Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
bs_bs_banner Austral Entomology (2014) 53, 1–17 Invited Review Sexual selection and genital evolution Leigh W Simmons* Centre for Evolutionary Biology, School of Animal Biology (M092), The University of Western Australia, Crawley, WA 6009, Australia. Abstract Male genitalia show patterns of divergent evolution, and sexual selection is recognised as being responsible for this taxonomically widespread phenomenon. Much of the empirical support for the sexual selection hypothesis comes from studies of insects. Here, I synthesise the literature on insect genital evolution, and use this synthesis to address the debate over the mechanisms of selection most likely to explain observed patterns of macroevolutionary divergence in genital morphology. Studies of seven insect orders provide evidence that non-intromittent genitalia are subject to sexual selection through their effects on mating success, while intromittent genitalia are subject to selection through their effects on fertilisation success. However, studies that use quantitative methods to analyse the form of selection are necessary to identify the mechanisms of sexual selection involved. Phylogenetic analyses from diverse taxonomic groups confirm that divergence in male genital morphology can be predicted from variation in the opportunity for sexual selection. Much debate revolves around the importance of female choice and sexual conflict in the evolution of male genitalia, the resolution of which lies in economic studies of mating interactions and in recognising sexual selection as a continuum between male competition, sexual conflict and female choice. The species isolating lock-and-key hypothesis is frequently dismissed as unimportant in genital evolution because in part of a perceived lack of variation in female genitalia across species. Increasingly, however, studies report species-specific variation in female genital morphology and its coevolutionary divergence with male genital morphology. Contemporary views recognise a continuum between female choice that enforces species isolation and female choice that targets variation in male quality within populations, placing lock-and-key processes into the realm of sexual selection. Distinguishing between species-isolating and directional forms of female choice will require studies that examine both the tempo and mode of divergence, both within and among species. Key words coevolution, female choice, genitalia, sexual conflict, sexual selection, species isolation. I NTRODUCT IO N Male genitalia are widely recognised as being the most variable and divergent of all morphological structures. Indeed, male genitalia are considered one of the most important diagnostic traits in insect systematics (Tuxen 1970), and there are entire groups whose classification is based solely on the structure of male genitalia (Eyer 1924; Dirsh 1956). Hypotheses for why genitalia should exhibit such divergent evolution date back to Dufour (1848) who proposed that genital morphology provides a prezygotic reproductive isolating mechanism whereby species-specific male genitalia represent a key to ‘unlock’ species-specific female genitalia. In contrast, Mayr (1963) argued that because genitalia are generally internal structures, they should be protected from natural selection and free to diverge at random because of *[email protected] © 2013 Australian Entomological Society pleiotropic effects of selection acting on general morphology. Waage’s (1979) pioneering work on damselfly genitalia revealed the remarkable dual function of male genitalia, whereby the male uses his intromittent secondary genitalia to remove rival sperm from the sperm stores of recently mated females before delivering his own ejaculate. This discovery raised the intriguing possibility that sexual selection might be a significant driving force behind the evolution of male genitalia (Lloyd 1979). In his influential syntheses of the literature, Eberhard (1985, 1996, 2010) has championed the hypothesis that sexual selection is responsible for both rapid and divergent evolution of male genital morphology. Moreover, he has argued that sexual selection through Fisherian female choice offers the most general explanation. Male genitalia are proposed to stimulate females during copulatory courtship, and females choose males cryptically by increasing offspring production or preferentially storing and using sperm from males better equipped to provide the appropriate stimulation. Eberhard (1985) doi:10.1111/aen.12053 2 L W Simmons rejected the pleiotropy hypothesis on the firm grounds that in many species, where males possess secondary structures involved in copulation, such as the secondary intromittent organs of dragonflies or the gonopods of millipedes, it is these secondary copulatory structures that exhibit patterns of rapid and divergent evolution, while the primary genitalia do not. Eberhard (1985) also argued that Dufour’s lock-and-key hypothesis cannot provide a general explanation for genital evolution because female genitalia are generally simple in their morphology, lacking any obvious structures that can act as ‘locks’. Whether the lock-and-key hypothesis can ever be rejected outright is debatable (Shapiro & Porter 1989). Indeed, sexual selection via female choice can be an important species-isolating mechanism (Ryan & Rand 1993; Higgie & Blows 2007; Ritchie 2007) so that lock-and-key and sexual selection arguments for genital evolution are not exclusive, a point to which I shall return later in this review. There are three principal mechanisms by which male genital morphology might be under sexual selection (Simmons 2001). As Eberhard (1985) first proposed, male genitalia may be subject to selection via female choice. They may also be involved in the avoidance of sperm competition through their role in the physical removal or displacement of rival sperm, and thus subject to sexual selection via malecontest competition. Furthermore, male and female interests over fertilisation events often may differ. Female interests may lie in the use of sperm acquired from males providing greater copulatory stimulation, while male interests lie in removing or displacing any rival sperm from the female’s reproductive tract. Divergent male and female interests can generate sexual conflict as males and females struggle in an evolutionary arms race to maximise their own fitness (Parker 1979; Arnqvist & Rowe 2005). Distinguishing between these mechanisms of sexual selection is not easy, and there has been considerable debate over which sexual selection force might be more important in genital evolution (Eberhard 2004a, 2006, 2010). Nonetheless, regardless of the precise mechanisms involved, sexual selection is now widely acknowledged as being a significant force driving the evolution of animal genitalia (Hosken & Stockley 2004), and much of the evidence for the sexual selection hypothesis comes from studies of insects. Here, I offer a synthesis of the literature on the evolution of insect genitalia and use this literature to address the debate over the mechanisms of selection that are most likely to explain observed patterns of macroevolutionary divergence in insect genitalia and animal genitalia more generally. Empirical studies have adopted two approaches with which to explore the role of sexual selection in genital evolution, within species studies of variation in male genital morphology and its effect on individual reproductive success, and comparative analyses of genital morphology across species. Arnqvist (1997) advocated a within-species approach that focused on exploring patterns of phenotypic and genetic variation in genital morphology and their effects on male fitness. Therefore, I start this review with an overview of my own research on a single species, the dung beetle © 2013 Australian Entomological Society Onthophagus taurus, which has proved a useful model system for studying the evolution of genital morphology. A broader review of the literature on single species studies follows, which has provided evidence that sexual selection can act on male genital morphology. Finally, I review evidence from comparative analyses that sexual selection acting on male genitalia can account for the broad patterns of macroevolutionary divergence we see in genital morphology. I conclude by discussing current controversies over which mechanisms most likely account for the evolutionary divergence of animal genitalia. ON T H OPH AGIN E DUN G BEET L ES : A MODEL S YS T EM F OR S T UDIES OF GEN ITAL EVOL UT ION Typical of insects generally, male genitalia have proven to be remarkably useful morphological traits for dung beetle classification (Tarasov & Solodovnikov 2011). The aedeagus consists of two segments, the phallobase and the articulating parameres (Fig. 1). The sample of aedeagi shown in Figure 1 illustrates that among species, there is considerable variation in the relative lengths of the aedeagal segments and in the shape of the parameres. Moreover, encased within the phallobase are two groups of sclerites associated with the endophallus, the lateral sclerites that form the lamella copulatrix and the accessory sclerites. While homologous sclerites can be found among onthophagines, the shape and complexity of these sclerites are highly