Rapidly Evolving Genes of Drosophila: Differing Levels of Selective Pressure in Testis, Ovary, and Head Tissues Between Sibling Species
http://www.100md.com
分子生物学进展 2005年第9期
Department of Biology, McMaster University, Hamilton, Ontario, Canada
E-mail: singh@mcmaster.ca.
Abstract
Investigations of rapidly evolving sex- and reproduction-related genes are expected to reveal important information about the process of speciation and species divergence. We screened testis, ovary, and head tissues to identify and characterize rapidly evolving genes (REGs) between closely related species. The results show differential patterns of evolution of genes expressed in reproductive and nonreproductive tissues. (1) There is a differential distribution of REGs in the Drosophila genome, with most REGs localized in the testis, followed by ovary, and then head. (2) Sequence analysis indicates that differential selective pressures are driving the rapid evolution of genes expressed in sex and nonsex tissues. Testis REGs from our data, on average, yielded higher rates of nonsynonymous substitutions relative to transcripts in ovary and head, indicating stronger selective pressures on the male reproductive system. (3) We identified REGs in the testis, ovary, as well as in head tissue that show evidence of evolving under positive selection. Identification of rapidly evolving sex genes is important for detailed investigations of cryptic female choice, sexual conflict, and faster male evolution and is pertinent to our understanding of the process of species divergence and speciation.
Key Words: speciation ? sexual selection ? sex- and reproduction-related genes ? rapidly evolving genes
Introduction
About 30% of the Drosophila genome has been shown to be rapidly evolving (Werman, Davidson, and Britten 1990; Schmid and Tautz 1997). This fraction of the Drosophila genome still remains to be characterized systematically, although some interesting trends have been revealed. Two-dimensional electrophoresis studies have suggested that a large proportion of this rapidly evolving fraction may be localized in the reproductive tissues (Coulthart and Singh 1988). In addition, many proteins in testis and ovary showed higher divergence over nonreproductive tissue proteins (Civetta and Singh 1995). Numerous recent comparative DNA studies have shown that several sex- and reproduction-related genes (SRR) evolve rapidly and often adaptively between closely related species (see Singh and Kulathinal 2000; Swanson et al. 2001a, 2004; Swanson and Vacquier 2002). This trend has been extended to transcriptional patterns of sex-biased genes between closely related species (Ranz et al. 2003). Male-biased genes show higher variation in expression patterns (Meiklejohn et al. 2003; Ranz et al. 2003). Whole-genome comparisons of Anopheles gambiae and Drosophila melanogaster (Parisi et al. 2003) and more recently of two Drosophila species diverged about 35 MYA (Powell 1997) also support this trend. Together, these data reveal, at the molecular level, an important trend that had previously been reported at the morphological level (Eberhard 1985)—differential divergence of sex traits over nonsex traits. This differential divergence of sex versus nonsex genes conforms to evolution by sexual selection (Darwin 1871; Eberhard 1985).
The challenge remains, however, to understand how rapidly evolving genes (REGs) relate to important phenomena such as species divergence and speciation (Singh and Kulathinal 2000; Wu and Ting 2004). How do REGs affect reproductive isolation and hybrid sterility or inviability? The higher divergence of male and female genes could result from coadaptation (Miller and Pitnick 2002) or conflict (Gavrilets 2000) driven by interacting male and female reproductive molecules. On the other hand, the higher divergence of male genes over female genes could imply male-driven evolution. The first step to resolve this issue is to identify genes that are rapidly evolving in both male and female reproductive systems and then to characterize their functions. It is also important to identify REGs in nonreproductive tissues such as head or brain, which may contribute to sexual behavior or affect other sensory stimuli.
We have conducted a survey to systematically identify, isolate, and investigate REGs in Drosophila species that have diverged for different lengths of time. We used complete cDNAs from testis, ovary, and head cDNA libraries to identify REGs and then sequenced candidate REGs from each tissue for further analysis. This allows us not only to dissect the rapidly evolving fraction of the Drosophila transcriptome but also to comprehensively study patterns of sequence evolution of REGs expressed in different tissues. We focused on genes that are evolving rapidly between closely related species of Drosophila, with an operational definition of an REG as a gene that shows relatively high divergence between two species separated for the least amount of time. In this contribution, we particularly focused on the sibling species pair D. melanogaster and Drosophila simulans, diverged about 2.5–5 MYA (Powell 1997). We asked the following questions: (1) Do reproductive tissues have larger proportions of REGs? (2) If so, are there differences in the proportion of REGs between the male and female reproductive systems? (3) Are they evolving neutrally or in response to selection? And most importantly, (4) What are these REGs? How do they affect the reproductive biology of Drosophila?
Materials and Methods
Screening for REGs
We used a modification of the original screen of Schmid and Tautz (1997) to isolate fast-evolving genes from Drosophila. We procured D. melanogaster adult testis cDNA library from Justin Andrews (National Institutes of Diabetes and Digestive and Kidney Diseases of the National Institute of Health, Bethesda, Maryland, NIH), adult ovary cDNA library from Tulle Hazellrigg (Columbia University), and adult head cDNA library from Richard Dearborn (Harvard University) (all cDNA libraries were normalized). cDNA libraries from each tissue were plated at low density, and we randomly picked out clones for screening. Phagemids containing the cDNA inserts were in vivo excised with ExAssist helper phage (Stratagene, La Jolla, Calif.). The cDNA inserts were polymerase chain reaction (PCR) amplified using the T3 and T7 universal primers in pBluescript vectors. PCR-amplified inserts were random prime labeled with -P32, using the Random Prime DNA Labeling Kit (Roche Applied Science, Indianapolis, Ind.). -P32–labeled cDNAs were then hybridized at high stringency (precalibrated, see below) to genomic targets from related drosophilid species.
