Rapid evolution of male-biased gene expression
Colin D. Meiklejohn*†, John Parsch‡, Jose ´ M. Ranz*, and Daniel L. Hartl*
*Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138; and‡Department of Biology II, Section of Evolutionary
Biology, University of Munich, Luisenstrasse 14, 80333 Munich, Germany
Edited by Eviatar Nevo, University of Haifa, Haifa, Israel, and approved June 17, 2003 (received for review February 5, 2003)
A number of genes associated with sexual traits and reproduction
evolve at the sequence level faster than the majority of genes
coding for non-sex-related traits. Whole genome analyses allow
this observation to be extended beyond the limited set of genes
that have been studied thus far. We use cDNA microarrays to
demonstrate that this pattern holds in Drosophila for the pheno-
type of gene expression as well, but in one sex only. Genes that are
male-biased in their expression show more variation in relative
expression levels between conspecific populations and two closely
monomorphic expression patterns. Additionally, elevated ratios of
interspecific expression divergence to intraspecific expression vari-
evolution may be due in part to natural selection. This finding has
implications for our understanding of the importance of sexual
dimorphism for speciation and rates of phenotypic evolution.
microarray ? intraspecific variation ? interspecific variation ? cDNA
important evolutionary consequences. For example, differential
selection coefficients between the two sexes can lead to stable
genetic polymorphisms or a decline in population mean fitness
(1). It can also drive accelerated rates of phenotypic evolution,
as many morphologies associated with sex and reproduction
diverge more rapidly than other phenotypes (2). Molecular
techniques that provide rapid and quantitative measures of
genotypic and phenotypic variation have extended this pattern to
include accelerated rates of evolution among proteins with
sexual or reproductive functions (3, 4). Since then, most data
supporting this observation have come from homologous nucle-
otide sequences of genes that are associated with sex or repro-
duction. In ciliates, green algae, diatoms, angiosperms, fungi,
and at least four animal phyla, unusually high ratios of nonsyn-
onymous to synonymous substitutions (dN?dS) between species
have been documented in sex-related genes (reviewed in ref. 5).
Some of these genes also show high levels of intraspecific
differentiation (5). In Drosophila, much of this work has focused
on genes that are expressed in testes or accessory glands (e.g.,
refs. 6 and 7), although a high dN?dShas also been observed for
genes expressed in females and components of the sex determi-
nation pathway (8).
Protein coding sequences provide a natural context for study-
ing rates of evolution, as the effect of a given nucleotide
substitution on the polypeptide is predictable, and comparison
between neighboring synonymous and nonsynonymous sites
controls for mutation rate. Because of the lack of an analogous
context for regulatory sequences, the rates and patterns of
evolution in regions of the genome controlling gene expression
are less well understood. Thus, it is not known whether the rapid
rates of evolution among genes associated with sex and repro-
duction holds for gene expression as well. Because a large
proportion of important phenotypic evolution may be the result
of changes in gene expression (9, 10), understanding rates and
patterns of regulatory change within and between species is
nisogamous reproduction is common in many animal and
plant species and can produce a number of conflicts with
critical for a comprehensive picture of biological evolution.
Given the pattern seen for amino acid sequences and morphol-
ogies, we would predict that genes associated with sex should be
evolving faster at the level of gene regulation as well. Indeed,
much of the divergence among proteins in the male reproductive
tract of Drosophila may be attributable to large changes in
protein levels, which is likely due in part to changes in gene
expression (3). To test this prediction, we obtained gene expres-
sion data for ?1?3 of the genome from adult males of eight
strains of Drosophila melanogaster, and from adult males and
females of one strain of D. melanogaster and one strain of
Drosophila simulans. By analyzing intra- and interspecific ex-
pression differentiation within males and the sex-specificity of
evolves more rapidly than in females. Genes that are male-biased
in their expression have on average more intra- and interspecific
divergence in expression than genes with female-biased expres-
sion. Furthermore, comparison of intra- and interspecific dif-
ferentiation suggests that at least some of the excess in diver-
gence among male-biased genes (MBGs) is due to differential
selective pressures acting on the expression of different sex-
biased classes of genes.