divergent (Fig. 2) offering a strong phylogenetic signal for classification of the group. Indeed, Tarasov and Solodovnikov (2011) found that genital sclerites offered significantly fewer homoplasious (and therefore phylogenetically informative) traits than did nongenitalic morphological traits. Onthophagine dung beetles thus offer an excellent model system for exploring the evolutionary mechanisms of genital divergence. Functional morphology of male genitalia Any study of selection acting on morphological traits requires an understanding of the functional morphology of those traits (Fairbairn et al. 2003; Jagadeeshan & Singh 2006). During courtship, the male Onthophagus taurus mounts the female from behind and courts by drumming on her elytra and flanks with his fore legs while probing the ventral surface of her terminal segments with the extruded aedeagus. If receptive, the female will open her genital tract by raising her pygidium, allowing the male to anchor the distal hooks of the parameres into pits located on its interior surface. Once genital contact is established, the endophallus is everted into the female’s genital tract (Werner & Simmons 2008). The endophallus is held in situ by the fit of the paramere hooks into the female’s genital pits, the inflated endophallus horn into the female’s rectum and the grasping of the soft tissue of the bursa copulatrix by the lamella copulatrix (Fig. 3). The axial, subaxial and frontolateral sclerites form a functional unit that Sexual selection and genital evolution 3 Fig. 1. Male aedeagus morphology of Onthophagus sp. The aedeagus of O. avocetta is shown with its associated endophalic sclerites highlighted in different colours (from Tarasov & Solodovnikov 2011). Aedeagus outlines from Zunino (1979). AcS, accessory sclerites; AMS, additional medial sclerite of lamella copulatrix; Blp, basolateral paramerite; Br, bristle; En, endophallus; IL, inferior left lobe of lamella copulatrix; IR, inferior right lobe of lamella copulatrix; LC, Lamella copulatrix; PhFS, phallobase frontal side; PhRS, phallobase rear side; PIS, paramere inferior side; PSS, paramere superior side; SL, superior left lobe of lamella copulatrix; SR, superior right lobe of lamella copulatrix. delivers the ejaculate to the bursa copulatrix. The sperm and seminal fluid are contained within a tubular spermatophore, which is moulded by the axial, subaxial and frontolateral sclerites, with its opening positioned within the spermathecal duct (Fig. 3). The superior right peripheral sclerite does not appear to interact directly with the female being located inside the endophallus. However, it may impart structural support for the inflated endophallus and/or aid in maintaining the position of the endophallus during copulation (Werner & Simmons 2008). Selection on male genitalia Quantitative methods for measuring the strength and form of selection acting on morphological traits have been available for some time (Arnold & Wade 1984a, b), yet with some exceptions (Bertin & Fairbairn 2005), few studies of genital evolution have used this method to explore patterns of selection. Estimates of the strength and form of selection acting on the aedeagus of O. taurus via a male’s ability to engage the female in copula have been conducted, following a landmarkbased geometric morphometric approach to capture variation in aedeagus size and shape. Selection analyses revealed significant directional sexual selection acting on the shape but not the size of the aedeagus (Simmons et al. 2009; Simmons & Garcia-Gonzalez 2011). Specifically, males with long, thin parameres and a long phallobase are more successful in engaging the female in copula than males with short, stout aedeagus segments (Fig. 4a). Aedeagus morphology was found to have no significant impact on a male’s fertilisation success following copulation and thus appears to be subject to precopulatory sexual selection only (Fig. 4b). In contrast, variation in the morphology of the endophallus sclerites was found to have a significant effect on competitive fertilisation success (House & Simmons 2003). Specifically, House and Simmons (2003) found that males with a large frontolateral peripheral sclerite and a small lamella copulatrix tended to have greater defensive paternity success (first male to mate), while males with a large axial and small superior right peripheral sclerite tended to have greater offensive (second male to mate) paternity success. These data can tell us that individually, the genital sclerites can influence paternity outcome when females mate multiply. However, because of the morphological complexity of the genital sclerites and their functional integration, these data cannot © 2013 Australian Entomological Society 4 L W Simmons Fig. 2. Male genital sclerite morphology of Onthophagus sp. The accessory sclerites of the endophalus are shown in detail for: (a,b) O. taurus; (c,d) O. avocetta; (e,f) O. tricornis; and (g,h) O. mouhoti. Colours indicate homologous structures among species (from Tarasov & Solodovnikov 2011). A, axial sclerite; FLP, frontolateral peripheral sclerite; InL, internal lobe; ExL external lobe; LP, lateral process; MP, medial peripheral sclerite; SA, subaxial sclerites 1–3; SRP, superior right peripheral sclerite. inform us of the strength or form of sexual selection acting on the integrated functional unit. Fortunately, multivariate approaches are available to assess selection acting on complex suites of traits (Lande & Arnold 1983; Phillips & Arnold 1989). Thus, Simmons et al. (2009) estimated multivariate selection acting on the endophallus sclerites in both defensive and offensive contexts, revealing non-linear selection on four of five axes of variation from a canonical analysis of the matrix of non-linear selection gradients. Visualisation of the fitness surface in Figure 5 clearly shows that, collectively, the genital sclerites of the endophallus are subject to strong stabilising selection. The strength of stabilising selection was found to be greatest in the defensive context (Simmons et al. 2009). © 2013 Australian Entomological Society Evolutionary response to selection on male genitalia Unequivocal support for a role for sexual selection in the evolutionary divergence of male genital morphology requires evidence that the patterns of selection observed within generations can generate changes in genital morphology across generations. The technique of experimental evolution offers a powerful approach for testing evolutionary hypotheses (Kawecki et al. 2012) and has been applied recently to the hypothesis that sexual selection can drive the evolutionary divergence of male genital morphology using the O. taurus model system (Simmons et al. 2009; Simmons & Garcia-Gonzalez 2011). Sexual selection and genital evolution 5 Fig. 3. Schematic representation of the Onthophagus taurus endophallus fully engaged with female genital structures: (a) left view; (b) right view (from Werner & Simmons 2008). 1, superior right peripheral sclerite; 2/3, axial and subaxial sclerites; 4, lamella copulatrix; 5, frontolateral peripheral sclerite; bp, bristle plate; en, endophallus; ov, oviduct; pm, paramere of the aeadeagus; pyg, pygidium; rec, rectum; sd, spermathecal duct; sde, spermathecal duct extension; sp, spermatheca. a Probability of mating b P2 Aedeagus shape (PC2) Second male’s aedeagus shape (PC2) Fig. 4. Selection acting on the Onthophagus taurus aedeagus. (a) cubic spline (with 95% confidence bands) visualisation of directional selection gradient acting on aedeagus shape via a male’s ability to engage the female in copula (negative scores on the principal component axis describes a long thin aedeagus) (from Simmons et al. 2009). (b) There is no significant relationship between the shape of a male’s aedeagus and his offensive paternity success (P2) once copulation is achieved (standardised gradient = −0.03 ± 0.06, F1,25 = 0.42, P = 0.525; LW Simmons unpublished data). Simmons and colleagues established six experimental populations of beetles, randomly assigning three to breed polygamously and three to breed monogamously. In polygamous populations, male and female beetles were allowed to interact and mate freely before females produced their broods (Simmons et al. 2009). Thus, there was the opportunity for sexual selection to act on these populations, and indeed, molecular screening of putative parents and the offspring emerging from these populations confirmed that there was a significant positive Bateman gradient – a measure of the opportunity for sexual selection based on the relationship between number of mates and relative male fitness (Simmons et al. 2004; Simmons & García-González 2008). In monoga- mous populations, a single male and female were allowed to mate before the female produced her brood, so that there was no opportunity for sexual selection to operate. All other aspects of the populations’ life-histories were tightly controlled. Simmons and colleagues allowed their populations to evolve under these monogamous and polygamous mating regimes for 19 generations before quantifying variation in male genital morphology, finding significant divergence in the shape, but not size of the aedeagus (Fig. 6). The aedeagus evolved to be longer and thinner in populations subject to sexual selection, exactly the divergence we should