GENETICS | INVESTIGATION
Conservation of Regional Variation in Sex-Specific
Sex Chromosome Regulation
Alison E. Wright,1 Fabian Zimmer, Peter W. Harrison, and Judith E. Mank
Department of Genetics, Evolution and Environment, University College, London WC1E 6BT, United Kingdom
ABSTRACT Regional variation in sex-specific gene regulation has been observed across sex chromosomes in a range of animals and is
often a function of sex chromosome age. The avian Z chromosome exhibits substantial regional variation in sex-specific regulation,
where older regions show elevated levels of male-biased expression. Distinct sex-specific regulation also has been observed across the
male hypermethylated (MHM) region, which has been suggested to be a region of nascent dosage compensation. Intriguingly, MHM
region regulatory features have not been observed in distantly related avian species despite the hypothesis that it is situated within the
oldest region of the avian Z chromosome and is therefore orthologous across most birds. This situation contrasts with the conservation
of other aspects of regional variation in gene expression observed on the avian sex chromosomes but could be the result of sampling
bias. We sampled taxa across the Galloanserae, an avian clade spanning 90 million years, to test whether regional variation in sexspecific gene regulation across the Z chromosome is conserved. We show that the MHM region is conserved across a large portion of
the avian phylogeny, together with other sex-specific regulatory features of the avian Z chromosome. Our results from multiple lines of
evidence suggest that the sex-specific expression pattern of the MHM region is not consistent with nascent dosage compensation.
KEYWORDS sex-biased gene expression; male hypermethylated region
S
EX chromosomes exhibit extensive sex-specific gene regulation in many animals, largely resulting from two independent forces. First, the reduced gene content on the sex-limited
Y or W chromosome observed in many species (Bachtrog 2013)
leads to a reduction in gene dose in the heterogametic sex for
X- or Z-linked genes. Because gene expression is often a function of gene dose, this dosage effect (Zhang et al. 2013) means
that many X- or Z-linked loci show differences in gene expression between males and females. Variation in gene dose is
often thought to be detrimental, and different mechanisms of
dosage compensation have evolved to address the differences
in sex chromosome dose in some organisms (Mank 2013).
Dosage compensation mechanisms in several organisms show
extensive regional variation, with some regions exhibiting less
complete compensation than other regions on the therian
(Carrel and Willard 2005) and stickleback (Schultheiß et al.
2015) X chromosomes.
Copyright © 2015 by the Genetics Society of America
doi: 10.1534/genetics.115.179234
Manuscript received June 10, 2015; accepted for publication July 27, 2015; published
Early Online August 4, 2015.
Supporting information is available online at www.genetics.org/lookup/suppl/
doi:10.1534/genetics.115.179234/-/DC1
1
Corresponding author: Department of Genetics, Evolution and Environment, Darwin
Building, University College, London WC1E 6BT, UK. E-mail: [email protected]
Second, independent of gene dose differences, sex chromosomes are also inherited unequally between the sexes,
leading to distinct evolutionary forces relative to the autosomes (Rice 1984; Charlesworth et al. 1987). In particular,
conflicting selection on males and females results in unequal
sex-specific selection pressures acting on the sex chromosomes. This, in turn, influences rates of sequence and expression evolution (Vicoso and Charlesworth 2009; Meisel and
Connallon 2013). Older sex chromosome regions, where recombination between the Z-W orthologs was halted earlier,
have been exposed to these selection pressures for a greater
period of time. This cumulative nature of sex-specific selection
can lead to regional differences in sex-specific gene regulation
based on regional age (Wright et al. 2012; Wang et al. 2014).
The avian Z chromosome, which is conserved across all
extant birds, is particularly interesting in the study of regional
differences in sex-specific gene regulation. All avian species
examined thus far show incomplete dosage compensation
(Ellegren et al. 2007; Itoh et al. 2007; Wolf and Bryk 2011;
Wright et al. 2012; Moghadam et al. 2013; Uebbing et al.
2013; Wang et al. 2014). As a result, most of the Z chromosome exhibits male-biased expression (Ellegren et al. 2007).
However, there is extensive variation in the degree of male
bias in expression across the chromosome. Previous work in
Genetics, Vol. 201, 587–598 October 2015
587
Figure 1 Taxonomic distribution of MHM region. Shown are the evolutionary
relationships and relative branch lengths (from birdtree.org) of taxa in which
presence (purple) or absence (black) of the MHM region has been examined.
Studies used a combination of expression, methylation, and sequence orthology data. We found that sex-specific regulatory variation in the MHM region is
conserved across the Galloanserae (species names in this study are highlighted
in gray), and this, together with preliminary evidence in the plover (highlighted
in light purple), suggests that the MHM regulatory pattern was lost secondarily in the passeriform birds. 1Itoh et al. (2007); 2Wang et al. (2014);
3Uebbing et al. (2013); 4Wolf and Bryk (2011); 5Moghadam et al.
(2013); 6this study; 7Melamed and Arnold (2007); 8Mank and Ellegren
(2009); 9Teranishi et al. (2011); 10Itoh et al. (2011).
the chicken identified the male hypermethylated (MHM) region, located between 25 and 35 Mb on the Z chromosome, as
a region with extreme sex-specific regulatory effects (Melamed
and Arnold 2007). In addition to hypermethylation in males,
which decreases average expression in this sex, the MHM locus
encodes a female-specific noncoding RNA (ncRNA) that accumulates around the site of transcription in female chickens
(Teranishi et al. 2001). There is also evidence for femalespecific acetylation of histones in the chromatin surrounding
this area, which is also typically associated with increased
gene expression in females (Bisoni et al. 2005).
The regulatory landscape of the MHM region is interesting
for several reasons. The pronounced deficit of male-biased
genes, together with the role of ncRNAs, such as Xist, in dosage
compensation mechanisms in other species (Deng and Meller
2006; Rens et al. 2010), leads to the suggestion that the MHM
region might represent a region of nascent dosage compensation (Melamed and Arnold 2007). Given that it is situated
within the oldest portion of the avian sex chromosomes (Zhou
et al. 2014), it may be that selection for dosage compensation
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A. E. Wright et al.
has simply had longer to act on this region, in a similar way to the
more complete dosage compensation on the older regions of the
therian X chromosome (Carrel and Willard 2005). In contrast,
on the basis of coexpression patterns, others have suggested that
the MHM locus is a regulator of DMRT1 (Yang et al. 2010b;
Caetano et al. 2014), the major avian sex-determining gene
(Smith et al. 2009), which is located nearby on the Z chromosome. Therefore, the distinct sex-specific regulatory pattern of
genes in this region instead may reflect an important role in
sex-specific fitness and sexual differentiation.