Materials and Methods
Fly Strains and cDNA Preparation. Eight strains of D. melanogaster
(three laboratory strains: Canton S, Oregon R, and Hikone R;
an isofemale strain derived from St. Louis; and four lines derived
from Zimbabwe: Zim53, Zim30, Zim29, and Zim2) were raised
on standard medium at 25°C. Adult males were collected up to
24 h after eclosing, separated from females, and allowed to age
an additional 3–4 days. Total RNA was extracted by using
TRIzol reagent (Invitrogen) followed by chloroform extraction
and isopropanol precipitation. Poly(A) RNA was purified by
using the Oligotex Direct mRNA kit (Qiagen, Valencia, CA) and
confirmed to be of high quality with a 2100 Bioanalyzer (Agilent
Technologics, Palo Alto, CA). Two micrograms of poly(A) RNA
was used as a template for SuperScript II reverse transcriptase
(Invitrogen) in the presence of amino-allyl dUTP (Sigma).
Cyanine-3 or cyanine-5 fluorochromes (Amersham Pharmacia)
were incorporated after reverse transcription. Purification of
cDNA and hybridizations were done following a published
protocol (11). Labeled cDNAs were competitively hybridized to
is published as supporting information on the PNAS web site,
www.pnas.org, with the following number of replicates per
strain: Canton S, 13; Oregon R, 5; Hikone R, 3; St. Louis, 3;
Zim53, 11; Zim30, 5; Zim29, 3; Zim2, 3.
cDNA Microarrays. A total of 5,928 clones from the Drosophila
Gene Collection version 1.0 (12) were amplified by PCR with
universal primers, and the products were confirmed by gel
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: MBGs, male-biased genes; FBGs, female-biased genes; UBGs, unbiased
genes; OBGs, ovary-biased genes.
‡To whom correspondence should be addressed. E-mail: email@example.com.
August 19, 2003 ?
vol. 100 ?
electrophoresis. Added to these was a set of 177 separately
amplified controls, each of which was replicated from 1 to 16
times on the array. The PCR products were purified and
mechanically spotted onto polylysine-coated glass slides (11).
The results from these hybridizations have been deposited to the
Gene Expression Omnibus (40) under accession nos. GPL356
Statistical Analysis. Relative gene expression levels were deter-
mined with a Bayesian method (Bayesian analysis of gene
expression levels, BAGEL) (13) from the normalized ratio data
(Supporting Materials and Methods, which is published as sup-
porting information on the PNAS web site). This method
estimates a normalized relative expression level for each strain
and a single variance parameter across all strains from the
Cy5?Cy3 ratios, on a gene-by-gene basis. It also calculates
credible intervals from the stationary distribution of the Markov
chain used to obtain the posterior distribution of the parameters
(eight mean expression levels and one variance). For all pairwise
intraspecific comparisons, unless otherwise stated, the threshold
chosen for statistical significance was P ? 0.01. This threshold
signifies that the relative expression value for a given strain was
greater than (or less than) that of another strain in ?99% of the
samples taken from the posterior distribution.
Sex-biased expression was defined by analysis of a parallel set
of experiments comparing gene expression in adult males and
females from a lab strain of D. simulans and the D. melanogaster
strain Canton S (14). The significance threshold for sex-biased
expression was defined by nonoverlapping 95% credible inter-
vals, which was determined to be equivalent to P ? 0.00025 by
using a randomization approach (Supporting Materials and Meth-
ods). We define MBGs and female-biased genes (FBGs) as those
with significantly different expression between the sexes (in the
same direction) in both D. melanogaster and D. simulans. Un-
biased genes (UBGs) are defined as those clones that show no
significant expression between the two sexes in either species.
For a given gene, we describe intraspecific expression poly-
morphism (Se) by the coefficient of variation of the relative
expression levels among all eight strains. Similarly, the kurtosis
for a given gene’s expression (Ke) was calculated from the eight
D. melanogaster expression values. Interspecific expression di-
the mean of all eight D. melanogaster expression levels and the
single D. simulans expression level (14) within a given sex.
