MBE Advance Access originally published online on January 22, 2007
Molecular Biology and Evolution 2007 24(4):929-938; doi:10.1093/molbev/msm009
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Research Articles |
Rapid Evolution of Outer Egg Membrane Proteins in the Drosophila melanogaster Subgroup: A Case of Ecologically Driven Evolution of Female Reproductive Traits
Department of Biology, McMaster University, Hamilton, Ontario, Canada
E-mail: singh{at}mcmaster.ca.
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Abstract |
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Although sexual selection has been predominantly used to explain the rapid evolution of sexual traits, eggs of oviparous organisms directly face both the challenges of sexual selection as well as natural selection (environmental challenges, survival in niches, etc.). Being the outermost membrane in most insect eggs, the chorion layer is the interface between the embryo and the environment, thereby serving to protect the egg. Adaptive ecological radiations such as divergence in ovipositional substrate usage and host-plant specializations can therefore influence the evolution of eggshell proteins. We can hypothesize that proteins localized on the outer eggshell may be affected to a greater degree by ecological challenges compared with inner eggshell proteins, and therefore, proteins localized in the outer eggshell (chorion membrane) may evolve differently (faster) than proteins localized in the inner egg membrane (vitelline membrane). We compared the evolutionary divergence of vitelline with chorion membrane proteins in species of the melanogaster subgroup and found that chorion proteins as a group are indeed evolving faster than vitelline membrane proteins. At least one vitelline membrane protein (Vm32E), specifically localized on the outer eggshell, is also evolving faster than other vitelline membrane proteins suggesting that all proteins localized on the outer eggshell may be evolving rapidly. We also found evidence that specific codons in chorion proteins cp15 and cp16 are evolving under positive selection. Polymorphism surveys of cp16 revealed inflated levels of divergence relative to polymorphism in specific regions of the gene, indicating that these regions are under strong selection. At the morphological level, we found notable difference in eggshell surface morphologies between specialist (Drosophila sechellia and Drosophila erecta) and generalist species of Drosophila. We do not know if any of the chorion proteins actually interact with spermatozoids, therefore leaving the possibility of rapid evolution through gametic interaction wide open. At this point, however, our results support previous suggestions that divergences in ecology, particularly, ovipositional substrate divergences may be a strong force driving the evolution of eggshell proteins.
Key Words: speciation • gametic surface proteins • sex and reproduction related genes • ecological diversification • positive selection • chorion proteins • vitelline proteins
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rapid evolution of sex and reproduction related genes are now frequent and common observations across a wide variety of taxonomic groups (Clark et al. 2006
). Rapid evolution, particularly, of gametic surface proteins can profoundly influence the process of fertilization, specifically in the nature of sperm–egg interactions (Swanson and Vacquier 1998) and also has serious implications in the process of speciation (Singh and Kulathinal 2000; Swanson and Vacquier 2002). To date, several reproductive proteins, both male and female, have been reported to be evolving rapidly in a wide variety of taxa and some are evolving adaptively as well (Coulthart 1988; Swanson et al. 2004; Jagadeeshan and Singh 2005; Panhuis and Swanson 2006; Turner and Hoekstra 2006). Sexual selection via gametic interaction (competition/coevolution) has been predominantly used to account for such concerted evolution of male and female gametic proteins (Swanson and Vacquier 2002). However, in oviparous organisms, female gametes are directly exposed to both the challenges of sexual selection as well as natural selection (i.e., environmental/ecological challenges, survival in niches, etc.).
The egg occupies a crucial stage of any insect life cycle and is intricately linked to successful reproductive strategies. As a result of divergences in females' ovipositional substrate usage, eggs are laid in different microhabitats and are exposed to varied challenges, such as mechanical support on substrates, desiccation, plant toxins, pathogen invasions, predation, etc. (Hinton 1981; Kambysellis et al. 1995). Drosophila species endemic to the Hawaiian Islands exemplify dramatic diversification in reproductive strategies, particularly, in ovipositional substrate and host-plant specialization (see Kambysellis 1975, 1993). Such ecological diversifications and species-specific ovipositional substrates usage have been suggested to have an effect on egg morphology (Lachaise et al. 1982; Lachaise and Tsacas 1983; Kambysellis 1993; Kambysellis et al. 1995). In fact, the importance and the utility of gross egg morphology and eggshell surface diversity as a taxanomic character had raised considerable interest in the past (see Mayr 1963).