Furthermore, older regions of the Z chromosome show
elevated levels of male-biased expression in the chicken
(Wright et al. 2012). Although the regions of the sex chromosomes stopped recombining independently across the
avian phylogeny (Zhou et al. 2014), the degree of male bias
is also associated with sex chromosome age in distantly related avian species (Wang et al. 2014), suggesting that the
effect is broadly conserved. This is in sharp contrast to the
MHM region, where sex-specific regulation has been observed only in the chicken (Teranishi et al. 2001; Melamed
and Arnold 2007). Studies in zebra finches, flycatchers,
crows, emus, and ostriches (Itoh et al. 2010; Wolf and Bryk
2011; Uebbing et al. 2013; Wang et al. 2014) failed to recover
regional variation in male-biased gene regulation in this portion of the Z chromosome. Therefore, it has yet to be established whether this region is a chicken-specific regulatory
feature of the Z chromosome or is more broadly conserved.
Here we assessed regional variation in gene regulation
across the avian Z chromosome in six species spanning 90
million years. Our results support the growing body of evidence that incomplete compensation is a central feature of
avian sex chromosome evolution (Mank 2013) and that
broadly consistent patterns of masculinization in gene expression accumulate over time across the Z chromosome.
More important, we show that the MHM region is conserved
across a wide range of avian evolutionary history. Furthermore, our analysis suggests that the MHM region is not a region of nascent dosage compensation.
Materials and Methods
Transcriptome assembly
We previously assembled adult gonad and spleen transcriptomes across six avian species. Detailed methods for the assembly are described elsewhere (Harrison et al. 2015; Wright
et al. 2015), and Illumina reads have been deposited in the
NIH Short Read Archive (PRJNA271731). Briefly, we obtained
RNA sequencing (RNA-seq) data from the adult spleen and
gonad of captive populations of Anas platyrhynchos (mallard
duck), Anser cygnoides (swan goose), Numida meleagris (helmeted guineafowl), Pavo cristatus (Indian peafowl), Meleagris
gallopavo (wild turkey), and Phasianus colchicus (common
pheasant) at the start of their first breeding season. RNA was
sequenced on an Illumina HiSeq 2000 at the Wellcome Trust
Centre for Human Genetics, University of Oxford, resulting in,
Figure 2 Density plots of male:female expression ratio. Density plots of log2 male:log2 female RPKM ratio for genes expressed in the adult gonad (A)
and spleen (B) for each species calculated using kernel density estimation. Density plots for autosomal genes are shown in purple, and median
autosomal log2 male:log2 female expression ratios are shown by a solid purple line. Density plots for Z-linked genes and median expression ratios
are shown in green. For all species, the autosomal density plot is centered near zero (shown by a black dotted line), indicating that autosomal gene
expression is typically unbiased. In contrast, the density plots of Z-linked genes are shifted to the right, indicating that Z-linked genes are typically male
biased. The width of the plot indicates the variation in sex-biased gene regulation, which is greater in the gonad than in the spleen.
on average, 26 million 100-bp paired-end reads per sample.
Data were quality assessed using FastQC v0.10.1 and trimmed
with Trimmomatic v0.22 (Lohse et al. 2012). The Trinity
method (Grabherr et al. 2011) was used to assemble de novo
transcriptomes for each species separately, and expression levels of genes were obtained using RSEM v1.1.21 (Li and Dewey
2011). The isoform with the highest expression level in each
Trinity contig cluster was selected for further analysis, and
ribosomal RNA (rRNA) was removed. We also imposed a minimum expression threshold of 2 FPKM (fragments per kilobase
of exons per million mapped reads) in at least half of any of the
tissues from either sex (Supporting Information, Table S1).
Identification of reciprocal orthologs and
chromosomal location
To maximize our ability to characterize variation in gene
regulation across the Z chromosome and male MHM region,
located at 25–35 Mb (Teranishi et al. 2001; Melamed and
Arnold 2007), we identified pairwise reciprocal orthologs between each species and Gallus gallus (chicken). Full details of
this approach are described in Harrison et al. (2015). Briefly,
chicken cDNA was obtained from Ensembl v73 (Galgal4/
GCA_000002315.2) (Flicek et al. 2013), the longest isoforms
were extracted, and orthologs were identified using BLASTN
v2.2.27+ (Altschul et al. 1990) with a minimum percentage
identify of 30% and an E-value cutoff of 1 3 10210. Reciprocal
orthologs were identified using the highest BLAST score. Because avian genomes exhibit an unusual degree of stability
(Stiglec et al. 2007) and the Z chromosome is highly conserved
across birds (Skinner et al. 2009; Vicoso et al. 2013), chromosomal location was assigned from chicken.
Quantifying regulatory variation across the avian
Z chromosome
Reads from each sample were separately mapped back to the
Trinity contigs using RSEM v1.1.21 (Li and Dewey 2011). Read
counts were extracted from RSEM for each tissue and species
separately and normalized using the trimmed mean of M-values
(TMM) in edgeR (Robinson et al. 2010). A minimum expression filter of 2 RPKM (reads per kilobase of exons per million
mapped reads) in at least half of either sex was applied separately
to each tissue. Because our filtering criteria permit sex-limited
Variation in Sex Chromosome Regulation
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Table 1 Proportion of dosage-compensated genes on the Z chromosome
Spleen
Species
Gonad
No. of Z-linked
genes
Proportion
compensated
Proportion
sex biased
No. of Z-linked
genes
Proportion
compensated
Proportion
sex-biased
421
408
431
430
421
414
0.710
0.706
0.548
0.519
0.589
0.558
0.017
0.017
0.019
0.030
0.033
0.063
494
470
500
507
504
504
0.277
0.215
0.264
0.239
0.260
0.234
0.480
0.568
0.498
0.556
0.512
0.530
Goose
Duck
Guineafowl
Peafowl
Turkey
Pheasant
Chromosomal location is based on synteny with chicken reciprocal orthologs. Dosage compensation is defined as log2 male:log2 female RPKM ratio # 0.5 and $ 20.5. Sexbiased genes were defined as female biased (P , 0.05 and log2 male:log2 female RPKM ratio # 21) and male biased (P , 0.05 and log2 male:log2 female RPKM ratio $ 1).
contigs, we added a small integer (1) to all RPKM values to
allow log2 transformation of genes with zero expression.
A moving average of sex-biased expression (log2 male 2
log2 female RPKM) was calculated across the Z chromosome
for each species using previously optimized parameters
(Mank and Ellegren 2009). Specifically, a moving average
with a window of 30 genes and a shift of 1 gene was calculated using the running function from the gtools package
v.3.4.2 in R (R Core Team 2014). Confidence intervals for
median expression of genes located on the Z chromosome
and within the MHM region (at 25–35 Mb) (Melamed and
Arnold 2007) were calculated using bootstrapping with 1000
repetitions. Wilcoxon tests were used in R to specifically test
whether median expression of the MHM region is significantly
different from the whole Z chromosome.
In order to identify the genes driving the distinct expression
pattern of the MHM region, the most strongly female-biased
genes in the MHM region were removed sequentially until there
was no significant difference in median sex bias (log2 male 2 log2
female RPKM) between the MHM region and the entire Z chromosome. This was conducted using a one-tailed Wilcox test.
Moving averages were recalculated using this filtered data set.
Sequence evolution of genes in the MHM region
For each species, each contig was BLASTed against the orthologous chicken protein sequence using BLASTX v2.2.30+
(Altschul et al. 1990) with a minimum percentage identify of
30% and an E-value cutoff of 1 3 10210. Coding frames were
extracted using BLAST outputs, and contigs with no valid
protein-coding sequence were excluded.
