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Genome-Wide Patterns of Expression in Drosophila Pure Species and
Hybrid Males
Pawel Michalak and Mohamed A. F. Noor
Department of Biological Sciences, Louisiana State University
One of the most fundamental questions for understanding the origin of species is why genes that function to cause
fertility in a pure-species genetic background fail to produce fertility in a hybrid genetic background. A related question
is why the sex that is most often sterile or inviable in hybrids is the heterogametic (usually male) sex. In this survey, we
have examined the extent and nature of differences in gene expression between fertile adult males of two Drosophila
species and sterile hybrid males produced from crosses between these species. Using oligonucleotide microarrays and
real-time quantitative polymerase chain reaction, we have identified and confirmed that differences in gene expression
exist between pure species and hybrid males, and many of these differences are quantitative rather than qualitative.
Furthermore, genes that are expressed primarily or exclusively in males, including several involved in spermatogenesis,
are disproportionately misexpressed in hybrids, suggesting a possible genetic cause for their sterility.
Introduction
The formation of new species is often associated with
reduced fertility or viability of hybrid progeny (Coyne and
Orr 1989, 1997; Sasa, Chippindale, and Johnson 1998;
Presgraves 2002; Price and Bouvier 2002). Dobzhansky
(1936) proposed that hybrid sterility and inviability may
arise as pleiotropic by-products of evolution in geographically separate lineages: alleles that increase fitness in
pure-species genetic backgrounds may fail to interact
properly when brought together in hybrid backgrounds. A
distinctive feature of hybrid sterility and inviability is that
they require epistasis: nonadditive interactions between
alleles at different loci (Dobzhansky 1937; Muller 1940;
Johnson and Porter 2000; Turelli and Orr 2000). Several
types of gene interactions may create hybrid dysfunctions:
1. Amino acid differences accumulated in divergent
populations may render proteins incapable of interacting properly, thereby producing unfit phenotypes (e.g.,
Rawson and Burton 2002).
2. Post-transcriptional processes such as mRNA splicing
or mRNA stability may be disrupted (e.g., Braidotti and
Barlow 1997).
3. Genes may be inappropriately overtranscribed or
undertranscribed (misregulated) in hybrids relative to
pure species (see below).
Both theoretical and empirical studies suggest that
failures in the regulation of gene expression may contribute
to hybrid dysfunctions (see review in Orr and Presgraves
2000). For example, Johnson and Porter (2000; see also
Porter and Johnson [2002]) modeled the evolution of
regulatory genetic pathways and found that binding
strength of proteins to promoter regions can provide
biologically plausible hybrid sterility. An empirical example is provided by the overexpression of Xmrk-2 oncogene
(Xiphophorus melanoma receptor kinase) that is associated
with tumor formation and hybrid lethality in crosses
between swordtails (Xiphophorus helleri) and platyfish
(X. maculatus) (Schartl 1995; Schartl et al. 1999). To
Key words: gene expression, hybrid sterility, reproductive isolation, DNA microarrays.
E-mail: [email protected].
Mol. Biol. Evol. 20(7):1070–1076. 2003
DOI: 10.1093/molbev/msg119
Ó 2003 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
1070
evaluate the role of gene expression in hybrid sterility,
technological advances can now enable researchers to
examine expression patterns of hundreds or thousands of
genes in hybrids relative to nonhybrids simultaneously.
Genome-wide expression profiling can rapidly identify whether qualitative failures in gene expression are associated with hybrid male sterility, and if so, what genes or
genetic pathways are responsible. Two hypotheses may
predict the identity and the location of genes that are
misexpressed in hybrids, and hence possibly associated
with sterility. First, genes expressed predominantly in
males and the male germ line, and thus often subject to
accelerated microevolutionary divergence (see review in
Singh and Kulathinal 2000), may be most likely to be
deregulated in hybrids. In Drosophila, it has been shown
that male reproductive proteins, such as accessory gland
proteins (Acps), can be twice as divergent between species
as nonreproductive proteins (e.g., Civetta and Singh 1995).