Differences in the distributions of these statistics are reported as
the arithmetic mean of the statistic across all genes within a given
sex-bias class (e.g., S?eM, S?eF, and S?eUfor the mean expression
polymorphism of MBGs, FBGs, and UBGs, respectively).
for each gene were recoded into discrete expression states by
assigning to different states all strains for which the 95% credible
intervals were nonoverlapping. Strains whose 95% credible
assigned to all of those states. The number of different tran-
scriptional states found among the eight strains for a given gene
was then tabulated.
Details regarding fluorescence ratio acquisition from microar-
ray hybridizations, signal normalization and data quality control,
assessment of false positive rates, and statistical analysis of
previously published microarray data (15) are given in Support-
ing Materials and Methods.
The comparison scheme used here to obtain transcription pro-
files from adult males of eight strains of D. melanogaster is shown
in Fig. 3. The strains were chosen to represent the range of
Populations from Africa show significant differentiation from
non-African populations in nucleotide variation (17) and mating
behavior (18). Of the 4,905 clones selected for analysis, 2,289
showed differences that were significant between at least one
pair of strains, whereas 297 are expected by chance (see Sup-
genome is detectably differentially regulated between males
from interfertile populations of D. melanogaster. Pairs of strains
showed from 218 to 928 genes with significantly different
expression, where on average only 26 are expected by chance
(Table 6, which is published as supporting information on the
PNAS web site). This degree of differentiation in expression
profile between strains is much higher than has been previously
reported for Drosophila (16), and may reflect differing experi-
mental designs and statistical methods as well as the inclusion in
this study of the Zimbabwe strains. However, this level of
variation is similar to the proportion of differentially expressed
genes detected between two strains of Saccharomyces cerevisiae
(19). Although the Zimbabwe strains do show evidence of
differentiation from the Cosmopolitan strains in global gene
expression (unpublished results), there are a surprisingly small
number of genes that show fixed differences between the two
groups (Table 7, which is published as supporting information on
the PNAS web site).
The intraspecific comparisons were combined with informa-
tion on interspecific divergence in gene expression between D.
melanogaster and its sibling species, D. simulans (14). The
combined data set consisted of 4,759 clones common to both
experiments. As expected, the degree of differentiation among
strains is smaller than the range of variation in transcription
profiles seen between species. The variance in the distribution of
log2ratios across all genes between males of D. melanogaster and
D. simulans is 0.208. Within D. melanogaster, the most divergent
pair of strains has a variance in log2ratios of 0.159, which is
1.30, P ? 0.001). Based on the distributions of log2 ratios,
intraspecific differentiation ranges from 23% to 77% of inter-
specific divergence across the elements on these arrays.
There is a strong effect of sex-biased expression on intraspe-
cific variation in gene expression, and this effect is reversed
between MBGs and FBGs. Among genes that show significantly
different expression between at least one pair of strains, there is
a significant overrepresentation of MBGs and an underrepre-
sentation of FBGs (Table 1). The strength of this effect increases
as the stringency of the threshold chosen for statistical signifi-
Table 1. Overrepresentation of MBGs among genes with polymorphic expression within
Significance level of polymorphismMBGsFBGsUBGs
G (2 df)
P ? 0.05
P ? 0.01
192 (P ? 0.001)
351 (P ? 0.001)
Subsets of genes include those that exhibit at least one pairwise difference between any two strains at the
significance level indicated. G, G test of independence.
Meiklejohn et al.
August 19, 2003 ?
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no. 17 ?
cance is increased. By comparison with genes whose expression
is not sex biased, the effect of sex bias on intraspecific transcrip-
tional variation can be shown to be the result of both a reduction
in variation among FBGs and an increase among MBGs; S?eMand
S?eFare both significantly different from S?eU(Table 2).