The typical Drosophila eggshell is comprised of 2 major proteinaceous layers: the inner vitelline layer and the outer chorion layer (Margaritis et al. 1980). A thin waxy layer separates the vitelline and chorion layers. The chorion layer is complex and substructured into 3 distinct membranes; the inner chorionic membrane, the endochorion, and the outer exochorion (see Margaritis et al. 1980). Although a number of major and minor chorion components had been identified (Petri et al. 1976), the most well studied are; cp36 and cp38, which are expressed early in eggshell formation (Parks et al. 1986) and cp15, 16, 18, and 19, which are expressed in later stages of eggshell formation (Spradling and Mahowald 1980; Spradling 1981; Griffin-Shea et al. 1982). The vitelline membrane on the other hand is a relatively simple, single, continuous layer without substructures (Margaritis et al. 1980). Vm26Aa, Vm26Ab, Vm32E, and Vm34Ca have been identified and studied (Petri et al. 1976; Griffin-Shea et al. 1982; Spradling 1993).
Being the outermost membrane in insect eggs, the chorion layer is the interface between the embryo and the environment, thereby serving to protect the egg (Spradling and Mahowald 1980). Kambysellis (1975, 1993) found dramatic variation in egg surface morphology of Hawaiian drosophilids and proposed that such modifications in chorion structure are likely to be adaptations to species-specific ovipositional substrate usage and host-plant specialization. For instance, one of the most striking differences observed in the eggs of Hawaiian Drosophila were in the nature of the dorsal appendages between closely related species. Eggs of species that oviposited in deep cracks of rotting trees have long filaments, eggs of species that oviposited on leaves or fruit have shorter filaments, and those laid on flowers had no filaments at all (Kambysellis 1975, 1993). Kambysellis (1993) also found dramatic differences in chorion ultrastructure between species of Hawaiian Drosophila. Eggs of species that oviposited on rotting trees had thicker outer endochorions, and eggs of species that oviposited on leaves or flowers have thinner outer endochorions (Kambysellis 1993). Striking differences in chorion surface morphology has been reported in several other taxa as well, such as Lissocephala species (African fig flies), Stator limbatus species complex (seed beetles; Morse and Farrell 2005), and some Lepidopterans (Regier et al. 2005). These reports collectively emphasize an important interplay between ecological diversification, female breeding behavior, and the evolution of egg morphology.
In view of such adaptive ecological radiations and dramatic egg morphology modifications, the evolutionary history of genes contributing to eggshell formation is worth investigating. It may not be unreasonable to expect that proteins localized in the outer layer of the eggshell are likely to evolve faster than proteins localized in the inner layer. There have been reports of chorion genes being highly divergent in Hawaiian Drosophila, and some are evolving more rapidly than others (Martinez-Cruzado et al. 1988; Fenerjian et al. 1989; Martinez-Cruzado 1990). However, at the molecular level, we are still unclear as to what selective forces drive the rapid divergence of eggshell proteins and what the consequences of such rapid divergence may be. In the present study, we examined the evolutionary history of chorion and vitelline membrane proteins. We asked the question " are proteins localized in the outer eggshell (chorion layer) evolving faster than proteins localized in the inner eggshell (vitelline layer)?" Accordingly, we show that chorion proteins as a group are indeed evolving more rapidly than vitelline membrane proteins. In addition, we show that "all" proteins (be they chorion or vitelline) that are localized in the outer egg membrane are evolving faster. Maximum likelihood methods identified several sites in two of the most rapidly evolving chorion proteins (cp16 and cp15) to be evolving under positive Darwinian selection. Subsequent polymorphism analysis of cp16 (which was also previously identified as a rapidly evolving gene; Jagadeeshan and Singh 2005) revealed that specific regions of this gene are evolving in response to selection. Through a morphological survey, we also found species-specific ultrastructural changes present in species of the melanogaster subgroup that have specialized host-plant adaptations. Together, these results strongly indicate that in addition to sexual selection, ecological diversification is a major player in shaping the evolution of egg membrane proteins in Drosophila.