Calibration of Hybridization Stringency Using Gene of Known Divergence
To calibrate stringency conditions, we used transformer cDNA (tra, obtained from John Belote, Syracuse University) as a reference probe for an REG (Kulathinal et al. 2003). PCR-amplified tra cDNA was random prime labeled with -P32 and hybridized to the genomic panel at increasing temperatures, starting at 42°C. The temperature at which tra probe failed to hybridize to the species most closely related to D. melanogaster was taken as the predetermined stringency temperature for further assays. In this case, tra hybridized inefficiently to Drosophila yakuba and completely failed to hybridize to Drosophila virilis at 54°C.
Screening Libraries for REGs
-P32–labeled cDNAs from each tissue were hybridized to a panel of genomic DNA from D. melanogaster (native species), D. simulans, D. yakuba, and D. virilis (diverged from D. melanogaster approximately 2.5 MYA, 7–14 MYA, and 40 MYA, respectively) (Powell 1997). Hybridizations were done in 6 x sodium chloride, sodium phosphatase, and ethylenediamine tetra acetic acids 50% formamide, 100 mg/ml of denatured Salmon sperm DNA, and 5 x Denhardt's reagent. All hybridizations were done at a standard (42°C) and at predetermined temperature (54°C) for 10–16 h. Following hybridizations, membranes were washed in 1 x standard saline citrate (SSC) and 0.5% sodium dodecyl sulfate (SDS) twice at room temperature for 20 min each and then in 0.2 x SSC and 0.1% SDS at 60°C for 35 min. The underlying principle of this technique is that under high stringency conditions (or calibrated predetermined conditions, see below), native D. melanogaster probe DNA will fail to hybridize or will hybridize inefficiently to orthologs that have diverged rapidly.
Obtaining Signal Ratios
Exposed negatives were scanned and analyzed in NIH Image (Scion Corp., Frederick, Md.). Signal ratio intensities were calculated as the difference between the intensity of hybridization of the D. melanogaster probe to D. melanogaster target (a) relative to the hybridization intensity of the D. melanogaster probe to the species target (b).
Signal ratio intensity = [(a – b)/a]. Signal ratio intensity obtained ranged from 0 to 1, with 0 representing high sequence identity and 1 indicating extensive sequence divergence.
Blast Searches
Prior to sequencing, clones used as probes were partially sequenced using T3 primers and Blast searches were performed to the D. melanogaster genome to obtain identity of the cDNA. Owing to long gene lengths and PCR-amplification difficulties, complete coding sequences could not be obtained for some of the genes. Missing fragments were obtained from the National Center for Biotechnology Information (NCBI) trace archive (http://www.ncbi.nlm.nih.gov/Traces/trace, sequencing done and deposited by Genome Sequencing Center, Washington University, St. Louis, Mo.). Drosophila melanogaster sequences were used as queries in Blast searches to the D. simulans and D. yakuba tracer database. Retrieved tracer sequences were aligned using ClustalX ver. 1.8 as well as RevTrans 1.0 and were manually assembled.
Sequence Analysis of Candidate REGs
Clones were ranked according to signal ratio divergence between D. melanogaster and D. simulans. A subset of the top 10 candidate REGs from each tissue was chosen for sequencing. Primers were designed from coding regions using the annotated D. melanogaster genome (release 3). Sequences were aligned using ClustalX ver. 1.8 and RevTrans 1.0 (Wernersson and Pedersen 2003). Tajima-Nei's distance estimates (total number of substitutions per site, Tajima and Nei 1984) were calculated using MEGA ver. 2.1 (Kumar et al. 2001). Pairwise estimates of dN and dS as well as maximum likelihood tests of positive selection were implemented using PAML (Yang 2002). To detect sites evolving under positive selection, likelihood ratio tests were done to compare data fit to models M7 (where 0 < < 1) versus M8 (which contains the additional site class > 1). Drosophila yakuba sequences for PAML analysis were obtained from the NCBI trace archive (http://www.ncbi.nlm.nih.gov/Traces/trace, sequencing done and deposited by Genome Sequencing Center, Washington University).
Results
Differential Distribution of REGs Among Tissues
A total of 50 clones from D. melanogaster testis cDNA library, 50 clones from ovary cDNA library, and 42 clones from head cDNA library were randomly isolated and screened for REGs using high stringency Southern dot blots. Some clones were mitochondrial genes and a few were defective (containing no cDNA sequences) and eliminated from further analysis, collapsing the total analyzable sample to 41 testis, 40 ovary, and 30 head cDNAs. Previous results have shown that a large fraction of D. melanogaster embryonic cDNAs (54%) were rapidly evolving and their homologs could not be detected in the D. virilis genome (Schmid and Tautz 1997), a species that has diverged from the former about 40 MYA. In our study, about 59% of the adult testis cDNA, 35% of ovary cDNAs, and 40% of head cDNAs did not hybridize to D. virilis, indicating that most of the REGs may be localized in the testis. Our interest lay in dissecting these rapidly evolving fractions in even closer relatives of D. melanogaster, such as D. simulans and D. yakuba (diverged from D. melanogaster 2.5 MYA and 6–14 MYA, respectively). Only a small fraction of D. melanogaster homologs are undetectable in the more closely related species D. yakuba (4%); however, it is noteworthy that this fraction was isolated from the testis and not from the other tissues. We further dissected hybridization results by analyzing the intensity of cross-hybridization (signal ratio intensities) between D. melanogaster cDNA probes and their homologs in related species.