Not only do MBGs on average have higher levels of expression
polymorphism than FBGs or UBGs, but this variation is distrib-
uted differently among the eight strains than it is for the other
two classes. The mean kurtosis (K?e), for the expression levels of
MBGs among the eight strains is significantly different from K?e
for FBGs or UBGs; K?eMis more platykurtic, and the two other
classes tend toward leptokurtosis (Table 2). A similar result is
observed by assigning expression levels to discrete states, anal-
ogous to transcriptional alleles. The distributions of number of
states for sex-biased and unbiased genes are shown in Fig. 1.
expression alleles than do the FBG or UBG classes, in which the
majority of genes have a single expression state (male-biased vs.
female-biased: G ? 388, df ? 3, P ? 0.001). The distribution of
expression states among FBGs is also significantly different from
that of UBGs (G ? 52.8, df ? 3, P ? 0.001). This result is not
due to differences in ability to discriminate between states
among the sex-bias classes. The average 95% credible interval
across all eight strains is very similar for MBGs and FBGs (0.432
and 0.452, respectively), but is significantly higher in the UBGs
(0.536; female-biased vs. unbiased: ts? ?7.68, df ? 2481, P ?
0.001). If this were responsible for the differences in transcrip-
tion state distributions, it would result in an excess of mono-
morphic genes in the UBG class relative to the FBG class, but
in fact the opposite result is observed (Fig. 1).
The interspecific hybridizations (14) allow a parallel set of
observations to be made regarding the influence of sex-biased
expression on interspecific divergence in gene expression. The
results are consistent with the intraspecific data; D?eis signifi-
cantly greater among MBGs than UBGs or FBGs (Table 3).
These experiments also provide information on expression di-
vergence for gene expression in females, as well as in males.
Although the same pattern of increased divergence among
MBGs relative to UBGs and FBGs exists for these genes when
they are expressed in females, it is not nearly as strong, nor is it
statistically significant (Table 3). Interestingly, MBGs are sig-
nificantly more divergent when they are expressed in males than
when they are expressed in females. Although not significant,
this pattern holds for UBGs, suggesting that gene expression in
males in general, and not just expression of MBGs, may be
Given DNA sequence data, deviations from the neutral
expectation in the ratio of divergence to polymorphism have
been used to infer the past activity of natural selection at a locus
(20, 21). Although gene expression changes do not have a simple
relationship with nucleotide sequence changes, a positive rela-
tionship between intra- and interspecific variation is predicted
for neutrally evolving polygenic characters (22, 23), and has been
empirically demonstrated for morphological traits that are pre-
sumably under selection as well (24). Elevated ratios of inter- to
intraspecific variation in phenotypes associated with male re-
production have been used to infer the importance of directional
selection on these characters relative to other types of morphol-
ogies (25). Thus, although we may not know a priori the neutral
ratio of divergence to polymorphism for a given gene’s expres-
sion, differences in this ratio between groups of genes may
indicate disparate selective pressures acting on these groups. Fig.
2 shows Deplotted against Sefor the three classes of sex bias. All
three classes show a weak positive correlation between Seand De,
and the correlation coefficient is significantly different between
all three classes, indicating a different relationship of covariation
between Seand Defor the three classes of sex bias.
The majority of sex-biased regulation in Drosophila has pre-
viously been shown to be the result of expression in germ-line
tissues (15, 26); thus, transcription in testes and ovaries is most
here, and in large part, it is genes expressed in the testes that are
evolving rapidly and genes expressed in the ovaries that are
evolving slowly. To confirm this conjecture, published data
directly comparing D. melanogaster expression profiles of males
and females, dissected testes and ovaries, and gonadectomized
males and females (15) were analyzed and integrated with the
results presented here. In addition to providing an independent
identification of MBGs and FBGs, these data allow the descrip-
tion of analogous classes of genes defined by significantly
different expression between testes and ovaries and between the
somatic tissues of males and females.
