Genetica (2007) 129:71–81
DOI 10.1007/s10709-006-0034-1
ORIGINAL PAPER
Gene expression divergence and the origin of hybrid dysfunctions
Daniel Ortı́z-Barrientos Æ Brian A. Counterman Æ
Mohamed A. F. Noor
Received: 21 June 2004 / Accepted: 20 June 2005 / Published online: 17 October 2006
Ó Springer Science+Business Media B.V. 2006
Abstract Hybrids between closely related species are
often sterile or inviable as a consequence of failed
interactions between alleles from the different species.
Most genetic studies have focused on localizing the
alleles associated with these failed interactions, but the
mechanistic/biochemical nature of the failed interactions is poorly understood. This review discusses recent
studies that may contribute to our understanding
of these failed interactions. We focus on the possible contribution of failures in gene expression as
an important contributor to hybrid dysfunctions.
Although regulatory pathways that share elements
in highly divergent taxa may contribute to hybrid
dysfunction, various studies suggest that misexpression
may be disproportionately great in regulatory pathways containing rapidly evolving, particularly malebiased, genes. We describe three systems that have
been analyzed recently with respect to global patterns
of gene expression in hybrids versus pure species, each
in Drosophila. These studies reveal that quantitative
misexpression of genes is associated with hybrid dysfunction. Misexpression of genes has been documented
in sterile hybrids relative to pure species, and variation
in upstream factors may sometimes cause the over- or
under-expression of genes resulting in hybrid sterility
or inviability. Studying patterns of evolution between
species in regulatory pathways, such as spermatogenesis, should help in identifying which genes are more
D. Ortı́z-Barrientos (&) Æ B. A. Counterman Æ
M. A. F. Noor
Department of Biological Sciences, Louisiana
State University, 107 Life Sciences Building,
Baton Rouge, LA 70803, USA
e-mail: danielo@interchange.ubc.ca
likely to be contributors to hybrid dysfunction. Ultimately, we hope more functional genetic studies will
complement our understanding of the genetic disruptions leading to hybrid dysfunctions and their role in
the origin of species.
Keywords Hybrid sterility Æ Hybrid inviability Æ
Gene expression Æ Speciation Æ Transcription Æ
Microarray
Introduction
‘‘The view generally entertained by naturalists is
that species, when intercrossed, have been specially
endowed with the quality of sterility, in order to prevent the confusion of all organic forms. . . . It must,
however, be confessed that we cannot understand,
excepting on vague hypotheses, several facts with
respect to the sterility of hybrids’’. (Darwin 1859).
Naturalists have long recognized that matings between members of different species often result in the
formation of sterile or inviable hybrids. Since
the merger of Mendelism with evolutionary theory in
the 1930’s and 1940’s, researchers realized that this
hybrid unfitness must necessarily involve incompatible
interactions between the two parental genomes of hybrids. However, the mechanistic nature of these interactions remains very poorly understood even today.
Dobzhansky and Muller pointed out that single
underdominant loci or chromosome rearrangements
are unlikely to cause most hybrid unfitness (Dobzhansky
1934; Muller 1940, 1942). If one species has an ancestral allele and its relative has a novel allele, then the
novel allele must have initially arisen via mutation and
appeared in the second species (or its ancestor) as a
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heterozygote. If the new allele exhibited strong
underdominance with the ancestral allele, as by causing
sterility or inviability, the first heterozygous individual
would never have reproduced, and the new allele
would have been eliminated in the generation after it
was formed. As such, strongly underdominant loci or
rearrangements cannot be the cause of hybrid unfitness
unless special assumptions are invoked. Instead,
Dobzhansky and Muller posited that interactions
between alleles at different loci were more likely to be
responsible for the interactions leading to hybrid
unfitness.
Genetic studies of hybrid sterility and inviability
have generally supported the Dobzhansky–Muller
formulation. F1 hybrid sterility and inviability are
sometimes associated with interactions between the
two sex chromosomes or between one sex chromosome
and one or more autosomes (e.g., Johnson et al. 1992;
Pantazidis et al. 1993; Davis et al. 1994; Noor et al.
2001; Orr and Irving 2001; Slotman et al. 2004). How
common are these general types of interactions in
causing hybrid dysfunctions? What is the mechanism of
Dobzhansky–Muller incompatibilities? What evolutionary forces drive the evolution of alleles causing
incompatibilities? There exists a wide gulf between
identifying chromosomal regions associated with
incompatibilities and understanding their mechanistic
or biochemical nature.