expect given the directional selection acting on aedeagus shape within © 2013 Australian Entomological Society L W Simmons Aedeagus shape (PC2±SE) 6 Fig. 5. Stabilising selection acting on the endophallus sclerites of the Onthophagus taurus aedeagus. Relative fitness is the fertilisation success of males mating in the defensive role of firts to mate, standardised to the population average fertilisation success. The axes m1 and m5 are the strongest positive and negative eigenvectors from a canonical analysis of the matrix of standardised quadratic and correlational selection gradients. Axis m1 describes variation in the size and shape of the superior right peripheral sclerite and the subaxial sclerites, while axis m5 describes variation in the size and shape of the axial and frontolateral peripheral sclerites, and the lamella copulatrix (from Simmons et al. 2009). the source population (Fig. 4a). In contrast, no divergence was found in the morphology of the endophallus sclerites (Simmons et al. 2009). Again, this is to be expected from the pattern of stabilising selection acting on the genital sclerites (Fig. 5). Experimental evolution thus provides unequivocal evidence that sexual selection can generate a divergence in genital morphology among populations of the same species. But can divergence occur among natural populations of beetles and contribute to speciation? There is evidence to suggest that it can. O. taurus originally was limited to a Turanic-EuropeanMediterranean distribution but was introduced into the eastern states of North America and into Australia in the late 1960s and early 1970s. Analyses of native and exotic populations of O. taurus have revealed substantial divergence in aedeagus shape among these allopatric populations (Pizzo et al. 2008). Moreover, the same axes of shape variation distinguishes native O. taurus populations from their sympatric closely related sister species O. illyricus (Pizzo et al. 2008), and it is interesting to note that more broadly, differences in aedeagus morphology among Onthophagus sp. are characterised by variation in the relative length and girth of the aedeagus segments (Fig. 1). © 2013 Australian Entomological Society Enforced monogamy Sexual selection Fig. 6. Evolutionary divergence in the shape of the Onthophagus taurus aedeagus across three populations subjected to enforced monogamy and three populations subjected to sexual selection for 19 generations of experimental evolution. Positive scores on PC2 characterise an aedeagus that is long and thin, while negative scores characterise an aedeagus that is short and stout (from Simmons et al. 2009). Coevolution of female genitalia Most hypotheses for the evolutionary divergence in male genital morphology predict a coevolutionary divergence in female genital morphology or the sensory traits that directly recognise and respond to stimulation generated by male genitalia. Thus, the lock-and-key hypothesis would predict that changes in female genital morphology should be closely tracked by changes in male genital morphology. A classic theoretical prediction underlying models of female choice is that the female preference – the morphological, behavioural or sensory mechanism by which females choose among males – should coevolve with the preferred traits in males (Andersson 1994). Likewise, correlated evolution of male and female traits is expected from the cycles of antagonistic coevolution that characterise sexual conflict (Arnqvist & Rowe 2005). The parameres of the O. taurus aedeagus and the genital pits on the internal surface of the female’s pygidium are mechanically coupled during copulation (Fig. 3), and this mechanical fit might be expected to affect their mutual evolution. Simmons and Garcia-Gonzalez (2011) quantified variation in the morphology of the female genital pits among their experimentally evolving populations. They found that in populations subject to sexual selection, the genital pits became smaller and their Sexual selection and genital evolution 7 Fig. 7. Evolutionary divergence in the configuration of the pygidial genital pits of Onthophagus taurus among three populations subjected to enforced monogamy and three populations subjected to sexual selection for 19 generations of experimental evolution. The thin plate splines come from a geometric morphometric analysis of landmarks placed around the edge of the pygidium and the genital pits, and show how positive scores on the relative warp axis describe pits that are reduced in size and more moved away from the pygidium edge (for more details, see Simmons & Garcia-Gonzalez 2011). position on the internal wall of the pygidium moved further away from the pygidium edge, while the reverse was true for populations subject to enforced monogamy (Fig. 7). Thus, sexual selection appears to favour both the internalisation of the genital pits in females, which would make them more difficult for males to reach, and the elongation of the aedeagus in males that we know makes it easier for them to reach the genital pits and engage the female in copulation (Fig. 4a). Quantitative genetic analyses of male and female genital configuration have also revealed significant genetic correlations between male and female genital morphology, a genetic architecture that is expected of traits undergoing concerted evolution (Simmons & Garcia-Gonzalez 2011). Finally, studies of allopatric populations of O. taurus confirm that correlated divergence in male and female genital morphology occurs among natural populations, and studies of Onthophagus sp. in the O. fracticornis-similis-opacicollis species complex have confirmed that concerted evolution of male and female genital traits characterises speciation in this complex (Macagno et al. 2011). GE NITAL MO RPHO LO G Y G E NE RAL LY AFFE CTS MALE FIT NE SS There is now evidence from seven orders of insect that variation in male genital morphology can be a significant predictor of male reproductive success and therefore be the focus of sexual selection (Table 1). The earliest evidence that variation among males in their genital morphology might predict male reproductive success came from experimental studies of tortoise beetles Chelymorpha alternans (Rodriguez 1995; Rodriguez et al. 2004). The males of Chelymorpha sp. possess an elongated genital flagellum that is threaded through the length of the female’s spermathecal duct to the entry of the spermatheca where sperm are stored. The male then deposits a spermatophore in the bursa copulatrix and sperm migrate from the spermatophore along the length of the genital flagellum to the spermatheca. Rodriguez and colleagues shortened the flagellum of some males experimentally, finding that this manipulation resulted in a reduction in the number of sperm stored, an increase in the number of sperm dumped by the female following copulation, and a decrease in the proportion of offspring sired (Rodriguez 1995; Rodriguez et al. 2004). Experimental manipulations of the size or length of genital traits, either by their partial of complete ablation, is a frequently used method for assessing the impact of male genital traits on mating and/or insemination success (Table 1). Such approaches, however, tell us little about how selection acts on genital morphology. By analogy, although removing the legs of a long distance runner would show us that legs are required to run, it would tell us little about how stride length contributes to running speed. Nonetheless, such approaches can sometimes be useful in identifying the functional mechanisms by which male genitalia might impact fitness. In Callosobruchus maculatus, for example, the ablation of genital spines (or their shortening via artificial selection) has the effect of reducing the ability of males to perforate the female’s internal genital tract walls, a process that is necessary for seminal fluid proteins to pass into the female’s circulatory system where they act on her reproductive physiology in such a way as to increase the copulating male’s paternity (Hotzy et al. 2012). In contrast, ablating genital spines has no impact on a male’s ability to prolong copulation or his ability to transfer ejaculate to the female (Hotzy et al. 2012). Arguably, the best evidence of the potential for selection to act on variation in male genital morphology comes from studies that have examined how natural variation in male genital size or shape affects male reproductive success. A number of correlational studies have shown us that variation in male genital morphology can influence either male mating success, the duration of copulation, the number of sperm transferred to the female’s sperm storage organs or paternity success (Table 1). In the Australian mantid genus © 2013 Australian Entomological Society © 2013 Australian Entomological Society Mating success Mating success Paternity Copula duration & mating speed Paternity Sperm transfer Sperm transfer and paternity Mating success Paternity Aedeagal spicule Genital sclerite length Genital spines Copulatory piece length Genital flagellum length Aedeagus shape Genital sclerites Genital clasper asymmetry Aedeagus length Mating success Copula duration Paternity Paternity Sperm transfer Mating success Genital length Genital width Genital sclerites Genital sclerites Processus gonopori length Genital claspers Paternity