlid
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
the estimates mentioned above, an autosomal lineage-specific
rate of 2.5 3 109, the combined rate of Z–W divergence may be
3.8 3 109 (2.6 1 1.2 3 109). This rate was used to estimate
divergence times between gametologs on the basis of estimates
of dS.
RESULTS
The most recent chicken genome assembly contains
eight genes on the W chromosome that have homologous sequences on chromosome Z (ATP5A1, CHD1,
HINT, KCMF1, NIPBL, SMADS, SPIN, and UBAP2). Two
additional genes, ZFR and ZNF532, have recently been
reported to map to the chicken W chromosome
(Wahlberg et al. 2007). Moreover, from chicken microarray data (Ellegren et al. 2007) we identified two
genes, MIER3 and hnRNPK, which consistently show
strong female-biased expression across tissues, indicative of W-linkage, confirmed by PCR with W-specific
primers (supplemental Figure 1). A closely related gene
copy on the Z chromosome was also identified for the
four latter genes by BLAST searches (percentage amino
acid identity of 95.6% for ZFR, 89.6% for ZNF532, 92.0%
for MIER3, and 99.3 for hnRNPK). In analogy with the
nomenclature for gametologous gene pairs on the avian
sex chromosomes (Ceplitis and Ellegren 2004), we
refer to these gene pairs as ZFRW/ZFRZ, ZNF532W/
ZNF532Z, MIER3W/MIER3Z, and hnRNPKW/hnRNPKZ.
The conservation between gametologs is generally
high, with dN (the non-synonymous substitution rate)
in the range of 0.003–0.068 (Table 1, and dN/dS of
0.01–0.32). The only exception is HINT for which the Wlinked gametolog HINTW is known to have evolved
rapidly under positive selection (Ceplitis and Ellegren
2004). Silent divergence (dS) ranges from 0.12 to 0.57
(Table 1). Divergence estimates cluster in three dif-
Evolutionary Strata on the Chicken Z
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TABLE 1
Data on gametologous genes shared between the chicken Z and W chromosomes
Gene pair
ZNF532Z/ZNF532W
SMAD2Z/SMAD2W a
ATP5A1Z/ATP5A1W
UBAP2Z/UBAP2W
ZFRZ/ZFRW
NIPBLZ/NIPBLW a
MIER3Z/MIER3W
hnRNPKZ/hnRNPKW
SPINZ/SPINW
HINTZ/HINTW
CHD1Z/CHD1W
KCMF1Z/KCMF1W
Ensembl ID (Z/W)
ENSGALG00000002852/
ENSGALG00000014697/
14184
ENSGALG00000014644/
ENSGALG00000013809/
ENSGALG00000003235/
ENSGALG00000003605/
14715, 22678, 21294
ENSGALG00000014721/
ENSGALG00000012591/
ENSGALG00000014916/
ENSGALG00000000428/
ENSGALG00000014642/
ENSGALG00000015391/
. . . 14003
. . . 10056,
Position
on Z
(kb)
Gene
length
(bp)
dS
dN
Divergence
time
(MYA)
747
1,290
2,974
1,326
0.210
0.213
0.065
0.005
55
56
...
...
...
...
1756
5785
14545
13312,
1,938
6,876
9,104
10,720
1,192
3,166
3,069
5,320
0.202
0.215
0.180
0.198
0.033
0.068
0.024
0.033
53
57
47
52
...
...
...
...
...
...
14584
14366
14641
22685
15278
14441
16,837
39,553
42,572
44,169
50,156
52,658
604
1,281
444
327b
3,567
1,144
0.378
0.281
0.269
0.569
0.502
0.301
0.061
0.004
0.003
0.317
0.050
0.040
99
74
71
150
132
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
genes), 0.28–0.36 (four genes), and 0.50–0.52 (two
genes) (Figure 1). These genes’ dS values differ significantly. In post hoc tests, the group located in the Z region
from 1–11 Mb (stratum 3) have significantly lower
divergence than the group located more distally (stratum 2) (t ¼ 5.09, P ¼ 0.0009, two-tailed). The third
group (stratum 1), despite being located among the
latter genes, have a significantly higher dS and thus
being the earliest region to have lost recombination (t ¼
5.42, P ¼ 0.0056). We therefore conclude that the
different regions stopped recombining at three different times.