Although chromosomal rearrangements, failed
interactions between proteins, and various epigenetic
mechanisms certainly contribute to many cases of
hybrid dysfunctions, we devote our efforts here to
review data and theory suggesting that divergence in
regulatory pathways and disruptions in gene expression
profiles may also contribute to hybrid sterility and
inviability. We review a few recent theoretical models
supporting the possible role of evolutionary changes in
regulatory genetic pathways causing hybrid sterility.
We identify broad empirical evidence for rapid
evolutionary divergence in gene expression between
species and expression disruptions in hybrids, and we
conclude with some prospects and future questions to
be addressed.
Disruptions in gene expression in hybrids
Several mechanisms could explain how genes fail to
interact properly in hybrids. For example, the rapid
evolution of an upstream transcription factor in one
species may lead to failed interaction with the promoter or enhancer of its downstream target gene in a
second species. Also, relaxed selection on pathway
branches may lead to disruptions in gene expression in
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hybrids by allowing the accumulation of mutations in
one species that fail to properly interact in the hybrid
genetic background. Below, we describe these and
other interactions of regulatory elements that may
cause disruption in gene expression in hybrids.
Transcription factor/ binding site divergence
Johnson and Porter (2000) have mathematically
explored the hypothesis that regulatory evolution is a
major source of epistatic variation for hybrid dysfunction. They found that hybrids can have low fitness
where natural selection has independently altered the
binding affinity between transcription factors and
DNA binding sites. They modeled reduction in fitness
as a function of both the number of interacting loci in a
genetic regulatory pathway and the complexity of the
match between transcription factors and binding sites.
They found that the frequency that hybrid fitness
reduction occurred was positively correlated with the
number of loci in a regulatory pathway and the complexity of binding site interactions. Finally, the
homogenizing effects of gene flow might prevent or
delay the accumulation of regulatory genetic incompatibilities. If selection is sufficiently strong, however,
hybrid incompatibility still occasionally evolves despite
moderate levels of gene flow. (Porter and Johnson
2002). If divergent populations come into contact, both
new and compensatory advantageous mutations may
spread into both species. Gene flow forces populations
to evolve towards the same compensatory genotypes.
The paradox of high levels of genetic variability
in regulatory pathways
When species hybridize, the likelihood of an improper
interaction is a function of the binding strength
between the transcription factor and its binding
site (Johnson and Porter 2000). Highly conserved
transcription factors are likely to match their sites in
the other genetic background. Paradoxically, transcription factors exhibiting high intraspecific variability
may also be likely to bind their respective sites in
hybrids. The reason is that the binding strength within
species of some transcription factors may be already
low, due to high levels of genetic variability, and
developmental pathways may be adapted to respond
to such low binding affinities. Therefore, diverged
genomes that come into contact in hybrids may still have
transcriptions factors with sufficient binding strengths
for proper developmental interactions. If we assume
that expression variability within species is reflected
in expression divergence between species, then
Genetica (2007) 129:71–81
fast-evolving cis-regulatory sites can handle nontrivial
levels of mismatch to regulatory proteins, and cisregulatory divergence should not be a good explanation for hybrid dysfunctions except at very high levels
of evolutionary divergence. This correlation between
intraspecific expression variation and interspecific
expression variation has been observed in Drosophila
(Meiklejohn et al. 2003), primate, and mouse species
(Khaitovich et al. 2004). However, recent studies of
genome-wide patterns of expression in pure-species
and sterile male hybrids (Michalak and Noor 2003;
Ranz et al. 2004) suggest that hybrids have disruptions
in the expression of many genes, particularly those with
sex-biased expression.
Other mechanisms
Gene loss or gene duplication may contribute to gene
expression disruption in hybrids via relaxed selection.
Eliminating an upstream gene in a pathway of one
species may allow the accumulation of mutations in
downstream targets that are not longer compatible
with regulatory elements in a second species. This situation is likely to occur in highly branched pathways or
where there is an alternative pathway responsible for
the phenotype (Zufall and Rausher 2004). Similarly,
gene duplicates preserved by complementary degenerate mutations (Force et al. 1999; Lynch and Force
2000) may provide new functions or highly diverged
gene copies that could fail to properly interact in a
hybrid genome.
Allelic variation for enhancers and their respective
genes may also contribute to certain hybrid dysfunctions such as hybrid breakdown. Consider a case where
both the enhancer and the gene each have two alleles.
If there is one combination of alleles from the
enhancer and the gene that works best (i.e. increases
fitness), natural selection may create strong linkage
disequilibrium between these alleles. If two species
bear different combinations of enhancer-gene alleles,
F1 individuals will likely have normal phenotypes but,
upon backcrossing to one of the parental species,
recombination between the enhancer of ‘species one’
and the gene from ‘species two’ may produce unfit
offspring. This may occur because transcription factors
from the parental species may not recognize the foreign enhancer. This effect would be more pronounced
in later backcrossing or intercrossing generations, as
more recombination events would take place, resulting
in hybrid breakdown.