The coding regions of their corresponding genes exhibit
a significant excess of nonsynonymous over synonymous
substitutions, suggesting that positive Darwinian selection
accelerated their divergence (e.g., Swanson et al. 2001).
Second, X-chromosomal genes may be misexpressed in hybrids more than autosomal genes, corresponding with suggestions of a disproportionate X-effect in hybrid sterility
(Charlesworth, Coyne, and Barton 1987; Coyne and Orr
1989; True et al. 1996).
To evaluate the potential for gene misexpression to
cause sterility in hybrids, we must first ascertain whether
and how much gene misexpression is detectable in hybrids
and the nature of the misexpressed transcripts. In a previous
study, Reiland and Noor (2002) documented a handful of
transcripts misexpressed in hybrids of Drosophila pseudoobscura and D. persimilis relative to pure species, but
their differential display technique had very low resolution
for detecting quantitative differences in gene expression.
Here, we apply much higher resolution techniques, microarrays and real-time fluorescent quantitative PCR, to examine differences in gene expression between two
Drosophila species (D. simulans and D. mauritiana) and
their sterile F1 hybrids. Infertility in male hybrids between
these species is associated with spermiogenic, or postmeiotic, failure at the cytological level (Wu et al. 1992).
We evaluate whether genes with male-specific patterns
Gene Expression and Hybridization in Drosophila 1071
of expression, or those located on the X chromosome,
are disproportionately disrupted in hybrids, testing the
hypotheses laid out above, and we discuss the implications
of our findings with regard to Haldane’s rule (Haldane
1922).
Methods
Flies
Stocks were reared in uncrowded cultures at 248C
with a 12-h light-dark cycle on standard sugar-yeast-agar
medium. Drosophila simulans flies were taken from the
Florida City (FC) line, an isofemale line collected in 1985
in Florida City. Drosophila mauritiana were taken from
the SYN stock, a wild-type line synthesized by combining
six isofemale lines collected on Mauritius in 1981. All F1
hybrids were produced by crossing D. simulans females
with D. mauritiana males.
Oligonucleotide Microarray Assays
For each replicate, 5 lg of total RNA were obtained
from 23 seven-day post-eclosion adult Drosophila simulans, D. mauritiana, and F1 hybrid males with the
QIAGEN RNeasy kit. A total of 14 Affymetrix GeneChip
Drosophila Genome array chips were used: 5 3 D.
simulans, 4 3 D. mauritiana, 5 3 F1 hybrids. The
GeneChip Sample Cleanup Module (QIAGEN) was used
according to the Affymetrix standard hybridization protocol. Double-stranded cDNA was synthesized with a T7(dT)24 primer; cRNA was synthesized and biotin-labeled
in an in vitro transcription reaction using the ENZO
BioArray HighYield RNA Transcript Labeling Kit. The
final concentration of cRNA in the fragmentation mixture
was 0.5 lg/lL. The target cRNA was hybridized to arrays
that allow one to monitor the relative abundance of more
than 14,000 mRNA transcripts. Patterns of hybridization
were detected in an Affymetrix scanner, and the results
were analyzed using Affymetrix Microarray Suite (MAS)
5.0 software. The software Detection algorithm calculates
the Discrimination Score from each probe pair (Perfect
Match vs. Mismatch), assesses probe saturation, calculates
a Detection P value from the one-sided Wilcoxon Signed
Rank test, and assigns a Present, Marginal, or Absent Call
in relation to a threshold value Tau ¼ 0.015. All transcripts
were scaled to a target intensity value of 10,000.
Expression differences obtained from this assay may
be confounded by sequence differences between these
species and D. melanogaster. Hence, we focus on those
transcripts that show little difference in hybridization between the two pure species assayed but a large difference between the pure species and the F1 hybrid males.
As the F1 hybrids were produced from the same strains as
the pure species evaluated, they would possess no sequence difference from them, and differences in hybridization between these pure species and these F1 hybrids
must reflect differences in transcript abundance.
