Genetica (2014) 142:23–41 DOI 10.1007/s10709-013-9751-4 Role of sexual selection in speciation in Drosophila Akanksha Singh • Bashisth N. Singh Received: 20 June 2013 / Accepted: 14 December 2013 / Published online: 22 December 2013  Springer Science+Business Media Dordrecht 2013 Abstract The power of sexual selection to drive changes in the mate recognition system through divergence in sexually selected traits gives it the potential to be a potent force in speciation. To know how sexual selection can bring such type of divergence in the genus Drosophila, comparative studies based on intra- and inter-sexual selection are documented in this review. The studies provide evidence that both mate choice and male–male competition can cause selection of trait and preference which thereby leads to divergence among species. In the case of intrasexual selection, various kinds of signals play significant role in affecting the species mate recognition system and hence causing divergence between the species. However, intrasexual selection can bring the intraspecific divergence at the level of pre- and post-copulatory stage. This has been better explained through Hawaiian Drosophila which has been suggested a wonderful model system in explaining the events of speciation via sexual selection. This is due to their elaborate mating displays and some kind of ethological isolation persisting among them. Similarly, the genetic basis of sexually selected variations can provide yet another path in understanding the speciation genetics via sexual selection more closely. Keywords Sexual selection
Intra- and inter-sexual selection
Frequency dependent sexual selection
Speciation
Drosophila A. Singh
B. N. Singh (&) Genetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221005, Uttar Pradesh, India e-mail: bnsingh@bhu.ac.in; bashisthsingh2004@rediffmail.com A. Singh e-mail: singh31bhu@gmail.com Introduction Sexual selection has received increasing attention as a potential factor in speciation. Although natural selection may often play an important role in the divergence of populations undergoing speciation (Turelli et al. 2001; Maan and Seehausen 2011), sexual selection plays an equally important role in the process of speciation (Schluter 2001). If sexual traits have some or the other adaptive value, they may also be naturally selected apart from being sexually selected. Therefore, an organism is subject to both natural and sexual selection simultaneously but strong sexual selection is more likely to evolve premating isolation. The idea of sexual selection as a driver of reproductive isolation received theoretical support only in the early 1980s (Lande 1981; West-Eberhard 1983) which is perhaps surprising given that sexual selection frequently shapes the very characters involved in mate preferences and reproductive isolation. In recent years behavioural ecologists have shown increased interest in sexual selection in females as well as males. This selection results from differential mating success among individuals within a population. Competition for fertilization occurs through direct competition between members of the same sex (i.e. premating male–male competition) or through cryptic female choice (i.e. postcopulatory mechanisms biasing fertilization which involves sperm competition). Also, competitive interactions within a sex may favour the evolution of diverse, elaborate ornamental traits (Andersson 1994). Zahavi’s (1975) handicap principle provides evidence to the above mentioned statement of cryptic female choice, that females which select males with the most developed characters are those with best genotypes of the male population. The rapid divergence via sexual selection is brought about through a parallel change in mate preference 123 24 and secondary sexual traits within a population and this might lead to prezygotic isolation between populations. Hence it indicates that sexual selection has the power to drive rapid divergence and generate reproductive isolation (Panhuis et al. 2001; Ritchie 2007; Snook et al. 2009; Maan and Seehausen 2011). Also, there is evidence of reinforcement of gametic isolation in Drosophila through sexual selection (Matute 2010). He has reported the first case of reinforcement of postmating prezygotic isolation, which has apparently evolved in natural populations of D. yakuba sympatric with the sister species D. santomea. Interest in speciation continues to grow, as evidenced by an increasing rise in citations of speciation studies over the last many years (Questiau 1999; Panhuis et al. 2001; Turelli et al. 2001; Orr et al. 2004; Wu and Ting 2004; Ritchie 2007; Sobel et al. 2009; Maan and Seehausen 2011). In the genus Drosophila, extensive work has been done in the field of speciation. In these studies, the major focus has been to understand the role of natural selection in causing species divergence (Nanda and Singh 2012). Therefore, it was still unclear whether only natural selection has the power to drive such type of divergence in sexual traits, or there is a role of sexual selection. This was answered when studies were done in Drosophila providing evidence that not only natural selection but also sexual selection plays a probable role in enhancing divergence (Ritchie 2007). There is increasing evidence that genes involved in reproduction, specifically those showing sex-biased expression evolve rapidly and are often subject to positive selection and hence adaptive evolution (Swanson et al. 2001; Proschel et al. 2006). However, what has received much less attention is exactly how this rapid evolution and divergence is related to reproductive isolation via sexual selection. The finding that reproductive genes in males and females are often subject to positive selection and adaptive evolution suggests a major role for sexual selection in any resulting divergence (Clark et al. 1995; Proschel et al. 2006). But some questions still remain unanswered as far as the process of speciation is concerned: (1) How sexual selection (inter and intrasexual selection) influence reproductive isolation? (2) Whether it acts directly (i.e. at the level of sexual traits) or indirectly (i.e. at level of reproductive proteins or both?) (3) Whether reproductive proteins of Drosophila play any kind of role in causing divergence among species? and (4) If yes, then which proteins (proteins formed from male biased genes or female biased genes) show higher level of species divergence? The answers to these questions may provide a way to understand the different levels at which sexual selection might play a role in causing divergence. The rapid divergence between populations (allopatric or sympatric) might be due to the prevention of gene flow between them. The potential effect of sexual selection on 123 Genetica (2014) 142:23–41 speciation are especially evident in allopatric populations since sexual selection can drive the evolution of signalling and preference traits in arbitrary divergent directions even in the absence of environmental differences (Lande 1981; West-Eberhard 1983; Parker and Partridge 1998; Rice 1998; Gavrilets 2000). However, experimentally evolving populations of D. melanogaster (Wigby and Chapman 2006) and D. pseudoobscura (Bacigalupe et al. 2007) showed no such mating discrimination, indicating that sexual selection is not an obligate promoter of reproductive isolation in allopatric populations. In sympatric populations, however, isolation may lead to divergence via its direct effect on traits that are involved in mate recognition (Panhuis et al. 2001). This involves a wide range of modalities like chemical, visual, tactile and auditory signals (Spieth and Ringo 1983; Ritchie et al. 1999; Rundle et al. 2005). It is important to point out that the rapid change between populations as a result of sexual selection can also play an indirect role in speciation by increasing the overall rate of change within isolated populations (Ligon 1999), this indirect role might be more important than its direct role. Darwin (1871) noted that elaborate secondary sexual characters tended to occur in groups that also had high species richness suggesting that sexually selected ornamentation and preference is a potent source of selection and sexual communication can indirectly cause sexual isolation. Several workers still debate on the use of secondary sexual traits in same sex competition thus pointing to their role in sexual selection (Stockley and Bro-Jorgensen 2011). In the 1980s researchers began to emphasize how mate choice (female and male choice) could cause divergence between populations thereby leading to speciation. This led to the natural conclusion that sexual selection within populations may lead to sexual isolation between populations. Lande (1981, 1982) showed in his model that as long as there is genetic variation for a male trait and a female preference for that trait, there will be assortative mating which generates positive covariance between the two. West-Eberhard (1983) also presented a highly influential view on the issue suggesting that social evolution including both intra and inter sexual selection could cause speciation (Coyne and Orr 2004). Analysis of the frequency of papers published on sexual selection and speciation shows significant upturn from the seminal West Eberhard and Lande papers (Ritchie 2007). This and subsequent theory has shown that it is possible for sexual isolation to evolve as female preference and male traits drift along this line (Uyeda et al. 2009). Comparative evidence suggests, however, that postmating effects