Post-transcriptional modifications like alternative
splicing increase protein diversity and influence tissue
distribution in a variety of taxa (Modrek and Lee
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2002), and these, too, may provide a setting for hybrid
dysfunction. Although originally considered a rare
event, alternative splicing has been shown to be a relatively common phenomenon (Brett and Pospisil 2002;
Boue et al. 2003). Conservation of alternative spliced
forms between species appears to depend on whether
exons appear in the majority (>50%) of the transcripts
(major form) or not (minor form). Minor forms of
alternative exons are poorly conserved between some
species. For example, only 25% of minor exons are
conserved between humans and mice (Modrek and Lee
2002 but see; Nurtdinov et al. 2003), a similar result to
that observed when comparing humans and rats (Boue
et al. 2003). As such, minor forms of exons, by providing a rapidly evolving element to gene expression,
might be candidates for failed interactions between
species.
The most likely contributors to failed regulatory
interactions leading to hybrid dysfunction seem to
be rapidly evolving genes, those genes which bear
sequences that have evolved faster than expected
under neutrality. These genes usually show an excess of
non synonymous substitutions over synonymous
substitutions compared to the average gene. Rapidly
evolving genes, particularly those involved in transcription factor/ binding site interactions (Johnson
and Porter 2001) are likely contributors to hybrid
dysfunctions. Recent work on speciation has begun to
explore patterns of gene expression divergence both
between species and their hybrids.
Patterns of gene-expression divergence between
species
Sequence and expression divergence between
species
Gene expression patterns may be similar between taxa,
yet sequence divergence in regulatory sequences may
be great (Wray et al. 2003). For example, Romano and
Wray (2003) found ‘‘dramatic divergence in promoter
sequence’’ in a study of Endo16 gene expression patterns in sea urchins, yet the overall expression pattern
was highly conserved among the different species. A
similar result was found in a study of CpG island
promoters among homologous mouse and human
genes, in which species specific promoters were highly
diverged, but maintained similar cell specific expression patterns (Cuadrado et al. 2001). A functional assay
of enhancer conservation between Caenorhabditis
briggsae and C. elegans also revealed similar gene
expression patterns in the two species despite extensive
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sequence divergence of enhancers (Ruvinsky and
Ruvkun 2003). Similar results have been found in
comprehensive analyses of sequence divergence and
gene expression profiles of gene regulatory networks
between taxa (Hinman et al. 2003; Ihmels et al. 2004).
These studies have revealed high conservation of the
overall network architecture (in terms of the genes
involved and their interactions) and overall expression
output, despite considerable diversification at the
sequence level among individual components, especially cis-regulatory factors.
Although regulatory pathways conserved between
taxa may still contribute to hybrid dysfunction, various
studies suggest that misexpression may be restricted to
regulatory pathways containing rapidly evolving genes
and controlling developmental systems like spermatogenesis. We review some of the recent findings from
Drosophila because of the large number of studies in
this genus and the greater familiarity of these studies to
the authors.
Rapid divergence of gene expression between
Drosophila species
Sex-biased expression evolution
After the initial work on gene expression in Drosophila
(White et al. 1999), other studies have looked at patterns of gene expression at different levels of organization in different organisms and have consistently
found that sex-related genes evolve faster, have an
unusual distribution across the genome and have highly
divergent patterns of expression. For example, Parisi
et al. (2003) identified genes with sex-biased expression
and compared their expression patterns in somatic and
germ cells. After examining the distribution of sexbiased genes across chromosomes, they identified a
relative paucity of genes with preferential expression in
males located on the X chromosome for both somatic
and germ cells. In addition, Parisi et al. (2003) also
examined movement and loss of sex-biased genes
between Drosophila and Anopheles and found that the
degree of male-biased expression was inversely related
to the probability of conservation: homologs of highly
male-biased genes in Drosophila typically could not be
identified in the Anopheles genome. This comparison,
albeit between highly divergent species noted that
male-biased genes tend to evolve faster than other
genes.
Studies of gene expression between closely related
species have revealed the same pattern. Ranz et al.
(2003) identified evolutionary changes in sex-specificity
of gene expression between D. melanogaster and
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D. simulans, and they documented that half of the 4776
genes surveyed increased, decreased, or lost sex-biased
expression. They also found that male-biased genes
showed significantly greater divergence in expression
than either female-biased or non-sex-biased genes.
