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MBE Advance Access originally published online on June 6, 2006
Molecular Biology and Evolution 2006 23(9):1707-1714; doi:10.1093/molbev/msl033
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Research Article

Gene Regulation Divergence Is a Major Contributor to the Evolution of Dobzhansky–Muller Incompatibilities between Species of Drosophila

Wilfried Haerty and Rama S. Singh

Department of Biology, McMaster University, Hamilton, Ontario, Canada

E-mail: singh{at}mcmaster.ca.


   Abstract
TOP
 Abstract
Introduction
 Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
The Dobzhansky–Muller model denotes incompatible gene interactions between diverging populations/species and is recognized as the basis of postzygotic reproductive isolation. Little is known about the molecular nature of such gene interactions. We have carried out comparative gene expression analyses in the testes of 3 closely related species of the Drosophila melanogaster subgroup and their hybrids (all of which are sterile). We show that in hybrids 1) a higher proportion of male-biased genes (i.e., genes with a higher level of expression in males) are underexpressed (or not expressed) compared with non–sex-biased genes, 2) the majority of the underexpressed genes appear to be under stabilizing selection by virtue of showing similar levels of expression in the parental species, and only a small proportion of genes show signs of directional selection, 3) very few of the misexpressed genes are shared between species pairs, suggesting that there may not be a "common" set of "speciation genes," and 4) expression of non–testes-specific genes is observed in the testes of interspecific hybrids, and the number of such genes is positively correlated with divergence time. These results suggest that gene regulation divergence of sex- and reproduction-related genes is a major contributor to the evolution of Dobzhansky–Muller incompatibilities between species of Drosophila.

Key Words: gene regulation • hybrid male sterility • stabilizing selection • sex-biased genes • Haldane's rule • Dobzhansky–Muller incompatibilities


   Introduction
TOP
 Abstract
 Introduction
Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
Postzygotic reproductive isolation by hybrid sterility or inviability is thought to arise between isolated populations by accumulation of different combinations of neutral or positively selected allelic substitutions. According to the Dobzhansky–Muller model (Dobzhansky 1936Go; Muller 1942Go), in interspecific hybridizations, gene interactions will be disrupted by different incompatible allele combinations that are fixed in diverged species, consequently leading to hybrid sterility or inviability (Orr 1997Go; Johnson and Porter 2000Go). Previous studies have focused on coding sequences' divergence driven by positive selection between species as a main driver of the Dobzhansky–Muller incompatibilities (Ting et al. 1998Go; Presgraves et al. 2003Go; Barbash et al. 2004Go). However, at the transcriptional level, studies focusing on one or a small number of genes have highlighted the divergence of regulatory elements between species (Shaw et al. 2002Go; Carroll 2005Go). Such studies have pointed out the importance of genic incompatibilities that could lead to hybrid sterility (Wittkopp et al. 2004Go; Landry et al. 2005Go). The Drosophila melanogaster subgroup species are a suitable model to study the nature of incompatibilities because interspecific hybrids are sterile and present severe defects in their germ line, reproductive organs, and gametogenesis (Kulathinal and Singh 1998Go; Hollocher et al. 2000Go). Because reproductive traits are those most commonly affected in hybrids, we expect the expression patterns of sex-biased and sex-related genes to be strongly disrupted in comparison to other gene categories.

In this study, we compared testes gene expression patterns in 3 different hybrids from crosses between Drosophila simulans females and D. melanogaster, Drosophila mauritiana, and Drosophila sechellia males. Although our study is restricted to genes expressed in the testes, we were able to classify genes as male biased, female biased, and non–sex biased in expression (using Andrews et al. 2000Go; Jin et al. 2001Go; Ranz et al. 2003Go; Parisi et al. 2004Go). Our results show a massive breakdown of male-biased gene expression among closely related species, suggesting that hybrid incompatibilities can accumulate rapidly over short divergence times. Most importantly, the majority of genes that show a breakdown in expression in hybrids do not show any expression-level divergence between the parental species, which strongly suggests that these genes are under stabilizing selection. This also indicates that species are accumulating divergent cryptic genetic changes in coding sequences as well as in regulatory regions that are revealed only in the case of genetic perturbations such as hybridizations.