promote speciation. The first model to explicitly address the influence of sexual conflict on speciation, concerned with conflict over mating at a parapatric Genetica (2014) 142:23–41 secondary contact (Parker and Partridge 1998, Partridge and Parker 1999). This proposed that the effect of conflict on speciation depended on which sex gained the upper hand in determining the outcome. If female preference predominated a mating system, speciation was more likely, but if male competition overcame female preference then speciation would be less likely. This is one of a few models which argue that strong sexual selection can sometimes inhibit speciation (Panhuis et al. 2001). The covariance between traits including relevant behaviours such as male morphology, courtship song or genitalia and preferences have been predicted to be the major evolutionary forces that can cause behavioural isolation (Panhuis et al. 2001; Coyne and Orr 2004; Hosken and Stockley 2004). Evidences such as the one describing 20 sister pairs of passerine birds showing divergence due to differences in their plumage colour provide evidence that sexual selection may lead to speciation, in various species taxa (Barraclough et al. 1995). Divergence also occurs by differential mating in plant species, according to the lengths of nectar spurs (Hodges and Arnold 1995). However, studies on sexual selection with major emphasis on Drosophila have not been discussed much yet. Here we provide our perspectives on exciting new research of how sexual selection unaided by ecological divergence can drive reproductive isolation in Drosophila. Broadly, we discuss certain aspects of intersexual selection like role of different types of stimuli which are the important components of mate recognition system that may lead to speciation. In addition to this, we also describe how pre- and post-copulatory intrasexual selection might play a role in intraspecific divergence. Further, we discuss the relationship between rapid reproductive protein evolution and reproductive isolation in the light of sexual selection. Major emphasis has been laid on how sexual selection play efficient role in divergence of certain traits in Hawaiian Drosophila. Keeping these points in view, the main aim of this review is to give an account of sexual selection operating to bring about reproductive isolation (pre- and postcopulatory) and thereby its role in speciation. In addition to this, how sexual selection plays a role in postzygotic reproductive isolation thereby leading to speciation will also be discussed. Intersexual selection as a mode in driving species divergence Intersexual selection is an evolutionary process in which choice of a mate depends on attractiveness of its traits. This selection generates or exaggerates precopulatory traits that improve a male’s mating success. (Darwin 1871; Andersson 1994). Paterson (1980) proposed that every species 25 possesses its own distinct specific mate recognition system that controls the exchange of sensory information sent and received by both sexual partners during courtship. Patterns of mate choice can be altered by changing the costs of choosiness without altering the preference function. However, adaptive male mate choice can lead to an important yet unappreciated cost of sex and sexual selection (Long et al. 2009). Sexual selection arises due to non random variation in the mating success of individuals, which often results from variations in the components of the mate recognition system. The evolution of a new mate recognition system can cause sexual isolation and hence speciation (Ritchie 2007). However, some questions are still unanswered, especially those concerned with variation in mating behaviour that may provide a way to understand divergence of sexually selected traits. The answers to these questions can be categorised into five broad areas of interest. Variation in mating behaviour and costs of choosiness could: • • • • • Influence the rate and direction of evolution by sexual selection. Provide information about the evolutionary history of female mating preferences. Help to explain inter specific differences in the evolution of secondary sexual characters by relating different tactics of mate choice to ecological factors (time and energy costs of sampling which had a potential constraint on optimal mate choice, predation risk which affects female choosiness, territory or resource quality), social or morphological factors (interactions between males, variability in male phenotypes, female–female competition and female mate copying). Provide information about the level of benefits gained from mate choice. Provide a mechanistic account of the emergence of mate choice (Jennions and Petrie 1997). According to the first point, increased variability in preference might decrease the intensity of female-driven directional selection. For Fisherian traits (traits which are selected do not necessarily increase survival) increased cost may still lead to the evolution of multiple preferences (Pomiankowski and Iwasa 1993). However, for traits indicating viability only a preference for a single trait is stable if assessment of additional traits increases costs disproportionately (Iwasa and Pomiankowski 1994). The second point may provide evidence that the Fisherian model dealing with the evolution of ornaments predicts considerable heritable variation in female mating preference, both within and between populations (Lande 1981). Turner and Burrows (1995) suggested that the genetic bases of preferences may lead to different speciation rates 123 26 among lineages. The Fisherian model was validated by the work of Sharma et al. (2010) who provided evidence that evolution of a trait occur due to genetic variation in female preference. In contrast to the Fisherian models, female choice for males with ‘viability genes’ requires neither genotypic nor phenotypic variation among females (Grafen 1990). However, polygenic models obviously assume a heritable basis to preference, and generally predict coevolution of preference and preferred trait (Bakker 1993). Thomas Getty (2006) in his paper has shown that application of the handicap principle to signalling in sexual selection is not a valid generalization. Although some of the signalling systems, with additive costs and benefits, have solutions that resemble sports handicaps, the signalling in sexual selection has multiplicative costs and benefits, and solutions that do not resemble the sports handicap. Thirdly, most attempts to explain variation in ornamentation among species invoke strong natural selection against the elaboration of male traits (Balmford et al. 1993; Winquist and Lemon 1994). Factors that inhibit female choice will reduce selection for elaborate male traits. Variation in the opportunity for mate choice may also affect other features of mating behaviour. For example, Slagsvold et al. (1988) suggested that polygyny in pied flycatchers (Ficedula hypoleuca) is partly due to high female sampling costs leading to limited searches for unpaired males. Sullivan (1994) has also noted that given severe time constraints on female choice, females are likely to use static morphological traits that are quickly assessed. Hence information on the duration over which females assess males may explain some interspecific variation in male ornamentation. Fourthly, given phenotypic plasticity in female mating preferences it is possible to manipulate the costs of choosiness and examine the effect on mate choice. This should provide information about the benefits associated with discriminatory mating (Jennions and Petrie 1997). Fifthly, once the importance of variability in female mating preferences is recognised, a more mechanistic account of mate choice should emerge. As Ryan (1994) has noted, knowledge of mechanism provides a stronger base when explaining why certain traits have evolved (Haines and Gould 1994). In Drosophila, the diversity of mating behaviour in various species and basic similarity between some species emphasize that mating behaviour has gone through evolutionary changes. Variation of different signals by which two sexes exchange and their predominance during mating can contribute to the appearance of the premating isolation (Butlin and Ritchie 1994). Similarly, Ruedi and Hughes (2008) studied variation in mating behaviour of D. melanogaster and emphasized the genetics of courtship behaviour and various mechanisms underlying sexual selection. Species interactions causing selection on mating traits play 123 Genetica (2014) 142:23–41 important role in generating species divergence. This variation may arise due to genetic differences in developmental trajectories or proximate environmental factors (Edward and Chapman 2013). Numerous correlational and experimental studies from many taxa now confirm that males with increased ornamentation or possessing certain attributes have a mating advantage (Bradbury and Andersson 1987; Ryan and Keddy-Hector 1992; Andersson 1994; Moller 1994; Johnstone 1995). In order to maintain female mating preference three explanations have been proposed: (a) preferences may be directly selected due to direct benefits which increase female survival or fecundity (Reynolds and Gross 1990), (b) preferences may be maintained by indirect selection due to genetic benefits that increase offspring fitness (Andersson 1994), (c) preferences may be maintained as pleiotropic effects of natural selection on female sensory systems in contexts other than mate choice such as foraging or predator evasion (Arak and Enquist 1995). For