The variation in signal ratio intensities revealed some very interesting trends (fig. 1a–c and table 1). Comparing the more closely related species, there is a higher proportion of cDNAs with high signal ratios in testis and ovary compared to head, indicating that there are more highly diverged genes in reproductive tissues than in nonreproductive tissue (fig. 1 and table 1). Between the sibling species D. melanogaster and D. simulans, the difference between signal ratio distribution of the cDNAs of testis and ovary was not significant; however, signal ratio distribution of each reproductive tissues was significantly higher than that of head (fig. 1a and table 1). This divergence trend is amplified in the D. melanogaster and D. yakuba comparison (fig. 1b), and the differences in the distributions between tissues are significant (table 1). Further away in the divergence clock, in the D. melanogaster–D. virilis comparison, the shift in signal ratios is more obvious with about 59% of genes in the testis and 35% in ovaries and 40% of head transcripts showing signal ratios of 1, indicating a complete lack of hybridization (fig. 1c).
FIG. 1.— Testis, ovary, and head cDNAs signal ratio distribution between species pairs. 0 indicates high sequence identity, and 1 indicates extensive sequence divergence and lack of cross-hybridization. (a) Drosophila melanogaster versus Drosophila simulans (divergence time: 2.5 MYA). (b) Drosophila melanogaster versus Drosophila yakuba (divergence time: 7–14 MYA). (c) Drosophila melanogaster versus Drosophila virilis (divergence time: 40 MYA).
Table 1 Mann-Whitney Sum Rank Test of Variation in Signal Ratio Distribution
Higher Divergence of Reproductive Genes
Hybridization assays are crude estimates of divergence and are subject to variability due to several factors underlying the technique. More importantly, they do not reveal the patterns of sequence evolution, which are important for our objectives. We calculated Tajima-Nei's genetic distance (total number of substitutions per site, Tajima and Nei 1984) for the subset of genes sequenced and compared these to signal ratio estimates. There was low correlation between divergence estimated through signal ratios and actual sequence divergence (R2 = 0.49), probably due to several variabilities in the technique, but also largely due to deletions and substitutions in the 3'- and 5'-untranslated regions present in the cDNAs, which were not present in PCR sequences because we focused mainly on coding sequence divergence. Nevertheless, the rank order of genes did not change drastically, and sequence divergence estimates reflected the same trend as the hybridization results. Testis REGs, on average, had significantly higher Tajima-Nei's distance estimates than ovary and head REGs (Mann-Whitney sum rank test, P < 0.05). Ovary and head REGs, on the other hand, did not differ significantly (fig. 2). As a general comparison, most genes in all tissues had Tajima-Nei's estimates higher than Adh, a relatively slower evolving gene, and all genes had lower estimates than Acp26Aa, which is one of the most REGs in the Drosophila genome (Tsaur, Ting, and Wu 1998; fig. 2). These results from our data set indicate that testis transcripts incur a higher proportion of substitutions per site relative to the transcripts of head and ovary.
FIG. 2.— Tajima-Nei's genetic distance measures between Drosophila melanogaster and Drosophila simulans for candidate REGs. Acp26Aa and Adh were not part of this data set and were used as examples of an REG and a slowly evolving gene, respectively, to compare distance estimates.
Differences in Rates and Patterns of Sequence Evolution Among Tissues
REGs can be evolving neutrally or under positive selection. It is important to identify the different evolutionary forces that underlie the evolution of REGs in order to understand how they affect the biology of the organism. To test for evidence of selection, we further dissected sequence evolution patterns by estimating the proportions of synonymous (dS) and nonsynonymous (dN) substitutions of REGs in the three tissues between D. melanogaster and D. simulans (table 2 and fig. 3). High dN/dS ratios (excess of nonsynonymous substitutions) are considered to indicate directional evolution (Lee, Ota, and Vacquier 1995; Pamilo and O'Neill 1997). On average, testis REGs showed higher dN/dS ratios compared to the REGs in ovary and head (Wilcoxon's rank test < 0.05); there was no significant difference between ovary and head REGs. As a class, testis REGs, on average, had dN estimates that were approximately two times higher than dN estimates for both ovary and head REGs (dNtestis = 0.0303 ± 0.009, dNovary = 0.0147 ± 0.006, dNhead = 0.0149 ± 0.007), results that were significant according to Mann-Whitney sum rank tests (P < 0.005). There was no significant difference in the rates of synonymous substitutions between REGs in the three tissues. These data indicate accelerated evolution of testis genes and that male genes are probably under stronger selective pressures.
Table 2 The dN/dS Estimates, Tissue Expression, and Predicted/Known Functions of REGs
FIG. 3.— dN/dS estimates for candidate REGs in testis, ovary, and head between Drosophila melanogaster and Drosophila simulans. Acp26Aa and Adh are not part of this data set and were used as examples of an REG and a slowly evolving gene, respectively, to compare dN/dS estimates.
Positive Selection Drives the Rapid Evolution of Candidate REGs in Testis, Ovary, and Head
We implemented maximum likelihood analysis to identify REGs that are evolving under positive selection. Drosophila yakuba sequences were obtained through the NCBI trace archive; some were PCR amplified. The neutral model could not be rejected for most candidate REGs in all tissues. However, the most REGs in each tissue (from our data set) showed evidence of evolving under positive selection (table 3). CG18869 in the testis showed a significantly better fit to M8 (P < 0.001, table 3). The function of this gene remains uncharacterized. In the ovary, CG6533 (chorion protein 16, cp16) showed a significantly better fit to M8 (P < 0.001, table 3), indicating that this gene has sites evolving under positive selection. cp16 encodes for a structural component of the egg in all insects and belongs to a family of chorion protein genes which have been reported to show significant divergence in sequence as well as expression patterns. In addition, further analysis indicates that this gene shows significant differences in the proportions and patterns of dN between species of the melanogaster subgroup, indicating different selective forces/functional constraints between closely related species (unpublished data). CG4716, expressed specifically in the head, had positively selected amino acids (P < 0.001). The function of CG4716 is unknown. The log ratio test for CG9284 was surprising because none of the sites were detected to be evolving positively, although this gene shows the highest dN/dS ratio between D. melanogaster and D. simulans. Given that the dN/dS is not significantly >1.0, this gene may be evolving rapidly and neutrally. However, log ratio tests must be treated with caution, particularly for small proteins (CG9284 is 69 codons long) (Anisimova, Bielawski, and Yang 2001). In addition, it is important to note that only three species were used; therefore, these results must be treated with caution. Before concluding that these genes are evolving neutrally or positively, additional taxon sampling as well as polymorphism studies will be essential to improve accuracy (Anisimova, Bielawski, and Yang 2002).