We aligned coding sequences with orthologous chicken
sequences using PRANK v140603 (Loytynoja and Goldman
2005). Genes were removed if the aligned regions were fewer
than 33 amino acids in length. We used the one-ratio model
test (model = 0, NSsites = 0) in the CODEML package in
PAML v4.8 (Yang 2007) to identify the contribution of purifying selection to coding sequence evolution of Z-linked genes.
For each pairwise comparison, we compared the one-ratio
model where v is fixed to equal 1 (expected dN/dS under neutral evolution) to the model where v is estimated, specifying
the following pairwise gene tree (focal Galloanserae species,
chicken). P-values were calculated using the resulting likelihood values with one degree of freedom and corrected for
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multiple tests using the qvalue function in R (R Core Team
2014). The proportions of genes evolving with a significant
contribution of purifying selection were compared between
the Z chromosome and the MHM region using chi-squared
tests. Finally, we calculated median dN/dS for Z-linked and
MHM region genes, and 95% confidence intervals (CIs) were
calculated using bootstrapping with 1000 replicates. Genes
were removed from the analysis if dS . 2, thereby excluding
genes subject to mutational saturation (Axelsson et al. 2008).
Functional gene analysis
We tested whether the MHM region is enriched for gene
function terms compared to the whole Z chromosome using
GOrilla (Eden et al. 2007; Eden et al. 2009). Mouse reciprocal
orthologs were identified using BioMart (Ensembl v80) for
chicken genes. The target list was comprised of MHM-linked
orthologs and the background list of Z-linked orthologs. P-values
were corrected for multiple tests with the Benjamini-Hochberg
method (Benjamini and Hochberg 1995).
Synteny of avian Z chromosome
We assessed synteny of the Z chromosome using the method
described in Dean et al. (2015). Proteomes were obtained from
Ensembl v73 and v79 for chicken and Anolis carolinensis, respectively; the longest isoforms were extracted, and orthologs
were identified using reciprocal BLASTP v2.2.27+ (Altschul
et al. 1990) with an E-value cutoff of 1 3 10210. Synteny between chicken and Anolis was quantified using MCScanX
(Wang et al. 2012) with default values. MC ScanX returns collinear blocks that represent syntenic regions where gene order
is preserved.
Data availability
Illumina reads used in this study are deposited in the NIH
Short Read Archive (PRJNA271731)
Results
We previously assembled adult gonad and spleen transcriptomes across six species within the Galloanserae lineage
(Harrison et al. 2015; Wright et al. 2015). The Galloanserae,
which include both the Galliformes (landfowl) and Anseriformes (waterfowl), form a monophyletic clade that originated 90 million years ago (Van Tuinen and Hedges 2001),
Figure 3 Moving average of
male-biased expression across
the Z chromosome. Moving averages of log2 male:log2 female
RPKM ratio for genes expressed
in the adult spleen for each of
the six Galloanserae species. Positional information on the Z chromosome was taken from chicken
reciprocal orthologs. Moving averages were calculated using consecutive windows of 30 genes,
moving along the Z chromosome
one gene at a time. The two black
vertical dashed lines depict the
boundaries of the MHM region
defined by Melamed and Arnold
(2007).
making it one of the oldest within Aves (Figure 1). Major
genomic rearrangements are infrequent in the avian genome
(Stiglec et al. 2007), potentially resulting from a lack of active
transposons (Toups et al. 2011), and previous work has revealed that Z-chromosome synteny is highly conserved across
the Galloanserae (Skinner et al. 2009). Therefore, we used the
chicken to identify reciprocal pairwise orthologs for each of our
study species, with which we assigned chromosomal location.
This resulted in sequence and expression data for, on average,
421 Z-linked and 7634 autosomal orthologs in the spleen and
497 Z-linked and 9083 autosomal orthologs in the gonad for
each species.
Our results are consistent with previous work demonstrating incomplete dosage compensation in many avian species
(Ellegren et al. 2007; Itoh et al. 2007; Naurin et al. 2011; Wolf
and Bryk 2011; Wright et al. 2012; Moghadam et al. 2013;
Wang et al. 2014). Z-chromosome expression is male biased in
both the gonad and spleen across all of our species (Figure 2
and Figure S1). Specifically, median female expression of the Z
chromosome is significantly lower than female autosomal
expression and male Z-chromosome expression (Table S2 and
Table S3). Furthermore, the proportion of Z-linked dosagecompensated genes (defined as log2 male:log2 female RPKM
ratio # 0.5 and $ 20.5) is higher in the spleen relative to the
gonad (Table 1), consistent with previous findings that geneby-gene (i.e., local) mechanisms of dosage compensation are
most effective in somatic tissue (Mank and Ellegren 2009).
Additionally, we found a much greater proportion of Z-linked
sex-biased genes (defined as P , 0.05 and log2 male:log2
female RPKM ratio # 21 or $ 1) in the gonad relative to the
spleen (Table 1). Our results therefore contribute to the increasing body of evidence that incomplete dosage compensation is a central tenet of avian sex chromosome evolution
(Mank 2013).
Regional variation in Z-chromosome masculinization
To compare regional sex-specific regulation across our study
species, we plotted a moving average of sex-biased expression
across the Z chromosome (Figure 3 and Figure S2). The Z
chromosome is present most often in males and therefore is
Variation in Sex Chromosome Regulation
591
Table 2 Masculinization of the avian Z chromosome
Spleen log2 expression
Gonad log2 expression
Species
Median male bias of
old Z region (95% CI)
Median male bias of
young Z region (95% CI)
Median male bias of
old Z region (95% CI)
Median male bias of
young Z region (95% CI)
Goose
0.409 (0.380–0.472)
0.578 (0.371–0.709)
Duck
0.412 (0.352–0.450)
Guineafowl
0.524 (0.463–0.575)
Peafowl
0.520 (0.480–0.559)
Turkey
0.461 (0.374–0.524)
Pheasant
0.497 (0.402–0.552)
0.243 (0.214–0.289)
P < 0.001
0.292 (0.229–0.340)
P < 0.001
0.425 (0.380–0.491)
P = 0.009
0.464 (0.430–0.498)
P = 0.024
0.396 (0.323–0.454)
P = 0.082
0.398 (0.345–0.466)
P = 0.034
0.320 (0.132–0.603)
P = 0.130
0.355 (0.159–0.645)
P = 0.134
0.517 (0.260–0.766)
P = 0.061
0.569 (0.338–0.814)
P = 0.142
0.563 (0.348–0.780)
P = 0.017
0.478 (0.289–0.710)
P = 0.003
0.578 (0.324–0.819)
0.713 (0.446–0.921)
0.686 (0.487–0.931)
0.774 (0.567–1.018)
0.867 (0.608–1.021)
The 95% confidence intervals (CIs) were calculated by bootstrapping with 1000 repetitions. Significant differences in log2 median expression between the old and young
regions of the Z chromosome are shown in bold and were assessed using one-tailed Wilcox P-values. Male bias is defined as log2 male:log2 female RPKM ratio.
subject to increased masculinizing selection. However, the avian
Z and W chromosomes diverged in a stepwise process, and there
is a considerable difference in the age of these strata (Wright
et al. 2012, 2014; Wang et al. 2014; Zhou et al. 2014). Given
the difference in age across these regions, we expected cumulative exposure to masculinizing selection to differ across the Z
chromosome (Wright et al. 2012).