many years, mate choice has been considered to be expressed predominantly in females resulting in selection on male displays or courtship characters. However, this perspective has now undergone both theoretical and empirical revision (Edward and Chapman 2011). A comprehensive set of tests revealed fitness benefits of male mate choice in D. melanogaster (Edward and Chapman 2012). The phenomenon of intersexual selection was further revealed through studies on sexual isolation reported by Vishalakshi and Singh (2006a) between two sibling species, D. ananassae and D. pallidosa and the results suggest that there is preferential mating between males and females of the same species in these two sibling species. Similarly sexual isolation among three sibling species, D. melanogaster, D. simulans and D. mauritiana was studied (Carracedo et al. 2000). The results show asymmetrical mating preferences i.e. D. mauritiana males mate with both D. melanogaster and D. simulans females and females of D. mauritiana discriminate strongly against males of these two species, and D. simulans males mate with D. melanogaster females but the reciprocal cross is difficult (Watanabe and Kawanishi 1979; Carracedo and Casares 1985; Coyne 1989). Taylor et al. (2009) reviewed the findings of a series of investigations on the fitness consequences of female preference in D. simulans and compared them with its sibling species D. melanogaster and found stark differences in the fitness consequences of mating with preferred males in the two species. This provides evidence for the existence of assortative mating in D. melanogaster and D. simulans. Jennings and Etges (2010) studied premating sexual isolation between D. mojavensis and D. arizonae which are the most important members of the repleta species group. Such findings were further supported by the studies on sexual isolation done by Banerjee and Singh (2012) in four species of the D. bipectinata complex. Genetica (2014) 142:23–41 Selection may act on various components of mating behaviour including rapprochement or pair formation and courtship behaviour (Alexander et al. 1997). It is thus crucial to understand how evolution has influenced the genes that shape courtship leading to species divergence. One important aspect of this investigation is to evaluate the biological role of each signal that is used for species discrimination. In this perspective, the co-evolution of male and female sexual signals and receptors suggests how these may provide heretofore neglected insight into the mechanism by which isolating barriers may emerge. Heterosexual courtship in different species of the melanogaster complex involves a series of behaviours prior to mating. It is thought that sexual selection operated over millions of years on preexisting neuronal pathways, recruiting them for sexual behaviours and producing the similarity of behavioural elements common to all members of the genus Drosophila (Spieth and Ringo 1983). Sturtevant (1915) first described the courtship of D. melanogaster and attempted to identify the stimuli involved. During courtship both partners exchange signals that belong to multiple sensory modalities. These are courtship signals and comprise chemical, visual, acoustic or tactile stimuli (Liimatainen and Jallon 2007). These stimuli function to inform the female of species identity of the male and to stimulate the female beyond her acceptance threshold for accepting the male in copulation (Spieth and Ringo 1983). Mature virgin females vary in their acceptance threshold, but the male often has to repeat his courtship elements often numerous times before the female is willing to copulate. The female outcome of courtship appears to be dependent upon the physiological state of the female and the temporarily summed effect of the male stimuli. However, Bretman et al. (2011) revealed the robust mechanisms by which males of D. melanogaster assess their socio-sexual environment to precisely attune responses through the expression of plastic behaviour. When the multiple cues (auditory, olfactory, tactile and visual) were experimentally removed then the males of D. melanogaster were unable to detect the rivals which shows the importance of different types of mating signals in recognition. Thus different types of stimuli that are key components of species mate recognition system are discussed below: Visual stimuli The requirement for the perception of visual stimuli for success in mating behaviour is variable within the genus Drosophila (Grossfield 1971, 1996) and this variability can further act as an element in isolation that thereby leads to speciation. During courtship, some visual stimuli are dynamic (locomotor activity, wing displays and motion) whereas others are static (colors, shapes). The level of 27 interspecific variability can be observed in different species of this genus. The comparison of the level of insemination suggests that D. melanogaster can mate in the dark whereas D. simulans and D. affinis tend to be inhibited in the dark (Spieth and Hsu 1950). A strong light-effect was noted between (and sometimes within) the four species of the melanogaster complex. Investigators have used various mutations of D. melanogaster to assess the role of visual stimuli in courtship (Spieth and Ringo 1983). Such mutants can be divided into two classes (1) those which have an increased amount of black pigment in the exoskeleton, and (2) those which have reduced amount of red (pterin) or brown (ommochrome) pigments in the primary and secondary pigment cells of the compound eye. Both classes of mutants have reduced visual acuity (Spieth and Ringo 1983). Crossley (1970) observed that wild type D. melanogaster males and ebony mutants court under darkness in a similar manner. Males of D. auraria can mate in both light and darkness but white-eyed mutants, which can perceive light but lacking visual acuity refuse to mate under light but readily mate in darkness (Grossfield 1972). The light dependent species apparently either remain immobile during darkness or have a key element of their courtship which is dependent upon visual stimuli. The loss of visual stimuli might decrease the mating ability. Species like D. subobscura, D. auraria (Isono et al. 1995) show almost no mating ability in the dark whereas D. affinis shows positive response for mating in the presence of light (McRobert and Tomkins 1987). However, partial isolation exists between two species of D. auraria complex (D. auraria and D. triauraria), when both are exposed to light but the same is not true in the dark as both exhibit complete isolation (Oguma et al. 1996). Thus, it is quite clear that visual stimuli often impart its effect in Drosophila mating system. However, mutations affect such behavioural aspects. For example there is inhibition of mating of whiteeyed mutants in light conditions in Drosophila and this could be explained by several hypotheses that eye pigment deficient mutants exhibit a deficit of optomotor response or there occurs some neurobehavioral disruption produced by faulty visual input. Similar type of study was done by Chatterjee and Singh (1988) in D. ananassae when they found that white -eyed males are more successful in mating in the dark than in light. However, such type of mating deficit in the dark was also found in red-eyed males due to their reduced locomotor activity. In certain species of Drosophila i.e. D. suzuki and D. biarmipes, males possess dark black patch on their wings which serve as a visual stimulus to the female during courtship. Removal of black shade in wings reduces mating success in males. Such type of study was done by Singh and Chatterjee (1987a) in D. biarmipes suggesting that visual stimuli play an important role in the mating behaviour of D. biarmipes. These types 123 28 of visual signals may cause differential mating and hence play an important role in mate recognition system and thereby lead to species divergence. Acoustic stimuli Variation in courtship songs is thought to contribute to reproductive isolation in various species of Drosophila. It influences female receptivity during courtship and species recognition (Ewing and Bennet-Clark 1968; Liimatainen et al. 1992; Tomaru and Oguma 1994). These songs consist of two elements: the sine song and the pulse song. The sine song of D. melanogaster consists of humming sound that is reminiscent of flight sound. The pulse song exhibits inter and intra specific variations among the sibling species of the D. melanogaster subgroup like melanogaster, simulans, mauritiana, erecta, yakuba and teissieri, in the number of cycles per pulse, pulse repetition rate and in the duration of interpulse interval (IPI) (Spieth and Ringo 1983). The male pulse songs are species specific. Interspecific variations arise due to involvement of volume and quality of sound produced (Ewing and Bennet-Clark 1968; Chang and Miller 1978). While sympatric sibling species typically have songs that differ significantly in the mean IPI e.g. melanogaster and simulans, pseudoobscura and persimilis, allopatric pairs have similar songs i.e. ananassae and athabasca, pseudoobscura and ambigua (Spieth and Ringo 1983). Species recognition is often based on variation in the IPI or pulse frequency (Bennet-Clark and Ewing 1969; Ewing and Bennet-Clark 1968; Ritchie et al. 1999). Song evolution in this group does not always show phylogenetic trends (Alonso-Pimentel et al. 1995; Etges 2002) suggesting that courtship songs in Drosophila species may often evolve too rapidly to discern clear pattern of evolution as in the