Table 3 Likelihood Ratio Test of Positive Selection in Testis, Ovary, and Head REGs
Discussion
Higher divergence of sex traits over nonsex traits indicates higher selective pressures on the sexual machinery (Eberhard 1985; Coulthart and Singh 1988). Assuming that a majority of genes expressed in reproductive tissue contribute to sexual and reproductive functions (which include development of the sex tissues), we would expect to find a higher proportion of REGs in reproductive tissues. It is likely that not all genes with a reproductive function evolve rapidly. It is important, therefore, to identify genes that are rapidly evolving and to understand what is driving their evolution, as well as how they affect the reproductive biology of Drosophila. As we had expected, our results show that there is a higher proportion of highly divergent genes in the reproductive system compared to the nonreproductive system (fig. 1), implying stronger selection on genes in the reproductive system. Importantly, our data indicate a difference in the proportion of REGs in the male and female sex tissues—we found a higher proportion of REGs in the testis compared to the ovary, implying stronger selection on the male reproductive system. Although our data set is small to arrive at a conclusive and general trend between tissues, our results are supported by findings from the study of Ranz et al. (2003), which showed a similar trend in expression divergence, as well as studies done at the protein level (Civetta and Singh 1995).
The divergence trend observed from our hybridization data offers a window to observe the nature of changes that may occur during speciation events. The trend (fig. 1) supports our hypothesis that SRR may be preferentially involved in the process of reproductive isolation. Higher divergence of testis and ovary genes between sibling species diverged only 2.5 MYA (fig. 1a and b) leads us to propose the notion that in the initial stages of speciation, reproductive genes diverge much faster than nonreproductive genes, which may play a crucial role in establishing reproductive isolation (Singh 1990). Higher divergence of nonreproductive traits is observed only in comparisons of species that have diverged for longer lengths of time (fig. 1c). Our results are also interesting in view of the nature of inherent differences between the testis and ovary transcriptomes, which differ from each other as much as each of them differs from somatic tissue (Andrews et al. 2000; Parisi et al. 2004). Testis and head express a large battery of unique and highly expressed transcripts relative to the ovary (Andrews et al. 2000). In addition, restricted function range of testis transcripts and a broader functional range of ovary transcripts (Parisi et al. 2004) may influence the manner in which these genes evolve. It must be noted that the functions of most of the REGs identified in our study are unknown and several genes are expressed both in sex and nonsex tissues (see table 1); therefore, their pertinent role as sex genes remains obscure.
Identification of REGs in male and female reproductive tissue is a starting point to address some important questions regarding evolution by sexual selection at the molecular level. Divergence of sex molecules can be driven through (1) the higher divergence of male reproductive molecules and (2) interacting male and female molecules—coadaptation or conflict? In concordance with the first scenario, the preponderance of REGs in the testis along with its high rate contributes to explain the higher instances of male hybrid sterility relative to female sterility (Wu and Davis 1993; Wu, Johnson, and Palopoli 1996) as well as the faster breakdown in the male sexual machinery in hybrids. All testis REGs in our data show higher proportions of both silent and amino acid–changing substitutions, implying that testis proteins retain high flexibility in protein fold structures, therefore conserving their respective functions between the sibling species. However, there are those testis REGs (such as CG11869) that evolve rapidly under positive selection, implying that they confer selective advantages. It is particularly important to know the functions of such testis proteins in order to understand the functional basis of their rapid adaptive divergence and how they relate to hybrid phenotypes.
Under the second scenario, high divergence of both ovary and testis genes may constitute coevolution or conflict as a result of interacting male and female proteins (Metz and Palumbi 1996; Metz, Robles-Sikisaka, and Vacquier 1998; Swanson et al. 2001b). Several male proteins that are transferred into the female after copulation are toxic to the female and may alter female longevity or reproductive behavior (Chen et al. 1988; Lung et al. 2002). Females may then produce proteins to counter this deleterious effect to protect themselves, resulting in an arms race between male and female reproductive molecules (Civetta and Clark 2000). On the other hand, variation in one sex molecule may instigate change in the corresponding sex molecule if their physical interaction is essential. The positively selected chorion protein (cp16) is interesting in this regard. Studies addressing conflict and coevolution at the molecular level are rare, and in order to test these hypotheses effectively, we need to identify all REGs in ovary and testis that interact with each other, particularly those that show evidence of positive selection (see Metz and Palumbi 1996; Metz, Robles-Sikisaka, and Vacquier 1998).
REGs in head that show evidence of evolving under positive selection are interesting and unveil opportunities to identify genes that possibly affect sexual behavior. Conversely, these genes may influence nonsexual sensory functions between species due to differences in their ecological traits. Considerable variation within and between species exists in visual and acoustic stimuli in relation to courtship and mating (see Greenspan and Ferveur 2000, for a review). Identifying such genes will be crucial in investigations of the genetic basis of behavioral differences that lead to premating isolation (Huttunen, Vieira, and Hoikkala 2002). Overall, our results support the notion that sexual selection is a strong driving force driving genetic change between species (Carson 1997).
Supplementary Material
Sequences are deposited in GenBank under accession numbers DQ062756–DQ062811.
Acknowledgements
We thank Justin Andrews, Richard Dearborn, and Tulle Hazelrigg for kindly supplying cDNA libraries. This work was supported by funds from the National Sciences and Engineering Research Council of Canada to R.S.S.