The patterns of rapid expression evolution seen among MBGs
relative to FBGs described above are also seen in the whole
fly experiments of Parisi et al. (15). MBGs are overrepresented
relative to FBGs among genes with polymorphic expression
when the experiments of Parisi et al. (15) are used to determine
sex-biased expression (data not shown). This same pattern is also
found among genes with testis or ovary-biased expression (Table
4). Interestingly, this discrepancy is not observed for sex-biased
genes when assayed in gonadectomized adults, because somat-
ically MBGs and somatically FBGs show virtually identical
Table 2. Amounts and distribution of expression polymorphism
is influenced by sex-biased expression
P(M vs U)
P(U vs F)
P values were calculated from Wilcoxon rank-sum test.
female-enriched, and non-sex-biased genes. Relative gene expression levels
were coded into discrete expression states as described in Materials and
Frequency distributions of gene expression states for male-enriched,
Table 3. Interspecific divergence is accelerated among MBGs
when expressed in males
P(M vs U)
P(U vs F)
Expression in males
Expression in females
P values were calculated from a Wilcoxon rank-sum test.
www.pnas.org?cgi?doi?10.1073?pnas.1630690100Meiklejohn et al.
4). Furthermore, both of these classes appear to be overrepre-
sented relative to genes with no somatic sex-biased expression,
suggesting that sex-biased expression may be evolving relatively
rapidly in somatic tissues as well as the gonads, but in both sexes.
The summary statistics S?e, K?e, and D?eshow similar patterns for
gene expression variation in the gonads and soma as was
described above for whole fly extractions (Table 5). Although
greater intra- and interspecific variation is seen among genes
with no sex-biased expression in the gonads compared with
ovary-based genes (OBGs), this difference is not as significant as
the difference between UBGs and FBGs seen from whole body
extractions (compare Tables 2, 3, and 5). Among genes with
somatic sex-biased expression, there is a greater S?eand D?eamong
both male-biased and female-biased genes than among genes
with no somatic sex bias (Table 5). These patterns are also
observed in the distributions of transcription states for testis-
based genes, OBGs, somatically MBGs, and somatically FBGs
A large proportion of the transcriptional differences observed
between D. melanogaster and D. simulans involves the loss, gain,
or reversal of sex-biased expression (14). Examination of intra-
and interspecific expression variation in these genes does not
reveal as clear a pattern as that observed for genes retaining an
ancestral sex bias in both D. melanogaster and D. simulans. This
is most likely due to a diversity of selective forces acting on genes
with rapidly evolving sex bias. However, such genes do appear to
be more variable in their expression within D. melanogaster than
those that have retained the ancestral sex bias, as shown by a
greater S?eamong genes that are sex biased in one species only
than among those that are sex biased in both species (male-
biased in one species only, S?e ? 0.180, P ? 0.05, Wilcoxon
rank-sum test; female-biased in one species only, S?e? 0.122, P ?
0.001, Wilcoxon rank-sum test). However, among genes with a
novel sex bias, there is still a correlation between Seand sex bias,
as genes that are male-biased in D. melanogaster or D. simulans
only have a S?ethat is significantly greater than genes that are
The data presented here indicate that rates of both intraspecific
and interspecific differentiation of gene expression in Drosophila
are correlated with sex-biased expression, and that this differ-
ence is largely a function of gene expression in testes and ovaries.
Furthermore, among somatically expressed genes, sex-biased
expression in both sexes appears to evolve more rapidly than
sexually monomorphic expression. Analogous results have come
from morphological studies in Drosophila that documented a
higher rate of intra- and interspecific divergence among mor-
phologies associated with male reproduction than nonreproduc-
tive morphologies (25). These conclusions are not the result of
nucleotide sequence divergence within or between species caus-
ing spurious inferences of changes in gene expression. Data from
competitive hybridizations using genomic DNA extracted from
D. melanogaster and D. simulans indicate that sequence diver-
gence between these two species has a small effect on hybrid-
ization signal intensity that is within the range of experimental
error associated with cDNA hybridizations (14). Furthermore,
across the clones on these arrays, a greater number of MBGs are
found in D. simulans than in D. melanogaster (14), which cannot
be the result of sequence divergence. Consistent with our results
on rates of expression evolution, a subset of male germ-line
genes in Drosophila are known to be enriched for sequences with
no detectable homologs in other eukaryotic genomes (26),
suggesting that MBGs may be on the whole younger than other
classes of genes. The accelerated rate of evolution among MBGs
may therefore extend further back in time than the comparison
between D. melanogaster and D. simulans, and is consistent with
often driven by the creation of new genes (e.g., refs. 27 and 28).