Real-Time Polymerase Chain Reaction
For the real-time polymerase chain reaction (RTPCR), 250 ng of total RNA prepared as described above
were reverse transcribed in a reaction bearing 5 mM
MgCl2, 50 mM KCl, 10 mM Tris–HCl, 1 mM dNTPs, 20
U RNasin, 20 U reverse transcriptase (SuperScript II), 2.5
lM of the target primer, and 50 nM of 18SrRNA reverse
primer (Applied Biosystems [ABI]). All real-time PCR
primers and fluorescent probes were designed using Primer
Express 2.0 software (ABI), and their sequences are
available by request. All target probes were FAM-labeled
(Biosearch Technologies), and the normalizer probe (18S
rRNA) was VIC-labeled (ABI). Pre-developed ABI TaqMan Assay Reagents and ABI standard protocols were
used to prepare the RT-PCR reaction mixture. The probes
contain a reporter dye at the 59 end of the probe and
a quencher dye at the 39 end of the probe. During the
reaction, the reporter dye and quencher dye are separated,
resulting in increased fluorescence of the reporter. All
PCRs consisted of an initial 2 min at 508C and 10 min at
958C, 40 cycles of 958C for 15 s and at 608C for 1 min.
Applied Biosystems Prism 7000 SDS software was used
for visualization and quantification of the amplification
products.
Statistical Analysis
Standard one-way analyses of variance (ANOVAs)
were used for detecting differences in expression between
each pure species individually and the F1 hybrids. In addition, we used Bayesian regularized t-tests (Baldi and
Long 2001; Long et al. 2001) to compare microarray expression signals between pure species and F1 hybrids. In
this analysis, a prior estimate of variance within groups is
estimated by the weighted average of a prior estimate of
the variance for that gene (obtained from the local
weighted average of the variance of other genes) and the
experimental estimate of the variance for that gene. This
leads to the desirable property of the Bayesian approach
converging toward the t-test as the number of replicates
increases and prior variance tends to zero. The mean
difference in threshold cycle number (CT) was tested with
a post hoc Duncan test after the 2-way repeated measures
ANOVA analysis, in which genotype (D. simulans, D.
mauritiana, and hybrids) effect was significant (P , 0.05).
There were two levels of replications: two independent
reverse transcriptase reactions and two PCR replicates of
each sample within the RT reaction. All results were
repeatable when different cycle thresholds were tested and
were largely independent of whether or not normalization
for 18SrRNA was applied.
Results
Microarray Analysis
The mean expression of 435 genes in this assay
differed significantly between F1 hybrids and either D.
simulans or D. mauritiana (P , 0.05, ANOVA; fig. 1).
The number decreased to 37 underexpressed and 14 overexpressed genes when the analysis was confined to those
transcripts which differed significantly in F1 hybrids from
both D. simulans and D. mauritiana analyzed separately
(ANOVA, P , 0.05, see table 1 in the online Supplementary Material; all raw microarray data can be found
1072 Michalak and Noor
exclusively in testes and thus presumably are responsible
for spermatogenesis: CG2206, Mst84Da, Mst84Db,
Mst84Dc, Mst84Dd (Andrews et al. 2000; Jin et al.
2001; Swanson et al. 2001; Parisi et al. 2003), the RNA
binding CG14718, cytosol aminopeptidase (CG13340),
and complex homolog subunit 6 (CH6). Although statistically significant overexpression or underexpression of
transcripts was observed, most differed in expression by
a factor of two or less in hybrids relative to pure species.
This observation may reflect truly quantitative differences
or tissue-specificity of the disruptions in gene expression.
No qualitative differences (e.g., tenfold or greater) in
expression were noted between pure species and hybrids.
Only one underexpressed gene, Mst84Dc, exhibited higher
than fourfold underexpression in hybrids relative to both
pure species, and this was the only gene with significant
misexpression detected when a Bayesian regularized t-test
was applied to the full data set (posterior P , 0.05) (Baldi
and Long 2001; Long et al. 2001). For all subsequent
analyses on the microarray data, we performed statistical
tests on (1) all genes assayed on the chips and (2) only
those genes which were detectably present based on
Present Call in Affymetrix MAS 5.0 software in all 9 purespecies chips.