D. willistoni group (Gleason and Ritchie 1998). When mating sound was studied in six D. affinis subgroup species (affinis, algonquin, athabasca, azteca, narragansett and tolteca), the results showed similar pattern as that of D. athabasca which is a widespread North American species and consists of three semi species with different courtship songs (Miller et al. 1975). Most affinis subgroup species possess both low and high pulse repetition courtship sounds. Differences in courtship and mating sounds between D. affinis subgroup members seem generally substantial and are most likely to be sufficient for one’s recognition of these species and semispecies. It has been reported that D. narragansett and D. tolteca show distinctive courtship sound patterns i.e. low and high pulse repetition sounds was found to be present in D. tolteca whereas D. narragansett shows only high pulse repetition sound. Absence of interspecific mating between D. algonquin and D. affinis clearly indicates differences in 123 Genetica (2014) 142:23–41 their sound patterns (Chang and Miller 1978). However, inter- and intra-population divergence was also observed in D. montana for male courtship song (Klappert et al. 2007). But keeping all these facts under consideration, courtship song may not be the only factor responsible for species discrimination. In the affinis group it is evident that closely related species show distant similarity with respect to courtship sound whereas in D. athabasca, courtship sound is found to be very similar to distantly related, D. azteca. Experiments carried out with song stimulation show that females are able to recognize homospecific males through discrimination via IPI, rhythm and pulse length (Kyriacou and Hall 1982; Isoherranen et al. 1999). D. simulans and D. mauritiana show difference in IPI hence females of both species show discriminating nature in selecting their mates. Kyriacou and Hall (1982) studied the inheritance pattern of IPI through segregational analysis and found that IPI gene is said to be located on X chromosome adjacent to per gene. This was further put into confirmation when per gene of D. simulans was transferred to per null D. melanogaster then D. melanogaster confers a characteristic D. simulans song rhythm. However, this may not be the case always as IPI of D. auraria complex is controlled by each autosome (Tomaru and Oguma 1994). The contribution of the non A/ diss gene to evolutionary variation has been shown in numerous experiments with D. virilis whose song differs from that of D. melanogaster in many parameters like long and short pulse etc. (Aspi and Hoikkala 1995; Isoherranen et al. 1999). The average IPI shows little variability in D. melanogaster suggesting that this acoustic parameter is under very strong selection (Ritchie and Kyriacou 1994, 1996). However, there was positive response after one generation when artificial selection for IPI phenotype was done. Similarly, Watson et al. (2007) provided the first description of the song of D. santomea. They reported that D. yakuba and D. santomea had the largest difference in IPI between any species of the melanogaster group. They state that the IPI of secondary and primary song types differed significantly between species with D. santomea having much shorter IPI than D. yakuba. Divergence in the IPIs is large and considerably larger than between other sibling species of the melanogaster group. This could indicate that songs also play considerable role in causing sexual isolation. However, Saarikettu et al. (2005) managed to break down sexual isolation between D. montana and D. lummei by playing back artificial D. lummei song modified to have the IPI typical of D. montana to D. montana females. Similarly Li et al. (2012) studied copulatory song in three species of the Drosophila montium subgroup i.e. D. lini, D. ogumai and D. ohnishii through the analysis of F1 and backcross generations. D. lini and D. ogumai produce similar high frequency sine song but a third species D. ohnishii repels males of the other two Genetica (2014) 142:23–41 species (Wen et al. 2011). Therefore, from these evidences it is understood that acoustic signal divergence plays a very specific role in reproductive isolation via sexual selection (Wilkins et al. 2012). Chemosensory stimuli Behavioral change may often be the initial trigger for population divergence (Mayr 1946; Butlin and Ritchie 1994) and altered recognition, processing and response to chemical cues are expected to be involved in many behavioural changes. Consequently, understanding the role and relationship of chemosensory evolution to behaviour is just for understanding speciation. Chemosensory reception which includes olfaction and gustation may have a large role so that differences in pheromones function as mating signals and can influence sexual isolation (Smadja and Butlin 2008). Chemical communication is brought by cuticular hydrocarbons which act as courting contact signals. Variability in CHC occurs due to differences in chain length (presence or absence of double bonds). It has been known that insects frequently employ chemical signals during courtship. It is not surprising that chemosensory speciation is documented in many insects e.g. bees (Vereecken et al. 2007); beetles (Peterson et al. 2007); and walking sticks (Nosil et al. 2007). Divergence of CHC among species suggests its role in species recognition and speciation in Drosophila (Etges and Jackson 2001). In D. melanogaster group, D. simulans and D. mauritiana exist in sexually dimorphic forms and when asymmetrical reproductive isolation occurs, it is found that males of sexually dimorphic species court females of all species, whereas monomorphic species males will court only conspecific females. Hence it was predicted that sexual isolation occurs through differences in female CHCs (Coyne et al. 1994; Coyne 1996). Several types of quantitative and qualitative differences in CHC blend and thereby give rise to premating isolation between D. virilis and D. novamexicana (Doi et al. 1996), D. serrata and D. birchii (Howard et al. 2003) and D. santomea and D. yakuba (Mas and Jallon 2005). However, in D. pseudoobscura and D. persimilis there is no role of CHCs in sexual isolation but mate discrimination in sympatric populations relies on olfaction (Ortiz-Barrientos et al. 2004). Between populations, divergence has been observed in D. mojavensis (Etges and Jackson 2001). In D. melanogaster, the divergence in CHC is likely to occur in different populations particularly when African and Carribean populations are compared with the rest of the world populations (CHC 7, 11-HD). Studies on sexual isolation in the Drosophila species have focussed on the cosmopolitan (M) and Zimbabwe (Z) races of D. melanogaster. The former race occurs throughout the world whereas the latter occurs only 29 in Zimbabwe, Zambia and Botswana. In some parts of Africa such as Zimbabwe, individuals of both races appear to be sympatric (Hollocher et al. 1997; Fang et al. 2002; Takahashi and Ting 2004). These races show marked but asymmetrical sexual isolation since Zimbabwe type females has discriminating power for cosmopolitan males whereas reciprocal mating occurs readily (Wu et al. 1995; Hollocher et al. 1997). This kind of selection by Zimbabwe females towards cosmopolitan males is a kind of female discrimination which may give rise to partial sexual isolation among the population. Intrasexual selection in species divergence Intrasexual selection in contrast to intersexual selection occurs when members of the same sex of a species compete with each other in order to gain opportunity to mate with members of the opposite sex e.g. the male–male competition for females. In the genus Drosophila, pairing and copulation are synchronous. Bateman (1948) studies intrasexual selection in the form of sexual isolation at the level of subspecies, geographic races and mutants. Usually, males are in the central arena where intrasexual selection occurs whereas females are often those which exert choice. Darwin (1871), however, was unable to explain such type of sex difference but this was rather an important aspect of intrasexual selection. Intrasexual selection has been categorized into pre and post copulatory intrasexual selection in order to study species divergence in Drosophila more closely. Precopulatory sexual selection is based on an individual’s ability to physically dominate a rival. Rendel (1944) provide evidence about the role of intrasexual selection in D. subobscura using wild and mutant forms of Drosophila. These findings revealed that the males court both type of females but it is the female which show discriminating nature towards either of the males. Such type of difference takes place due to difference between the two sexes which leads to differential mating tendency. Similarly, Tan (1946) observed such type of sex difference in D. pseudoobscura. Likewise, aristapedia mutant in Drosophila reduces the mating ability of females whereas Bare Curly mutants enhance the mating ability of females. Intrasexual selection plays potent role in causing sexual isolation between two subspecies of D. virilis i.e. D. v. virilis and D. v. americana. This was experimentally proved when males were confined with females of the opposite subspecies it shows discrimination (Stalker 1942). The above finding was further supported by the work done by Singh and Chatterjee (1987b) in D. ananassae. They studied the variation in the mating propensity and fertility in five laboratory strains of D. ananassae, established from single