Complementary to this research, Meiklejohn et al.
(2003) observed greater variation among D. melanogaster populations in male-biased genes than in femaleor non-sex-biased genes. This evidence of rapid
divergence of sex-biased expression and functional
clustering of highly diverged male-biased genes suggests sex-specific selection may play a major role in the
fast evolution of gene expression in Drosophila. Similar observations of rapid divergence (faster than neutral) of male reproductive genes have been
documented in DNA sequence in several organisms
(Swanson and Vacquier 2002), and these observations
suggest a potential pathway for the evolution of
incompatibilities in hybrid males evolving before those
in hybrid females.
More recently, Nuzhdin et al. (2004) analyzed the
expression patterns of 6, 707 transcripts from male
genotypes of D. melanogaster and D. simulans and
found that 26% of genes differed significantly in
expression. Their use of heterozygous D. simulans lines
was more appropriate for identifying within species
variability for gene expression patterns, compared to
highly inbred lines, as used in other studies (e. g.,
Rifkin et al. 2003). Nuzhdin et al. (2004) compared
divergence in transcript levels to DNA sequence
divergence, identifying several genes with rapid rates
of evolution between the two sister species and using
intra- vs. interspecific variation for transcript levels.
Consistent with the earlier observations, the genes
bearing the greatest DNA sequence and expression
changes between species were often testis-specific
genes and male accessory gland proteins. In addition,
they observed a positive correlation between expression changes and the rate of nonsynonymous substitution (r = 0.42) but not between expression changes
and the rate of synonymous substitution (r = 0.05).
Nuzhdin et al. (2004) suggested that this correlation
with nonsynonymous substitution rate arises from
similar selection regimes (i. e. relaxed selection or
strong directional selection) acting on the sequence
and expression levels for many genes.
Late versus early developmental pathways
One recent study has suggested particular parts of
regulatory pathways may be particularly prone to rapid
expression evolution. Rifkin et al. (2003) compared
expression patterns for 12, 866 transcripts between
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Drosophila simulans, D. yakuba, and four strains of D.
melanogaster. Transcript abundance levels were measured during two developmental stages during metamorphosis for each strain and species. Relative
differences in transcript abundance between developmental stages varied between species for 27% of the
transcripts studied. Functional classification of these
diverged transcripts revealed an overrepresentation of
genes involved late in developmental pathways versus
early in the pathway. Targets of regulatory factors were
rapidly evolving lineage-specific expression patterns,
however the regulatory factors were highly conserved
between species. This evidence suggests ‘late genes’ or
regulatory factor targets represent a functional class of
genes responsible for some of the rapid gene expression divergence observed between the species. Cork
and Purugganan (2004) review an array (no pun intended) of studies supportive of and contradicting this
conclusion. This is an area clearly needing further
study.
In sum, recent studies on sequence and expression
divergence of regulatory pathways are revealing several features of regulatory evolution important to our
understanding of hybrid dysfunction: first, male-biased
have been repeatedly shown to evolve faster than most
other categories of genes. Second, the rapid evolution
of male-specific genes might be associated with chromosomal patterns of gene distribution (e. g. the feminization of the X chromosome in certain taxa). Third,
there is evidence that particular parts of a regulatory
pathway may evolve faster than others. Some studies
suggest that upstream regulators are under strong
balancing selection, while others advocate that a high
level of genetic variability is common at these levels of
the pathway. Finally, closely related species differ in
gene expression profiles, and there appears to be a
positive correlation between expression divergence
and non-synonymous rates of substitution. What is the
relationship between these observations and hybrid
dysfunction? Now we turn to current studies examining
genome-wide patters of expression in hybrids and their
parents.
Genome-wide studies of expression disruption in
species hybrids
Systems of study
Many studies have investigated gene expression differences between pure species and hybrids in the
context of studying the evolution of development or
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differences in form between taxa (e.g., Dickinson and
Carson 1979; Swalla and Jeffery 1996; Nielsen et al.
2000; Skaer and Simpson 2000). Hybrid disruptions
were documented in some of these studies, such as the
loss of cell-type specific expression of CyIII actin in
hybrids of Heliocidaris sea urchin species (Nielsen
et al. 2000). However, until very recently, almost no
studies examined genome-wide patterns of expression
in pure-species and hybrids, particularly with the aim
of identifying possible genetic underpinnings of hybrid
dysfunctions. For example, Zeng and Singh (1993) used
two-dimensional gel electrophoresis to examine
expression of 1,000 testis proteins in Drosophila simulans, D. sechellia, and their hybrids. They noted that
very few proteins were expressed in hybrids at levels
above or below both of the pure species, but specific
proteins could not be isolated or identified.