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Materials and Methods |
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Fly stocks
Drosophila melanogaster: 0231.0 was obtained from the Tucson stock centre, Arizona, D. melanogaster: Z34 and Z83 are Zimbabwean strains acquired from Alberto Civetta (University of Winnipeg), D. melanogaster: Loua, Kronenbourg, Madibou, Primus20, and Primus25 are African strains collected in Congo and were kindly provided by Pierre Capy (CNRS-Gif). Drosophila simulans: 0251.2 was obtained from the Tucson stock centre, Arizona. Drosophila simulans: Marrakech (collected in Africa) and St Martin (collected on the island of St. Martin) were kindly provided by Pierre Capy (CNRS-Gif). Drosophila mauritiana: gif was obtained from Pierre Capy (CNRS-Gif), D. mauritiana: 0.241.1, 0241.3, 0241.6, 0241.7, and 0241.8 were obtained from Tucson stock centre, Arizona. Drosophila sechellia: gif was provided by Pierre Capy (CNRS-Gif). Drosophila sechellia: Robertson (originally maintained by Huge Robertson, University of Illinois) was obtained from Jerry Coyne (University of Chicago), D. sechellia: 0248.1, 0248.25, 0248.3, 0248.5, 0248.6, 0248.7, and 0248.8 (collected on Cousin Island) were obtained from Tucson stock centre, Arizona. Drosophila yakuba: 0261.01, Drosophila santomea: 0271.00, Drosophila erecta: 0224.01, and Drosophila teissieri: 0257.00 were obtained from the Tucson stock centre, Arizona.
DNA Preparation and Polymerase Chain Reaction Amplification
Sequences of all chorion and vitelline proteins used in the divergence analysis were obtained by polymerase chain reaction (PCR) amplification from D. melanogaster (loua, CNRS-Gif), D. simulans (0251.2), D. mauritiana: gif (CNRS-Gif), D. sechellia: gif (CNRS-Gif), D. yakuba (0261.01), D. santomea (0271.00), D. erecta (0224.01), and D. teissieri (0257.00). DNA was extracted from 10 flies of each species which were homogenized in 0.1 M Tris HCl (pH 9.0), 0.1 M EDTA, and 1% SDS and then incubated at 70 °C for 20 min. DNA samples were then extracted using a standard phenol–chloroform extraction protocol. Extracted DNA was redissolved in 30 µl of Tris-EDTA buffer and stored at –20 °C until PCR amplification. Primers for chorion and vitelline genes for amplification from species of the melanogaster clade (species mentioned above) were designed from D. melanogaster coding sequence. To amplify chorion and vitelline genes from species of the yakuba clade, primers were designed from D. yakuba. For this task, D. melanogaster chorion and vitelline coding sequences were used in Blast searches to the D. yakuba and D. erecta trace archive (http://0-www.ncbi.nlm.nih.gov.library.vu.edu.au/Traces/trace.cgi?) to obtain the orthologous D. yakuba and D. erecta coding sequences. These sequences were useful in detecting conserved regions to design primers for the yakuba clade species. For polymorphism analysis of cp16, DNA was extracted from a single female fly from the strains mentioned above. cp16 sequences for D. simulans:166, :167, :w501, :NC48S, and :MD106TS were downloaded from the Drosophila Populations Genomics Project database (http://dpgp.org/syntenic_assembly/; sequencing done and deposited by the Genome Sequencing Centre, Washington University, St Louis).
Polymorphism and Divergence Analysis
All sequence alignments were done using ClustalX ver. 1.8 as well as RevTrans 1.0 (Wernersson and Pederson 2003). For polymorphism analysis of cp16, we performed tests of neutrality using Tajima's D (Tajima 1989), test of Fu and Li (without an outgroup, Fu and Li 1993), test of Fay and Wu (Fay and Wu 2000), as well as the MacDonald–Kreitman test (McDonald and Kreitman 1991). All these tests were done using the DnaSp ver 4.10 software package (Rozas et al. 2003). A Sliding window analysis of 100 bp with a step size of 50 was also done using DnaSp (Rozas et al. 2003).