females collected 123 30 from different geographical localities and the results clearly indicate the presence of intraspecific variations which is due to the difference in sexual activity of males, that means males are more subject to intrasexual selection. Similar type of results were obtained from the study done by Singh and Sisodia (1995) in the laboratory strains of D. bipectinata where variation in mating propensity occurs due to difference in the sexual activity of both the sexes. Therefore, these evidences suggest that though the discrimination is sometimes found in males but it is more or less restricted to females. This is further put into relevance by the study of mating ability of homo- and hetero-karyotypes of D. ananassae from natural populations and the study suggests that chromosomal polymorphism in D. ananassae may have a partial behavioural basis and males are inherently more subject to intrasexual selection (Singh and Chatterjee 1986). However, the fact that males are more subject to intrasexual selection was still debated by the study of Singh and Singh (1999) for the mating success on six wild type strains of D. ananassae. The results clearly implicate that the variation among the strains for the mating success in different geographical strain is due to variation in receptivity of females than sexual activity of males which reveals that females also play significant role in intrasexual selection. Such intra-specific variation is due to variation in their genetic constitution. Similarly, divergence in body size, underlie the evolution of incipient reproductive isolation between a set of D. melanogaster populations which provide an example how selection acts on body traits (Ghosh and Joshi 2012). Therefore, keeping this in view, Vishalakshi and Singh (2008) studied whether there is any relationship between mating success and size and asymmetry of different morphological traits, using two geographical strains of D. ananassae. The results suggest that the size of the sexual trait is a more reliable indicator of individual quality in sexual selection rather than fluctuating asymmetry (FA) in D. ananassae. The mating system of a species was considered by Darwin (1871) to be an important element in determining sexual selection. The only mating system in which intra-sexual selection is ineffective is strict monogamy with numerical equality of both sexes. However, post copulatory (post mating) sexual selection is also considered as a driving force where differential selection by females (cryptic female choice) (Eberhard 1996) or competition between males (sperm competition) leads to rapid divergence between species. Piscedda and Rice (2012) have clearly provided evidence that the post copulatory process bears potential to drive evolution of promiscuous mating system similar to that of female mate choice. One of the important aspects of post copulatory sexual selection is female remating since it determines the patterns of sexual selection and sexual conflict. Female 123 Genetica (2014) 142:23–41 remating has been studied in various species of Drosophila under both natural and laboratory conditions (Singh et al. 2002). Female remating is fundamental to evolutionary biology as it determines the patterns of sexual selection via sperm competition. The patterns of remating may provide an insight into the phylogenetic relationship shared by the four closely related members of the D. bipectinata complex (Singh and Singh 2013). Sperm competition is another aspect of intrasexual selection and is considered as an outcome of female remating. In this context sperm competition can also drive selection of offensive and defensive male traits. In one way forces will favour males that may affect the storage of another male’s sperm or that can use his own sperm in such a way that his own fertilization success is maximized. While the other way round males that are able to prevent or reduce subsequent competition from sperm of other males gains an advantage. Hence the impact of sperm competition on male fitness (Gromko and Pyle 1978) exhibits an excellent example of sexual selection. Sperm competition offers a unique opportunity to study adaptations shaped by the interacting forces of natural, sexual and antagonistic selection (Rice 1996). The occurrence of sperm competition in Drosophila is depicted through the proportion of progeny produced by second male in double mating experiments (Singh et al. 2002). This approach has been used to quantify genetic variation underlying sperm competition and thereby elucidate the dependence of different male competitive abilities on the genotypes of the females with which they mate in order to discern the potential role of sperm competition in species isolation (Civetta 1999). Manier et al. (2013 a, b) studied species specific sperm precedence mechanism in D. simulans and D. mauritiana by expressing GFP or RFP in sperm heads of these sister species. This experimental approach illustrates how sperm precedence mechanism can be used to predict the mechanisms and extent of reproductive isolation between populations and species. Similarly, rapid evolution of reproductive traits has been attributed to sexual selection arising from interaction between sexes. At the post-copulatory level, intra- and inter-specific size coevolution between male sperm and female sperm storage organs have been documented in Drosophila (Pitnick et al. 1999, Miller and Pitnick 2002). In the extreme case, it has been suggested that females may mate with a number of males and then select the sperm that will be used to fertilize the eggs (Eberhard 1996). The selective pressure arising from sperm competition has led to numerous adaptations to assist males in gaining fertilization. These adaptations may be (1) pre- and post-copulatory guarding behaviour, (2) mating plugs, (3) chemical or physical characteristics of the ejaculate which reduce receptivity to remating, (4) sperm displacement, and (5) sperm precedence. The foremost problem which is faced at the time of sperm Genetica (2014) 142:23–41 competition is that which sperm should get access to fertilize the eggs of female. Sperm competition may be intense when the sperm of several males is stored simultaneously within specialized storage organs of the female reproductive tract before fertilization. It has also been conjectured that males have evolved to produce large quantities of sperm in order to confuse or confound female’s cryptic system of selection (Wigby and Chapman 2004). Recent investigations on sperm precedence mechanisms in three closely related species of Drosophila revealed how postcopulatory sexual selection leads to divergence in male and female reproductive traits (Manier et al. 2010, 2013c; Lupold et al. 2011). In many insect species, costs of multiple mating are offset via direct benefits due to: (1) the replenishment of sperm stores, and (2) nutrient donations found in the ejaculate from each of the mates. However, evolutionary maintenance of polyandry in insects can be understood as direct benefits. This could be explained in D. melanogaster whereby males exposed to rivals subsequently mate for longer and thus accrue fitness benefits under increased competition (Bretman et al. 2009). Similar type of study in D. melanogaster revealed that remating by small bodied low fecundity females resulted in the production of daughters of relatively higher fecundity, whereas the opposite pattern was observed for large–bodied females. This shows the direct and indirect benefits of polyandry on the fitness of an organism (Long et al. 2010). Role of sexual conflict in speciation In sexual reproduction there are two individuals who may actually have no genetic interest in each other’s future, the parents, but who nevertheless have a joint genetic interest in the other individual or group of individuals. A conflict that expresses courtship acceptance or refusal usually arises because each parent’s fitness is generally maximized if it invests less and the other parent invests more than would maximize the other parent’s fitness. This conflict has been referred to as ‘‘the battle of the sexes’’. Most of the conflict between mates is the result of post-copulatory sexual selection that is sperm competition and cryptic female choice (Eberhard 1996). Traditionally post copulatory sexual selection generates sexual conflict through three discrete processes: (1) there may be conflict over how many gametes are dedicated to each mate (Parker 1970). This type of conflict will generate selection for traits such as copulatory plugs (Polak et al. 2001), antiaphrodisiacs and mate guarding by males, which invest in gametes monopolized through direct intervention of mate behaviour, (2) conflict may evolve through physiological tradeoffs between traits contributing to reproductive success, 31 this is common because one sex invests predominantly in offspring while the other sex (males) invests predominantly in fertilization opportunities (Bateman 1948) and (3) sexual conflict may arise when traits that are adaptive for one sex in reproductive competition have negative effects on the opposite sex for example the toxicity of male seminal fluid proteins in Drosophila (Chapman et al. 1995). Any deviation from monogamy increases sexual conflict because an individual’s lifetime reproductive interests will not coincide (Rice and Holland 1997). Therefore, sexual conflict should increase with multiple mating as does the potential for sperm competition as a result, sperm competition should enhance sexual conflict and thus lead