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How new species are formed remains one major question in evolutionary biology. The focus of speciation research in recent years has gradually shifted from broad geography-based models of sympatry versus allopatry toward understanding the mechanisms that give rise to reproductive isolation (RI) potentially resulting in speciation (Rice and Hostert 1993; Schluter 2001; Coyne and Orr 2004; Rundle and Nosil 2005; Butlin and Ritchie 2009; Fry 2009; Butlin et al. 2012) . Experimental evolution and laboratory selection approaches are particularly useful for understanding the mechanistic bases of evolutionary processes and such methods are being employed increasingly to study the evolution of RI (Reviewed in Rice and Hostert 1993; Ritchie 2007). While several laboratory studies have shown that partial RI can evolve as a correlated response to divergent selection on behaviors (Del Solar 1966; Hurd and Eisenberg 1975; Markow 1981; Lofdahl et al. 1992) or life-history traits (Miyatake and Shimizu 1999), or adaptation to different environments (Kilias et al. 1980; Rice and Salt 1988; Dodd 1989; Boake et al. 2002; Rundle et al. 2005; Vines and Schluter 2006; Dettman et al. 2007, 2008), studies providing evidence of the mechanisms underlying the correlated evolution of RI are meager (reviewed in Coyne and Orr 2004; Fry 2009). In the melonfly Bactrocera cucurbitae, selection for slow and fast preadult development led to changes in circadian clock period that, in turn, led to the evolution of RI due to changed phase of the circadian mating rhythm (Miyatake and Shimizu 1999). Divergent adaptation to nutritional (Rundle et al. 2005: Drosophila serrata) aspects of different environments can directly result in evolutionary shifts in display and reception of visual or chemical signals involved in mate recognition/choice. Such results provide evidence for the involvement of “classic” sexual selection in mediating RI. In yeast and Neurospora, adaptation to different environments has been shown to result in RI via genetic incompatibilities (Dettman et al. 2007, 2008).
In this study, we focus on the possible role of mating success and sexual conflict, both likely mediated by divergent body size evolution, in creating reproductive barriers between populations of Drosophila melanogaster selected for rapid preadult development and their ancestral controls. Sexual selection has long been thought to be an important driver of speciation, because it directly acts on traits related to mate recognition and reproductive success, and species typically show considerable divergence in such traits (West-Eberherd 1983; Panhuis et al. 2001; Ritchie 2007). The degree and precise form of sexual selection can differ among populations undergoing divergent adaptation, and this has been thought to cause reproductive traits to diverge between populations, potentially leading to the formation of reproductive barriers (Panhuis et al. 2001; Arnqvist and Rowe 2005; Rundle and Nosil 2005; Ritchie 2007). However, most evidence in support of the role of sexual selection in speciation comes from comparative data, and clear empirical support for this view is largely lacking (Panhuis et al. 2001; Kraaijeveld et al. 2011; but see Boughman 2001). It is also increasingly realized that divergent sexual selection, in the sense of phenotypic variation being correlated with differential mating success, can result in myriad direct and indirect ways from divergent ecological adaptation (Ritchie 2007; Maan and Seehausen 2011).
Another phenomenon related to sexual selection is sexual conflict, which arises when traits that increase fitness in one sex simultaneously impose fitness costs on the opposite sex (Chapman et al. 2003). Such conflicts can give rise to sexually antagonistic coevolution, where adaptive change in one sex leads to counter-adaptation in the other (Chapman et al. 2003). Sexual conflict can also bring about rapid changes in reproductive traits and has been thought to play a role in generating RI between diverging populations (Parker and Partridge 1998; Rice 1998; Arnqvist et al. 2000; Gavrilets 2000; Gavrilets et al. 2001). Again, as in the case of sexual selection, there is a paucity of clear empirical data linking sexual conflict and RI (Panhuis et al. 2001; Gavrilets and Hayashi 2005; Ritchie 2007; Kraaijeveld et al. 2011; Maan and Seehausen 2011). Although laboratory selection has been fruitfully deployed to study the evolutionary consequences of sexual selection and sexual conflict in general (Rice 1996; Blows 2002; Rundle et al. 2005; Prasad et al. 2007; Ritchie 2007; Morrow et al. 2008; Edward et al. 2010; García-González 2011), empirical evidence for links between sexual selection/conflict and evolution of RI is inconsistent. Increasing the level of sexual conflict led to the evolution of RI in some studies (Martin and Hosken 2003; Hosken et al. 2009), but not in others (Wigby and Chapman 2006; Bacigalupe et al. 2007; Gay et al. 2009).