Male-Specificity of Misexpression
FIG. 1.—Volcano plots of gene expression difference (A) between
pure D. simulans and F1 hybrids, and (B) between D. mauritiana and F1
hybrids. Each point represents a transcript from the microarray assay. The
log10 fold change (ratio of mean expression values of pure species and F1
hybrids) is shown on the x-axis and the log10 P values from ANOVA
significance tests are shown on the y-axis.
at http://www.biology.lsu.edu/webfac/mnoor/alldata.xls).
None of these genes were significantly misexpressed if
a conservative Bonferroni correction was applied, but
we were nonetheless able to confirm many of them via
real-time PCR (see below). To estimate the extent of
misexpression after eliminating genes not detectably
expressed in pure-species males and those which fail to
hybridize to the D. melanogaster array, we limited the
analysis in two manners: (1) those transcripts which were
detectably present based on Present Call in Affymetrix
MAS 5.0 software in all 9 pure-species chips and (2) those
transcripts for which the standard deviation of the
expression level (calculated as Signal Call) within purespecies replicates was less than 25% of the mean assayed
expression level (following Leemans et al. 2000). Limiting
the data set according to (1) resulted in 10 underexpressed
candidate gene and 1 overexpressed (CG11266, splicing
factor) candidate gene from a total of 692 genes assayed.
According to grouping (2), we identified 8 underexpressed
candidate genes and 1 overexpressed candidate gene
(CG11266) from a total of 394 genes. Transcripts
designated as underexpressed in both groupings included
several that previously had been shown to be expressed
We assigned transcripts from our microarray analysis
to two sets: male-specific transcripts (according to combined lists from Andrews et al. 2000; Jin et al. 2001;
Swanson et al. 2001; Parisi et al. 2003; from Jin et al. 2001
and Parisi et al. 2003, we took only those transcripts that
differed between sexes by more than 100%) and all other
genes. We then divided each set into two complementary
subsets: one consisting of genes differing significantly in
expression between both pure species and hybrids
(ANOVA, P , 0.05 for both D. simulans and D. mauritiana, 51 genes in total; see Supplementary Material online) and the rest with no significant difference. We found
that statistically significant misexpression tends to occur
much more frequently in the male-specific group for both
grouping criteria used (fig. 2). Indeed, based on grouping
criterion (2), 11 genes exhibited statistically significant
differences in expression between pure species and
hybrids, and 6 of the 11 were male-specific. In contrast,
only 34 of the 665 genes not exhibiting significant
differences in expression between pure species and hybrids
were expressed primarily in males.
We also compared the mean expression level in hybrids relative to pure species between male-specific genes
and all other genes (fig. 3). The difference was highly
significant for both species and for both grouping criteria
used: (1) D. simulans, Student t-test, t ¼ 15.751, df ¼
13964, P , 0.0001; D. mauritiana, Student t-test, t ¼
3.371, df ¼ 13964, P ¼ 0.0008; (2) D. simulans, t ¼ 10.444,
df ¼ 674, P , 0.0001; D. mauritiana, t ¼ 4.175, df ¼ 674,
P , 0.0001). A similar result was obtained when fold
change values were replaced with log10 of P values
from respective ANOVA contrasts (Mann-Whitney U test,
P , 0.05).
Gene Expression and Hybridization in Drosophila 1073
FIG. 2.—Frequency (numbers in bars) of statistically significant
misexpression (black), as detected by ANOVA (P , 0.05) of the
microarrays, among male-specific genes (open) and all other genes
(dotted) in F1 hybrids relative to both D. simulans and D. mauritiana.
Genes were defined as male-specific according to the criteria of Andrews
et al. 2000, Jin et al. 2001, Swanson et al. 2001, and Parisi et al. 2003.
Overexpressed genes were included in the analysis, but only one of those
occurred among male-specific genes. (A) All transcripts: chi-square ¼
51.55, df ¼ 1, P , 0.0001. (B) When the comparison is limited to
transcripts detectably present (‘‘P’’ in Affymetrix MAS 5.0 software) in
all 9 pure-species chips, misexpression in hybrids is ;19 times more
frequent among male-specific genes (chi-square ¼ 47.50, df ¼ 1, P ,
0.0001).