Estimates of divergence for all chorion and vitelline were calculated in terms of dN and dS using the Phylogenetic Analysis by Maximum Likelihood software package (PAML; Yang 2000), which implements the method of (Yang and Nielsen 2000) that corrects for transversion/transition as well as codon usage biases. We also used PAML, which employs likelihood ratio tests, to infer if the data fits a neutral model of evolution versus alternative selection models. Evidence for positive selection is done by comparing data fit to models in which no codons have a dN/dS > 1.0 (neutral models) to models in which a subset of codons are allowed to have a dN/dS > 1.0 (selection models) (Nielson and Yang 1998;Yang et al. 2000). Significance of tests are calculated using the negative of twice the difference in log likelihood obtained through likelihood ratio tests from the models tested: –[2 log(L0) – log(L1)] and compared with a chi-square distribution (degrees of freedom = differences in the number of estimated parameters between the models used). Variations in dN/dS across branches were modeled by using discrete distributions (Models M0 vs. M3) contained in codeml.exe. We also implemented codeml.exe to calculate the likelihood of data fit to a nearly neutral model M1 (where 0 < dN/dS < 1.0 and no codons have a dN/dS ratio > 1.0), relative to a selection model M2 (where a subset of sites can have dN/dS > 1.0) as well selection models based on ß-distributions (models M7 vs. M8). Sites under positive selection were identified using the Bayesian Empirical Bayes approach (Yang 2005) implemented in both the selection model (M2) as well as the ß-distribution models (M8).
Scanning Electron Microscopy
Egg surface morphology across species of the melanogaster subgroup was surveyed using electron microscopy. Stage 14 eggs (i.e., mature eggs ready to be oviposited) from all species of the melanogaster subgroup (except Drosophila orena) were collected, fixed in 3% gluteraldehyde for a minimum of 18 hours and washed twice in 0.5 M Cacodylate buffer. Eggs were then directly mounted on aluminum specimen stubs, gold coated (to 6 nm) and viewed under the SEM. Although sample orientations are variable, all images are taken at the same magnification (30 µm) except for D. teissieri and D. erecta (at 25 µm).
Phyletic System
We used all species of the melanogaster subgroup in our analysis, except D. orena (see Powell 1997; Tamura et al. 2004 for divergence times). Drosophila melanogaster and D. simulans are human commensals, cosmopolitan in distribution with several sympatric habitats (Lachaise and Silvain 2004). Yet, these species exploit different ecological niches and differ significantly in their ecophysiology for certain traits (see Chakir et al. 1995; Chakir et al. 2002; David et al. 2004; Gibert et al. 2004; Lachaise and Silvain 2004). Drosophila sechellia is a strict island endemic and exhibits strong preference and host specificity to Morinda citrifolia fruits (Lachaise et al. 1988, Jones 2005), which are apparently toxic to other Drosophila species (see R'Kha et al. 1991). Drosophila mauritiana and D. simulans are opportunistic generalists and although they exploit distinct niches, they share regions of sympatry (Lachaise et al. 1988). Drosophila yakuba, D. teissieri, and D. erecta are restricted to Afrotropical regions with various degrees of niche overlap (Cariou et al. 2001). Drosophila santomea has been suspected to show a preference for Ficus chlamydocarpa (see Markow and O'Grady 2005 for a review). Drosophila erecta, like D. sechellia, has a special relationship with Pandanus spp. Pandanus fruiting is seasonal, and therefore, D. erecta behaves as a seasonal specialist, feeding and ovipositing on Pandanus spp. fruits (Rio et al. 1983; Lachaise et al. 1988).
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Results |
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Molecular Divergence: Faster Evolution of Genes Encoding Outer Egg Membrane Proteins
We examined the evolutionary history of genes encoding the Drosophila egg membrane proteins (chorion and vitelline proteins) in all known species of the melanogaster subgroup (except D. orena). Our intention was to expand our knowledge of the evolutionary forces driving the evolution of female reproductive traits, in this case, the egg. Using molecular data, we can test as well as expand on Kambysellis' (1975, 1993) predictions that divergences and specializations in ovipositional substrate usage are major evolutionary forces shaping the evolution of the egg. In which case, proteins localized in the outer membrane (chorion) are expected to evolve faster than proteins localized in the inner membrane (vitelline).