Mating success Mating success Mating success Sperm storage, female remating Sperm storage, ovulation, female remating Copula duration & paternity Length of virgae Genital clasper length & asymmetry Genital spine length Genital spine length Clasper Median cercal hooks Lateral cercal teeth Sperm transfer Dorsal left complex & right phalomere Correlational Correlational Correlational Ablation Ablation Correlational Ablation Ablation Correlational Correlational Ablation & selection Ablation Ablation Correlational Correlational Correlational Correlational Correlational Correlational Correlational Ablation Correlational Correlational Ablation Correlational Correlational Evidence Koshio et al. (2007) Xu and Wang (2010) Otronen (1998) Grieshop and Polak (2012) Kamimura and Polak (2011); Polak and Rashed (2010) Blanckenhorn et al. (2004) Briceño and Eberhard (2009b) Briceño and Eberhard (2009a) Wenninger and Averill (2006) Sakurai et al. (2012) Hotzy et al. (2012) Takami (2003) Rodriguez (1995); Rodriguez et al. (2004) Simmons et al. (2009) House and Simmons (2003); Simmons et al. (2009) Bertin and Fairbairn (2005) Gagnon and Turgeon (2011) Danielsson and Askenmo (1999) Arnqvist and Danielsson (1999) Tadler (1999); Tadler and Nemeschkal (1999) Moreno-Garcia and Cordero (2008) van Lieshout (2011); van Lieshout and Elgar (2011) Holwell et al. (2010) Tsuchiya and Hayashi (2008) Córdoba-Aguilar (1999, 2002) Córdoba-Aguilar (2009) Source Studies are categorised as ‘ablation’ if they involved the surgical reduction or removal of male genital features, or ‘correlational’ if they examined the effect of natural variation in measures of male genital traits. Diptera Dryomyza anilis Drosophila ananassae Drosophila bipectinata Sepsis cynipsea Glossina marsitans Glossina pallidipes Lepidoptera Elcysma westwoodii Ephestia kuehniella Sperm displacement Sperm displacement Sperm displacement Affect Spiny lateral horns of adeagus Aedeagus width Aedeagus width Trait Studies on the fitness consequences of variation in male genital morphology for male mating and fertilisation success Odonata Calopteryx cornelia Calopteryx haemorrhoidalis Hetaerina americana Mantodea Ciulfina klassi Dermaptera Euborellia brunneri Hemiptera Aquarius remigis Gerris gillettei Gerris lacustris Gerris lateralis Lygaeus simulans Stenomacra marginella Coleoptera Anomala orientalis Callosobruchus chinensis Callosobruchus maculatus Carabus insulicola Chelymorpha alternans Onthophagus taurus Species Table 1 8 L W Simmons Sexual selection and genital evolution Ciulfina, for example, the ventral left complex and the right phallomere of the male’s genitalia are involved both in physically opening the genital tract of the female and in the insertion of the spermatophore (Holwell & Herberstein 2010). In Ciulfina klassi, quantitative variation in the shapes of these structures are correlated with the number of sperm transferred by the male (Holwell et al. 2010). The studies in Table 1 also show us that, as seen in dung beetles, different genital structures can influence different stages of reproduction. Thus, in Gerris water striders, the genitalia consist of both nonintromittent and intromittent structures. The length of the nonintromittent structure is under sexual selection in both natural and experimental populations, in which males with a longer pygophore and proctiger have a greater mating success (Preziosi & Fairbairn 2000; Fairbairn et al. 2003; Bertin & Fairbairn 2005, 2007). The intromittent phalotheca has three genital sclerites – the ventral, lateral and dorsal sclerites – the shape of which influence a male’s competitive paternity success when females have mated with more than one male (Arnqvist & Danielsson 1999; Danielsson & Askenmo 1999). Some studies identified in Table 1 can inform us of the functional morphology of male genitalia. For example for the damselfly Calopteryx haemorrhoidalis, Córdoba-Aguilar (1999, 2002) showed how aedeagus width influenced the number of rival sperm that were ejected from the female’s sperm storage organs and ultimately removed from her reproductive tract at the end of copulation. The male’s aedeagus stimulates the female’s sensory system that controls egg fertilisation and laying. Stimulation of the reproductive tracts of target species basal in the phylogeny to Calopteryx haemorrhoidalis with the Calopteryx haemorrhoidalis aedeagus likewise induced sperm ejection, even though conspecific aedeagi were unable to do so. This example strongly suggests that the Calopteryx haemorrhoidalis aedeagus has evolved to exploit the sensory system of females (Córdoba-Aguilar 2002) and that the selective mechanism involves the engagement in sperm competition by males (removal of rival sperm), the action of female choice (sensory perception and expulsion of sperm by females) and the potential for sexual conflict (Córdoba-Aguilar 2005). Nonetheless, it is not clear if selection on aedeagus width in Calopteryx haemorrhoidalis is directional for increasing aedeagus width or stabilising for some optimal species-specific width. Thus, while there is now good evidence that male genital morphology can influence male reproductive fitness, it is also clear that different genital traits can be subject to different episodes of sexual selection, either precopulatory or postcopulatory. Importantly, few studies in Table 1 have characterised the form of sexual selection acting on genital morphology. This is unfortunate because without an understanding of the form of selection, we are unable to distinguish between putative mechanisms underlying genital evolution. If we are to move forward, future studies will need to adopt the quantitative framework of Lande and Arnold (1983) and Arnold and Wade (1984b) for characterising the relative strengths of linear and non-linear sexual selection acting on male and female genitalia. 9 COMPARAT IV E AN ALYS ES OF GEN ITAL EVOL UT ION AMON G S PECIES Divergence in male genital morphology If sexual selection is generally responsible for the evolution of male genital morphology, we should expect to see macroevolutionary patterns of divergence in genital shape and size that depend on the opportunity for and strength of sexual selection. Thus, in monogamous species, where females mate with only a single male, the opportunity for sexual selection to act on genital morphology during and after copulation is limited, so we should expect monandrous taxa to exhibit far less evolutionary divergence in genital morphology than polyandrous taxa. This expectation seems to be upheld by studies of Heliconius butterflies (Eberhard 1985), apoid bees (Roig-Alsina 1993) and ischnuran damselflies (Robinson & Novak 1997; Simmons 2001). Of course, monandrous species can diverge in genital morphology, as for example in the bumblebee genus Bombus (Richards 1927b). Male contest competition for females can be particularly intense in monandrous species, where males struggle to gain and maintain genital contact with females at emergence or feeding sites, or in mating swarms (Thornhill & Alcock 1983). Sexual selection is likely to act strongly on male genital traits that facilitate coupling and/or decrease the probability of take-overs by rival males prior to insemination, and sexual selection can favour the evolution of complex genital traits that allow males to monopolise access to monandrous females (Richards 1927a; Thornhill & Alcock 1983; Simmons 2001). Nonetheless, we see in Table 1 that some genital traits are subject to sexual selection via mating success, while others are subject to postcopulatory sexual selection. Thus, we should expect intromittent genitalia to show particularly strong patterns of divergence because of mating system differences because it is only in polyandrous taxa that postcopulatory sexual selection also will operate. Indeed, among species in the grasshopper genus Melanoplus, non-intromittent genitalia exhibit significantly less divergence than do intromittent genitalia (Márquez & Knowles 2007). Arnqvist (1998) provided strong evidence for the role of sexual selection acting on genital divergence in his phylogenetically controlled comparative analysis of insects from four orders: Ephemeroptera, Diptera, Lepidoptera and Coleoptera. Arnqvist (1998) calculated a morphometric distance, as the mean distance in multivariate trait space for a species from the generic mean genital configuration. He then calculated a distance ratio, as the mean distance for polyandrous genera divided by the mean distance for monandrous genera for 19 phylogenetically independent clades. On average, polyandrous clades were twice as divergent in genital morphology than monandrous clades, as might be expected given the greater opportunity for sexual selection in polyandrous species. In contrast, non-genital morphological traits did not differ in their degree of divergence among monandrous and polyandrous clades. This study provides strong evidence that sexual selection is responsible for the © 2013 Australian Entomological Society 10 L W Simmons evolutionary divergence in male genitalia generally. Patterns of genital divergence among closely related species have also been linked to more fine-grained variation in mating system. In their comparative analysis of ground beetles belonging to the subgenus Ohomopterus (genus Carabus), Takami and Soto (2007) documented significant positive correlated evolution between the length of the male copulatory piece and spermatophore size, and significant negative correlated evolution between copulatory piece length and copulation duration. Within species the length of the copulatory piece determines the speed of copulation, the correct placement of the spermatophore within the female reproductive tract and ultimately insemination success (Takami 2003). Likewise, male genital titillators exhibit correlated evolution with copulation duration across species of bushcrickets (Tettigoniidae) (Vahed et al. 2011). Species with male genital titillators have longer periods of genital coupling prior to spermatophore transfer than species without titillators, and titillator complexity influences the relationship between copulation duration and spermatophore size. Such patterns are difficult to interpret without knowledge of the functional morphology of genital traits, but they do show us that the evolutionary divergence in male genital morphology can be tightly linked with the evolution of mating behaviour. Comparative studies across species of water striders, where we have a detailed understanding of the functional morphology of genital traits and selection acting on those traits, have offered far greater insight. Rowe and Arnqvist (2012) conducted a comparative analysis of the shape and complexity of both non-intromittent and intromittent male genitalia across 15 water strider species from the genus Gerris. Their analysis yielded four important findings. First, genital shape and complexity exhibited a greater evolutionary divergence than general morphological traits, and intromittent genital traits exhibited greater divergence than non-intromittent traits. Second, measures of complexity generally exhibited a strong phylogenetic signal, while measures of shape did not, suggesting that shape may be evolving more rapidly than complexity. Third, the divergence in intromittent and non-intromittent genital traits was uncorrelated, suggesting that these different genital traits are subject to different selection pressures. Indeed, Rowe and Arnqvist’s analysis strongly supported findings from the single-species studies of waterstriders highlighted in Table 1, that intromittent genitalia are subject to postcopulatory sexual selection, while non-intromittent genitalia are subject to precopulatory sexual selection. Both groups of genital traits exhibited correlated evolution with female mating rate, indicating that stronger sexual selection generated greater divergence in intromittent and nonintromittent genitalia. However, there was positive correlated evolution between non-intromittent genitalia and sexual size dimorphism, a proxy for the intensity of precopulatory sexual selection (Arnqvist & Rowe 2002) but no correlated evolution between non-intromittent genitalia and the sizes of male reproductive accessory glands and testes, a widely used proxy for © 2013 Australian Entomological Society the level of postcopulatory sexual selection (Simmons & Fitzpatrick 2012). This pattern makes sense, given what we know about selection acting on non-intromittent genitalia during premating struggles in this genus (Preziosi & Fairbairn 2000; Fairbairn et al. 2003; Bertin & Fairbairn 2005, 2007). The reverse pattern was true for intromittent genital traits; there was no correlated evolution between the shape of intromittent genital traits and sexual size dimorphism, but significant positive correlated evolution between the shape of intromittent genitalia and male reproductive accessory gland size. Again, this pattern is consistent with what we know about the influence of genital sclerites on male fertilisation success in this genus (Arnqvist & Danielsson 1999; Danielsson & Askenmo 1999). These macroevolutionary patterns among water striders, together with the differences in selection pressures on intromittent and non-intromittent genital traits found in single-species studies of dung beetles and water striders, support the general expectation that different genital traits can be subject to very different sexual selection pressures (Simmons et al. 2009). Coevolutionary divergence in female genital morphology Eberhard (1985, 2010) has argued that the lock-and-key mechanism cannot provide a general explanation for the divergent evolution of male genitalia because females do not possess species-specific genitalia that could constitute the required ‘lock’. However, this claim may be more apparent than real. There is an extraordinary amount of data on male genital morphology that has been derived from the taxonomic literature (Eberhard 1985, 2004a,b). In contrast, female genital structures have been inadequately studied by taxonomists perhaps because they are frequently internal and require destructive sampling. Not surprisingly, studies that have relied largely on the taxonomic literature have failed to find evidence for widespread coevolutionary divergence of male and female genitalia (Eberhard 2004b, 2006). However, there is now a growing body of evidence to suggest that females do possess species-specific genital traits and that female genitalia can exhibit patterns of divergent evolution that match those seen in male genitalia. In the scarab beetle Phyllophoga hirticula, female genitalia are more complex than male genitalia (Polihronakis 2006) and show greater divergence among populations (Polihronakis 2009). Among sepsid flies the internal female genitalia appear to be evolving faster than either molecular or behavioural characters (Puniamoorthy et al. 2010), and both male and female genital traits contribute significant phylogenetic signals for the classification of the hemipteran family Lophopidae (Soulier-Perkins 2001). In Lepidoptera, signa (sclerotised teeth within the female reproductive tract) are associated with polyandrous mating systems, and the evolution of monandry is associated with an evolutionary loss of the signa (Sánchez et al. 2011). Importantly, there is growing evidence for the widespread coevolutionary divergence of male and female genital morphology (Table 2). Sexual selection and genital evolution 11 Table 2 Studies of coevolution between male and female genital morphology Taxon Odonata Calopteryx spp. Enallagma spp. Hemiptera Gerris gillettei Gerris incognitus Gerris spp. Coridromius spp. Coleoptera Callosobruchus spp. Male trait Female trait Method Source Aedeagus width Terminal abdominal cerci Sensillum number Mesostigmal plates Species comparison Phylogenetic analysis Córdoba-Aguilar (2005) McPeek et al. (2009) Non-intromittent genital length Non-intromittent genital size and shape Non-intromittent genital shape Intromittent genital complexity Connexival spines Connexival spine size and shape Connexival spine shape Paragenitalia complexity Population comparison Population comparison Gagnon and Turgeon (2011) Perry and Rowe (2012) Phylogenetic analysis Phylogenetic analysis Arnqvist and Rowe (2002) Tatarnic and Cassis (2010) Genital spines Phylogenetic analysis Rönn et al. (2007) Experimental evolution Onthophagus taurus Aedeagus shape Copulatory duct connective tissue Genital pits Onthophagus taurus Onthophagus spp. Diptera Drosophila spp. Drosophila spp. Aedeagus shape Aedeagus shape Genital pits Genital pits Population comparison Species comparison Simmons and Garcia-Gonzalez (2011) Macagno et al. (2011) Macagno et al. (2011) Basal processes Epandrial posterior lobe Phallic spike Phallic hook Phallic spines Cercal teeth Aedeagal filament length Genital cavities Oviscapt pouch Oviscapt furrow Vulval shield Uterine shield Ventral vaginal shield Spermathecal duct length Species comparison Phylogenetic analysis Kamimura (2012) Yassin and Orgogozo (2013) Species comparison Ilango and Lane (2000) Phlebotomine flies Comparative studies are categorised based on whether they involved appropriate phylogenetic control for common ancestry among species (phylogenetic analysis) or not (species comparisons). Within species, studies are categorised based on whether they involved population comparisons or laboratory experimental evolution. Single species studies of the dung beetle O. taurus revealed how internal female genital structures that interact with the male’s aedeagus can diverge in response to experimental manipulation of sexual selection pressures (Fig. 7) (Simmons & Garcia-Gonzalez 2011). A recent detailed examination of the female reproductive tracts of nine species in the Drosophila melanogaster subgroup discovered five female genital traits that are species-specific in form and that exhibit significant coevolution with distinct male genital features (Yassin & Orgogozo 2013). Likewise, in Gerris water striders, female abdominal spines exhibit significant coevolutionary divergence with male non-intromittent genitalia involved in grasping females during copulatory attempts (Arnqvist & Rowe 2002). Patterns of coevolutionary divergence are seen both among species and among populations within species (Table 2), and they are not limited to insects (Brennan et al. 2007; Kuntner et al. 2009; Evans et al. 2011; Burns et al. 2013). The coevolution of male and female genitalia can be facilitated if they share a common genetic architecture, as seems to be the case for dung beetles (Simmons & Garcia-Gonzalez 2011) and ground beetles (Sasabe et al. 2010). Clearly, female genital morphology is an important yet neglected aspect of studies that aim to determine the causes of divergent evolution in male genital morphology. Greater efforts need to be made in characterising female genital structures, exploring how female genitalia interact with male genitalia during copulation, and in determining patterns of phenotypic and genotypic variation in female genitalia and their covariation with male genitalia. DIS T IN GUIS H IN G BET W EEN MECH AN IS MS OF S EL ECT ION Early studies of genital evolution used patterns of allometric scaling to infer the form of selection acting on male genital morphology. Directional sexual selection is thought to generate positive allometric scaling of traits under selection, a pattern often associated with male secondary sexual traits (Petrie 1988; Wilkinson 1993; Simmons & Tomkins 1996; Wilkinson & Taper 1999). In contrast, stabilising selection favours intermediate values of a trait irrespective of body size, which will reduce trait size variation and generate negative allometry (Eberhard et al. 1998). Indeed, strong stabilising selection is expected to generate patterns of canalised growth, in which traits are protected from environmental perturbations that affect adult body size (House & Simmons 2007). In addition to their divergent evolution, a second striking pattern is that insect genitalia generally exhibit negative allometric scaling (Eberhard et al. 1998; Hosken et al. 2005; Eberhard 2009), suggesting that genital size is subject to widespread stabilising selection: Eberhard et al.’s (1998) ‘one-sizefits-all’ hypothesis. Single species studies have found that male genitalia exhibit canalised patterns of growth (Dreyer & Shingleton 2011). In the dung beetle O. taurus for example, sexually selected head horns showed plasticity in size and allometry in response to variation in nutritional environment, but genital traits were highly invariant (House & Simmons 2007). In the hemipteran treehopper Enchenopa binotata, genotype-by-environment interactions were found to be © 2013 Australian Entomological Society 12 L W Simmons strongest in sexual signals and weakest in genital traits, which were again highly canalised (Rodríguez & Al-Wathiqui 2011, 2012). Theoretically, these general patterns could arise from widespread stabilising selection acting on male genitalia. However, inferring mechanisms of selection from patterns of allometric variation is problematic (Bonduriansky & Day 2003; Bonduriansky 2007). Studies of water striders, Aquarius remigis, show that nonintromittent genitalia exhibit negative allometry and low levels of variation despite strong and persistent directional sexual selection (Bertin & Fairbairn 2007). Experimental evolution studies offer conflicting results. Experimental manipulation of sexual selection in dung beetles, O. taurus, did not generate divergence in the size of either non-intromittent or intromittent genital traits, or in their allometric scaling (Simmons et al. 2009). A similar study of seed beetles, Callosobruchus maculatus, did find an evolutionary reduction in the allometric scaling of genital spines in populations from which sexual selection had been experimentally removed via enforced monogamy (Cayetano et al. 2011). While this result suggests that sexual selection maintained elevated allometric scaling, the form of selection acting on genital spines remains unclear. Collectively, these studies show that patterns of allometric scaling alone can be unreliable for inferring the mechanisms of selection acting on genital traits. Much effort has been spent in attempting to distinguish between mechanisms of sexual selection. Eberhard’s (1985) original hypothesis was based on traditional Fisherian female choice, whereby females favour males with certain genitalic forms because they are more ‘attractive’ during copulatory courtship, and by favouring such males, females will produce more attractive sons and daughters with the preference for genital stimulation. The hypothesis has its foundation on indirect benefits of female choice. Sexual conflict (Parker 1979) has become widely recognised as an important mechanism of sexual selection acting on male genitalia (Arnqvist & Rowe 2005). Male traits, evolved in the context of sperm competition or female genital stimulation, often can be damaging to females. For example, the intromittent genitalia of sepsid flies and seed beetles can rupture the internal reproductive tract of the female, leading to a reduction in female lifespan (Crudgington & Siva-Jothy 2000; Blanckenhorn et al. 2002). Such mating costs can favour counter adaptations in females, such as the thickening of the female reproductive tract wall to prevent genital damage in seed beetles (Rönn et al. 2007). Under the right conditions, male and female counteradaptations can generate sustained cycles of sexually antagonistic coevolution between male and female genital morphology (Parker 1979). The empirical evidence presented in this review of insect genital evolution is consistent with most models of sexual selection; female choice, sperm competition and sexual conflict models all predict that variation in genital morphology should influence paternity, that multiple mating by females – and thus the opportunity for these mechanisms of sexual selection to act – should be positively correlated with the degree of genital divergence, and that there should be a correlated evo© 2013 Australian Entomological Society lution of male and female genital morphology. None of these mechanisms of sexual selection are mutually exclusive (Simmons 2001); for example, female choice represents a key component of sexual conflict (Arnqvist & Rowe 2005). Nonetheless, Eberhard suggests that the current evidence for sexual conflict being generally important for genital evolution is relatively weak. He based this argument on three qualitative surveys of insect genital morphology. First, when classifying insect species as either protected (species where females control mating activity) or unprotected (species where males control mating), he found that there was no difference in the proportion of species with species-specific male genitalia (Eberhard 2004a). This is not a strong test because female choice in ‘protected’ species could favour genital divergence of equal magnitude to sexual conflict in ‘unprotected’ species. Second, he found little evidence of an evolutionary divergence of female traits that might interact with male traits (Eberhard 2004b). However, as noted earlier, detailed studies of the functional morphology of female genital traits are rare. Those that have recently appeared are revealing considerable variation in female genital traits and their coevolution with male genital traits. Even among species of insects in which sexual conflict is likely to be strong, Eberhard (2006) found male genital morphology was only weakly divergent and genital traits tended to be less complex than found among insects generally. Largescale surveys such as these are certainly useful in revealing pattern, but they can provide very little evidence of process. A key distinction between classic female choice and sexual conflict mechanisms lies in the costs and benefits to females from their interactions with males, and as such economic studies are required to distinguish between them (Fricke et al. 2009). The importance of economic studies can be illustrated with two examples. Single species studies of the dung beetle O. taurus have shown us how male and female genital traits exhibit a coevolutionary divergence under sexual selection that could be due to sexual conflict or female choice alone (Simmons & Garcia-Gonzalez 2011). Under sexual conflict, genital pits are expected to evolve in such a way as to minimise male mating ability because of the costs of multiple mating for females, while the males’ aedeagus evolves to more effectively engage females in costly copulation. In contrast, a process of female choice alone predicts that by choosing among males, females mate only with the best males that can convey their mating ability (sexy sons) and other potential genetic qualities to their offspring, providing females with a net fitness gain. Two lines of evidence suggest that for these beetles, female choice is the most likely mechanism of sexual selection. First, among experimentally evolving populations, sexual selection improved offspring condition (Simmons & García-González 2008). Second, and most importantly, females exposed to attractive males mated more frequently, and both mating frequency per se and mating with attractive males in particular increased the viability of offspring produced at no apparent cost to female fecundity or longevity (Simmons & Holley 2011). These data show that repeated Sexual selection and genital evolution mating interactions are beneficial for females and that sexual selection acting on male and female genitalia is most likely to reflect female choice. In contrast, work on water striders shows that mating is costly for females (Rowe 1994; Arnqvist & Rowe 1995) and that the coevolution of male grasping genitalia and female antigrasping spines is most likely the consequence of antagonistic coevolution (Rowe & Arnqvist 2012). As noted repeatedly, it is highly unlikely that different