Body size, an important life-history trait ontogenetically linking the preadult and adult stages in holometabolous insects, is strongly correlated with preadult development time in D. melanogaster (Chippindale et al. 1997; Prasad and Joshi 2003). Selection for rapid development in D. melanogaster has repeatedly been shown to result in the correlated evolution of smaller body size (Zwaan et al. 1995; Nunney 1996; Chippindale et al. 1997; Prasad et al. 2000). Moreover, body size is also known to play a significant role in sexual selection and sexual conflict in Drosophila. Large females are generally more fecund (Stearns 1992; Roff 2002) and are often preferred by males (Andersson 1994; Byrne and Rice 2006). Bigger size typically confers greater competitive ability in male-male competition in D. melanogaster (Partridge and Farquhar 1983; Partridge et al. 1987a,b; Markow 1988; Markow and Ricker 1992), and large males are also often preferred by female flies (Ewing 1961; Markow 1986; Partridge et al. 1987a; Pitnick 1991). However, the preference for larger males can also give rise to sexual conflict because mating with large males reduces female lifespan and egg-production rates (Pitnick and García-González 2002; Friberg and Arnqvist 2003; Taylor et al. 2008). Given that both development time and body size in Drosophila can evolve in response to a variety of selection pressures in both laboratory and natural habitat (reviewed by Prasad and Joshi 2003), we focus here on the influence of development time and body size evolution on reproductive traits in laboratory populations of D. melanogaster to examine whether mating success and sexual conflict may be mediating RI in this system.
We studied a set of four laboratory populations of D. melanogaster that have been selected for rapid development for over 300 generations, and have also evolved reduced body size relative to the four ancestral control populations (Fig. 1; Prasad et al. 2000, 2001; Ghosh-Modak 2009). The selected populations have also evolved reduced lifespan and fecundity, preadult larval competitive ability, changes in larval behavioral traits, and decreased resistance to biotic and abiotic stresses during both larval and adult stages (Prasad et al. 2000, 2001; Prasad 2004; Shakarad et al. 2005; Ghosh-Modak 2009; Ghosh-Modak et al. 2009). We tested for RI between the selected populations and their ancestral controls, and found evidence for two complementary asymmetric pre and postmating barriers to effective reproduction between selected and control populations. We found no evidence for any direct effect of the large life-history divergence between selected and control populations on postzygotic RI through genetic incompatibility resulting in hybrid breakdown. We discuss our results in the light of sexual selection and possible sexual conflict in these populations, and show how the likely mechanism of the evolution of RI in this study underscores the subtlety with which natural selection and sexual selection can interact to yield isolation.
Figure 1. Laboratory populations of Drosophila melanogaster selected for rapid preadult development and early reproduction for 370 generations have undergone 25% reduction in development time and >50% reduction in body size compared to their ancestral controls. Flies from control populations (Joshi Baseline [JB]) shown on top, and those from selected populations (Faster reproducing, Early reproducing, JB-derived [FEJ]) shown below.