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Accepted for publication May 12, 2005.(Santosh Jagadeeshan and R)
E-mail: singh@mcmaster.ca.
Abstract
Investigations of rapidly evolving sex- and reproduction-related genes are expected to reveal important information about the process of speciation and species divergence. We screened testis, ovary, and head tissues to identify and characterize rapidly evolving genes (REGs) between closely related species. The results show differential patterns of evolution of genes expressed in reproductive and nonreproductive tissues. (1) There is a differential distribution of REGs in the Drosophila genome, with most REGs localized in the testis, followed by ovary, and then head. (2) Sequence analysis indicates that differential selective pressures are driving the rapid evolution of genes expressed in sex and nonsex tissues. Testis REGs from our data, on average, yielded higher rates of nonsynonymous substitutions relative to transcripts in ovary and head, indicating stronger selective pressures on the male reproductive system. (3) We identified REGs in the testis, ovary, as well as in head tissue that show evidence of evolving under positive selection. Identification of rapidly evolving sex genes is important for detailed investigations of cryptic female choice, sexual conflict, and faster male evolution and is pertinent to our understanding of the process of species divergence and speciation.
Key Words: speciation ? sexual selection ? sex- and reproduction-related genes ? rapidly evolving genes
Introduction
About 30% of the Drosophila genome has been shown to be rapidly evolving (Werman, Davidson, and Britten 1990; Schmid and Tautz 1997). This fraction of the Drosophila genome still remains to be characterized systematically, although some interesting trends have been revealed. Two-dimensional electrophoresis studies have suggested that a large proportion of this rapidly evolving fraction may be localized in the reproductive tissues (Coulthart and Singh 1988). In addition, many proteins in testis and ovary showed higher divergence over nonreproductive tissue proteins (Civetta and Singh 1995). Numerous recent comparative DNA studies have shown that several sex- and reproduction-related genes (SRR) evolve rapidly and often adaptively between closely related species (see Singh and Kulathinal 2000; Swanson et al. 2001a, 2004; Swanson and Vacquier 2002). This trend has been extended to transcriptional patterns of sex-biased genes between closely related species (Ranz et al. 2003). Male-biased genes show higher variation in expression patterns (Meiklejohn et al. 2003; Ranz et al. 2003). Whole-genome comparisons of Anopheles gambiae and Drosophila melanogaster (Parisi et al. 2003) and more recently of two Drosophila species diverged about 35 MYA (Powell 1997) also support this trend. Together, these data reveal, at the molecular level, an important trend that had previously been reported at the morphological level (Eberhard 1985)—differential divergence of sex traits over nonsex traits. This differential divergence of sex versus nonsex genes conforms to evolution by sexual selection (Darwin 1871; Eberhard 1985).
The challenge remains, however, to understand how rapidly evolving genes (REGs) relate to important phenomena such as species divergence and speciation (Singh and Kulathinal 2000; Wu and Ting 2004). How do REGs affect reproductive isolation and hybrid sterility or inviability? The higher divergence of male and female genes could result from coadaptation (Miller and Pitnick 2002) or conflict (Gavrilets 2000) driven by interacting male and female reproductive molecules. On the other hand, the higher divergence of male genes over female genes could imply male-driven evolution. The first step to resolve this issue is to identify genes that are rapidly evolving in both male and female reproductive systems and then to characterize their functions. It is also important to identify REGs in nonreproductive tissues such as head or brain, which may contribute to sexual behavior or affect other sensory stimuli.
We have conducted a survey to systematically identify, isolate, and investigate REGs in Drosophila species that have diverged for different lengths of time. We used complete cDNAs from testis, ovary, and head cDNA libraries to identify REGs and then sequenced candidate REGs from each tissue for further analysis. This allows us not only to dissect the rapidly evolving fraction of the Drosophila transcriptome but also to comprehensively study patterns of sequence evolution of REGs expressed in different tissues. We focused on genes that are evolving rapidly between closely related species of Drosophila, with an operational definition of an REG as a gene that shows relatively high divergence between two species separated for the least amount of time. In this contribution, we particularly focused on the sibling species pair D. melanogaster and Drosophila simulans, diverged about 2.5–5 MYA (Powell 1997). We asked the following questions: (1) Do reproductive tissues have larger proportions of REGs? (2) If so, are there differences in the proportion of REGs between the male and female reproductive systems? (3) Are they evolving neutrally or in response to selection? And most importantly, (4) What are these REGs? How do they affect the reproductive biology of Drosophila?
Materials and Methods
Screening for REGs
We used a modification of the original screen of Schmid and Tautz (1997) to isolate fast-evolving genes from Drosophila. We procured D. melanogaster adult testis cDNA library from Justin Andrews (National Institutes of Diabetes and Digestive and Kidney Diseases of the National Institute of Health, Bethesda, Maryland, NIH), adult ovary cDNA library from Tulle Hazellrigg (Columbia University), and adult head cDNA library from Richard Dearborn (Harvard University) (all cDNA libraries were normalized). cDNA libraries from each tissue were plated at low density, and we randomly picked out clones for screening. Phagemids containing the cDNA inserts were in vivo excised with ExAssist helper phage (Stratagene, La Jolla, Calif.). The cDNA inserts were polymerase chain reaction (PCR) amplified using the T3 and T7 universal primers in pBluescript vectors. PCR-amplified inserts were random prime labeled with -P32, using the Random Prime DNA Labeling Kit (Roche Applied Science, Indianapolis, Ind.). -P32–labeled cDNAs were then hybridized at high stringency (precalibrated, see below) to genomic targets from related drosophilid species.