Here, we review three systems that have been analyzed recently with respect to global patterns of gene
expression in hybrids versus pure species, each in
Drosophila. In each case, consistent with the rapid
divergence in expression of male-specific genes we
described between species, genes having male-specific
patterns of expression appear to have been disproportionately disrupted in sterile hybrids. We explain
why this result is not as intuitive as it may appear. We
then describe studies of three putative hybrid sterility
or inviability genes that may have regulatory roles.
Drosophila simulans/D. mauritiana
The Drosophila simulans clade has been the focus of
intensive genetic studies because it is comprised of
three species that hybridize to form sterile hybrid
males and fertile hybrid females and because of
their close genetic relationship to the model species
D. melanogaster. Because of the sequence similarity to
D. melanogaster, many genetic tools, such as microarrays, can be applied to these species. Michalak and
Noor (2003) used commercial D. melanogaster oligonucleotide microarrays to examine patterns of expression in adult male D. simulans, D. mauritiana, and their
sterile F1 male hybrids. Because of sequence divergence, the authors could not survey expression of all
14, 000 transcripts, but even by a conservative measure,
692 genes were surveyed. Consistent with the study of
Zeng and Singh (1993) in a related species pair, only 10
of these 692 genes were significantly underexpressed
in the hybrids relative to pure-species. The observed
hybrid misexpression was concentrated in genes
preferentially expressed in males: 6 of the 10 underexpressed genes had male-specific expression (including some known to function in spermatogenesis),
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compared to 34 of the remaining 682 genes. However,
all hybrid underexpression was quantitative- differences in expression were all five-fold or less.
A cautionary note on these findings is warranted.
The results presented above are derived from the
hybridization of RNA from D. simulans and D. mauritiana to a D. melanogaster microarray chip. This
hybridization will be impacted both by transcript
abundance and by sequence mismatch between the
probe and mRNA. Hence, the 682 genes surveyed (and
presumably matching in sequence) may not be representative of the genome as a whole, as they are likely
genes conserved in sequence across millions of years of
evolutionary divergence. Nonetheless, the striking
pattern with respect to the male-specific vs. non-sexspecific genes surveyed is enlightening.
One could suggest that the observed misexpression
was a trivial consequence of the sterility of these hybrids. For example, if hybrid males never complete the
process of spermatogenesis or have misformed gonads,
then genes transcribed late in the process would surely
not have been expressed since their precursors were
absent. However, Drosophila spermatogenesis is
unusual in that virtually all transcription occurs
premeiotically (Fuller 1998). In contrast, the disruptions in spermatogenesis in hybrids of D. simulans
and D. mauritiana are postmeiotic (e.g., Wu et al.
1992). Indeed, testes in such hybrids often appear
morphologically normal. Of course, it is impossible to
definitively exclude some undetected premeiotic or
morphological anomalies causing both sterility and
misexpression. Nonetheless, with the information
available, it appears the opportunity for transcription
may have occurred in these hybrids, but the genes are
nonetheless underexpressed. As such, sterility may not
have caused underexpression, but the underexpression
may have contributed to sterility.
To test for a possible association between misexpression and sterility in these male hybrids, Michalak
and Noor (2004) backcrossed the fertile female hybrids
over five generations to D. simulans males, scoring
fertility and expression in the offspring ‘‘BC5sim’’
males. Assuming normal recombination, this backcrossing should leave only 3% of a haploid D. mauritiana genome, and half of the BC5sim males they
assayed were fertile. They surveyed expression of five
genes previously shown to be underexpressed in the F1
male hybrids. Michalak and Noor (2004) observed very
little variation in expression among the fertile BC5sim
males, but they observed extensive variation in
expression among the sterile males. Specifically, sterile
males expressed these transcripts at levels at or substantially below the levels observed in fertile males,
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suggesting an association between gene expression
disruption and hybrid sterility. Michalak and Noor
(2004) built further on this by identifying the DNA
segments from D. mauritiana present in these same
BC5sim males, hence mapping the divergent upstream
regulator(s) causing misexpression and sterility. They
noted that all the sterile males bore a region of the
X-chromosome from D. mauritiana that bears the
Odysseus gene (see section on OdsH below), while
fertile males all bore the D. simulans homologous
segment. All markers elsewhere in the genome surveyed were homozygous for the D. simulans allele.