We found that chorion proteins as a group are indeed evolving faster than vitelline membrane proteins (fig. 1a and b). Moreover, on average, the dN and dN/dS of chorion proteins was 2-fold to 3-fold higher than that of vitelline membrane proteins (Wilcoxon sum rank test, P < 0.001, statistical significance indicated by asterisks in fig. 2). In contrast, we did not find any significant differences in the proportions of synonymous substitutions between the two. These results indicate that the chorion and vitelline membranes have indeed evolved quite differently from each other at the protein level despite serving, presumably, the common primary purpose of protecting the egg. This trend holds true in comparisons of both, the species in the melanogaster clade as well as the yakuba clade (fig. 2). Consequently, the first important outcome of our divergence study is that chorion proteins are evolving faster than vitelline proteins. The second important outcome of our divergence study is related to the developmental expression profile of all proteins localized in the endochorion. Among the vitelline membrane proteins analyzed in this study, Vm32E shows the highest estimates of divergence. This result is rather striking especially considering the fact that Vm32E protein in particular has been shown to be localized in the endochorion (Pascucci et al. 1996; Andrenacci et al. 2001). This result now extends our previous conclusion in suggesting that perhaps all proteins localized on the outer eggshell are affected (although to varying degrees) by ecological specializations or ovipositional substrate specializations.
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Polymorphism Survey: Specific Regions of cp16 Are Under Strong Selection
Whether these molecular changes directly effect eggshell morphology requires further detailed functional investigations. Without protein structure information we are unable at this point, to model if and how protein structure or functioning is affected as a consequence of observed molecular changes. We can, however, closely examine the nature of observed molecular changes to infer if genes encoding the outer eggshell are evolving in response to selection. More importantly, it would be worthwhile to explore if the entire gene is under selection or if specific regions of genes are under selection. In this regard, polymorphism studies complement divergence studies in allowing us to make more proficient inferences about the nature of selective forces driving the evolution of genes (e.g., see Vermaak et al. 2005
; Panhuis and Swanson 2006). Polymorphism data is useful for testing deviations from neutrality in a single species, whereas divergence studies, particularly, those implementing maximum likelihood methods, effectively uncover codons that have experienced frequent positive selection in the species compared. Combining polymorphism and divergence data, we can perform sliding window analyses to determine if specific regions and codons in a gene are under selection. Such data will also be valuable in further tests of the affects of region specific changes on protein structure and function.
We surveyed polymorphism in cp16 among species of the melanogaster clade. cp16 was isolated as a rapidly evolving ovary-specific gene in a previous study and was also suggested to be evolving adaptively (see Jagadeeshan and Singh 2005). We performed a McDonald–Kreitman test, which tests the prediction that, as per the neutral evolutionary theory, the ratio of fixed replacement (Rf) to silent changes (Sf) between species (i.e., Rf:Sf) is expected to be more or less similar to the ratio of polymorphic changes (McDonald and Kreitman 1991). We did not find any significant deviation from neutrality using this test, nor were we able to detect any indications for recent adaptive sweeps or major demographic effects using Tajima's D (Tajima 1989) and Fu and Li's test (Fu and Li 1993; results not shown). In contrast, a sliding window analysis clearly reveals that divergence far exceeds polymorphisms in specific regions of the gene by virtue of showing inflated dN/dS values relative to Pi(a)/Pi(s) values (fig. 3). This result is strongly indicative of these regions evolving under positive selection. Moreover, the sliding window analysis was helpful in identifying species-specific regions, in D. sechellia and D. mauritiana that are under selection by virtue of showing inflated levels of dN/dS relative to polymorphisms (see fig. 3). These regions are toward the 3' end of the gene, which was also shown to be highly divergent between closely related Hawaiian Drosophila (Martinez-Cruzado 1990). These regions are in stark contrast to the 5' end of the gene, which shows both elevated levels of divergence and within species polymorphism (fig. 3). To compare, a brief sliding window analysis of dN and dS in cp15 (without polymorphism data) suggests that, as in the case of cp16, the 3' end of the gene has elevated proportions of dN relative to dS in the D. sechellia and D. mauritiana lineages (data not shown). The rest of gene does not show any apparent elevations in dN or dS. This suggests that both proteins may be subject to similar selection pressures, however, this speculation must be corroborated along with polymorphism data. Our polymorphism study with tests of neutrality was intended to identify recent selective events in a specific gene and was indeed useful in suggesting that specific regions of cp16 may be under selection (sliding window analysis). Although the tests failed to reject neutrality, the inflated peaks of divergence relative to polymorphism are strong indications of selection and demand further investigation.