to the evolution of characters that increases reproductive success in one sex, while they are costly to other. There are two arguments on the path of sexual conflict i.e. (1) The one who experience the strongest selection pressure will win the conflict and (2) those that are in a superior position to manipulate the other will win the evolutionary race. The male will also feel any costs to females from mating with males through reduced reproductive success which improves the competitive ability of sperm. Thus, selection for male paternity assurance is expected to be stronger than selection for female resistance to mating or male adaptations (Parker 1970). Hosken and Snook (2005) in their review suggest that sexual conflict generates sexually antagonistic evolution. Alteration of the operational sex ratio of adult Drosophila over just a few tens of generations can lead to altered ejaculate allocation patterns and the evolution of resistance in females to the costly effects of elevated mating rates. Manipulation of the relative intensity of intra- and inter-sexual selection can lead to replicable and repeatable effects on mating systems and reveal potential for significant contemporary evolutionary change (Edward et al. 2010). The co-evolution between males and females that can be caused by sexual conflict can result in several types of evolutionary dynamics (Parker 1970). One of these is reproductive isolation that might lead to speciation. Two contributions investigate this experimentally. Gay et al. (2009) test the prediction that under models of sexual conflict, larger rather than smaller population size may lead to more rapid reproductive isolation. Hosken et al. (2009) illustrate using data from two experimental evolution studies in flies that the experimental manipulation of sexual conflict may provide evidence for reproductive isolation in some species. Recently, Pitnick et al. (2001) demonstrated that sexual selection favours larger males which invest a greater production of their total energy produced in sperm production. While observing the greater reproductive success of females with monogamousline males they suggested that male and female reproductive success interest’s do not naturally counteract in D. melanogaster. However, studies carried out by Somashekar 123 32 and Krishna (2011) revealed that females of D. bipectinata prefer to mate with older males giving a very good example of intrasexual selection. In contrast to this, similar type of study revealed that attractive males do not sire superior daughters which contradict the good gene model (Taylor et al. 2010). There is increasing evidence that genes involved in reproduction, specifically those showing sex-biased expression evolve rapidly and are often subject to positive selection (Swanson et al. 2001; Proschel et al. 2006). For example male sperm competition success in D. melanogaster is associated with certain male genotypes of seminal accessory gland proteins (Acps) (Clark et al. 1995; Fiumera et al. 2005) and certain features of the evolution of these Drosophila Acps are also consistent with sexual selection. Hence sexual selection might explain the rapid evolution of reproductive protein in theory leading to speciation. The Acps of Drosophila which comprise the bulk of the nonsperm part of the male ejaculate, have been subjected to the most intense investigation (Swanson et al. 2001; Mueller et al. 2005) and it is estimated that about 10 % of them show some evidence of positive selection (Swanson et al. 2001). Acp genes in D. melanogaster do not appear to have homologues in D. pseudoobscura because in at least some cases D. melanogaster Acp genes lacking homologues in D. pseudoobscura show evidence of directional selection and hence adaptive evolution (Mueller et al. 2005). Moreover, there is accumulating data to show that non-homologous and sometimes very different, genes can encode very similar seminal fluid traits across species (Mueller et al. 2005; Begun et al. 2006; Braswell et al. 2006; Davies and Chapman 2006). A fundamental question regarding rapid reproductive protein evolution is whether such changes simply result in secondary isolating barriers. Alterations in reproductive proteins can indeed result in reproductive isolation and could theoretically cause speciation in allopatry or sympatry (Sainudiin et al. 2005). Furthermore, in Drosophila there is some experimental evidence that Acps are associated with reproductive isolation. This can be proved when a cross between male D. pulchrella and female D. suzukii is sterile even though sperm are transferred (Fuyama 1983). However, the cross can be made fertile if the female is given a dose of D. suzukii Acps. This suggests that the isolation is at least partly maintained by the actions of Acps. If reproductive isolation is associated with rapid evolution in reproductive proteins, there is also merit in asking how exceptionally labile novel reproductive genes evolve. It is evident that new Acp gene could be created by means other than gene duplication possibly from non coding regions of DNA (Begun et al. 2006). This was further revealed by Singh and Jagadeeshan (2012) who suggested that Drosophila sex and reproduction- 123 Genetica (2014) 142:23–41 related (SRR) genes evolve faster than non reproductive proteins unveiling the importance of SRR molecules in speciation research. When genes expressed in testis, ovary, and non reproductive tissues were screened for rates of evolution it became proportion of genes in reproductive tissue evolved more rapidly than genes expressed in non reproductive tissues. FA is often used as a measure of developmental instability resulting from perturbations in developmental pathways. This is explained by the work of Polak et al. (2004) who have demonstrated that the sex comb in D. bipectinata is subject to intraspecific sexual selection suggesting that the sex comb differences seen across populations throughout the species geographic range are at least in part the result of adaptive diversification driven by differential mating success. The data also indicates a potential constraint on the evolution of sex comb size driven by the combined effects of selection and a genetic interaction with comb asymmetry which could promote the maintenance of heritable genetic variation underlying expression of this sexual ornament. In D. ananassae it has been found that magnitude of FA differs significantly among morphological traits being lowest for non-sexual traits and highest for sexual traits suggesting that sexual traits are better indicator of developmental stress (Vishalakshi and Singh 2006b). Likewise, Therefore, a divergence trend of testis [ ovary [ somatic genes emerged suggesting male and female SRR genes evolve under different selective pressures. Frequency dependent sexual selection in Drosophila Natural selection is seldom constant and changes with abiotic and biotic factors in the environment. In the field of population genetics the maintenance of genetic variability in a given population is of foremost importance. Here the importance of phenomenon of frequency-dependent selection may be mentioned as it maintains the genetic variability in a population. It has been experimentally demonstrated that the selective value of a given genotype is often dependent on the function of its frequency in the population. Frequency dependence may be positive (i.e. in favour of the common type), or negative (i.e. in favour of the rare type). Rare-male mating advantage or minority male mating advantage is one of the interesting and best studied examples of this frequency-dependent selection. When two variants of the same species are present together the rare type is more successful in mating than the common type. The phenomenon of rare-male mating advantage is of considerable evolutionary significance as it plays an important role in the maintenance of high levels of genetic variability (Singh 1999). An important consequence of rare-male mating advantage is that it promotes outbreeding Genetica (2014) 142:23–41 because an occasionally visiting male from another population tends to be favoured by the female. The rare-male effects have been reported when Drosophila males differ at single loci affecting external somatic traits, when males come from different laboratory strains, are of different karyotypes, or carry different isozyme variants (Singh and Sisodia 2000). Petit (1951) was the first to report the occurrence of rare-male mating advantage in multiple choice mating between Bar eye and wild type Drosophila melanogaster. Ehrman (1966) demonstrated frequencydependent selection between populations of different geographic origin in D. pseudoobscura and D. paulistorum. Minority male mating advantages have so far been reported in 9 species of Drosophila: melanogaster, pseudoobscura, persimilis, willistoni, tropicalis, equinoxialis, funebris, ananassae and bipectinata (Singh 1999). Singh and Chatterjee (1989) studied this phenomenon in D. anannassae by using sepia and cardinal mutant stocks and wild type stock in order to detect rare-male effect and they found that both types of males are more successful in mating when they are in minority. Thus, the results provide evidence for the existence of a minority male mating advantage in D. ananassae. Likewise, density and frequency-dependent selection on the signed locus was studied in D. melanogaster. The results clearly revealed that at higher density the frequency of either of the two genotypes (wild type and mutant) is low, its viability increases. While, when