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As evident from the results of the female choice (Figs. 2b, 3b) and the group mating assays (Fig. 3a), the fast developing and small FEJ males obtain very few matings with either JB or FEJ females in presence of the JB males. This is not surprising given the well-known disadvantage of small body size in male-male competition for matings in Drosophila (Partridge and Farquhar 1983; Partridge et al. 1987a,b; Markow 1988; Markow and Ricker 1992). More interestingly, in absence of the male-male competition, FEJ males mated three times more often with FEJ females than with JB females (Figs. 2a, 3a). This could be due to two reasons – JB females might exercise a choice against FEJ males; and/or FEJ males show a preference for FEJ females over JB females. FEJ males were observed to court JB females in almost all the cases (although courtship was not quantified), but JB females were often seen to resist mating attempts by the FEJ males, which suggests the choice might be exercised by the JB females. Female Drosophila are known to preferentially mate with larger males (Ewing 1961; Markow 1986; Partridge et al. 1987a; Pitnick 1991), but the causal mechanism is not clearly known (Partridge 1988). It is possible that JB females avoid mating with the small FEJ males because of some innate size preference. Alternatively, FEJ males might be less attractive to JB females due to some other reason, such as differences in courtship song or pheromonal cues, or simply because they are not vigorous and active compared to JB males. It could also be that the small FEJ males are just not able to deal easily with mounting and copulating with the much larger JB females (e.g., see Maynard Smith 1956). With the present data, we cannot distinguish among these various possibilities, but the data clearly suggest the evolution of premating RI between FEJ males and JB females, driven by some form of sexual selection in the broad sense. Evolution of premating RI between FEJ males and JB females is supported by the finding that the longest mating latency is observed in this type of cross (Fig. 4a,b).
There is no possibility in our populations of the kind of circadian clock mediated RI seen in the fast and slow developing B. cucurbitae populations of Miyatake and Shimizu (1999). Such mating phase dependent isolation would be expected to be symmetric across both types of heterogamic mating. Moreover, although there is evidence for some effect of the eclosion circadian rhythm on development time in D. melanogaster populations sharing ancestry with those used in this study (Paranjpe et al. 2005), there is no clear circadian rhythm in mating exhibited by our populations, as they are housed under constant light (V. K. Sharma, pers. comm, 2012).
While there is no impediment to the other type of heterogamic mating between JB males and FEJ females, the results of the postcopulation female mortality assay (Fig. 5) indicate the existence of postcopulatory RI between JB males and FEJ females due to the high mortality suffered by the FEJ females in this type of cross. There is preliminary evidence suggestive of this barrier being driven by differing levels of sexual conflict between the JB and FEJ populations. Female flies are known to show reduced lifetime fitness as a consequence of mating (Partridge et al. 1987c; Partridge and Fowler 1990), mediated by harmful effects of both male courtship and male accessory gland proteins (Acps) transferred to the female's body during mating (Chapman et al. 1995; Wigby and Chapman 2005), and this fitness cost to females is known to rise with increased body size of their mating partners (Pitnick and García-González 2002; Friberg and Arnqvist 2003). The high mortality rate of FEJ females after mating with JB males (Fig. 5) could be due to various reasons. First, it is possible that the amount/composition of seminal fluid proteins transferred by JB males is more toxic than what the FEJ females have evolved to deal with over the course of a few hundred generations of laboratory evolution. Microarray data from whole adult flies show that many of the accessory gland proteins (Acps), including Acp76A, Acp36DE, Acp98AB, Acp26Aa, Acp76A, Acp53C14a, Acp36DE, Acp70A, Acp95EF, Acp53C14c, and Acp63F have undergone 1.5–7-fold downregulation in FEJ males compared to JB males (Satish 2010; K. M. Satish, P. Dey, and A. Joshi, unpubl. data). We speculate that the FEJ males have undergone an evolutionary reduction in Acp production, perhaps as a correlate of reduced body size, and female resistance to the toxic effects of Acps has also consequently reduced over the 370 generations of laboratory selection for rapid development. In the FEJs, conservation of energy reserves is important to fitness because the flies have very low lipid levels at eclosion due to the reduced third larval instar duration; however, they need to be relatively fecund on day 3 posteclosion