Calibration of Hybridization Stringency Using Gene of Known Divergence
To calibrate stringency conditions, we used transformer cDNA (tra, obtained from John Belote, Syracuse University) as a reference probe for an REG (Kulathinal et al. 2003). PCR-amplified tra cDNA was random prime labeled with -P32 and hybridized to the genomic panel at increasing temperatures, starting at 42°C. The temperature at which tra probe failed to hybridize to the species most closely related to D. melanogaster was taken as the predetermined stringency temperature for further assays. In this case, tra hybridized inefficiently to Drosophila yakuba and completely failed to hybridize to Drosophila virilis at 54°C.
Screening Libraries for REGs
-P32–labeled cDNAs from each tissue were hybridized to a panel of genomic DNA from D. melanogaster (native species), D. simulans, D. yakuba, and D. virilis (diverged from D. melanogaster approximately 2.5 MYA, 7–14 MYA, and 40 MYA, respectively) (Powell 1997). Hybridizations were done in 6 x sodium chloride, sodium phosphatase, and ethylenediamine tetra acetic acids 50% formamide, 100 mg/ml of denatured Salmon sperm DNA, and 5 x Denhardt's reagent. All hybridizations were done at a standard (42°C) and at predetermined temperature (54°C) for 10–16 h. Following hybridizations, membranes were washed in 1 x standard saline citrate (SSC) and 0.5% sodium dodecyl sulfate (SDS) twice at room temperature for 20 min each and then in 0.2 x SSC and 0.1% SDS at 60°C for 35 min. The underlying principle of this technique is that under high stringency conditions (or calibrated predetermined conditions, see below), native D. melanogaster probe DNA will fail to hybridize or will hybridize inefficiently to orthologs that have diverged rapidly.
Obtaining Signal Ratios
Exposed negatives were scanned and analyzed in NIH Image (Scion Corp., Frederick, Md.). Signal ratio intensities were calculated as the difference between the intensity of hybridization of the D. melanogaster probe to D. melanogaster target (a) relative to the hybridization intensity of the D. melanogaster probe to the species target (b).
Signal ratio intensity = [(a – b)/a]. Signal ratio intensity obtained ranged from 0 to 1, with 0 representing high sequence identity and 1 indicating extensive sequence divergence.
Blast Searches
Prior to sequencing, clones used as probes were partially sequenced using T3 primers and Blast searches were performed to the D. melanogaster genome to obtain identity of the cDNA. Owing to long gene lengths and PCR-amplification difficulties, complete coding sequences could not be obtained for some of the genes. Missing fragments were obtained from the National Center for Biotechnology Information (NCBI) trace archive (http://www.ncbi.nlm.nih.gov/Traces/trace, sequencing done and deposited by Genome Sequencing Center, Washington University, St. Louis, Mo.). Drosophila melanogaster sequences were used as queries in Blast searches to the D. simulans and D. yakuba tracer database. Retrieved tracer sequences were aligned using ClustalX ver. 1.8 as well as RevTrans 1.0 and were manually assembled.
Sequence Analysis of Candidate REGs
Clones were ranked according to signal ratio divergence between D. melanogaster and D. simulans. A subset of the top 10 candidate REGs from each tissue was chosen for sequencing. Primers were designed from coding regions using the annotated D. melanogaster genome (release 3). Sequences were aligned using ClustalX ver. 1.8 and RevTrans 1.0 (Wernersson and Pedersen 2003). Tajima-Nei's distance estimates (total number of substitutions per site, Tajima and Nei 1984) were calculated using MEGA ver. 2.1 (Kumar et al. 2001). Pairwise estimates of dN and dS as well as maximum likelihood tests of positive selection were implemented using PAML (Yang 2002). To detect sites evolving under positive selection, likelihood ratio tests were done to compare data fit to models M7 (where 0 < < 1) versus M8 (which contains the additional site class > 1). Drosophila yakuba sequences for PAML analysis were obtained from the NCBI trace archive (http://www.ncbi.nlm.nih.gov/Traces/trace, sequencing done and deposited by Genome Sequencing Center, Washington University).
Results
Differential Distribution of REGs Among Tissues
A total of 50 clones from D. melanogaster testis cDNA library, 50 clones from ovary cDNA library, and 42 clones from head cDNA library were randomly isolated and screened for REGs using high stringency Southern dot blots. Some clones were mitochondrial genes and a few were defective (containing no cDNA sequences) and eliminated from further analysis, collapsing the total analyzable sample to 41 testis, 40 ovary, and 30 head cDNAs. Previous results have shown that a large fraction of D. melanogaster embryonic cDNAs (54%) were rapidly evolving and their homologs could not be detected in the D. virilis genome (Schmid and Tautz 1997), a species that has diverged from the former about 40 MYA. In our study, about 59% of the adult testis cDNA, 35% of ovary cDNAs, and 40% of head cDNAs did not hybridize to D. virilis, indicating that most of the REGs may be localized in the testis. Our interest lay in dissecting these rapidly evolving fractions in even closer relatives of D. melanogaster, such as D. simulans and D. yakuba (diverged from D. melanogaster 2.5 MYA and 6–14 MYA, respectively). Only a small fraction of D. melanogaster homologs are undetectable in the more closely related species D. yakuba (4%); however, it is noteworthy that this fraction was isolated from the testis and not from the other tissues. We further dissected hybridization results by analyzing the intensity of cross-hybridization (signal ratio intensities) between D. melanogaster cDNA probes and their homologs in related species.
The variation in signal ratio intensities revealed some very interesting trends (fig. 1a–c and table 1). Comparing the more closely related species, there is a higher proportion of cDNAs with high signal ratios in testis and ovary compared to head, indicating that there are more highly diverged genes in reproductive tissues than in nonreproductive tissue (fig. 1 and table 1). Between the sibling species D. melanogaster and D. simulans, the difference between signal ratio distribution of the cDNAs of testis and ovary was not significant; however, signal ratio distribution of each reproductive tissues was significantly higher than that of head (fig. 1a and table 1). This divergence trend is amplified in the D. melanogaster and D. yakuba comparison (fig. 1b), and the differences in the distributions between tissues are significant (table 1). Further away in the divergence clock, in the D. melanogaster–D. virilis comparison, the shift in signal ratios is more obvious with about 59% of genes in the testis and 35% in ovaries and 40% of head transcripts showing signal ratios of 1, indicating a complete lack of hybridization (fig. 1c).