This pair of studies coupled a reverse-genetics
approach (genome-wide expression profiling) with a
forward genetics approach (genetic mapping) and a
correlational analysis to yield a hypothesis of the
expression disruptions in hybrids that may cause
sterility and the location of the divergent trans-acting
regulatory factor(s). While the evidence is still inferential that these expression disruptions are the cause of
sterility, the expression disruptions studied are now
candidates that can be further assayed through reversegenetics approaches such as RNAi or transformations.
This combined approach in a model system has the
potential to greatly elucidate the genes causing hybrid
dysfunctions as well as the explicit genetic or developmental consequences of their replacement. As much
of speciation genetics has focused on identifying the
genes causing hybrid dysfunctions (Noor 2003), the
latter question has very rarely even been asked.
Drosophila melanogaster/D. simulans
More recently, Ranz et al. (2004) examined gene
expression differences between D. melanogaster, D.
simulans, and their F1 hybrid females. These species
are substantially more divergent than D. simulans and
D. mauritiana: all hybrids are sterile or nearly so, and
one hybrid sex is inviable if ‘‘rescue’’ mutations are not
present in the hybridizing strains. Surviving female
hybrids possess severely atrophied gonads as well as
other morphological anomalies.
Using D. melanogaster cDNA microarrays, Ranz
et al. (2004) found that 69% (3083/4450) of transcripts
surveyed were significantly over- (1311) or underexpressed (1772) in female hybrids relative to females
of both pure species when whole bodies were surveyed.
This number is strikingly high, but it may partially
reflect the morphological anomalies and substantial
gonadal atrophy in hybrid females. To account for this,
Ranz et al. (2004) also surveyed expression of the
heads alone, and they found that 3% (145/4448) of
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transcripts surveyed were significantly over- (84) or
under-expressed (61).
Unsurprising given their gonadal atrophy, very
many of the genes found to be underexpressed in
hybrids (1003) were ones that are female-specific
in expression, including several known to function in
oogenesis. However, surprisingly, a disproportionately
large fraction of male-specific genes (518) was overexpressed in these female hybrids. This observation
may reflect a hybrid breakdown of the regulatory
mechanisms that normally repress transcription of
male-specific genes in females.
Drosophila pseudoobscura/D. persimilis
The experiments described above took advantage of
the only published Drosophila genome sequence at the
time: that of D. melanogaster. However, surveys of
genome-wide patterns of expression do not require a
published genome or a model system (see e.g., Crawford 2001). Taking advantage of the technique of differential display (Liang et al. 1993; Liang 2002),
Reiland and Noor (2002) surveyed patterns of gene
expression in adult male Drosophila pseudoobscura, D.
persimilis, and their sterile F1 male hybrids. Their
technique involves PCR amplification of cDNA using
short oligonucleotides, hence randomly scanning the
transcriptome for expression. This approach has the
advantage that transcripts surveyed do not have to
have been characterized previously, but it has the disadvantages of being much less sensitive than microarrays or real-time RT-PCR, having a higher error
rate, and requiring extensive additional work to identify specific transcripts.
Of 3312 transcripts surveyed by Reiland and Noor
(2002), only 28 appeared to be underexpressed in
hybrids relative to both D. pseudoobscura and
D. persimilis. Noor et al. (2003) later analyzed the most
repeatable of these transcripts putatively misexpressed
in hybrids, and they found it to be a large, male-predominant, antisense transcript of the TRAP100 gene.
The sense transcript of this gene produces a protein
that functions in regulating transcriptional initiation
during development, and is expressed similarly in pure
species as in hybrids. One would presume the antisense
transcript functions to regulate the production of this
protein, and therefore the protein may be expressed at
a higher level in females than in males. If true, the
deficiency of the antisense transcript in hybrids would
cause an overproduction of the protein, hence ‘‘feminizing’’ these sterile male hybrids. At present, this
model is highly speculative, as no Western blot was
performed to confirm that the protein is expressed at
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higher levels in females or hybrids. Nonetheless, the
results further illustrate the utility of a reverse genetics
approach for yielding testable hypotheses with regard
to the genetics of hybrid dysfunctions.
Cloned hybrid dysfunction genes
Variation in upstream factors, likely genic repressors,
may cause the overexpression of genes that cause
disruptions in hybrids. This role has been inferred
from three of four genes suggested to be involved in
hybrid inviability or sterility. This doesn’t appear true
for a fourth gene: Nup96 (Presgraves et al. 2003)
(Fig 1).
Xmrk-2
One of the earliest identified genes causing hybrid
dysfunctions was found in the southern platyfish,
Xiphophorus maculates. A fraction of backcross
hybrids from Xiphophorus maculates, and the
unspotted swordtails, X. helleri, have enlarged spots
on their dorsal fins that spontaneously develop malign
melanomas (Schartl 1995; Schartl et al. 1999). This
system fits well the expectation of the DobzhanskyMuller model of incompatibilities: it consists of an
X-linked ‘‘tumor gene’’ (Tu) whose activity is
suppressed by a regulatory locus on an autosome (R).