   Methods
TOP
 Abstract
 Introduction
 Methods
Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
Drosophila Species
Four-day-old virgin D. simulans females (14021-0251.2) were crossed with D. melanogaster (14021-0231.00), D. mauritiana (14021-0241.1), and D. sechellia (Cousin Island, Jean R. David Centre National de la Recherche Scientifique [CNRS], Gif sur Yvette, France) males. All the species were reared on standard cornmeal medium at 25 °C under a 12-h light/dark cycle. Hybrids and pure species males were collected at emergence and were maintained for 4 days in the same conditions as indicated before.

RNA Extraction and Hybridization
In order to obtain appropriate quantities of RNA for hybridization assays, total RNA from a pool of 200 testes, dissected without accessory glands, from 4-day-old individuals was extracted using the RNeasy kit (Qiagen, Mississauga, ON). One round of linear amplification (Van Gelder et al. 1990Go) using MessageIIAmp aRNA amplification kit (Ambion Inc, Austin, TX) was performed. Three independent replicates were prepared for each species and each type of hybrids.

RNA from a pool of 4-day-old D. melanogaster males (whole body) was extracted using Trizol (Invitrogen, Carlsbad, CA) in order to reach an appropriate quantity (5 mg) and used as reference RNA in all the hybridizations. Hybridizations of experimental and reference RNA were performed on 12Kv1 microarrays prepared by the Canadian Drosophila Microarray Centre (CDMC, http://www.flyarrays.com). The array contains more than 12,500 D. melanogaster cDNAs representing approximately 10,500 genes (Neal et al. 2003Go). All hybridizations were performed at the CDMC for the 3 different hybrids and the 4 parental species. Arrays were scanned using a ScanArray 4000 XL (GSI Lumonics/Packard Biochips, Billerica, MA); images were preprocessed and quantified using QuantArray v3.0 (PerkinElmer, Boston, MA).

Data Analysis
Preparation and normalization of the data were accomplished using GeneTraffic Duo (Iobion Informatics, La Jolla, CA). A spot was considered as valid if the ratio "probe intensity/background intensity" was higher than 2, if the probe intensity was greater than 2 times the local background, and if the covariance of the intensity between replicates for these spots was lower than 150%. Genes with less than two-thirds of their spots considered valid were removed. Only genes showing at least one log2 ratio greater than 0.5 in parents and/or hybrids were used for statistical analyses. A modified t-test (Significance Analysis of Microarray, Tusher et al. 2001Go) was used to identify genes showing significant changes in expression with the following parameters: 500 permutations of the data, a falsely significant call cutoff lower than 1 and a false discovery rate (FDR) lower than 0.05.