Recombination between the Z and W ceased independently
in Galliformes and Anseriformes for the youngest region of the
sex chromosomes (Wright et al. 2014), and we can therefore
divide the Z into the old conserved region (regions corresponding to 43–80 Mb on the chicken Z chromosome) and
young independently evolving portion (regions corresponding
to 0–43 Mb on the chicken Z chromosome). We excluded the
MHM region from this analysis. We found that the Z chromosome is masculinized in both the spleen and gonad, where
male-biased expression is greater in the older regions of the Z
chromosome across all species. This difference is significant in
the spleen across all species with the exception of the turkey
and significant in the turkey and pheasant gonad (Table 2).
Conservation of sex-specific regulation in the
MHM region
We uncovered the characteristic valley of male-biased expression associated with the MHM region, defined by previous studies (Melamed and Arnold 2007; Mank and Ellegren 2009) and
located at 25–35 Mb on the chicken Z chromosome (Figure 3),
in all six species. Specifically, in the spleen, we observed a pronounced valley of male-biased expression, resulting from a
deficit of male-biased genes and an excess of strongly femalebiased genes (Figure 3). In fact, male bias is significantly lower
in this region compared to the whole Z chromosome in all species (Table 3). Our results indicate that the MHM region is not
a chicken-specific feature of the Z chromosome but is conserved
at least within the 90-million-year-old Galloanserae (Figure 1).
In contrast, in the adult gonad, we did not observe the
characteristic MHM region male-biased expression valley
(Figure S2 and Figure S3). Male bias is also not significantly
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A. E. Wright et al.
lower in this region than in the whole Z chromosome in any of
the six Galloanserae species (Table 3), consistent with a previous study showing that the MHM region is less pronounced
in the adult gonad (Mank and Ellegren 2009). Sex-specific
regulation of gonadal expression is considerably more variable
than the spleen (Figure 2), and it is possible that this may mask
any distinct gene regulation pattern in the MHM region. However, our findings highlight the variable and tissue-biased
nature of gene regulation in this portion of the Z chromosome.
Is the MHM region a region of dosage compensation?
The MHM region has been proposed previously to represent
regional dosage compensation on the avian Z chromosome
because of the high concentration of female-biased genes
(Melamed and Arnold 2007). Here we used Ohno’s theory of
dosage compensation as a framework to explicitly test the
status of dosage compensation in this region (Ohno 1967).
Ohno’s theory predicts that hemizygosity of the Z chromosome
selects for hypertranscription of Z-linked genes in females to
compensate for the single Z chromosome. This balances expression between the sexes and between the Z chromosome
and autosomes.
Although Ohno’s theory suggests that female expression of
the MHM region should be higher than uncompensated
regions of the Z chromosome, we found no significant difference in female expression between the MHM region and the
whole Z chromosome in any species (Table 4). In contrast, in
the male spleen, we observed significantly lower expression of
the MHM region compared to the whole Z chromosome in four
of six species. Male downregulation is not consistent with
Ohno’s theory of dosage compensation, particularly without
hypertranscription in females (Ohno 1967). In the gonad, we
observe no significant difference in male or female expression
between the MHM region and Z chromosome (Table S4), consistent with our previous findings that male-specific regulation
in the MHM region is not pronounced in adult gonadal tissue.
We conclude that together the deficit of male-biased genes and
enrichment of strongly female-biased genes in this portion of
Table 3 Gene regulation of the MHM region
Spleen
Gonad
Species
Z chromosome log2 median
male bias (95% CI)
MHM region log2 median
male bias (95% CI)
Z chromosome log2 median
male bias (95% CI)
MHM region log2 median
male bias (95% CI)
Goose
0.319 (0.287–0.351)
0.457 (0.285–0.617)
Duck
0.325 (0.290–0.357)
Guineafowl
0.457 (0.408–0.491)
Peafowl
0.491 (0.457–0.510)
Turkey
0.396 (0.353–0.442)
Pheasant
0.419 (0.372–0.466)
0.268 (0.101–0.378)
P = 0.038
0.221 (0.096–0.266)
P = 0.007
0.314 (0.221–0.427)
P = 0.001
0.458 (0.293–0.502)
P = 0.049
0.214 (0.064–0.290)
P = 0.001
0.221 (20.026–0.418)
P = 0.002
0.301 (20.202–0.897)
P = 0.513
0.486 (-0.305–1.023)
P = 0.504
0.483 (-0.221–0.924)
P = 0.182
0.607 (0.222–1.148)
P = 0.573
0.791 (0.133–1.111)
P = 0.579
0.477 (0.032–0.960)
P = 0.288
0.485 (0.313–0.654)
0.585 (0.435–0.771)
0.663 (0.476–0.821)
0.695 (0.541–0.824)
0.665 (0.476–0.768)
95% confidence intervals were calculating by bootstrapping with 1000 repetitions. Significant differences in log2 median expression between the MHM and Z chromosomes
are shown in bold and were assessed using one-tailed Wilcox P-values. Male-bias is defined as log2 male: log2 female RPKM ratio.
the Z chromosome is not likely to represent regional dosage
compensation as defined by Ohno.
Architecture of the MHM region
We used an iterative approach to identify the genes underlying the characteristic valley of male-biased expression in the
spleen MHM region. For each species, we sequentially removed
the most strongly female-biased MHM genes until there was
no significant difference in sex bias (log2 male:log2 female
RPKM ratio) between the MHM region and Z chromosome
as a whole.
We found that a very low proportion of Z-linked genes
(,0.160 in all species) drives the distinct gene-regulatory
pattern we observed (Table S5). Removing these few genes not
only restores male bias in this region to the Z-chromosome
median (Figure 4) but also eliminates any significant difference
between male expression of this region and the Z chromosome
(Figure 5). In contrast, female expression of the MHM region
remains statistically indistinguishable from the Z chromosome,
reinforcing our earlier finding that the MHM region is downregulated in males (Table S5).
Together we identified 16 genes that are female biased in
one or more of our species and contribute to the MHM region
valley of male-specific regulation (Table S6). Specifically, they
cluster between 25 and 28 and 31 and 34 Mb on the chicken Z
chromosome. Of these 16 genes, two have been identified previously as strongly female biased in the chicken (RFLB and
ENSGALG00000018479) (Mank and Ellegren 2009; Nätt
et al. 2014), and half underlie the MHM regulatory valley
in two or more of our species. Taken together, our results indicate a common architecture of the MHM region regulatory
landscape.