Both the Tu gene and suppressors alleles (R) are
absent in X. helleri leading to the overexpression of Tu
and consequently to the development of exaggerated
macromelanophores in backcross offspring.
The molecular basis of this interaction has been
elucidated in some detail as the genes responsible for
the Tu phenotypes have been isolated. The Tu phenotype is controlled by a growth factor receptor named
Xiphophorus melanoma receptor kinase (Xmrk)
(Witbrodt et al. 1989). This gene has two copies, one of
which is expressed in all Xiphophorus fish and is not
responsible for causing tumors (Adam et al. 1991;
Zechel et al. 1992; Woolcock et al. 1994). In contrast,
the second copy, Xmrk-2, represents the gene responsible for the Tu phenotype and is only present in the
sex chromosomes of some Xiphophorus species. The
duplicates are very similar in sequence but differ in
their promoters. This difference, in turn, affects their
expression patterns (Adam et al. 1993): Xmrk-1 is
expressed at low levels in all tissues while Xmrk-2 is
expressed at high levels in hybrids with melanomas
(Wittbrodt et al. 1989). This system not only brings to
light the molecular mechanism responsible for hybrid
dysfunction in Xiphophorus, but also suggests how
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Fig. 1 Types of disruptions in
gene expression that may lead
to hybrid dysfunction. Each
panel represents the potential
parent for a hybrid individual.
Each species has undergone
evolution at different levels of
their genetic machinery,
possibly leading to failures in
interactions when the two
genomes come together in
hybrids
gene duplication may be involved in creating interactions disrupting gene expression in hybrids.
Hmr
A very well developed system for studying hybrid
inviability occurs in hybrids between D. melanogaster
and its sibling species (Hutter et al. 1990). In this
group, the increase in dose of the gene Hybrid male
rescue (Hmr) reduces viability in their hybrids
(Barbash et al. 2000). Hmr, a gene with two major
DNA binding protein motifs, is expressed through out
development and encodes a protein with homology to
a family of MYB-related DNA binding transcriptional
regulators and is one of the most rapidly evolving
genes in Drosophila (Barbash et al. 2003). Hmr also
seems to be a good inferential example for compensatory evolution because in spite of the many deletions,
insertions and substitutions, the gene has an intact
open reading frame and is normally expressed in both
D. melanogaster and its sibling species. In addition,
Hmr has functionally diverged between D. melanogaster,
D. simulans and D. mauritiana (Barbash et al. 2004).
This functional divergence is consistent with the
Dobzhasky–Muller model of genetic incompatibilities,
and appears to have resulted from ancient positive
selection in both lineages.
OdsH
Genetic studies of hybrid male sterility identified a
region of the Drosophila mauritiana X-chromosome
123
that confers sterility in a D. simulans autosomal genetic
background (Coyne and Charlesworth 1986). Perez
et al. (1993) further localized this gene, named it
Odysseus, and found that it also must interact with
other D. mauritiana segments to cause hybrid sterility
(Perez and Wu 1995). Ting et al. (1998) genotyped
introgressions between these species to map the sterility effect to a rapidly evolving testis-specific
homeobox gene, called OdsH (for Ods-site homeobox
gene). This gene is expressed in both fertile and sterile
male hybrids, but the transcript is highly localized to
the distal ends of testes in sterile males while it is more
homogeneously expressed across the testes in fertile
males (Sun et al. 2004). Homeobox genes generally
function in transcriptional regulation during development, and the mapping of disruptions in gene expression to the OdsH region by Michalak and Noor (2004)
supports a possible role for divergence in this gene (or
other(s) closely linked to it) in causing both misexpression and sterility.
Expression profiling as a complementary approach to
studying speciation
Some of the progress described above regarding use of
microarrays and other new genome-wide expression
profiling approaches may suggest they are excellent
avenues to supplement standard forward-genetic
mapping. However, it must be remembered these new
approaches are no holy grail: they cannot solve the
speciation problem. Microarrays in particular have
Genetica (2007) 129:71–81
become exceedingly popular as a means for addressing
any questions in many biological fields, including evolutionary biology. However, they merely score a new
phenotypic trait: amount of particular transcripts in
individuals. The connection of this trait to either
genotype or morphological/ physiological/ behavioral/
ecological phenotype is unclear. Identifying a large
difference in expression of a particular gene or set of
genes between fertile pure-species males and sterile
hybrid males does not necessarily implicate these genes
or their expression disruptions in causing hybrid sterility. Similarly, some hybrid dysfunctions may not be
associated with any expression differences. Hence, we
would be ill-advised to abandon established (and successful!) methodologies such as QTL mapping, deletion mapping, and the use of recombinant inbred lines
in favor of expression approaches.