mechanisms of sexual selection will operate in isolation. Rather, dung beetles and water striders are likely to represent cases that lie at either extreme of a pure female choice-sexual conflict spectrum, making attempts to identify one general mechanism of sexual selection acting on insect genitalia biologically unrealistic. Arguably, the same is true of debates over the importance, or lack thereof, for species isolating lock-and-key mechanisms in genital evolution. Eberhard (1985, 2010) dismisses lockand-key on the grounds that tight morphological fits between male and female structures are not a general feature of insect genitalia. Nonetheless, Eberhard et al.’s (1998) one-size-fitsall hypothesis arguably is congruent with a species isolation mechanism in which stabilising selection favours a speciesspecific male genital size to fit the species-specific size of the female genital tract. Moreover, tight morphological fits need not characterise a female’s lock, which could be sensory in nature (Patterson & Thaeler 1982; Eberhard 1985) or could involve only those parts of the female reproductive tract that actually contact male genital traits. Cryptic traits within the internal genital tract of females are being found increasingly, and these traits are highly divergent and coevolving with male genital traits (e.g. Yassin & Orgogozo 2013). Eberhard (2010) also cites a lack of character displacement among males in zones of sympatry of closely related species as evidence against a general role of species isolation mechanisms in genital evolution. Nonetheless, evidence of this type is mixed. In stag beetles (Kawano 2003) and rhinoceros beetles (Kawano 2002), there is greater genital character displacement in sympatry than allopatry. Among Carabus ground beetles, variation in the size of the copulatory piece imposes mechanical isolation between species (Sota & Kubota 1998). However, Nagata et al. (2009) suggest that character displacement is unlikely in these species as there is little indication of secondary contact between divergent forms of Carabus arrowianus or evidence for greater divergence between populations close to the species boundaries. Similarly, in Ciulfina mantids, geographic variation in male genital morphology is unrelated to the location of populations relative to species contact zones (Holwell 2008). In contrast, Bath et al. (2012) found evidence that genital morphology imposed asymmetric reproductive isolation between the Australian neriid flies Telostylinus lineolatus and Telostylinus angusticollis, while Richmond et al. (2012) report less genital variation within species than among species of the diverging Drosophila mojavensis species cluster. Clearly, some species or species groups show evidence of genitalia being involved in reproductive isolation while others may not. As Eberhard (2010) concedes, the possibility that a 13 lock-and-key mechanism applies in some cases cannot be discarded without careful study. Contemporary views recognise a continuum between female choice that enforces species isolation and female choice that targets variation in male quality within populations (Endler 1989; Ryan & Rand 1993; Endler & Houde 1995; Castellano & Cermelli 2006; Higgie & Blows 2007). That is, rather than being an alternative to sexual selection, a species isolating lock-and-key mechanism should be viewed as one end of the continuum of female choice. The question is how to identify these modes of female choice and their relative strengths. Patterns of stabilising selection acting on genitalia and coevolutionary divergence between male and female genital traits are both expected from a mechanism of selection driven by species isolation. Stabilising female choice is expected to result in very slow evolutionary divergence between speciation events and a punctuated pattern of evolutionary change. In contrast, directional female choice should generate rapid and continuous patterns of divergence among species (McPeek et al. 2008). We often see claims in the literature that male genital morphology is undergoing both rapid and divergent evolution, but what evidence is there that genital evolution has been rapid? The answer is very little. Few studies have actually quantified both the tempo and mode of genital evolution. In their study of Enallagma damselflies, McPeek et al. (2008) contrasted the divergence in secondary male genital structures – the terminal cerci used to grasp the female during copula – to models of Brownian motion and punctuated equilibrium, finding that the cerci were diverging following a model of punctuated equilibrium. Correlated divergence in female traits grasped by males – mesostigmal plates – showed similar patterns of punctuated evolution (McPeek et al. 2009), supporting a role of speciesisolating female choice in this group of damselflies. In contrast, a similar approach using Timema stick insects supported a model of continuous evolutionary divergence and directional sexual selection (Arbuthnott et al. 2010). Approaches such as these offer great potential for distinguishing between putative mechanisms of sexual selection. But what options are there for estimating the rates of divergence in single species studies? Techniques are available for estimating the rates of evolutionary divergence in morphological traits among isolated populations of single species, but these have not yet been widely adopted in studies of genital evolution. Contrasts between genetic divergence at neutral loci – FST – and divergence in quantitative traits – QST – can provide an indication of the rates of divergence in morphological traits (Brommer 2011; but see Merilä & Crnokrak 2001; McKay & Latta 2002; Whitlock 2008; Edelaar et al. 2011; Lamy et al. 2012). When FST equals QST, random genetic drift is sufficient to explain morphological divergence. When QST exceeds FST, morphological traits can be inferred to be diverging more rapidly than would be expected from neutral divergence and thus subject to directional selection, and when FST exceeds QST, morphological traits are diverging more slowly than would be expected from neutral genetic divergence and as would be expected from strong stabilising selection. © 2013 Australian Entomological Society 14 L W Simmons Only one study of genital evolution has adopted this approach so far, and that was a study of Australian millipedes Antichiropus variabilis (Wojcieszek & Simmons 2012). In these millipedes, the males secondary genitalia – the gonopods – afford a tight fit with the genital tract of the female (Wojcieszek et al. 2012), and they are under stabilising selection within populations (Wojcieszek & Simmons 2012). Among isolated populations of these millipedes, male genitalia have diverged more slowly than would be expected from neutral divergence (Wojcieszek & Simmons 2012). These patterns suggest strongly that the male genitalia are under species-isolating female choice. Indeed, in experimental crosses between populations, male genitalia contribute to a mechanical reproductive isolation (Wojcieszek & Simmons 2013). Traits that evolve rapidly are expected to provide a lower phylogenetic signal than traits that evolve slowly (Losos 1999). Thus, across 41 cladistic analyses of insects, Song and Bucheli (2010) found that genital traits were just as phylogenetically informative as non-genital traits, suggesting that genital traits might not be subject to a particularly rapid divergence. Does this mean that species-isolating female choice is of greater general importance in insect genital evolution? The answer is probably no. Rowe and Arnqvist (2012) show that the strength of phylogenetic signal may depend on how genital traits are characterised. Thus, among Gerris water striders, they found that genital complexity – measured as the presence/absence of substructures typical of cladistic analyses – showed a strong phylogenetic signal, while quantitative variation in the shape of genital structures did not (Rowe & Arnqvist 2012). This suggests that different genital features may be subject to different rates of evolutionary divergence and are perhaps subject to different forms of female choice. CONCLUDIN G RE MARKS Considerable evidence now exists that sexual selection is responsible for a general and widespread divergence in male genital morphology. Nonetheless, as a recent experimental evolution study of Drosophila simulans illustrates, it is important to recognise that natural selection will also contribute to the evolution of genitalia and sometimes oppose sexual selection (House et al. 2013). Research attention now focuses more on distinguishing between the mechanisms of sexual selection involved rather than whether sexual selection is important. Sexual selection should be viewed as a continuum between male contest competition, sexual conflict and pure female choice, and it is unrealistic to expect that one particular mechanism of sexual selection will prove to be generally more important than another. Moreover, it is time to recognise that female choice often acts as a species-isolating lock-and-key mechanism in addition to imposing directional selection on male genitalia within populations. Different genital traits – intromittent or nonintromittent, size, shape, complexity – can be subject to different tempos and modes of selection even within the same © 