because of the selection regime (Prasad 2004). In general, the FEJs appear to have evolved a syndrome of reduced energy expenditure, relative to the JB controls (Prasad et al. 2001). This might have led to a reduced energy expenditure for Acp production in males, and female resistance in females in the FEJs, resulting in FEJ females paying a fatal cost upon mating with the large JB males. In D. melanogaster, variation in female resistance to male harm was documented by Linder and Rice (2005), and female resistance evolved in experimental evolution studies manipulating the levels of sexual conflict (Holland and Rice 1999; Wigby and Chapman 2004; Lew et al. 2006). Reproductive traits in Drosophila including Acp levels are known to undergo rapid evolutionary change (Swanson et al. 2001; Swanson and Vacquier 2002; Wolfner 2002; Begun and Lindfors 2005; Panhuis et al. 2005), and heritability for male harm has been documented in the seed beetle Callosobrachus maculates (Gay et al. 2011). Thus, it is possible that the level of sexual conflict in the FEJs has settled down at a lower level of antagonism as a result of male-female coevolution, perhaps driven by a combination of energy requirements for early fecundity and reducing size over the course of their laboratory evolution. However, we are yet to assign a definitive cause for death of FEJ females upon mating with JB males. It is possible that the small-sized FEJ females also suffer from mechanical injury while being courted or during mating with large JB males, although we did not see any evidence of gross injury in the dead females in the cumulative postcopulation mortality assay.
We found no evidence for postzygotic RI, as hybrids between FEJ and JB populations were as viable as the JBs, and also nearly as fertile as the JBs (Figs. 5a,b, 6b). The development time of the hybrids, however, was intermediate between the FEJs and JBs (Fig. 7a). Thus, despite the considerable evolutionary restructuring of most aspects of the preadult and adult life-history, and many related traits, in the FEJs (Prasad et al. 2000, 2001; Joshi et al. 2001; Prasad 2004), a restructuring that has resulted in substantially reduced preadult survivorship (Fig. 6a,b), there does not seem to be any intrinsic genetic incompatibility between the FEJ and JB development that would reduce hybrid viability. Duration of all preadult developmental stages, starting from embryonic development to pupal duration have been significantly reduced in FEJs compared to the JBs (Ghosh-Modak 2009), and there is evidence for large-scale changes in the temporal profile of gene expression during development in the FEJ populations (Satish 2010; K. M. Satish, P. Dey, and A. Joshi, unpubl. data). Despite such differences in the FEJs and JBs, the hybrids were viable and fertile, and their development time was intermediate, suggesting that the kinds of genetic difference needed to generate genomic incompatibilities may be rather more extensive than often thought to be the case, as also suggested by Rice and Hostert (1993). Our results, thus, support the widely held view that prezygotic isolation often evolves much faster than postzygotic isolation (Kilias et al. 1980; Coyne and Orr 1989, 1997; Rice and Hostert 1993; Coyne and Orr 2004; Vines and Schluter 2006).
Our study shows that long-term directional selection for rapid development has led to some degree of RI between selected populations of D. melanogaster and their ancestral controls, most likely a consequence of the correlated evolution of greatly reduced body size in the selected FEJ populations. In Drosophila, both development time and body size respond readily to various kinds of selection, and also show plastic responses to various environmental factors like temperature and crowding (reviewed by Prasad and Joshi 2003). Thus, RI mediated by changes in development time and/or body size could in principle be a reasonably common outcome of divergent ecological adaptation in this genus, suggesting that early stages of ecological speciation can be a by-product of differential life-history evolution, even in the absence of major differences in habitat or resource use between populations. The manner in which the body size differences appear to have mediated RI between fast developing FEJs and the JB controls is also interesting in that it seems to involve two complementary and asymmetric isolating mechanisms. Small FEJ males obtain few matings with large JB females, giving rise to unidirectional, premating RI mediated by mating success (sexual selection). Conversely, small FEJ females suffer greatly increased mortality following mating with large JB males, resulting in unidirectional viability-selection-driven postmating RI. This exemplifies the view that the manner in which life-history, sexual selection, and natural selection interact in the course of ecological speciation can be both subtle and complex (Ritchie 2007; Maan and Seehausen 2011).