One interpretation of these results is that mutations affecting
the expression of MBGs on average experience greater (i.e.,
product moment correlation coefficients for each class are all significantly
r ? 0.162, P ? 0.001) and from each other (MBG vs. UBG, z ? 2.717, P ? 0.007;
Seand Defor male-biased, female-biased, and unbiased genes. The
Table 4. Overrepresentation of testis-biased genes and
somatically sex-biased genes among genes with polymorphic
expression within D. melanogaster
Significance level of
P ? 0.05 (%)
P ? 0.01 (%)
Significant departures from independence were determined by a G test
with 1 df. Both sMBGs and sFBGs are overrepresented among polymorphic
genes relative to genes with no somatic sex bias, of which 80% are polymor-
phic at P ? 0.05 and 47% are polymorphic at P ? 0.01. These results do not
sex-biased expression. TBGs, testis-biased genes; sMBGs, somatically MBGs;
sFBGs, somatically FBGs.*, P ? 0.001.
Meiklejohn et al.
August 19, 2003 ?
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no. 17 ?
either more positive or less negative) selection coefficients than
UBGs or FBGs. A larger positive average selection coefficient
would result in the fixation of a greater number of beneficial
mutations affecting gene expression of MBGs, whereas less
negative selection coefficients would result in a smaller fraction
of deleterious mutations contributing to intraspecific variation
affecting the expression of MBGs. Both of these hypotheses are
suggested in the greater correlation between Se and De seen
among MBGs (Fig. 2). Differences in this relationship between
the sex-biased classes are largely due to an elevated De?Seratio
among the most extremely male-biased genes. The 324 genes
with the most significantly testis-biased expression (P ? 0.001)
have a higher average De?Sethan the 290 corresponding OBGs
(Wilcoxon rank sum test, P ? 0.04). Circumstantial evidence for
the role of positive selection in the differentiation among MBGs
D. simulans (Table 3). If relaxed selection is driving the diver-
gence of MBGs, the fact that gene expression among MBGs is
less divergent in females than it is in males requires that the
neutral mutations that fix and cause changes in expression have
their effects in males only, because their regulation is more
conserved in females.
An alternate interpretation of these data are that there is a
fundamentally different relationship between fold-change in
expression and effect on fitness between the sex-biased classes of
genes. Because gene regulation in male gametogenesis appears
to be highly specialized in both mammals and insects (29), we
might expect the evolution of gene expression in testes to be
unusual. However, this explanation does not address the differ-
ences observed here between FBGs and UBGs (i.e., Tables 1
It is important to remember that the intraspecific compar-
isons presented here include gene expression data from males
only. However, preliminary results indicate that the relation-
ship between sex bias and rates of intraspecific expression
evolution seen here for gene expression in males holds for
expression in females as well. Assessment of expression pro-
files in virgin females of Canton S and Zim2 using the same
methods described above reveals a significant overrepresen-
tation of MBGs among those genes with significant differences
between these two strains. Furthermore, in these females
S?eM? S?eU? S?eF, and these differences are highly significant
(J.M.R., unpublished data). This finding is in agreement with
the results from the interspecific comparisons, which show that
FBGs show similar or reduced amounts of interspecific dif-
ferentiation than MBGs when these genes are expressed in
females (Table 3).
The literature documenting elevated rates of evolution
among genes associated with reproduction has included nu-
merous examples of genes with functions in both males and
females (4, 5). This is in contrast to the results presented here,
which show that the expression of FBGs evolves more slowly
than that of both MBGs and UBGs, although this pattern is not
as extreme as the accelerated evolution observed for male-
biased expression. Some of the studies that have found rapidly
evolving genes associated with female reproduction have
focused on a small sample for which there was an a priori
expectation of positive selection (e.g., ref. 30, but see ref. 4).