Subsequent to the formation of the two oldest clusters
or strata (strata 1 and 2), a chromosomal inversion in
Figure 1.—Distribution of gametologous genes on the
chicken Z chromosome and the corresponding Z–W divergence (dS) with 95% confidence intervals.
the lineage leading to the chicken seems to have erased
the orderly pattern to give a nonperfect correlation
between gametologous divergence and position on the
Z chromosome. Critical to this interpretation is the
distal location and relatively moderate divergence of
KCMF1Z/KCMF1W. One potential alternative possibility would be that this gene pair has undergone gene
conversion that homogenized the sequences of otherwise independently evolving Z and W chromosomes
copies. Geneconversion betweendifferentiated sexchromosome gametologs seems rare but has been reported
for several mammals (Pecon Slattery et al. 2000; Ross
et al. 2005).
GC content and recombination rate tend to be
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; Backström 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
HINTZ (see discussion), have higher GC3 than their
W-linked gametologs. This suggests that interchromosomal 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
recombine. The high GC of HINTW is in accordance with
the fact that while HINTZ is a single-copy gene, HINTW
has been amplified on the avian W chromosome to become a multicopy gene (in the chicken there are 40
copies, Hori et al. 2000) and undergoes frequent intrachromosomal gene conversion on the W (Backström
et al. 2005). HINTW became amplified after the original
HINT gene ceased to recombine, hence all HINTW
copies are orthologs to HINTZ.
Divergence estimates for gametologous genes in the
three different evolutionary strata on the chicken Z
chromosome can be used to estimate when recombination between homologs ceased. As diverging sex chromosomes are differentially affected by male and female
mutation rates, we assumed a molecular clock that took
into account sex-specific mutation rates for the Z and
the W chromosome lineage (3.8 3 109/site/year; see
materials and methods). This gives estimates of
divergence times for stratum 1 of 132–150 MYA; for
stratum 2, 71–99 MYA; and for stratum 3, 47–57 MYA,
respectively (Table 1).
DISCUSSION
The evolutionary history of genes shared between the
chicken Z and W chromosomes is consistent with a
process of sex chromosome evolution including arrest of
recombination at different times. Moreover, the consistent observation of a Z-linked gametolog for genes on
the chicken W chromosome suggests that the proteincoding content of avian W largely derives from a degenerating protosex chromosome, without the addition
of autosomal sequences, through processes that include
translocation or transposition. The consecutive expansions of the nonrecombining region on the avian Z
chromosome mirrors the situation in both mammals
(Lahn and Page 1999; Sandstedt and Tucker 2004;
Ross et al. 2005) and dioecious plants of the genus Silene
(Filatov 2005; Nicolas et al. 2005; Zluvova et al. 2005;
Bergero et al. 2007), two systems with male heterogamety. The observation of this pattern in highly divergent
phylogenetic lineages of male as well as female heterogamety suggests that progressive restriction of recombination generating evolutionary strata is an inherent
theme of sex chromosome evolution (Charlesworth
et al. 2005). Two possible, but not mutually exclusive,
mechanisms for such segmental steps of sex chromosome divergence are inversions on Y (W) and recombination restriction without inversions. Neither of these
alternatives can currently be tested, as the poor assembly
of the repeat-rich chicken W chromosome precludes
gene-order analysis, and no antagonistic effects of W
genes are known. When a W chromosome physical map
becomes available, comparisons with the Z gene order
should reveal any inversions that have occurred.
We cannot exclude the possibility that additional strata
exist in those regions where we lack data from gametologous genes (17–39 and 53–75 Mb, respectively). Moreover, it is theoretically possible that the heterogeneity in
divergence estimates seen within each inferred stratum
reflects the existence of additional strata within these
regions. However, the stochastic variation in divergence
estimates from finite sequences coupled with mutation
rate variation on a local scale, as previously demonstrated
for the avian sex chromosomes (Berlin et al. 2006), seem
to be more likely explanations to such heterogeneity. In
any case, the interpretation of three evolutionary strata
on the chicken Z chromosome should be seen as a
minimum number. Moreover, although divergence estimates cluster in three different intervals, we cannot
formally distinguish between a process of discrete events
of cessation of recombination and a process of more
gradual recombination restriction.
In mammals, the evolutionary strata identified on the
human X chromosome are also seen on the mouse X
chromosome (Sandstedt and Tucker 2004). However,
these strata are not ordered linearly along the mouse X,
similar to what we observe for the chicken Z. This has
been explained by X chromosome rearrangements in
the rodent lineage subsequent to the split from primates, breaking up the strict correlation between divergence of X–Y gametologs and physical position on the
X (Sandstedt and Tucker 2004). A similar explanation
is also reasonable for birds. While the body of evidence
suggests that the gene content of the avian Z chromosome has remained conserved during the evolution of
extant birds (Shetty et al. 1999; Backström et al. 2006;
Itoh et al. 2006), comparative mapping indicates that
intrachromosomal rearrangements have occurred several
times during avian Z chromosome evolution (Shibusawa
et al. 2004a,b; Backström et al. 2006; Griffin et al. 2007),
Evolutionary Strata on the Chicken Z
including within Galliformes (chicken and its allies).