Because RNA from D. simulans, D. mauritiana, and D. sechellia was hybridized on microarrays spotted with D. melanogaster cDNA, a hybridization bias due to sequence divergence between D. simulans clade species and D. melanogaster may be expected (Gilad et al. 2005Go). In order to limit the effects of such a bias, a previous study, using the same species and D. melanogaster Affymetrix arrays, restricted the analysis to genes with similar level of expression between parental species but significantly different between hybrids and parental species (Michalak and Noor 2003Go). Here, such a bias was estimated by looking at possible correlation between sequence divergence and expression difference between D. melanogaster and D. simulans. Sequence divergence was estimated for 286 genes, chosen randomly from genes showing nonsignificant and significant expression differences between these species in our data set. Drosophila melanogaster coding sequences were downloaded from Flybase (http://flybase.bio.indiana.edu/), and D. simulans genes were obtained from the National Center for Biotechnology Information (NCBI) trace archive (http://www.ncbi.nlm.nih.gov/Traces/trace). Sequences were aligned using ClustalX 1.8 (Thompson et al. 1997Go) and RevTrans 1.4 (Wernersson and Pedersen 2003Go). Rates of synonymous and nonsynonymous substitutions were estimated using PAML (Yang and Nielsen 2000Go). Rates of divergence of 68 genes from the study of Betancourt and Presgraves (2002)Go were added to the data set, leading to a final sample size of 354 genes. No significant linear correlation was observed between the absolute value of the observed difference between the mean expression levels of both species and sequence divergence (R2 = –0.0025, P = 0.73; fig. S1, Supplementary Material online) for this sample of genes. Because D. sechellia has special ecological conditions (living on Morinda citrifolia) compared with D. mauritiana and D. simulans, we may expect to observe greater differences in some regions of the genome. Therefore, the same analysis was performed on a sample of 167 genes from D. sechellia obtained from the NCBI trace archive. No significant correlation between sequence divergence (dN/dS) and expression difference between D. melanogaster and D. sechellia was observed for this data set (R2 = –7.254 x 10–5, P > 0.05). Even though no relationship was observed between sequence divergence and expression difference between D. simulans or D. sechellia and D. melanogaster, only genes significantly misexpressed in hybrids in comparison to both parental species were retained for further analysis. However, the analysis was not restricted to genes with similar level of expression between species as Michalak and Noor (2003)Go did.

Chromosomal distributions of misexpressed genes across the genome were estimated for each hybrid using the release 4.2.1 of the D. melanogaster genome. Proportions of over- and underexpressed genes were compared with the expected proportions of genes on each chromosome according to a random distribution of misexpressed genes across the genome using chi-square tests and Bonferroni correction.

Proportions of over- and underexpressed sex-biased and non–sex-biased genes classified according to criteria laid out in previous studies (Andrews et al. 2000Go; Jin et al. 2001Go; Ranz et al. 2003Go; Parisi et al. 2004Go) were compared using chi-square tests. Levels of misexpression among sex-biased and non–sex-biased genes were also investigated using Mann–Whitney tests. According to gene annotations from Flybase (Drysdale and Crosby 2005Go), genes were also classified as male specific, female specific, or involved in both reproductive functions if they are affecting reproductive processes (spermatogenesis, oogenesis, and meiosis).


   Results
TOP
 Abstract
 Introduction
 Methods
 Results
Discussion
 Supplementary Material
 Acknowledgements
 References
 
Breakdown in Gene Expression in Hybrids' Testes
Following the expectations of the Dobzhansky–Muller model, that is, accumulation of incompatibilities with divergence time (Orr 1997Go), hybrids sired by D. melanogaster males show the highest number of misexpressed genes in comparison to both parents (1,541 vs. 200 and 241 for hybrids sired by D. mauritiana and D. sechellia males, respectively, fig. 1). These results parallel the observations of strongly atrophied testes in hybrids sired by D. melanogaster males (Hollocher et al. 2000Go) (table 1). In case of hybrids sired by D. sechellia or D. mauritiana males, even though the numbers of misexpressed genes are similar (241 in D. sechellia hybrids vs. 200 in D. mauritiana hybrids), the majority of the genes (235/241) are underexpressed in hybrids sired by D. sechellia males, whereas in hybrids sired by D. mauritiana males, the numbers of underexpressed and overexpressed genes are similar (89 and 73, respectively). Moreover, hybrids sired by D. mauritiana males show a higher number of genes with an additive expression, that is, show an intermediate level of gene expression in comparison to the parents (38/200) in contrast to the hybrids sired by D. melanogaster (3/1,541) or D. sechellia males (4/241).


Figure 1
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  		FIG. 1.— Comparison of gene expression in parental and hybrid testes. (a) Drosophila simulans versus hybrid (D. simulans females x Drosophila mauritiana males). (b) D. mauritiana versus hybrid (D. simulans females x D. mauritiana males). (c) D. simulans versus hybrid (D. simulans females x Drosophila sechellia males). (d) D. sechellia versus hybrid (D. simulans females x D. sechellia males). (e) D. simulans versus hybrid (D. simulans females x Drosophila melanogaster males). (f) D. melanogaster versus hybrid (D. simulans females x D. melanogaster males). Misexpressed male-biased genes are represented in green, all other misexpressed genes in red, and non–misexpressed genes in gray.