The Analysis of X Chromosome Misexpression Linkage
We compared frequencies of significant misexpression (according to ANOVA, P , 0.05) within the group
of the X chromosome–associated transcripts against the
autosome-associated transcripts group. We did not observe a significant association of gene misexpression with
chromosome X for any comparison (2 3 2 chi-square test,
P . 0.05, see also table 2 in the online Supplementary
Material).
FIG. 3.—Mean fold change (6SE) between F1 hybrids and pure
species in male-specific gene expression (open) relative to all other genes
(dotted) in microarrays. Genes were defined as male-specific according to
the criteria of Andrews et al. 2000, Jin et al. 2001, Swanson et al. 2001,
and Parisi et al. 2003. (A) D. simulans. (B) D. mauritiana. Left two bars:
all transcripts; right bars: transcripts detectably present.
Real-Time PCR Analysis
We validated the putative underexpression of
Mst84Dc with quantitative fluorescent real-time PCR
(RT-PCR, TaqMan Applied Biosystems). In this assay,
we used a repeated measures ANOVA in which 12 bulkRNA samples from males (4 each from D. simulans, D.
mauritiana, and F1 hybrids) were analyzed, and each
sample was replicated in two reverse-transcription reactions followed by independent RT-PCR assays. The cDNA
from all F1 samples consistently amplified exponentially
after ;4 more PCR cycles than pure species samples,
indicated as higher threshold cycle (CT) values for hybrid
samples than for nonhybrid samples (fig. 4a). This delayed
amplification suggests a lower concentration of the transcript in hybrids than in pure species (table 1).
We extended the RT-PCR analysis and the ANOVA
design to three additional genes: Mst98Cb, CG5762, both
known to be among genes expressed predominantly in the
male germ line (Andrews et al. 2000; Jin et al. 2001;
Swanson et al. 2001; Parisi et al. 2003), and CG4792,
which was a randomly chosen negative candidate from our
microarray analysis and does not have a sex-specific
expression pattern (table 1). Both Mst98Cb and CG5762
consistently exhibited mean CT values in F1 hybrids
significantly higher by ;1.5 cycles than in nonhybrids,
and there was no significant difference between D. simulans and D. mauritiana (fig. 4b and c). The expression
level of our negative control, CG4792, which encodes an
RNA helicase-like enzyme, did not differ significantly
between hybrids and nonhybrids.
Discussion
For more than 60 years, it has been recognized that
hybrid sterility and other hybrid dysfunctions are caused
by failures in epistatic interactions between alleles at loci
derived from one species with alleles at different loci
derived from the other species (Dobzhansky 1937; Muller
1942; see review in Johnson 2000). Here, we have
established that dozens, perhaps hundreds, of genes are
subject to quantitative transcriptional deregulation in male
hybrids of Drosophila simulans and D. mauritiana. Hybrids are likely to suffer more from downregulation of
genes than from upregulation of genes, as the former is
several times more abundant than the latter in our data set.
1074 Michalak and Noor
Table 1
Repeated Measures ANOVA of Gene Expression, as
Measured with Real-Time PCR, for Mst84Dc, Mst98Cb,
CG5762, and CG4792 Genes
Source of Variation
Effect Mean
Square
df
F
P
Mst84Dc
Type effect
RT-reaction effect
Replicate effect
Type 3 RT-reaction
Type 3 replicate
RT-reaction 3 replicate
Type 3 RT 3 replicate
0.312
0.748
0.024
0.033
0.024
0.025
0.026
2
1
1
2
2
1
2
7.471
17.887
2.352
0.783
2.358
2.358
2.636
0.004
0.0005
0.143
0.472
0.123
0.123
0.099
Mst98Cb
Type effect
RT reaction effect
Replicate effect
Type 3 RT-reaction
Type 3 replicate
RT-reaction 3 replicate
Type 3 RT 3 replicate
0.095
0.407
0.001
0.009
0.001
0.0006
0.0009
2
1
1
2
1
2
2
13.200
56.288
1.096
1.311
1.088
0.457
0.684
0.0003
0.000001
0.309
0.294
0.311
0.640
0.517
CG5762
Type effect
RT-reaction effect
Replicate effect
Type 3 RT-reaction
Type 3 replicate
RT-reaction 3 replicate
Type 3 RT 3 replicate
0.184
0.000
0.0002
0.000
0.00006
0.000
0.000
2
1
1
2
2
1
2
8.530
0.000
2.727
0.000
0.712
0.00002
0.00001
0.002
0.9998
0.116
1.000
0.504
0.996
1.000
CG4792
Type effect
Replicate
Type-effect 3 replicate
0.003
0.0002
0.000
2
1
2
0.512
4.205
0.007
0.615
0.071
0.993
NOTE.—Type effect includes three categories: D. simulans, D. mauritiana, and
their F1 hybrids. Two separate reverse-transcriptase reactions were treated as an independent variable (RT-reaction effect), and Replicate represents duplications of a
sample.