Sequence evolution of genes in the MHM region
We used the CODEML package in PAML to identify the contribution of purifying selection acting on Z-linked genes and genes
located in the MHM region (at 25–35 Mb). We found that the
proportion of MHM genes evolving with a significant contribution of purifying selection was consistently higher than the Z
chromosome as a whole, and this difference was significant in
three of the six species Table 5. Of these, all the female-biased
genes we identified as underlying the distinct sex-specific
Table 4 The MHM region is downregulated in males
Z chromosome log2 median expression
MHM region log2 median expression
Species
Female (95% CI)
Male (95% CI)
Female (95% CI)
Male (95% CI)
Goose
3.545 (3.395–3.752)
3.820 (3.708–4.065)
Duck
3.636 (3.489–3.824)
3.982 (3.845–4.149)
Guineafowl
3.306 (3.177–3.435)
3.771 (3.647–3.886)
Peafowl
3.377 (3.178–3.523)
3.789 (3.612–4.008)
Turkey
3.114 (2.908–3.331)
3.509 (3.376–3.722)
Pheasant
3.142 (3.013–3.309)
3.508 (3.372–3.781)
3.146 (2.692–3.996)
P = 0.134
3.336 (2.753–3.642)
P = 0.098
2.965 (2.698–3.619)
P = 0.102
3.390 (2.964–3.809)
P = 0.861
2.664 (2.445–2.943)
P = 0.120
3.075 (2.575–3.423)
P = 0.621
3.434 (2.760–3.855)
P = 0.036
3.586 (3.174–3.974)
P = 0.024
3.074 (2.561–3.745)
P = 0.007
3.629 (3.095–4.028)
P = 0.441
2.903 (2.493–3.286)
P = 0.007
3.015 (2.728–3.717)
P = 0.094
The 95% confidence intervals (CIs) were calculated by bootstrapping with 1000 repetitions. Significant differences in log2 median expression between the MHM region and Z
chromosome are shown in bold and were assessed using one-tailed Wilcox P-values. Expression values are from spleen samples.
Variation in Sex Chromosome Regulation
593
Figure 4 Sex-specific gene regulation in the MHM region. A
deficit of male-biased genes and
a pronounced valley in the moving
averages of log2 male:log2 female
expression ratio (solid black lines)
are characteristic of the MHM region. A limited number of genes
drive the distinct expression pattern of the MHM region (listed in
Table S6), and dotted gray lines
show recalculated moving averages after the exclusion of these
female-biased genes. Expression
values are from the adult spleen.
Physical position of chicken
DMRT1 (ENSGALG00000010160)
and the locus encoding the
MHM region ncRNA are indicated. The dashed vertical lines
depict unbiased expression.
regulation of the MHM region are evolving under purifying
selection, with the exception of one gene in the pheasant
(Table S7).
Functional gene analysis
Of the 16 MHM genes we identified (Table S6), one is known
to play a role in sexual differentiation. Previous work has
shown that VLDL encodes a receptor that is important for the
transport of plasma lipoproteins, yolk synthesis, and avian
ovarian follicle development (Barber et al. 1991; Wang et al.
2013; Hu et al. 2014).
Given the strong female-biased expression of these MHM
genes in contrast to the male bias of the Z chromosome, we
conducted a GOrilla analysis (Eden et al. 2007, 2009) to explicitly test whether there was an enrichment of gene-function
terms in this region. We found no significantly enriched gene
ontology terms for the 16 MHM genes when compared to the Z
chromosome. There was significant enrichment in regulatoryregion nucleic acid binding [GO:0001067, P , 0.001, false
594
A. E. Wright et al.
discovery rate (FDR) q-value = 0.271] for the whole MHM
region (at 25–35Mb) when compared to the Z chromosome;
however, this was nonsignificant after multiple-testing
correction.
Synteny of the avian Z chromosome
The chicken sex chromosomes are syntenic with Anolis chromosome 2 (Alfoeldi et al. 2011; Vicoso et al. 2013). We used
MCScanX (Wang et al. 2012) to investigate the fine-scale
synteny of the MHM region and surrounding area. Consistent
with prior work (Wang et al. 2014), we found evidence of
intrachromosomal rearrangement within this region, with evidence of five colinear blocks, syntenic regions where gene
order is preserved, spanning approximately 50 Mb on Anolis
chromosome 2 (Figure 6). One of these colinear blocks, only
3.3 Mb in length, encompasses nine of the MHM genes responsible for the characteristic MHM gene regulation we identified
(Table S6), together with the putative avian sex-determining
gene DMRT1.
Figure 5 MHM region is downregulated in males. Male expression is shown in blue and female expression in red. Solid lines show moving averages of
log2 RPKM calculated using consecutive windows of 30 genes, moving along the Z chromosome one gene at a time. Dotted lines show recalculated
moving averages after the exclusion of the female-biased genes listed in Table S6. Solid horizontal lines depict median Z-chromosome expression.
Expression values are from the adult spleen, and the asterisk indicates that there was a significant difference in expression between the MHM region and
the entire Z chromosome before these female-biased genes were excluded.
Discussion
Regional variation in sex-specific gene regulation has been
observed across the chicken sex chromosomes (Melamed and
Arnold 2007; Mank and Ellegren 2009; Wright et al. 2012; Wang
et al. 2014). However, the extent to which certain features of
regional variation are species specific has not been clear. More
importantly, it was not clear whether some of this regional variation was related to dosage compensation. Here we present a
comparative analysis of Z-chromosome gene regulation across
six species to comprehensively assess conservation of regional
differences in sex-specific gene regulation.
Variation in dosage compensation has been documented
across the mammalian sex chromosomes (Carrel and Willard
2005), where X inactivation is less complete in the younger
portions of the human X chromosome [but see Yang et al.
(2010a)]. The MHM region of the chicken Z chromosome
exhibits an excess of female-biased genes (Mank and Ellegren
2009), and given the pronounced male bias of the Z chromosome as a whole, it has been proposed as a region of dosage
compensation in birds (Melamed and Arnold 2007; Melamed
et al. 2009). However, subsequent work on other avian species
found no evidence of regional MHM region regulatory variation
in the ratites (Wang et al. 2014) or passerines (Itoh et al. 2010;
Wolf and Bryk 2011; Uebbing et al. 2013), raising questions
about whether the MHM region was confined to the chicken
lineage. We found that the MHM region is not a chicken-specific
regulatory feature of the Z chromosome but conserved across
one of the oldest avian clades, the Galloanserae (Figure 1). Our
results are supported by other lines of evidence, including the
presence of MHM ncRNA within the turkey genome (Itoh et al.
2011) and hypermethylation across the MHM region in several
Galliform species (Teranishi et al. 2001). Further work is required to establish whether the MHM region is present outside
the Galloanserae, but preliminary evidence in the plover
(Moghadam et al. 2013) may suggest that the regulatory variation in this region is a prominent feature of Z-chromosome evolution across a large portion of the avian phylogeny. Given that
the plover lies outside the Galloanserae, this could indicate either that the MHM region regulatory pattern was lost secondarily in the passeriform birds or that it has evolved convergently.