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We implemented further analysis of divergence utilizing dN, dS and dN/dS information through maximum likelihood approaches. Divergence analysis, specifically using codon-based models can effectively identify specific codons that may have experienced "recurrent" selection in lineages (Swanson et al. 2004
; Nielsen 2005). We used PAML (Yang 2000) to test if any of the chorion (particularly cp16) or vitelline genes were evolving in response to selection. We found significant amounts of variation in dN/dS among sites in all chorion proteins (table 1, M0 vs. M3), except cp36 (which is also the least diverged among chorions). Among vitelline proteins, we found significant rate heterogeneity only in Vm32E (table 1, M0 vs. M3), which is also the most diverged vitelline gene. Interestingly, several codon sites in cp15 and cp16 were detected to be evolving under positive selection (table 1 and fig. 4). Table 2 shows the nature of amino acid changes that have occurred between species in sites that have detected to be evolving under positive selection. Replacements of neutral amino acids by charged amino acids may be of functional or structural relevance (see table 2), however, this must be corroborated with further detailed functional analysis. It would also be important to take into account the Universal Evolutionary Index for amino acid changes recently developed by Tang et al. (2004) to infer the evolutionary significance of amino acid changes reported in this study. The outcome for cp16 from the PAML model test analyses substantiates earlier reports (Jagadeeshan and Singh 2005) but more importantly, it complements results from the sliding window analysis providing addition evidence to show that specific regions and sites are evolving in response to selection in cp16.
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It must be noted here that the discrepancy in the outcomes of the polymorphism and divergence studies with respect to detecting signatures of selection may not be uncommon (see Neilson 2005 for a useful discussion). Few genes have shown significant outcomes of positive selection using the McDonald–Kreitman test (Moriyama and Powell 1996
; Fay et al. 2001). Although the McDonald–Kreitman test is robust to any demographic assumptions, it may lack the power to detect positive selection, particularly, in small genes, unless a large proportion of adaptive substitutions have occurred (see Moriyama and Powell 1996; Fay et al. 2002). In addition, it must be noted from figures 3 and 4 that only a few sites in specific regions of cp16 are under selection. This may also affect the outcomes of a robust test such as the McDonald–Kreitman test. Moreover, this test cannot distinguish recurrent selective events (see Nielsen 2005). At this point therefore, we are led by the outcomes of the sliding window analysis as well as maximum likelihood model tests to suggest that cp16 (and cp15) are indeed evolving rapidly and adaptively.
Ultrastructural Modifications of Eggshell Surface in Specialist Species
Outcomes of our molecular study on genes encoding eggshell proteins prompted us to investigate diversification at the morphological level. Although at this point, we cannot directly make any links between the molecular changes and morphological modifications, such investigations accrue necessary information that will help in eventually connecting the dots. Moreover, unlike in the case of Hawaiian drosophilids, we know little about the morphological diversity of the eggshell in the melanogaster subgroup. In parallel to their behavioral divergence, Hawaiian drosophilids have undergone far more extensive ecological diversification and host-plant specialization than species of the melanogaster subgroup. We, however, have 2 important ecological specialists among species of the melanogaster subgroup. Drosophila sechellia is endemic to the island of Seychelles, and its life history has been adapted and specialized to the M. citrifolia (Lachaise et al. 1988). Such adaptation and specialization to the M. citrifolia fruit (which is toxic to most other drosophilids, see Lachaise et al. 1988; Legal et al. 1992; Jones 2005) in D. sechellia comes with a cost of reduction in ovariole number along with other species-specific behavioral modifications (R'Kha et al. 1991). Drosophila erecta is another species in the yakuba clade, which exhibits seasonal specialization on Pandanus spp. mostly found in swampy or coastal habitats (Lachaise et al. 1988). Although D. santomea has been reported to be associated with figs (Markow and O'Grady 2005), we are not completely sure about the nature of its specialization. All other species are opportunistic generalists. Strikingly, we found notable eggshell morphology differences, particularly, in the surface ridges, of the 2 specialists, D. sechellia and D. erecta. Although the surface ridges of the other species are relatively similar in thickness (see fig. 5), D. sechellia has higher and thicker ridges (fig. 5). Drosophila erecta, on the other hand, has relatively smoother egg surface and much thinner ridges (fig. 5). Drosophila erecta eggs also differ from the other species in the fine surface morphology in having less porous surface. We are not entirely sure of the relevance of these modifications in either species, particularly, considering the fact that D. sechellia is known to oviposit on M. citrifolia fruit, but it is generally assumed that D. erecta also oviposits on Pandanus sp. fruits. It is possible that requirements of mechanical support or challenges of desiccation may be different for eggs on the 2 fruits. In previous reports, Kambysellis (1975, 1993) was able to identify specific morphological features (thicker chorion surface and variable ridge thicknesses) that were predominantly present in ovipositional substrate specialist species (in bark ovipositors). Perhaps, a closer look at the ecology of D. sechellia and D. erecta to corroborate their oviposition behavior would be helpful in making more proficient inferences regarding the functional relevance of these morphological variations.