The transcripts downregulated in hybrids seem to be
associated in large part with male reproduction, although
they are randomly distributed between the X chromosome
and autosomes.
One of the transcripts identified, Mst98Cb, has
been described in some detail previously (Schäfer et al.
1993, White-Cooper et al. 1998). Schäfer et al. (1993)
hypothesized it to function as a structural protein of the
sperm tail. Mst98Cb is transcribed prior to spermiogenesis
but not translated until after meiosis. As the spermatogenetic failure of these hybrids is postmeiotic, but transcription of Mst98Cb and the other genes identified is
premeiotic (as is most transcription involved in Drosophila
spermatogenesis; see Fuller [1998]), the misexpression in
hybrids may contribute to sterility rather than being a mere
by-product of possessing incompletely developed sperm.
Although we confirmed quantitative misexpression of
Mst98Cb and CG5762 in hybrids, this misexpression was
not statistically significant from the microarray analysis
alone. Hence, the RT-PCR assay succeeded in detecting
underexpression suggested by microarray analysis but not
confirmed with our statistical tests, suggesting that the
latter (especially the Bayesian approach) might increase
type II statistical error. This exemplifies the need for an
independent validation of microarray-based studies with
FIG. 4.—RT-PCR amplification plots for three genes: (A) Mst84Dc,
(B) Mst98Cb, and (C) CG5762. Each curve represents a single sample;
pure species in blue, F1 hybrids in red. PCR cycle numbers are shown on
the x-axis and normalized fluorescence intensity (Rn) reflecting cDNA
level is shown on the y-axis. (A) D. simulans was significantly different
from F1 (P ¼ 0.002, Duncan test) but not from D. mauritiana (P ¼ 0.312).
D. mauritiana differed from F1 (P ¼ 0.015). (B) D. simulans was
significantly different from F1 (P ¼ 0.0002) but not from D. mauritiana
(P ¼ 0.068). D. mauritiana was different from F1 (P ¼ 0.006). (C) D.
simulans differed from F1 (P ¼ 0.0009) but not from D. mauritiana (P ¼
0.070), and D. mauritiana was different from F1 (P ¼ 0.041).
more sensitive techniques such as RT-PCR (Rajeevan et al.
2001) and northern blots (Taniguchi et al. 2001).
Implications for Haldane’s Rule
If one sex of interspecies hybrids is sterile or inviable,
it is typically the heterogametic sex; a pattern referred to as
Gene Expression and Hybridization in Drosophila 1075
Haldane’s rule (see review in Orr 1997). In the past
decade, three theories have emerged that may partially or
completely explain Haldane’s rule in a wide variety of
taxa. First, if alleles conferring deleterious interactions in
hybrids are typically recessive, heterogametic hybrids,
which bear only a single X chromosome, will exhibit
sterility earlier in evolutionary divergence than homogametic hybrids (Muller 1942; Turelli and Orr 1995). This
theory has been supported by several recent observations
(see review in Orr 1997), although it does not appear to
completely explain hybrid sterility in Drosophila and
mammals (see Wu, Johnson, and Palopoli 1996). A
second, but not mutually exclusive, theory suggests that
hybrid male sterility in particular may evolve rapidly,
through either sexual selection or unique physiological
properties of spermatogenesis (sometimes called ‘‘faster
male’’: Wu and Davis 1993; Wu, Johnson, and Palopoli.