Our results are consistent with previous findings (Mank
2013) that birds lack a complete chromosome-wide mechanism of dosage compensation but do not support a role of the
Variation in Sex Chromosome Regulation
595
Table 5 Proportion of genes evolving under purifying selection
Figure 6 Synteny between the chicken Z chromosome and Anolis chromosome 2. Synteny between chicken and Anolis was quantified using
MC ScanX. Consistent with previous work, we found that the chicken Z
chromosome (GG Z) and MHM region (shown in light gray from 25–35 Mb)
are both syntenic with Anolis chromosome 2 (AC 2). We found that the
MHM region is comprised of five collinear blocks (shown in blue and
green), syntenic regions where gene order is preserved, spanning approximately 50 Mb on Anolis chromosome 2. One of these collinear blocks
(shown in blue) encompasses nine of the MHM region genes together with
the avian sex-determining gene DMRT1 (in red) (Table S6).
MHM region as an area of nascent dosage compensation
(Melamed and Arnold 2007). The classic model of dosagecompensation evolution (Ohno 1967) predicts that dosagecompensation mechanisms arise as a consequence of selection
to balance gene dose of the single X or Z chromosome in the
heterogametic sex with the two copies of interacting loci on
the autosomes. In female heterogametic species such as the
birds assessed here, this predicts increased expression in
females on the Z chromosome. However, instead of the expected upregulation in females, we observed downregulation
of the MHM region in males across all six of our species. We
therefore conclude that the distinct gene regulation in the
MHM region does not represent dosage compensation as
defined by Ohno. However, without knowing the ancestral
expression of genes in the MHM region, it is difficult to concretely reject female upregulation if the ancestral baseline
expression of the MHM region was lower than the Z chromosome as a whole.
Furthermore, even if dosage compensation evolves by
another mechanism and not via the specific set of regulatory
steps defined by Ohno, we would still expect equal expression
between males and females for most of the genes. Instead,
inconsistent with dosage compensation, we found that a small
proportion of female-biased Z-linked genes is responsible for
the characteristic MHM region valley of male-biased expression. All these genes were located on the ancestral avian protosex chromosome, and half are located on the same conserved
syntenic block as the major sex-determining gene DMRT1
(Smith et al. 2009). Interestingly, recent work suggests that
the MHM region is in the oldest stratum of the avian Z chromosome (Vicoso et al. 2013; Zhou et al. 2014), and the pronounced female-bias expression we observed, together with
previous work (Barber et al. 1991; Wang et al. 2013; Hu
et al. 2014), may indicate that some of these genes play a role
in female fecundity.
Our failure to observe pronounced sex-specific MHM region regulation in the gonad relative to the rest of the Z
chromosome might initially appear inconsistent with a role
596
A. E. Wright et al.
Species
MHM
region
Z chromosome
Chi-squared
statistic
P-value
Goose
Duck
Guineafowl
Peafowl
Turkey
Pheasant
1.000
1.000
0.959
0.940
0.904
0.923
0.882
0.874
0.861
0.818
0.833
0.841
6.186
7.254
3.803
4.754
1.767
2.481
0.013
0.007
0.051
0.029
0.184
0.115
Significant differences in the proportion of genes evolving under purifying selection
between the MHM region and Z chromosome are shown in bold.
of the MHM region in female reproduction. However, there are
many strongly female-biased genes in the MHM gonad, but
gonadal expression is typically more variable than somatic
tissue expression and may mask distinct gene regulation
patterns in this region. Therefore, further empirical work is
required to verify the role of the MHM region in sexual
differentiation and female reproduction.
Previous work has revealed that male-biased expression
accumulates on the chicken Z chromosome over time (Wright
et al. 2012), likely a result of cumulative exposure to malespecific selection. Consistent with previous work in the zebra
finch and ratites (Wang et al. 2014), we found that malebiased expression is greatest in the older regions of the Z chromosome across all species. Together our findings highlight
the complex nature of selective forces driving variation in gene
regulation across the avian Z chromosome.
Concluding Remarks
We present a comprehensive comparative analysis of regional
variation in sex-specific gene regulation across the avian Z
chromosome. We found that male bias accumulates on the
avian Z chromosome, where older regions are the most sex
biased. Additionally, we showed that the MHM region is not
chicken specific and is conserved across the Galloanserae. Our
evidence concordantly suggests that this region does not
represent regional dosage compensation on the avian sex
chromosomes. Instead, the MHM region may play an important role in sexual differentiation and female fecundity.
Acknowledgments
We thank Rebecca Dean, Stephen Montgomery, Natasha
Bloch, Vicencio Oostra, and two anonymous reviewers for
helpful comments on the manuscript. Sequencing was performed by the Wellcome Trust Centre for Human Genetics
Sequencing Hub and funded by the Wellcome Trust (grant
090532/Z09/Z) and the Medical Research Council (MRC)
Hub (grant G0900747 91070). The authors acknowledge the
use of the University College London (UCL) Unity SMP
Facility and the UCL Legion High Performance Computing
Facility (Legion@UCL). This work was funded by the European Research Council under the Framework 7 Agreement
(grant agreement 260233).
Author contributions: AEW and JEM designed the research
and collected the data. AEW, PWH, and FZ analyzed the
data. All authors wrote the manuscript.
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GENETICS
Supporting Information
www.genetics.org/lookup/suppl/doi:10.1534/genetics.115.179234/-/DC1
Conservation of Regional Variation in Sex-Specific
Sex Chromosome Regulation
Alison E. Wright, Fabian Zimmer, Peter W. Harrison, and Judith E. Mank
Copyright © 2015 by the Genetics Society of America
DOI: 10.1534/genetics.115.179234
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Figure'S1.'Boxplots'of'autosomal'and'Z8linked'expression.'
ACY
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B.#
Z&linked#(female:#dark#red,#male:#dark#blue)#and#autosomal#(female:#light#red,#male:#light#blue)#log2#RPKM#for#genes#expressed#in#the#adult#gonad#(A)#and#spleen#
(B)#are#shown#for#each#of#the#six#Galloanserae#species.#Boxes#show#the#median#and#interquarGle#range,#and#whiskers#extend#to#1.5x#the#interquarGle#range.#For#
all#species#and#both#Gssues,#there#is#a#significance#difference#between#Z&linked#and#autosomal#expression#in#females,#and#between#male#and#female#Z&linked#
expression#(Supplementary#Tables#2#and#3).#This#is#consistent#with#previous#findings#that#birds#lack#a#global#dosage#compensaGon#mechanism.#
##2#S1#######################################################################################################################################A.E#Wright#et#al.#
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Physical position on Z chromosome (Mb)
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Physical position on Z chromosome (Mb)
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Physical position on Z chromosome (Mb)
Figure'S2.'Moving'average'of'male8biased'expression'in'the'gonad'across'the'Z'chromosome.'