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Discussion |
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We are beginning to uncover that several female-specific sex and reproduction related proteins are evolving rapidly in Drosophila (Civetta and Singh 1995
; Jagadeeshan and Singh 2005; Panhuis and Swanson 2006). Rapidly evolving egg membrane proteins are not only implicate in gametic surface protein evolution and gametic incompatibility (Swanson and Vacquier 1998; Zigler et al. 2005), but also addresses the evolution of a unique female reproductive trait whose evolution can be driven by sexual selection, via sperm–egg interaction, and/or diversification in species ecology, via diversification of ovipositional behavior. The detail that emerges from our molecular and ultrastructural investigations is that the outer membrane (chorion) proteins show obvious signs of evolving under selection in comparison to the inner (vitelline) membrane proteins. This divergence trend strongly indicates that the evolution of the Drosophila egg membrane was primarily influenced by ecological factors. The alternative, sexual selection, has invariably been implicated to explain the evolution of gametic proteins (Swanson and Vacquier 2002). In this case, however, evolution driven by sperm–oocyte interaction appears unlikely. Very little is known about sperm–egg interaction in Drosophila. Sperm entry into the Drosophila egg is restricted to the microphyle area. Unless cp16 and cp15 are specifically localized to this region and also shown to interact with sperm proteins, we cannot implicate sperm–egg interaction (coevolution\or competition) as a causal factor driving the rapid evolution of these chorion proteins. Nonetheless, if sexual selection via sperm–oocyte interaction were indeed responsible, we would expect to see signatures of elevated rates of evolution in vitelline membrane proteins as well. This is not apparent from our study.
Results from our divergence as well as polymorphism study provide a brief snapshot of the evolutionary history of egg membrane proteins among members of the D. melanogaster subgroup. The strong selective peaks observed in species of the D. simulans clade suggest a unique evolutionary event, particularly, relating to the split between D. simulans and D. sechellia. Exactly how D. sechellia diverged from D. simulans is unclear, but the most common view is that of either founder-effect or migration/introduction followed by rapid adaptive ecological radiations (Lachaise et al. 1988; Powell 1997). The founder-flush theory of Carson and genetic-transilience theory of Templeton (Carson and Templeton 1984) invoke a model of positive selection and rapid adaptive radiation, which fit the cp16 polymorphism and divergence trend observed in our study of species of the simulans clade. This is particularly relevant to D. sechellia, an island endemic that has specialized host-pant interactions.
Although chorion surface formation and relevant modifications can be a result of divergence in expression patterns (Hatzopoulos and Regier 1987), rapid sequence divergence is also likely to play a role and must be further investigated, particularly, to determine if specific amino acid changes affect structural integrity of chorion proteins. The amino acids identified to be under selection in this study provide a starting point for such investigations. Nevertheless, the molecular changes as well as the ultrastructural modifications reported in this study present a strong case for some sort of ecologically driven evolution of the egg in Drosophila. Together these data support earlier reports (Kambysellis 1993; Kambysellis et al. 1995) in suggesting that adaptive ecological diversification may drive the evolution of the insect eggshell.
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Footnotes |
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Yoko Satta, Associate Editor
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References |
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