FIG. 1.— Testis, ovary, and head cDNAs signal ratio distribution between species pairs. 0 indicates high sequence identity, and 1 indicates extensive sequence divergence and lack of cross-hybridization. (a) Drosophila melanogaster versus Drosophila simulans (divergence time: 2.5 MYA). (b) Drosophila melanogaster versus Drosophila yakuba (divergence time: 7–14 MYA). (c) Drosophila melanogaster versus Drosophila virilis (divergence time: 40 MYA).
Table 1 Mann-Whitney Sum Rank Test of Variation in Signal Ratio Distribution
Higher Divergence of Reproductive Genes
Hybridization assays are crude estimates of divergence and are subject to variability due to several factors underlying the technique. More importantly, they do not reveal the patterns of sequence evolution, which are important for our objectives. We calculated Tajima-Nei's genetic distance (total number of substitutions per site, Tajima and Nei 1984) for the subset of genes sequenced and compared these to signal ratio estimates. There was low correlation between divergence estimated through signal ratios and actual sequence divergence (R2 = 0.49), probably due to several variabilities in the technique, but also largely due to deletions and substitutions in the 3'- and 5'-untranslated regions present in the cDNAs, which were not present in PCR sequences because we focused mainly on coding sequence divergence. Nevertheless, the rank order of genes did not change drastically, and sequence divergence estimates reflected the same trend as the hybridization results. Testis REGs, on average, had significantly higher Tajima-Nei's distance estimates than ovary and head REGs (Mann-Whitney sum rank test, P < 0.05). Ovary and head REGs, on the other hand, did not differ significantly (fig. 2). As a general comparison, most genes in all tissues had Tajima-Nei's estimates higher than Adh, a relatively slower evolving gene, and all genes had lower estimates than Acp26Aa, which is one of the most REGs in the Drosophila genome (Tsaur, Ting, and Wu 1998; fig. 2). These results from our data set indicate that testis transcripts incur a higher proportion of substitutions per site relative to the transcripts of head and ovary.
FIG. 2.— Tajima-Nei's genetic distance measures between Drosophila melanogaster and Drosophila simulans for candidate REGs. Acp26Aa and Adh were not part of this data set and were used as examples of an REG and a slowly evolving gene, respectively, to compare distance estimates.
Differences in Rates and Patterns of Sequence Evolution Among Tissues
REGs can be evolving neutrally or under positive selection. It is important to identify the different evolutionary forces that underlie the evolution of REGs in order to understand how they affect the biology of the organism. To test for evidence of selection, we further dissected sequence evolution patterns by estimating the proportions of synonymous (dS) and nonsynonymous (dN) substitutions of REGs in the three tissues between D. melanogaster and D. simulans (table 2 and fig. 3). High dN/dS ratios (excess of nonsynonymous substitutions) are considered to indicate directional evolution (Lee, Ota, and Vacquier 1995; Pamilo and O'Neill 1997). On average, testis REGs showed higher dN/dS ratios compared to the REGs in ovary and head (Wilcoxon's rank test < 0.05); there was no significant difference between ovary and head REGs. As a class, testis REGs, on average, had dN estimates that were approximately two times higher than dN estimates for both ovary and head REGs (dNtestis = 0.0303 ± 0.009, dNovary = 0.0147 ± 0.006, dNhead = 0.0149 ± 0.007), results that were significant according to Mann-Whitney sum rank tests (P < 0.005). There was no significant difference in the rates of synonymous substitutions between REGs in the three tissues. These data indicate accelerated evolution of testis genes and that male genes are probably under stronger selective pressures.
Table 2 The dN/dS Estimates, Tissue Expression, and Predicted/Known Functions of REGs
FIG. 3.— dN/dS estimates for candidate REGs in testis, ovary, and head between Drosophila melanogaster and Drosophila simulans. Acp26Aa and Adh are not part of this data set and were used as examples of an REG and a slowly evolving gene, respectively, to compare dN/dS estimates.
Positive Selection Drives the Rapid Evolution of Candidate REGs in Testis, Ovary, and Head
We implemented maximum likelihood analysis to identify REGs that are evolving under positive selection. Drosophila yakuba sequences were obtained through the NCBI trace archive; some were PCR amplified. The neutral model could not be rejected for most candidate REGs in all tissues. However, the most REGs in each tissue (from our data set) showed evidence of evolving under positive selection (table 3). CG18869 in the testis showed a significantly better fit to M8 (P < 0.001, table 3). The function of this gene remains uncharacterized. In the ovary, CG6533 (chorion protein 16, cp16) showed a significantly better fit to M8 (P < 0.001, table 3), indicating that this gene has sites evolving under positive selection. cp16 encodes for a structural component of the egg in all insects and belongs to a family of chorion protein genes which have been reported to show significant divergence in sequence as well as expression patterns. In addition, further analysis indicates that this gene shows significant differences in the proportions and patterns of dN between species of the melanogaster subgroup, indicating different selective forces/functional constraints between closely related species (unpublished data). CG4716, expressed specifically in the head, had positively selected amino acids (P < 0.001). The function of CG4716 is unknown. The log ratio test for CG9284 was surprising because none of the sites were detected to be evolving positively, although this gene shows the highest dN/dS ratio between D. melanogaster and D. simulans. Given that the dN/dS is not significantly >1.0, this gene may be evolving rapidly and neutrally. However, log ratio tests must be treated with caution, particularly for small proteins (CG9284 is 69 codons long) (Anisimova, Bielawski, and Yang 2001). In addition, it is important to note that only three species were used; therefore, these results must be treated with caution. Before concluding that these genes are evolving neutrally or positively, additional taxon sampling as well as polymorphism studies will be essential to improve accuracy (Anisimova, Bielawski, and Yang 2002).