Moving#averages#of#log2#male:#log2#female#RPKM#raGo#for#genes#expressed#in#the#adult#gonad#for#each#of#the#six#Galloanserae#species.#PosiGonal#informaGon#
on#the#Z#chromosome#was#taken#from#chicken#reciprocal#orthologs.#Moving#averages#were#calculated#using#consecuGve#windows#of#30#genes,#moving#along#
the#Z#chromosome#one#gene#at#a#Gme.#The#two#black#verGcal#doUed#lines#depict#the#boundary#of#the#MHM#region#defined#by#Melamed#and#Arnold#2007.#
##3#S1#######################################################################################################################################A.E#Wright#et#al.#
2.0
Male:female expression ratio
Male:female expression ratio
2.0
1.0
0.0
-1.0
1.0
0.0
-1.0
20
25
30
35
40
20
Physical position on Z chromosome (Mb)
30
35
40
2.0
Male:female expression ratio
Male:female expression ratio
2.0
1.0
0.0
-1.0
1.0
0.0
-1.0
20
25
30
35
40
20
Physical position on Z chromosome (Mb)
25
30
35
40
Physical position on Z chromosome (Mb)
2.0
Male:female expression ratio
2.0
Male:female expression ratio
25
Physical position on Z chromosome (Mb)
1.0
0.0
-1.0
1.0
0.0
-1.0
20
25
30
35
40
Physical position on Z chromosome (Mb)
20
25
30
35
40
Physical position on Z chromosome (Mb)
Figure'S3.'Sex8specific'regulaBon'in'the'MHM'region'is'less'pronounced'in'the'gonad.'
There#is#no#pronounced#valley#in#the#moving#averages#of#log2#male:#log2#female#gonad#RPKM#raGo#(solid#black#lines)#or#deficit#of#male&biased#genes#
characterisGc#of#the#MHM#region.#This#is#in#contrast#to#moving#averages#of#expression#data#from#the#spleen#(Figure#4).#
##3#S1#######################################################################################################################################A.E#Wright#et#al.#
Table!S1.!Details!of!Trinity!assembly!
Species!
Goose!
Duck!
Guineafowl!
Peafowl!
Turkey!
Pheasant!
No.!of!!Trinity!contigs!
655,375!
595,319!
579,484!
626,521!
662,634!
657,249!
No.!of!!filtered!Trinity!contigs!
44,077!
37,402!
45,437!
54,491!
50,720!
55,985!
!
!
!
!
!
!
!
!
Only!contigs!with!expression!above!2!FPKM!in!at!least!half!of!any!of!the!tissues!from!either!sex!were!
included!in!the!analysis.!rRNA!was!also!removed!(Wright!et!al.!Molecular!Ecology!2015;!Harrison!et!al.!
PNAS!2015).!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
S4!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!A.E!Wright!et!al.!
Table!S2.!Median!expression!in!the!spleen!
!
Species!
Goose!
Duck!
Guineafowl!
Peafowl!
Turkey!
Pheasant!
!
Z!chromosome!
No.!of!
Female!
contigs!
log2!RPKM!!
421!
3.545!
408!
3.636!
431!
3.306!
430!
3.377!
421!
3.114!
414!
3.142!
Male!
log2!RPKM!
3.820!
3.982!
3.771!
3.789!
3.509!
3.508!
Autosomes!
No.!of!
Female!
1
contigs !
log2!RPKM!
7721!
3.959!
7715!
4.196!
7725!
4.006!
7369!
3.998!
7681!
3.845!
7594!
3.935!
Male!
log2!RPKM!
3.945!
4.200!
3.980!
3.978!
3.807!
3.858!
PXvalue!
FZ:!MZ!
FZ:!Fautosomes!
0.001$
0.002$
<0.001$
<0.001!
<0.001!
<0.001!
<0.001!
<0.001!
<0.001$
<0.001!
<0.001!
<0.001!
Chromosomal!location!based!on!synteny!with!chicken!reciprocal!orthologs.!
Significant!differences!between!log2!median!expression!are!shown!in!bold!and!based!on!Wilcox!pXvalues.!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
S5!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!A.E!Wright!et!al.!
Table!S3.!Median!expression!in!the!gonad!
!
Species!
Goose!
Duck!
Guineafowl!
Peafowl!
Turkey!
Pheasant!
!
Z!chromosome!
No.!of!
Female!
contigs!
log2!RPKM!
494!
3.464!
470!
3.517!
500!
3.095!
507!
3.142!
504!
2.991!
504!
3.122!
Male!
log2!RPKM!
4.002!
3.998!
3.682!
3.796!
3.675!
3.816!
Autosomes!
No.!of!
Female!
contigs!
log2!RPKM!
9350!
3.944!
9178!
4.098!
8995!
3.827!
8777!
3.794!
9018!
3.802!
9182!
3.855!
Male!
log2!RPKM!
3.973!
4.121!
3.798!
3.696!
3.776!
3.891!
PXvalue!
FZ:!MZ!
FZ:!Fautosomes!
<0.001$
<0.001$
<0.001$
<0.001!
<0.001!
<0.001!
<0.001!
<0.001$
<0.001$
<0.001!
<0.001!
<0.001!
Chromosomal!location!based!on!synteny!with!chicken!reciprocal!orthologs.!
Significant!differences!between!log2!median!expression!are!shown!in!bold!and!based!on!Wilcox!pXvalues.!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
S6!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!A.E!Wright!et!al.!
Table!S4.!Expression!of!the!MHM!region!in!the!gonad!
!
Species!
!
!
Goose!
!
!
Duck!
!
!
Guineafowl!
!
!
Peafowl!
!
!
Turkey!
!
!
Pheasant!
!
!
Z!chromosome!log2!median!expression!
!
Female
Male!
(95%!CI)!
(95%!CI)!
3.464!
4.002!
(3.309X3.633)! (3.815X4.203)!
3.517!
(3.345X3.691)!
3.998!
(3.850X4.187)!
3.095!
(2.890X3.307)!
3.682!
(3.501X3.877)!
3.142!
(2.939X3.338)!
3.796!
(3.672X3.958)!
2.991!
(2.811X3.159)!
3.675!
(3.551X3.845)!
3.122!
(2.995X3.323)!
3.816!
(3.637X3.989)!
MHM!region!log2!median!expression!
Female!
Male!
(95%!CI)!
(95%!CI)!
3.095!
4.131!
(2.432X4.011)! (3.486X4.416)!
p=0.727!
p=0.963!
3.389!
3.960!
(2.889X3.848)! (3.306X4.441)!
p=0.303!
p=0.593!
2.970!
3.494!
(2.375X3.938)! (3.033X4.314)!
p=0.853!
p=0.642!
3.426!
3.786!
(2.790X3.691)! (3.564X4.553)!
p=0.995!
p=0.538!
3.027!
3.911!
(2.604X3.534)! (3.302X4.394)!
p=0.932$
p=0.515!
3.357!
4.002!
(2.905X3.807)! (3.302X4.648)!
p=0.395!
p=0.467!
95%!confidence!intervals!calculating!by!bootstrapping!with!1000!repetitions.!
Differences!in!log2!median!expression!between!the!MHM!and!Z!chromosomes!were!assessed!using!oneX
tailed!Wilcox!pXvalues.!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
S7!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!A.E!Wright!et!al.!
Table!S5.!A!handful!of!genes!drive!the!distinct!expression!pattern!of!the!MHM!region!
!
Species!
Goose!
!
No./Proportion!!
of!MHM!genes!!