 

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  		Table 1 Comparison of Male- and Female-Biased Gene Proportions between Significantly Over- and Underexpressed Genes in Testes in Comparison to Both Parents

 
In hybrids sired by D. melanogaster males, a greater proportion of significantly underexpressed genes are actually "nonexpressed" (log2 < 0, log2 > 0.5 in the parental species) compared with the proportion of underexpressed genes observed in the hybrids sired by D. mauritiana males (table 2, {chi}2 = 19.76, df = 1, P < 0.001, after Bonferroni correction) as well as in hybrids sired by D. sechellia males (table 2, {chi}2 = 7.16, df = 1, P < 0.05, after Bonferroni correction). It is important to note that although their divergence times are similar, hybrids sired by D. sechellia males have a significantly higher proportion of nonexpressed (absent) genes in the testes than hybrids sired by D. mauritiana males ({chi}2 = 19.76, df = 1, P < 0.001, after Bonferroni correction).


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  		Table 2 Proportions of Genes Underexpressed and Overexpressed in Hybrids Classified According to Their log2 Ratio

 
Among the group of overexpressed genes, the hybrids sired by D. melanogaster males have a greater proportion of "newly expressed" genes in the testes (log2 > 0.5 in hybrids but <0 in parental species, table 2) compared with the hybrids sired by D. mauritiana males ({chi}2 = 4.85, df = 1, P < 0.05). In hybrids sired by D. sechellia males, there are only 2 overexpressed genes, both of which are absent (not expressed) in the parental testes (log2 < 0).

Commonly Misexpressed Genes across Hybrids
We found that very few genes are commonly misexpressed in hybrids sired by D. mauritiana and D. sechellia males (16 genes, fig. 2). In fact, only 14 genes, in total, are commonly misexpressed in all 3 hybrids (fig. 2). However, a large number of genes that are misexpressed in hybrids sired by D. melanogaster males are also misexpressed in hybrids sired by D. mauritiana and D. sechellia males. These results may be explained by the large defects observed in the testicular atrophy observed in hybrids sired by D. melanogaster males (Hollocher et al. 2000Go).


Figure 2
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  		FIG. 2.— Number of misexpressed genes shared by the different hybrids. Shown are the total number of misexpressed genes (bold) and male-biased genes.

 
Sex-Biased Misexpression across Hybrids
We classified genes in our data set as male-, female-, or non–sex biased and compared their expression patterns across hybrids to examine if any particular class was preferentially misexpressed. We found that male-biased genes were predominantly underexpressed in hybrids sired by D. mauritiana ({chi}2 = 17.53, df = 1, P < 0.001) and D. melanogaster males ({chi}2 = 79.42, df = 1, P < 0.001, fig. 1 and table 1). Moreover, the proportions of underexpressed male-biased genes in hybrids sired by D. melanogaster (66.04%), D. sechellia (76.50%), and D. mauritiana (79.77%) males are similar among hybrids ({chi}2 = 0.787, 1.57, and 1.29; df = 1, P > 0.05 in comparisons between hybrids involving D. mauritiana and D. sechellia, D. mauritiana and D. melanogaster, and D. sechellia and D. melanogaster).

Among the groups of underexpressed genes, male-biased genes showed stronger breakdown scores than non–sex-biased genes in hybrids sired by D. melanogaster males (Mann–Whitney tests, P < 0.001, data not shown). However, no significant difference was observed in the hybrids sired by D. sechellia males (Mann–Whitney tests, P > 0.05, data not shown). Among the groups of overexpressed genes, male-biased and non–sex-biased genes were equally disrupted in hybrids sired by D. mauritiana and D. melanogaster males (Mann–Whitney tests, P > 0.05, data not shown). In hybrids sired by D. sechellia males, only 2 overexpressed genes were observed (supplementary data 2, Supplementary Material online).