Table 3 Likelihood Ratio Test of Positive Selection in Testis, Ovary, and Head REGs
Discussion
Higher divergence of sex traits over nonsex traits indicates higher selective pressures on the sexual machinery (Eberhard 1985; Coulthart and Singh 1988). Assuming that a majority of genes expressed in reproductive tissue contribute to sexual and reproductive functions (which include development of the sex tissues), we would expect to find a higher proportion of REGs in reproductive tissues. It is likely that not all genes with a reproductive function evolve rapidly. It is important, therefore, to identify genes that are rapidly evolving and to understand what is driving their evolution, as well as how they affect the reproductive biology of Drosophila. As we had expected, our results show that there is a higher proportion of highly divergent genes in the reproductive system compared to the nonreproductive system (fig. 1), implying stronger selection on genes in the reproductive system. Importantly, our data indicate a difference in the proportion of REGs in the male and female sex tissues—we found a higher proportion of REGs in the testis compared to the ovary, implying stronger selection on the male reproductive system. Although our data set is small to arrive at a conclusive and general trend between tissues, our results are supported by findings from the study of Ranz et al. (2003), which showed a similar trend in expression divergence, as well as studies done at the protein level (Civetta and Singh 1995).
The divergence trend observed from our hybridization data offers a window to observe the nature of changes that may occur during speciation events. The trend (fig. 1) supports our hypothesis that SRR may be preferentially involved in the process of reproductive isolation. Higher divergence of testis and ovary genes between sibling species diverged only 2.5 MYA (fig. 1a and b) leads us to propose the notion that in the initial stages of speciation, reproductive genes diverge much faster than nonreproductive genes, which may play a crucial role in establishing reproductive isolation (Singh 1990). Higher divergence of nonreproductive traits is observed only in comparisons of species that have diverged for longer lengths of time (fig. 1c). Our results are also interesting in view of the nature of inherent differences between the testis and ovary transcriptomes, which differ from each other as much as each of them differs from somatic tissue (Andrews et al. 2000; Parisi et al. 2004). Testis and head express a large battery of unique and highly expressed transcripts relative to the ovary (Andrews et al. 2000). In addition, restricted function range of testis transcripts and a broader functional range of ovary transcripts (Parisi et al. 2004) may influence the manner in which these genes evolve. It must be noted that the functions of most of the REGs identified in our study are unknown and several genes are expressed both in sex and nonsex tissues (see table 1); therefore, their pertinent role as sex genes remains obscure.
Identification of REGs in male and female reproductive tissue is a starting point to address some important questions regarding evolution by sexual selection at the molecular level. Divergence of sex molecules can be driven through (1) the higher divergence of male reproductive molecules and (2) interacting male and female molecules—coadaptation or conflict? In concordance with the first scenario, the preponderance of REGs in the testis along with its high rate contributes to explain the higher instances of male hybrid sterility relative to female sterility (Wu and Davis 1993; Wu, Johnson, and Palopoli 1996) as well as the faster breakdown in the male sexual machinery in hybrids. All testis REGs in our data show higher proportions of both silent and amino acid–changing substitutions, implying that testis proteins retain high flexibility in protein fold structures, therefore conserving their respective functions between the sibling species. However, there are those testis REGs (such as CG11869) that evolve rapidly under positive selection, implying that they confer selective advantages. It is particularly important to know the functions of such testis proteins in order to understand the functional basis of their rapid adaptive divergence and how they relate to hybrid phenotypes.
Under the second scenario, high divergence of both ovary and testis genes may constitute coevolution or conflict as a result of interacting male and female proteins (Metz and Palumbi 1996; Metz, Robles-Sikisaka, and Vacquier 1998; Swanson et al. 2001b). Several male proteins that are transferred into the female after copulation are toxic to the female and may alter female longevity or reproductive behavior (Chen et al. 1988; Lung et al. 2002). Females may then produce proteins to counter this deleterious effect to protect themselves, resulting in an arms race between male and female reproductive molecules (Civetta and Clark 2000). On the other hand, variation in one sex molecule may instigate change in the corresponding sex molecule if their physical interaction is essential. The positively selected chorion protein (cp16) is interesting in this regard. Studies addressing conflict and coevolution at the molecular level are rare, and in order to test these hypotheses effectively, we need to identify all REGs in ovary and testis that interact with each other, particularly those that show evidence of positive selection (see Metz and Palumbi 1996; Metz, Robles-Sikisaka, and Vacquier 1998).
REGs in head that show evidence of evolving under positive selection are interesting and unveil opportunities to identify genes that possibly affect sexual behavior. Conversely, these genes may influence nonsexual sensory functions between species due to differences in their ecological traits. Considerable variation within and between species exists in visual and acoustic stimuli in relation to courtship and mating (see Greenspan and Ferveur 2000, for a review). Identifying such genes will be crucial in investigations of the genetic basis of behavioral differences that lead to premating isolation (Huttunen, Vieira, and Hoikkala 2002). Overall, our results support the notion that sexual selection is a strong driving force driving genetic change between species (Carson 1997).
Supplementary Material
Sequences are deposited in GenBank under accession numbers DQ062756–DQ062811.
Acknowledgements
We thank Justin Andrews, Richard Dearborn, and Tulle Hazelrigg for kindly supplying cDNA libraries. This work was supported by funds from the National Sciences and Engineering Research Council of Canada to R.S.S.
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Accepted for publication May 12, 2005.(Santosh Jagadeeshan and R)