!
removed
1!(0.024)!
!
Duck!
!
4!(0.093)!
!
Guineafowl!
!
7!(0.156)!
!
Peafowl!
!
1!(0.024)!
!
Turkey!
!
7!(0.159)!
!
Pheasant!
!
6!(0.140)!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
MHM!region!log2!median!expression!after!gene!removal
SexXbias!
Female!
Male!
!
!
!
(95%!CI)
(95%!CI)
(95%!CI)
0.277!
3.070!
3.444!
(0.108X0.394)! (2.688X3.745)! (2.786X3.935)!
!
!
!
p=0.062
p=0.104
p=0.062
0.245!
3.342!
3.636!
(0.108X0.279)! (3.054X3.642)! (3.272X3.987)!
!
!
!
p=0.057
p=0.193
p=0.102
0.366!
3.005!
3.452!
(0.294X0.483)! (2.763X3.747)! (2.856X4.136)!
!
!
!
p=0.064
p=0.259
p=0.180
0.460!
3.358!
3.635!
(0.300X0.502)! (2.957X3.776)! (3.173X4.062)!
!
!
!
p=0.079
p=0.947
p=0.603
0.236!
2.565!
2.994!
(0.137X0.310)! (2.412X2.961)! (2.588X3.450)!
!
!
!
p=0.060
p=0.082
p=0.052
0.338!
3.075!
3.037!
(0.141X0.439)! (2.400X3.539)! (2.738X3.970)!
!
!
!
p=0.055
p=0.596
p=0.320
!
95%!confidence!intervals!calculating!by!bootstrapping!with!1000!repetitions.!
Differences!in!log2!median!expression!between!the!MHM!and!Z!chromosome!were!assessed!using!oneX
tailed!Wilcox!pXvalues.!
Expression!values!from!spleen!samples.!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
S8!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!A.E!Wright!et!al.!
Table!S6.!FemaleXbiased!genes!underlying!the!distinct!gene!regulatory!pattern!of!the!
MHM!region!!
!
Chicken!
Ensembl!Gene!ID!
Gene!name!
ENSGALG00000015028!
!
ENSGALG00000010187!
!
ENSGALG00000010158!
!
ENSGALG00000010166!
!
ENSGALG00000015008!
!
ENSGALG00000014995!
!
ENSGALG00000015101!
!
ENSGALG00000015109!
!
ENSGALG00000000438!
!
ENSGALG00000015052!
!
ENSGALG00000005441!
!
ENSGALG00000028142!
!
ENSGALG00000018479!
!
ENSGALG00000026194!
!
ENSGALG00000015036!
!
ENSGALG00000015027!
!
RFLB!
!
SLC1A1!
!
KANK1!
!
VLDL!
!
ZNF366!
!
PGM5!
!
Novel!gene!
!
TJP2!
!
ERMP1!
!
UHRF2!
!
NFIB!
!
PTPLAD2!
!
Novel!gene!
!
Novel!gene!
!
RIC1!
!
JAK2!
!
Chicken!
physical!position!
(chromosome:start)!
Z:!27.5!
!
Z:!26.9!
!
Z:!25.7!
!
Z:!26.4!
!
Z:!25.1!
!
Z:!25.4!
!
Z:!32.2!
!
Z:!34.4!
!
Z:!27.6!
!
Z:!27.8!
!
Z:!31.2!
!
Z:!34.1!
!
Z:!27.2!
!
Z:!26.4!
!
Z:!27.6!
!
Z:!27.4!
!
Contigs!driving!MHM!region!
expression!
(log2!sexXbiased!RPKM!in!spleen)!
Guineafowl! Pheasant! Duck!
X0.708!
X1.474!
X0.478!
Guineafowl! Turkey!
Goose!
X6.538!
X5.510!
X2.824!
Guineafowl! Pheasant!
X0.325!
X0.491!
Guineafowl! Pheasant!
X0.465!
X0.489!
Turkey!!
Pheasant!
X0.470!
X0.443!
Guineafowl! Duck!
X0.402!
X0.906!
Guineafowl! Turkey!
X0.084!
X0.227!
Guineafowl! Duck!
X0.236!
X0.392!
Pheasant!
X0.423!
Pheasant!
X0.670!
Turkey!
X0.134!
Turkey!
X0.252!
Peafowl!
X5.029!
Duck!
X0.744!
Turkey!
X0.130!
Turkey!
X0.112!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
S9!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!A.E!Wright!et!al.!
Synteny!
group!
1!
!
1!
!
1!
!
1!
!
2!
!
1!
!
3!
!
4!
!
1!
!
1!
!
3!
!
5!
!
X!
!
X!
!
1!
!
1!
!
Table!S7.!dN/dS!of!femaleXbiased!genes!underlying!the!distinct!gene!regulatory!
pattern!of!the!MHM!region!
!
Ensembl!Gene!ID!
!
ENSGALG00000015028!
!
ENSGALG00000010187!
!
ENSGALG00000010158!
!
ENSGALG00000010166!
!
ENSGALG00000015008!
!
ENSGALG00000014995!
!
ENSGALG00000015101!
!
ENSGALG00000015109!
!
ENSGALG00000000438!
!
ENSGALG00000015052!
!
ENSGALG00000005441!
!
ENSGALG00000028142!
!
ENSGALG00000018479!
!
ENSGALG00000026194!
!
ENSGALG00000015036!
!
ENSGALG00000015027!
!
!
!
RFLB!
!
SLC1A1!
!
KANK1!
!
VLDL!
!
ZNF366!
!
PGM5!
!
Novel!gene!
!
TJP2!
!
ERMP1!
!
UHRF2!
!
NFIB!
!
PTPLAD2!
!
Novel!gene!
!
Novel!gene!
!
RIC1!
!
JAK2!
dN/dS!
(Likelihood!Ratio)!
Guineafowl!
Pheasant!
0.380$
0.526!
(10.167)!
(3.365)!
Guineafowl!
Turkey!
X!
X!
Guineafowl!
0.173$
(120.339)!
Guineafowl!
0.078$
(87.763)!
Turkey!!
0.133$
(74.769)!
Guineafowl!
0.040$
(73.360)!
Guineafowl!
0.055$
(81.023)$
Guineafowl!
0.151$
(86.904)!
Pheasant!
0.200$
(61.210)$
Pheasant!
0.069$
(38.800)!
Turkey!
<0.001$
(30.744)!
Turkey!
0.209$
(15.364)!
Peafowl!
X!
Duck!
X!
Turkey!
0.091$
(134.645)!
Turkey!
0.042$
(96.626)!
Pheasant!
0.144$
(117.905)!
Pheasant!
0.108$
(85.556)!
Pheasant!
0.087$
(89.360)!
Duck!
0.060$
(114.033)$
Turkey!
0.026$
(85.615)$
Duck!
0.118$
(257.610)!
Duck!
0.283$
(27.883)$
Goose!
0.092$
(96.587)$
!
Genes!evolving!with!a!significant!contribution!of!purifying!selection!are!highlighted!in!bold.!
!
!
S10!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!A.E!Wright!et!al.!
!