No difference was observed in the proportion of overexpressed male-biased genes between hybrids sired by D. mauritiana (50.68%) and D. melanogaster males (49.25%, {chi}2 = 0.02, df = 1, P > 0.05). There is a higher proportion of overexpressed female-biased genes in hybrids sired by D. mauritiana and D. melanogaster males in comparison to underexpressed genes ({chi}2 = 9.58, df = 1, P < 0.01, table 1).

An examination of "sex-related" genes (i.e., genes known to affect sexual processes) expression in hybrids sired by D. melanogaster males confirmed previous reports; a significantly higher proportion of sex-related genes was observed to be underexpressed ({chi}2 = 4.12, df = 1, P < 0.05, table 3). Of these genes, male-specific genes were mostly underexpressed ({chi}2 = 15.86, df = 1, P < 0.001, table 3), and female-specific genes were predominantly overexpressed ({chi}2 = 10.32, df = 1, P < 0.01, table 3).


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  		Table 3 Number of Male- and Female-Specific Reproductive Genes Found among Misexpressed Genes in Hybrids Involving the Male Drosophila melanogaster

 
Chromosomal Distribution of Misexpressed Genes in Hybrids
We analyzed the distribution of misexpressed genes according to their chromosomal location. With the exception of the hybrids sired by D. mauritiana males ({chi}2 = 2.91, df = 1, P > 0.05), only underexpressed genes presented a significant departure from a random chromosomal distribution, showing significant underrepresentation on the X chromosome ({chi}2 = 14.01 and 16.87, df = 1, P < 0.001 and P < 0.001 in hybrids sired by D. sechellia and D. melanogaster males, respectively; fig. S2, Supplementary Material online). This was also true for male-biased genes ({chi}2 = 13.76 and 15.33, df = 1, P < 0.001 and P < 0.001, for the hybrids sired by D. melanogaster and D. sechellia males). In hybrids sired by D. mauritiana males, sex-biased genes are randomly distributed over the genome (see table S1, Supplementary Material online).

Stabilizing Selection on Gene Expression
From the comparisons of parental gene expressions, we noticed that the majority of the under- and overexpressed genes in hybrids have rather similar expression levels (FDR > 0.05, table 4, supplementary data 1, 2, and 3, Supplementary Material online) in the parental species (81.48%, 88.79%, and 73.44% for the hybrids sired by D. mauritiana, D. sechellia, and D. melanogaster males, respectively, table 4). We interpret this low between-species divergence to be the result of stabilizing selection on gene expression. Moreover, for the hybrid involving the D. mauritiana male, we found no significant differences in the proportions of genes under stabilizing selection among over- and underexpressed genes (86.30% and 77.52%, respectively, {chi}2 = 0.21, df = 1, P > 0.05). However, in hybrids sired by D. melanogaster males, a significantly lower proportion of underexpressed genes appear to be under stabilizing selection in comparison to overexpressed genes (67.17% and 87.36% for under- and overexpressed genes, respectively, {chi}2 = 10.1, df = 1, P < 0.01). Finally, a higher proportion of male-biased genes appear to be evolving under directional selection or random genetic drift (i.e., genes showing expression differences between species) in comparison to genes under stabilizing selection (66.83% vs. 54.32%, {chi}2 = 4.96, df = 1, P < 0.05).


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  		Table 4 Proportion of Misexpressed Genes in Hybrids Classified According to Gene Expression Difference in Parental Species

 

   Discussion
TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
Supplementary Material
 Acknowledgements
 References
 
Our hybridization results conform to the expectations of Dobzhansky–Muller model, that is, we find a strong increase in incompatibilities with divergence time (Orr 1997Go) because the number of misexpressed genes parallel the level of reproductive incompatibility observed between species. However, the rate of accumulation of misexpressed genes strongly differs among sex-biased and non–sex-biased genes. The number of misexpressed sex-biased genes is higher in hybrids sired by D. mauritiana (72.5%) and D. sechellia males (79.25%) in comparison to hybrids sired by D. melanogaster males (67.81%). Previous studies on gene expression in female or male hybrids from one species pair (Michalak and Noor 2003Go; Ranz et al. 2004Go) also observed an overabundance of misexpression of sex-biased genes. However, being limited to a single species pair, these studies did not shed light on the pattern of evolution of interspecies incompatibilities over time.