the frequency of either of the two genotypes is high its viability decreases which suggest the inverse relationship between frequency and adaptive value (Singh and Sisodia 2000). However, Markow et al. (1978) did not find a rare-male effect for the mutant sepia competing with a wild type in D. melanogaster. However, similar type of studies was done by Singh and Sisodia (1997) in D. bipectinata by using wild type and cut wing mutants and the results clearly illustrate that both type of males were more successful in mating when they are in minority. The genetic basis was given by Som and Singh (2004) by studying such type of selection on the alpha inversion in the left arm of the second chromosome (2L) in D. ananassae by using two strains: ST/ST standard gene arrangement and AL/AL alpha inversion in 2L. The results clearly illustrate the presence of minority male mating advantage and preferential mating found in the AL/AL strain which shows inversion karyotype also plays role in rare-male mating advantage. When similar study was carried out by Som and Singh (2005) in D. ananassae by using three pairs of wild type i.e. Mysore, Pune and Tirupati and three types of mutant strains i.e. yellow body colour, claret eye colour and cut wing and they found one sided rare-male mating advantages one for claret eye colour males and other wild type males (Tirupati). However, no advantage was found for rare males with Mysore and yellow body colour. Hence 33 this study provides evidence for minority male mating success and minority female mating disadvantage in D. ananassae. Rare-male mating advantage was also studied at an enzyme locus in D. pseudoobscura. The study revealed that Amy locus has an effect on the mating behaviour which includes some degree of rare-male mating advantage (Singh and Sisodia 2000). Therefore it is well proven from the above findings that a rare-male effect seems to occur for mutants, inversion karyotype, isozyme variants, and geographic strains, strains reared at different temperatures and having behavioural differences. In order to explain the rare-male effect, Ehrman and Spiess (1969) suggested sampling and habituation hypothesis. According to this hypothesis nature of cue is different for different male types. The females become conditioned against mating with the males that first court them during their unreceptive period after eclosion. Since these males would usually be the more frequent type, the rare-male type would gain mating advantage when the females become sexually active as they are able to break through the habituation by its slightly different cues. Thus from an evolutionary point of view rare-male mating advantage bears a great importance in the field of population genetics. Initially the rare genotype will increase in frequency if there are no other selective forces operating against it but as soon as this rare-male becomes common its advantage decreases. Thus as a result of frequency-dependent sexual selection, a balanced polymorphism can be maintained by sexual selection in the absence of heterosis in the heterozygotes. If such type of phenomenon is at all widespread in natural populations, it may play a considerable role in maintaining genetic diversity. Hence rare-male mating advantage is of great importance in genus Drosophila as the number of genes and chromosomal polymorphism in Drosophila is maintained by such frequency dependent selection (Singh and Sisodia 2000). Therefore, it is well understood from the above facts that the cause of rare-male effect is not yet fully resolved. Nonetheless, this effect is likely to play an important facet in the process of sexual selection and speciation. Sexual selection and speciation in Hawaiian Drosophila Recently, studies on sexual selection and speciation in the Hawaiian species have been enhanced, since Hawaiian Drosophila has stimulated considerable thought about the role of sexual selection in speciation. As far as we know the Hawaiian Drosophila have astonishing diversity i.e. they represent about 20 % of the described species in a world-wide distributed genus, despite the fact that Hawaiian Islands have such a small land area (Carson 1982). The closely related species are however not 123 34 ecologically different but they differ in secondary sexual traits such as courtship pheromones (Tomkins et al. 1993), the number of tibial bristles (Carson and Bryant 1979), head width (Boake et al. 1997), acoustic signals and courtship behavioural response (Hoikkala and Kaneshiro 1993). Several extraordinarily close and apparently newly evolved species pairs have been identified on the newest island, Hawaii. Among them the three best studied members of the genus in Hawaii, D. planitibia, D. silvestris and D. heteroneura inhabit cloud forest on the flanks of volcanoes on Maui (D. planitibia) and Hawaii (D. silvestris, D. heteroneura). The flies of Hawaii are highly suitable for investigation of the role of sexual selection in speciation due to two possible reasons. First they have elaborate mating displays and show some degree of ethological isolation. Second, the crosses between them are usually fertile (Craddock 1974; Ahearn and Templeton 1989). Ringo (1977) proposed that sexual selection was the main reason for the great diversity of this group. However, Templeton (1979) disagreed arguing that the sexual selection is generally stabilizing and hence could not lead to divergence. Hence the debate between the two raises the question that sexual selection is ever directional and specifically whether directionality is found in the Hawaiian Drosophila. To answer these questions, Kaneshiro (1976, 1980) proposed a hypothesis based on the studies done on the members of the planitibia group. He observed that there is existence of behavioural isolation among the four most recently evolved species of the planitibia group. Behavioural isolation is often asymmetrical with females of more ancestral species being unlikely to mate with males of the more derived species. This could lead to a pattern of asymmetrical behavioural isolation, with ancestral females being narrower in their preference (Boake 2005). Another model was put forward which states that the males do not provide resources to females in relation to mating and that females visit many males and compare their mating displays which helps them in finding the most appropriate mate. This shows how sexual selection influences the divergence of phenotype and hence leads to reproductive isolation. Ahearn et al. (1974) studied sexual isolation among three species of Drosophila i.e. D. heteroneura, D. silvestris and D. planitibia and found that D. heteroneura and D. silvestris are sympatric while D. planitibia shows allopatric distribution. In order to understand behavioural isolation among Hawaiian species, studies based on morphological traits such as head width, shows differential pattern between the two species. It was revealed that the females prefer males with a broad head, which are also more likely to win the fights. This shows that head width is sexually selected through both female mating preferences and male–male aggression and that selection is directed in 123 Genetica (2014) 142:23–41 favour of broader heads. However, no significant difference for the mating success was found for the two types of hybrid males with D. heteroneura females. This shows that head width was not involved in intraspecific mate choice. Also, secondary sexual traits and number of tibial bristles were compared across populations of D. silvestris and significant differences were found (Carson and Bryant 1979; Carson et al. 1982). Similarly, flies of Hawaiian region possess spectacular diversity in male foreleg modifications which thereby represent the results of sexual selection (Stark and O’Grady 2009). Recently, qualitative and quantitative chemical compositions of cuticular hydrocarbons (CHCs) in 138 flies belonging to 27 Hawaiian Drosophila species, picture winged and nonpicture-winged were analyzed regarding sexual dimorphism, differences in saturation, branching position and length of CHCs. The study shows significant variation in CHCs pattern i.e. new species show decrease in unsaturated hydrocarbons and gradual increase in branched compounds, monomethylalkanes and dimethylalkanes (Alves et al. 2010). However, Boake and Konigsberg (1998) studied in detail the genetics of sexual selection and speciation with major emphasis on genetic analysis of male sexually selected traits in D. silvestris. Among all the traits observed, wing vibration seems to play role in behavioural isolation. These examples clearly illustrate that Hawaiian Drosophila are excellent model to study the role of sexual selection in causing species divergence. Genetics of sexual selection What type of genetic changes brings about speciation is one of the most basic questions in biology. The concept of sexual selection involves identification of genes that are involved in the divergence of sexually selected traits. Usually, it has been observed that traits that are related to mating behaviour and fertilization have a direct role in species formation. Hence one will expect for high divergence between species in those genes whose products are linked to sexuality. Studies on the molecular evolution of genes may help us to understand the role played by different evolutionary forces during evolution. The rapidly accumulating number of gene sequences provides an opportunity to study the molecular nature of sex related gene evolution. These genes show an interesting pattern of high divergence