The scrambled location of gametologous genes on the
chicken Z chromosome, with respect to Z–W divergence,
is consistent with this and suggests at least one inversion
event that distinguishes the contemporary gene order in
the chicken from the ancestral organization of the chromosome. One possibility is that a fragment including
HINTZ, CHD1Z, and KCMF1Z was inverted after the
evolution of strata 1 and 2. However, there are several
alternative scenarios that cannot be excluded.
The estimated times of origin of evolutionary strata
on the chicken Z chromosome are of relevance for
temporal aspects of avian evolution. Highly differentiated sex chromosomes are characteristic of birds from
the dominant clade of contemporary bird lineages, the
Neognathae (everything but ratites and tinamous,
which belong to Paleognathae). In Paleognathae, the
Z and W chromosomes have largely remained recombining, including HINT, CHD1, and other genes that do
not recombine in the chicken (Nishida-Umehara et al.
2007). As we estimate the age of stratum 1 to 132–150
MYA, the Neognathae and Paleognathae in this view
should have diverged earlier than this. The estimated
divergence time between Neognathae and Paleognathae is, however, slightly later (120 MYA) (Van Tuinen
and Hedges 2001), but the discrepancy is small and is
probably due to the uncertainty of rate of the molecular
clock.
Our previous work showed that genes from stratum 1
(HINTZ/HINTW and CHD1Z/CHD1W) and stratum 2
(SPINZ/SPINW) had ceased to recombine prior to the
split of the majority of living Neognathae bird orders
(Ellegren and Carmichael 2001; Lawson Handley
et al. 2004), thought to have occurred prior to the
Cretaceous/Tertiary (K/T) boundary 65 MYA (Cooper
and Penny 1997; Kumar and Hedges 1998; Cracraft
2001; Clarke et al. 2005). With an estimated age of
stratum 2 of 71–99 MYA, this would suggest that the
major avian radiation occurred in late Cretaceous, not
long before the K/T boundary. Moreover, this is consistent with the dating of stratum 3 (47–57 MYA), as
previous work showed that two genes from this region
(ATP5A1 and UBAP2) still recombined when the major
avian lineages split (Ellegren and Carmichael 2001;
Lawson Handley et al. 2004).
One notable observation in the distribution of genes
on the chicken Z chromosome is that the incidence of
genes with a still active (not degenerated) gene copy on
the W is highest in the youngest stratum close to the PAR
at Zp. There are six gametologs out of a total of 155
genes in the first 11 Mb of the Z genome assembly
(approximately stratum 3) compared to a total of six out
of a total of 606 genes in the rest of the chromosome
(P ¼ 0.022, Fisher’s exact test) (the chicken W chromosome is not completely sequenced yet, these 12 genes
represent the gametologs known so far). It seems
reasonable to assume that the number of ‘‘surviving’’
1135
genes in the nonrecombining region of W will decrease
with the time since recombination stopped. The density
of remaining gametologs shared between Z and W
should then decrease with the age of the evolutionary
strata, as we observe. A similar pattern is seen on the
human X chromosome (Lahn and Page 1999).
The observed pattern of avian sex chromosome
organization is similar to that seen in male heterogametic animals and plants. Interestingly, the three most
well-studied systems with respect to the formation of sex
chromosomal strata represent three different temporal
scales of sex chromosome evolution. Mammalian sex
chromosome evolution was initiated 320–240 MYA
(stratum 1; but see Wallis et al. 2007, who place the
start at 210–180 MYA, and Potrzebowski et al. 2007)
and was followed by the formation of a second stratum
170–130 MYA. In contrast, in Silene sex chromosomes
evolved ,10 MYA. The avian sex chromosomes are
intermediate, with Z and W starting to diverge 150
MYA. The three systems thus offer possibilities for
comparative studies of sex chromosome evolution on
different time scales.
Niclas Backström, Judith Mank, Deborah Charlesworth, and two
anonymous reviewers are acknowledged for helpful comments.
Financial support was obtained from the Swedish Research Council.
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Communicating editor: D. Charlesworth