An unexpected finding in our study is that gene expression patterns in hybrids sired by D. mauritiana and D. sechellia males are strikingly different (fig. 1) and that very few misexpressed genes are shared between hybrids (fig. 2), despite the fact that these 2 species have been separated from D. simulans for roughly similar lengths of time (0.3 and 0.4 Myr, respectively; Kliman et al. 2000Go). The contrasting patterns of gene expression differences in hybrids sired by D. sechellia and D. mauritiana males suggest that in spite of the fact that they both produce sterile hybrid males with postmeiotic defects when crossed with D. simulans female (Kulathinal and Singh 1998Go), these siblings have experienced different genetic changes during the course of speciation. Because the suites of misexpressed genes strongly differ, different gene interactions must be involved in producing postzygotic reproductive isolation in either of the species with D. simulans.

Furthermore, few misexpressed genes are shared between hybrids sired by D. mauritiana and D. sechellia males (fig. 2), and the chromosomal distribution of underexpressed genes also differ. Confirming the observations of Michalak and Noor (2003)Go, we found no significant differences between observed and expected numbers of genes on the X chromosome. However, for the 2 remaining hybrids, a strong paucity of underexpressed genes was observed on the X. As somatically expressed male-specific genes are underrepresented on the X chromosome and because these genes are not inactivated in the soma (Swanson et al. 2001Go), the significant paucity of underexpressed genes on the X chromosome observed in the hybrids sired by D. sechellia and D. melanogaster males cannot be the consequence of the early condensation of this chromosome during spermatogenesis (Parisi et al. 2003Go; Wu and Xu 2003Go; Singh and Kulathinal 2005Go). As well, hybrids sired by D. melanogaster males that present premeiotic defects (Hollocher et al. 2000Go) showed a significant paucity of underexpressed genes on the X chromosome (fig. S2, Supplementary Material online). These results support the male sex drive hypothesis (see Singh and Kulathinal 2005Go), which invokes a mutation selection enrichment of male-biased genes on the autosomes regardless of the X inactivation although contradicting the Sexual Antagonism driving germline X Inactivation hypothesis (Wu and Xu 2003Go).

Finally, gene expression appears to be conserved between closely related species and also appears to be under stabilizing selection (Rifkin et al. 2003Go; Lemos et al. 2005Go; Rifkin et al. 2005Go) even for genes that are strongly up- or downregulated in hybrids. Stabilizing selection on gene expression and rapid sequence divergence are not contradictory in the context of gene networks, given that neutral mutations can accumulate rapidly (Gibson and Dworkin 2004Go). However, many sex- and reproduction-related genes have been shown to evolve under positive selection, most probably due to sexual selection (Civetta and Singh 1998Go; Kulathinal and Singh 2004Go; Jagadeeshan and Singh 2005Go). Directional selection on nucleotide sequence can trigger stabilizing selection on gene expression as a means of alleviating deleterious pleiotropic effects of, for example, quantitative variations in gene products (Rifkin et al. 2005Go). Gene interactions can minimize the effects of mutations through buffering, feedback, or compensation (Rutherford 2000Go; Kondrashov et al. 2002Go; Kulathinal et al. 2004Go; Proulx and Phillips 2005Go). Compensatory evolution was observed in Drosophila, where cis regulatory elements have been observed to evolve rapidly in sequence divergence between species of Drosophila without conferring significant changes in gene expression (Ludwig et al. 2000Go, 2005Go). Moreover, coevolution of cis and trans elements was demonstrated between more distant Dipteran species (Shaw et al. 2002Go). Therefore, misregulations in hybrids could be the result of incompatible interactions between cis and trans elements as shown by Landry et al. (2005)Go. Thus, in spite of the overall similarity of gene expression between parental species observed in this study, regulatory pathways appear to have evolved differently (True and Haag 2001Go) due to accumulation of different combinations of coevolving cis and trans elements. The interplay between directional selection on protein sequences and stabilizing selection on gene expression could be a driving force for regulatory elements evolution.