We argue instead that combining forward and reverse
genetic approaches may advance our understanding of
Dobzhansky–Muller incompatibilities more than either
approach alone. Forward genetics approaches localize
the regions of the genome bearing the genes causing
reproductive isolation, and can ultimately identify genes
involved (see above), but they tend to become less
powerful at finer scales. In contrast, genome-wide
expression profiling can supplement these approaches
by identifying a single (or few) strongest candidate gene
within these intervals for the final manipulative experiment needed for confirmation (e.g., Wayne and McIntyre 2002). Further, there exists abundant theoretical
and inferential experimental evidence that gene
expression disruptions are associated with hybrid
sterility and/ or inviability. At minimum, this combination of approaches can help explain the first-stage phenotypic consequences of the D-M incompatibilities that
ultimately lead to gross morphological or developmental disruptions in hybrids.
Microarrays in particular must be used judiciously,
and there are many factors to consider in their application. A wide range of papers and books are devoted
to this topic, and we refer the reader to these (e.g.,
Boake et al. 2002; Gibson 2002; Nadon and Shoemaker
2002; Causton et al. 2003).
Prospects and future questions to be addressed
Genome-wide patterns of expression in hybrids have
revealed a striking feature: the misexpression of genes
is often quantitative, yet this misexpression nonetheless appears to be repeatedly associated with hybrid
dysfunction. This observation could suggest that either
few changes between species are sufficient to cause
failed interactions, or closely related species may
79
withstand between species variation as a byproduct
of the inherent within species variation in gene
expression. Empirical data demonstrating that fast
evolving male-biased genes are preferentially misexpressed in species hybrids suggests that certain regulatory pathways may contribute more directly to hybrid
dysfunction than others. This may take place through
their pattern of transcription factor/binding site divergence between species.
However, the picture appears more complicated. A
question derived from the above discussion is why do we
detect so many sterility factors in certain species crosses?
Why do some species crosses with similar levels of F1
sterility differ so much in the number of factors contributing to hybrid dysfunction? One simple explanation
is the geographical setting of speciation. If isolated
populations come into contact, gene flow may purge and
homogenize regulatory changes that took place in each
species. As a consequence, the number of genetic
incompatibilities should be smaller than those found
between strictly allopatric species. Another is the drastically different rates of evolution of male versus female
incompatibilities, which may be consistent with studies
we have reviewed. Studies of cis-regulatory evolution
comparing species that have diverged in allopatry or
sympatry (e. g. secondary contact), and rates of evolution in male vs. females specific genes, should be helpful
to understand the forces driving the accumulation of
hybrid dysfunction factors.
Disruptions in gene expression between hybrids may
result from either failed interactions in upstream factors of a regulatory pathway or its downstream targets.
Studying patterns of evolution between species in
regulatory pathways such as spermatogenesis should
help clarify whether their early or late genes are more
likely to be contributors to hybrid dysfunction. Ideally
functional divergence tests should accompany the
hierarchical study of DNA divergence of regulatory
pathways between species.
The level of interconnection of proteins may contribute to failed interactions between proteins or with
DNA in species hybrids. If natural selection or other
forces accelerating the rate of change in the genome
are fundamental to the speciation process, it is more
likely that proteins not using most of their structural
space when interacting with other proteins or DNA
sequences, should be more prone to produce failed
interactions in hybrids. Nup96 may be an example of
such a protein since the nuclear pore to which it
belongs contains few interacting proteins (Daven
Presgraves, personal communication).
Theoretical studies on the role of mildly deleterious
mutations on speciation should be particularly fruitful
123
80
if connected to regulatory evolution and gene expression disruptions in hybrids. The idea that binding
affinity is critical to theoretical models of speciation
involving regulatory pathways, suggests that mildly
deleterious mutations may play a buffering role during
speciation. This idea may also underlie the observed
correlation of variation in gene expression within- and
between species. Evolving to new adaptive peaks in
enhancer sequences, or coding regions of enhancers,
may take a long time, while most new mutations could
be mildly deleterious. In such case, development might
just respond well enough and produce imperfect, but
viable organisms.
Acknowledgements We thank S. Dixon Schully for preparing
an early draft of one section of this paper. Funding was provided
by National Science Foundation grants 0211007 and 0314552.
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