between related species. Clark et al. (1995) studied the role of reproductive protein in the genus Drosophila and revealed that the male accessory gland proteins affect female postmating behaviour and sperm precedence. For example, Acp26A has shown high divergence between species (Aguade et al. 1992; Tsaur and Wu Genetica (2014) 142:23–41 1997) and this is most probably due to directional selection (Aguade 1998). This type of evolution has been also detected for Acp29 and Acp70A (Aguade 1999). Karotam et al. (1993) studied the divergence of Esterase-6 which is an ejaculatory duct protein between D. melanogaster and D. simulans. Similarly, gene called transformer (tra) which involves in sexual differentiation in Drosophila shows poor conservation between D. melanogaster, D. simulans, D. erecta, D. hydei and D. virilis (O’Neil and Belote 1992). However, certain sets of genes that have a direct as well as indirect role in courtship behaviour have been suggested to be involved in variations in mating preference like cacophony, fruitless, Voila, courtless, desat as well as per, nonA/dissonance and dissatisfaction. Also, sex specific transcripts of two loci, fruitless (fru) and double sex (dsx) determine male versus female identity (Williams and Carroll 2009). For sexual signal traits, such as wing song and pheromones, sex specific neuron development is critical (Kurtovic et al. 2007; Yamamoto 2008). Expression of the male dsx and fru variants are necessary for development of the region of the brain (P1) and associated dendrites that are associated with male multimodal sensory processing and courtship behaviour (Kimura et al. 2008). Role of fru in wing song demonstrated that neural commands for song are absent in females because they depend on neurons expressing male fru transcripts (Clyne and Miesenbock 2008). Similarly, genomic response to courtship song stimulation in female D. melanogaster provides novel insight into specific molecular changes in females in response to courtship song stimulation (Immonen and Ritchie 2012). Nanda and Singh (2012) reviewed the genetic basis of mate recognition between D. simulans and D. sechellia which revealed that the majority of quantitative trait loci responsible for both male mating behaviour and pheromone concentration are located on the third chromosome. In their review it has been shown that the genes affecting cuticular hydrocarbons that differ between D. simulans and D. sechellia may cause sexual isolation. Elicitation of male courtship by female D. melanogaster is strongly dependent on cuticular hydrocarbon (CHC) pheromones especially dienes which depend on femalespecific expression of the desaturase locus i.e. desatF (Shirangi et al. 2009). Disruption of expression of the desat1 locus in D. melanogaster has phenotypic effects on both the production of CHCs and mating decisions in both sexes (Grillet et al. 2006; Marcillac et al. 2005) suggesting possible pleiotropic control of trait and preference. Similarly, role of fru in wing song demonstrated that neural commands for song are absent in females because they depend on neuron expressing male free transcripts (Clyne and Miesenbock 2008). In the case of post copulatory sexual selection, female remating is proved to be an important aspect of sexual selection and its genetic control 35 has been proved to be yet another path in understanding the genetics of sexual selection. Similarly, various studies provide evidence that the genes on X-chromosome play role in affecting remating speed of females of D. melanogaster. However, chromosome substitution analysis, biometrical and planned comparison analysis, and recombination analysis of experiments for remating speed demonstrate the involvement of chromosome II which contribute significantly to the differences in remating speed in two selected lines i.e. fast and slow lines (Singh et al. 2002). Genes found in simulans clade of melanogaster i.e. Odysseus has been proved to influence sperm production and potentially sperm competition in D. simulans so post copulatory sexual selection may have driven its divergence and indirectly contributed to hybrid sterility (Sun et al. 2004). Similarly, results of Singh and Singh (2001) selection experiment in D. ananassae revealed that the remating speed, mating propensity and fertility are under polygenic control. Hence from all these findings it is quite obvious that genes play role in driving species divergence via sexual selection. Conclusion There is no doubt that sexual selection has the potential to play a major role in speciation. Examples from inter- and intra-sexual selection studies in Drosophila revealed that sexual selection has power to drive rapid divergence via choice and competition that may lead to reproductive isolation. But sexual selection may not be the only cause of speciation and more than one force may operate to bring about speciation. Any one study is insufficient to prove what forces have operated to bring about speciation. This problem stems from our inability to observe the whole process, forcing us either to infer the most probable future course of events (when the process of speciation is not yet complete) or to separate different possible histories. It has been revealed that sexual selection should be demonstrated directly from the effect of variation in the trait on mating success rather than simply being inferred from sexual dimorphism. However, divergence under sexual selection does not necessarily result in a substantial barrier to gene exchange. Usually, prezygotic isolation is the direct result of changes in sexually selected traits or evolutionary history. Although Darwin (1871) in his work on sexual selection ‘‘The Descent of Man, and Selection in Relation to Sex’’ appreciated the importance of mating preferences in sexual selection he did not clearly identify the evolution of mate choice as a key topic in its own right. It is now clear that the evolution of mate choice is one of the most important topics in sexual selection research. The Fisherian process is probably operating in some system, but we do 123 36 not know how ubiquitous it is. On the other hand, depending on the evolution and maintenance of genetic correlations between traits and preferences, the possibility remains that the Fisherian process explains very little with respect to the evolution of female preferences. However, special emphasis has been made in this review is to know how sexual selection causes rapid divergence among different species of Hawaiian Drosophila. This has been revealed by the fact that divergence in some of the traits may cause differences in female preferences which are sexually selected and propagate among the species. However, rapid radiation in the Hawaiian Drosophila can be explained by other evolutionary forces such as drift followed by natural selection, or that the sexually selected traits are not involved in species recognition. Thus sexual selection might not be as important in the origin of some of the Hawaiian Drosophila species pairs. While our understanding of the processes that facilitate reproductive isolation and the genetics of speciation has advanced enormously over the past few years (Coyne and Orr 2004), this has not been matched by an increased understanding of the molecular processes underlying reproductive isolation. It is exceptionally difficult to determine whether sexual selection is responsible for phenomenon associated with reproductive isolation solely by examining patterns of evolutionary change in traits. However, understanding rapid evolution of reproductive proteins has helped in solving this problem to a great extent. To determine the respective roles of selection and drift one has to determine whether the effect of divergence in a particular sequence benefits males, females or both. Such information would perhaps allow us to detect whether sexual selection is predominantly responsible. The role of sexual selection could also be revealed by associations between reproductive protein variants and mating systems. Thus future studies investigating how sexual selection might result in reproductive isolation need to consider whether reproductive traits are evolutionary constrained. Despite the triumph of modern sexual selection research, there are still many related topics that need to be addressed. For example, some models of the evolution of mate choice enjoy limited support and for most part we are not sure which model explains the majority of choice evolution within or between systems. Studies of factors determining intensity of sexual selection are still more confusing. We are still in the process of building connections between reproductive ecology and selection differentials. Finally, there seems to be a lack of connection between theory related to mate choice evolution and theory related to sexual selection intensity. Overall, our review on sexual selection elucidates how selection acts at the level of reproduction that may lead to evolution of different types of traits. However, still we are far from resolving 123 Genetica (2014) 142:23–41 many issues so the next several decades should be at least as exciting as the recent past in the field of speciation research. Acknowledgments Financial assistance in the form of Meritorious Fellowship to AS and UGC-BSR Faculty Fellowship Award to BNS from the University Grant Commission, New Delhi is gratefully acknowledged. We also thank two anonymous reviewers for their helpful comments on the original draft of the manuscript. 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