In view of these results, we envision a general scenario for the evolution of hybrid male sterility between species.

Stage 1: A rapid and "preferential" accumulation of male-biased gene substitutions, leading to downregulation of male-biased gene expression in hybrids as observed in the hybrids sired by D. mauritiana and D. sechellia males (table 1).

Stage 2: As genetic divergence proceeds, male-biased genes are more severely downregulated than non–sex-biased genes as observed in the hybrids sired by D. melanogaster males compared with the hybrids sired by D. sechellia males. The overall proportion of underexpressed, male-biased genes decreases with time due to the accumulation of non–sex-biased gene incompatibilities leading to downregulation of these genes in hybrids. Therefore, a negative correlation between the proportion of underexpressed male-biased genes and divergence time is expected. However, even though a significant difference in the proportions of non–sex-biased genes is observed between hybrids sired by D. mauritiana and D. melanogaster males (18/89 vs. 363/1,063, {chi}2 = 3.96, df = 1, P < 0.05) (table 1), the proportions of underexpressed male-biased genes are not significantly different between hybrids sired by D. mauritiana and D. melanogaster males. Therefore, more distant species should be used to test this hypothesis. At the same time, hybrid incompatibilities also lead to an increasing number of non–testes-specific genes being expressed in the testes, that is, genes not expressed in testis of parents are being expressed in hybrids. This consequently leads to a significant difference between the hybrids sired by D. mauritiana and D. melanogaster males (P < 0.05, table 2) and to the nonexpression of genes normally expressed in these organs (table 2).

Because gene expression is under strong stabilizing selection, differences between hybrids and parents would appear to be more the result of divergence in the gene regulatory systems (cis and trans) than coding sequences' differences (Ludwig et al. 2000Go; True and Haag 2001Go; Shaw et al. 2002Go; Landry et al. 2005Go; Ludwig et al. 2005Go). Therefore, during the initial stages of species divergence, even though some genes may be evolving under directional selection or random genetic drift and might have important effects on sexual isolation, the postzygotic reproductive isolation in these sibling species appears to involve mostly genes that are under stabilizing selection for gene expression. A significant effect of directional selection will of course be manifested between distantly related species (D. simulans and D. melanogaster).

In conclusion, the results presented here provide support for a rapid evolution of expression incompatibility between closely related species of Drosophila. Furthermore, even though our results represent only a subset of genes affected by Dobzhansky–Muller incompatibilities, the results show that 1) initially sex- and reproduction-related genes, especially those affecting male fertility, are the basic targets of Dobzhansky–Muller incompatibilities, 2) gene regulation divergence is a major contributor to this process, and 3) stabilizing selection on gene expression, in addition to directional selection, may also play an important part in this process. The next step of the investigation will make use of D. melanogaster, D. simulans, and soon-to-be-released D. sechellia genomes to analyze species divergence of regulatory elements to understand the basis of gene disruption in hybrids and to identify the different genetic pathways involved in this phenomenon.


   Supplementary Material
TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary Material
Acknowledgements
 References
 
Microarrays data that have been deposited in the Gene Expression Omnibus under the accession number GSE3673 [NCBI GEO] , table S1, figures S1 and S2, and 3 data files are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).


   Acknowledgements
TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
References
 
We thank Jean R. David, who provided the D. sechellia strain, Tim Westwood, and CDMC for their help in the completion of this work. We are grateful to Carlo Artieri, Pierre Capy, Santosh Jagadeeshan, Rob Kulathinal, and Richard Morton for their comments on the manuscript. This work was supported by a Natural Science and Engineering Research Council of Canada genomics grant to R.S.S.


   Footnotes
 
Marcy Uyenoyama, Associate Editor


   References
TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 

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Accepted for publication June 1, 2006.


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