1996). This theory is supported by the higher number of
factors conferring male sterility over female sterility in
genetic mapping studies of D. simulans and D. mauritiana
(e.g., True, Weir, and Laurie 1996) and the observation of
greater male sterility in mosquito species lacking a degenerate Y chromosome (Presgraves and Orr 1998). However, it predicts that Haldane’s rule should be weaker in
Lepidoptera, where males are homogametic, than in
Drosophila, and this prediction is not supported (Presgraves 2002). The final theory that may contribute to
Haldane’s rule is that the relative rate of evolution of sexchromosomal genes may exceed that of autosomal genes if
new advantageous mutations are typically recessive
(sometimes called ‘‘faster X’’: Charlesworth, Coyne, and
Barton 1987). This theory predicts an excess of hybrid
sterility factors on the sex chromosomes, which is
observed weakly (True et al. 1996), and generally higher
rates of evolution of sex-chromosomal genes, something
which is not detected in Drosophila (Betancourt, Presgraves, and Swanson 2002).
In our analyses, transcripts known to be predominantly male-specific in their expression were significantly
overrepresented among underexpressed genes in hybrids.
The male-specificity of misexpression is consistent with
the faster-male theory and the repeated observations of
rapid evolution of male reproductive proteins. The results
demonstrate that genes with male-specific patterns of expression are more prone to disruption in hybrids of these
species than genes without sex-specific expression. Indeed,
our results may be conservative because male-specific
genes are often among the most rapidly evolving (e.g.,
Singh and Kulathinal 2000; Swanson et al. 2001), so some
of the most divergent male-specific genes may not have
been surveyed here because of sequence divergence from
D. melanogaster.
The lack of a disproportionate number of Xchromosome genes being misexpressed in hybrids may
incorrectly appear to militate against the faster-X theory.
The transcripts misexpressed in hybrids are likely to be
downstream genetic targets of the changes that cause
hybrid dysfunctions. Although genes involved in spermatogenesis are likely to regulate other genes involved in
spermatogenesis or male secondary sexual traits, most Xlinked genes probably do not regulate exclusively other
X-linked genes (but see Boutanaev et al. 2002). As such,
our results do not refute the faster-X theory.
Prospective
Our study is one of very few that have attempted to
determine why genes might fail to interact properly in
hybrids, resulting in sterility (e.g., Reiland and Noor
2002). Although this study has not directly shown that the
observed quantitative disruptions in gene expression in
hybrids cause sterility, we have shown that many genes do
have disruptions in their expression in hybrids, including
several that contribute to spermatogenesis. This study has
further illustrated the utility of microarrays and other
technologies for the global analysis of gene expression, to
evolutionary studies. In addition, it has established a panel
of genes on which to focus further investigations of the
genetics of hybrid sterility. This panel constitutes a dramatic advance given that the past 70 years of study has
identified one only gene (Ting et al. 1998). Given that we
have now documented that gene misexpression does occur
in sterile species hybrids, clear next steps will include (1)
documenting transcriptional variation among various life
cycle stages and tissues and (2) comparing gene expression
in sterile and fertile males among multigenerational
backcross lines to determine whether the disruptions in
gene expression are directly associated with sterility.
Acknowledgments
This research was supported by National Science
Foundation grants 9980797, 0100816, 0211007; Louisiana
Board of Regents Governor’s Biotechnology Initiative
grant 005; and a Lalor Foundation fellowship to P.M. We
thank Jill R. Schurr from LSU Health Sciences in New
Orleans for assistance with the microarray assays; B.
Counterman, S. Dixon, D. Ortiz, and R. Staten for helpful
comments on the manuscript; K. Brown and R. Staten for
technical assistance; and M. Batzer for use of real-time
PCR machines.
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Diethard Tautz, Associate Editor
Accepted March 10, 2003