The low correlation between Seand Defor all three sex-bias
classes shows that the rate of expression evolution for any given
gene is likely to be idiosyncratic, and we observe a number of
FBGs with high levels of intra- and interspecific expression
variation. Nonetheless, assaying ?1?3 of the Drosophila ge-
nome indicates that rapid evolution of expression is far more
prevalent among MBGs than FBGs, and indicates the value of
data sets of this size.
One potential source of error in the above analyses is the
assumption of independence across genes in their intra- and
interspecific variation in expression. This assumption will be
violated when a single genetic locus influences variation in the
expression of multiple genes simultaneously, as will result from
coordinate regulation. In the most extreme scenario, all genes
expressed in the testes might appear to be up-regulated in a
given strain of D. melanogaster because of an increase in the
relative size of the testes in that strain. Such an extreme bias
can be ruled out by the large number of genes that show all
possible patterns of covariation among these strains, indicating
that at a broad scale there are many groups of independently
regulated genes. Further argument to this point can be made
by reference to the few experiments to date that have examined
the genetics of gene expression. Studies in S. cerevisiae (19),
mice, and maize (31) indicate that 35–80% of QTLs that
influence expression of a gene map to the gene itself, suggest-
ing cis-regulation. The fraction of cis-acting genetic factors
increases with more stringent statistical cutoffs (31), suggest-
ing that large changes in expression may more often be in cis,
whereas trans-acting mutations are more often of small effect.
Although these numbers are influenced by the power of each
experimental design, these data from three different biological
kingdoms suggest that a large fraction (?30%) of large effect
variants affecting gene expression are in cis, and that this may
be a phenomenon intrinsic to eukaryotic gene expression. This
does not address how the remaining fraction of variation in
gene expression is distributed across many unlinked factors of
small effect. Nonetheless, it is unlikely that these caveats could
affect the nature of our conclusions, or render them statisti-
cally insignificant. For example, all of the comparisons made
in Tables 1 and 4 remain significant at P ? 0.01, when the
numbers of genes are multiplied by a factor of 0.1 (as might be
appropriate if, on average, a single genetic variant were
responsible for changes in the expression of 10 downstream
genes). However, this issue will require population genetic
studies of the inheritance of global gene expression to be
It is tempting to speculate that MBG expression may be
related to the evolution of hybrid male sterility (32). The
genetic factors that influence male fertility appear to evolve
much faster at both intra- and interspecific levels than those
influencing female fertility or viability in either sex in Dro-
sophila. This rapidity is evident in the disproportionately high
amounts of genetic variation affecting male fertility observed
in mutation–accumulation lines (33) and the excess of hybrid
male sterility factors relative to hybrid female sterility factors
that have accumulated between closely related species (34–
Table 5. Expression polymorphism and divergence as a function of sex-biased expression in the gonads
P(T vs U)
P(U vs O)
P(M vs U)
P(M vs F)
P values were calculated from a Wilcoxon rank-sum test.
www.pnas.org?cgi?doi?10.1073?pnas.1630690100Meiklejohn et al.
36). None of these patterns is caused by an excess of loci
affecting male fertility, because mutagenesis screens indicate
that approximately seven times more genes influence viability
than male fertility, whereas similar numbers affect fertility in
the two sexes (37). A causal relationship between these
observations would require that the rapid evolution of gene
expression in MBGs leads to the misexpression of these genes
in sterile hybrid males. A recent study of gene expression in
hybrids between Drosophila mauritiana and D. simulans found
that MBGs were preferentially misexpressed in the sterile F1
males (38), lending support to this hypothesis. Together, these
patterns of gene expression and misexpression are consistent
with the idea that rapid evolution of male reproductive
characters contributes to Haldane’s rule (39) for hybrid male
sterility in Drosophila.
We thank the members of the Harvard Drosophila Microarray Consor-
tium and the Bauer Center for Genomics Research for help in creating
the arrays; the Hartl and Wakeley laboratories, A. M. Wilczek, and
especially J. P. Townsend for helpful discussions; and Rama Singh,
Mohamed Noor, and an anonymous reviewer for valuable comments on
Grant GM60035 (to D.L.H.) and a postdoctoral fellowship from the
Ministerio de Ciencia y Tecnología (to J.M.R.).
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