2013 Australian Entomological Society species. Few studies examine the rates of genital evolution despite the general claim that genitalia evolve rapidly. Progress in our understanding of genital evolution will be achieved from a greater focus on the functional morphology of female genitalia, an increased use of quantitative methods of assessing the strength and form of selection acting on both male and female genitalia, and an increased use of modern comparative methods for exploring tempos and modes of evolutionary divergence. What is clear is that the patterns of sexual selection acting on genital morphology are every bit as complex as the traits they produce. ACK N OW L EDGEMEN T S I thank David Hosken, Nic Tatarnic and Jon Evans for comments on earlier drafts and the Australian Research Council for financial support. REF EREN CES Andersson M. 1994. Sexual Selection. Princeton University Press, Princeton, New Jersey, USA. Arbuthnott D, Elliot MG, McPeek MA & Crespi BJ. 2010. Divergent patterns of diversification in courtship and genitalic characters of Timema walking-sticks. Journal of Evolutionary Biology 23, 1399– 1411. Arnold SJ & Wade MJ. 1984a. On the measurement of natural and sexual selection: applications. Evolution 38, 720–734. Arnold SJ & Wade MJ. 1984b. On the measurement of natural and sexual selection: theory. Evolution 38, 709–719. Arnqvist G. 1997. The evolution of animal genitalia: distinguishing between hypotheses by single species studies. Biological Journal of the Linnean Society 60, 365–379. Arnqvist G. 1998. Comparative evidence for the evolution of genitalia by sexual selection. Nature 393, 784–786. Arnqvist G & Danielsson I. 1999. Copulatory behavior, genital morphology, and male fertilization success in water striders. Evolution 53, 147–156. Arnqvist G & Rowe L. 1995. Sexual conflict and arms races between the sexes: a morphological adaptation for control of mating in a female insect. Proceedings of the Royal Society of London B 261, 123–127. Arnqvist G & Rowe L. 2002. Correlated evolution of male and female morphologies in water striders. Evolution 56, 936–947. Arnqvist G & Rowe L. 2005. Sexual Conflict. Princeton University Press, Princeton, New Jersey, USA. Bath E, Tatarnic N & Bonduriansky R. 2012. Asymmetric reproductive isolation and interference in neriid flies: the roles of genital morphology and behaviour. Animal Behaviour 84, 1331–1339. Bertin A & Fairbairn DJ. 2005. One tool, many uses: precopulatory sexual selection on genital morphology in Aquarius remigis. Journal of Evolutionary Biology 18, 949–961. Bertin A & Fairbairn DJ. 2007. The form of sexual selection on male genitalia cannot be inferred from within-population variance and allometry – a case study in Aquarius remigis. Evolution 61, 825–837. Blanckenhorn WU, Hosken DJ, Martin OY, Reim C, Teuschl Y & Ward PI. 2002. The costs of copulating in the dung fly Sepsis cynipsea. Behavioral Ecology 13, 353–358. Blanckenhorn WU, Kraushaar URS, Teuschl Y & Reim C. 2004. Sexual selection on morphological and physiological traits and fluctuating asymmetry in the black scavenger fly Sepsis cynipsea. Journal of Evolutionary Biology 17, 629–641. Bonduriansky R. 2007. Sexual selection and allometry: a critical reappraisal of the evidence and ideas. Evolution 61, 838–849. Bonduriansky R & Day T. 2003. The evolution of static allometry in sexually selected traits. Evolution 57, 2450–2458. Sexual selection and genital evolution Brennan PLR, Prum RO, McCracken KG, Sorenson MD, Wilson RE & Birkhead TR. 2007. Coevolution of male and female genital morphology in waterfowl. PLoS ONE 2, e418. Briceño RD & Eberhard W. 2009a. Experimental modifications imply a stimulatory function for male tsetse fly genitalia, supporting cryptic female choice theory. Journal of Evolutionary Biology 22, 1516– 1525. Briceño RD & Eberhard WG. 2009b. Experimental demonstration of possible cryptic female choice on male tsetse fly genitalia. Journal of Insect Physiology 55, 989–996. Brommer JE. 2011. Whither Pst? The approximation of Qst by Pst in evolutionary and conservation biology. Journal of Evolutionary Biology 24, 1160–1168. Burns MM, Hedin M & Shultz JW. 2013. Comparative analyses of reproductive structures in harvestmen (Opiliones) reveal multiple transitions from courtship to precopulatory antagonism. PLoS ONE 8, e66767. Castellano S & Cermelli P. 2006. Reconciling sexual selection to species recognition: a process-based model of mating decision. Journal of Theoretical Biology 242, 529–538. Cayetano L, Maklakov AA, Brooks RC & Bonduriansky R. 2011. Evolution of male and female genitalia following release from sexual selection. Evolution 65, 2171–2183. Córdoba-Aguilar A. 1999. Male copulatory sensory stimulation induces female ejection of rival sperm in a damselfly. Proceedings of the Royal Society of London B 266, 779–784. Córdoba-Aguilar A. 2002. Sensory trap as the mechanism of sexual selection in a damselfly genitalic trait (Insecta: Calopterygidae). American Naturalist 160, 594–601. Córdoba-Aguilar A. 2005. Possible coevolution of male and female genital form and function in a calopterygid damselfly. Journal of Evolutionary Biology 18, 132–137. Córdoba-Aguilar A. 2009. Seasonal variation in genital and body size, sperm displacement ability, female mating rate, and male harassment in two calopterygid damselflies (Odonata: Calopterygidae). Biological Journal of the Linnean Society 96, 815–829. Crudgington HS & Siva-Jothy MT. 2000. Genital damage, kicking and early death. Nature 407, 855–856. Danielsson I & Askenmo C. 1999. Male genital traits and mating interval affect male fertilization success in the water strider Gerris lacustris. Behavioral Ecology and Sociobiology 46, 149–156. Dirsh VM. 1956. The phalic complex in Acridoidea (Orthoptera) in relation to taxonomy. Transactions of the Royal Entomological Society of London 108, 223–356. Dreyer AP & Shingleton AW. 2011. The effect of genetic and environmental variation on genital size in male Drosophila: canalized but developmentally unstable. PLoS ONE 6, e28278. Dufour L. 1848. Anatomie générale des Dipteres. Annuare de Science Naturelle 1, 244–264. Eberhard W. 2006. Sexually antagonistic coevolution in insects is associated with only limited morphological diversity. Journal of Evolutionary Biology 19, 657–681. Eberhard W. 2009. Static allometry and animal genitalia. Evolution 63, 48–66. Eberhard W. 2010. Evolution of genitalia: theories, evidence, and new directions. Genetica 138, 5–18. Eberhard WG. 1985. Sexual Selection and Animal Genitalia. Harvard University Press, Cambridge, Massachusetts, USA. Eberhard WG. 1996. Female Control: Sexual Selection by Cryptic Female Choice. Princeton University Press, Princeton, New Jersey, USA. Eberhard WG. 2004a. Male–female conflict and genitalia: failure to confirm predictions in insects and spiders. Biological Reviews 79, 121–186. Eberhard WG. 2004b. Rapid divergent evolution of sexual morphology: comparative tests of antagonistic coevolution and traditional female choice. Evolution 58, 1947–1970. Eberhard WG, Huber BA, Rodriguez RLS, Briceño RD, Salas I & Rodriguez V. 1998. One size fits all? Relationships between the size and degree of variation in genitalia and other body parts in twenty species of insects and spiders. Evolution 52, 415–431. 15 Edelaar PIM, Burraco P & Gomez-Mestre I. 2011. Comparisons between QST and FST—how wrong have we been? Molecular Ecology 20, 4830–4839. Endler JA. 1989. Conceptual and other problems in speciation. In: Speciation and Its Consequences (eds D Otte & JA Endler), pp. 625–648. Sinauer, Sunderland, Massachusetts, USA. Endler JA & Houde AE. 1995. Geographic variation in female preferences for male traits in Poecilia reticulata. Evolution 49, 456–468. Evans JP, Gasparini C, Holwell GI, Ramnarine IW, Pitcher TE & Pilastro A. 2011. Intraspecific evidence from guppies for correlated patterns of male and female genital trait diversification. Proceedings of the Royal Society of London B 278, 2611–2620. Eyer JR. 1924. The comparative morphology of the male genitalia of the primitive Lepidoptera. Annals of the Entomological Society of America 17, 275–328. Fairbairn DJ, Vermette R, Kapoor NN & Zahiri N. 2003. Functional morphology of sexually selected genitalia in the water strider Aquarius remigis. Canadian Journal of Zoology 81, 400–413. Fricke C, Perry JC, Chapman T & Rowe L. 2009. The conditional economics of sexual conflict. Biology Letters 5, 671–674. Gagnon MC & Turgeon J. 2011. Sexual conflict in Gerris gillettei (Insecta: Hemiptera): intraspecific intersexual correlated morphology and experimental assessment of behaviour and fitness. Journal of Evolutionary Biology 24, 1505–1516. Grieshop K & Polak M. 2012. The precopulatory function of male genital spines in Drosophila ananassae [doleschall] (diptera: drosophilidae) revealed by laser surgery. Evolution 66, 2637–2645. Higgie M & Blows MW. 2007. Are traits that experience reinforcement also under sexual selection? American Naturalist 170, 409–420. Holwell G. 2008. Geographic variation in genital morphology of Ciulfina praying mantids. Journal of Zoology (London) 276, 108– 114. Holwell G, Winnick C, Tregenza T & Herberstein ME. 2010. Genital shape correlates with sperm transfer success in the praying mantis Ciulfina klassi (Insecta: Mantodea). Behavioral Ecology and Sociobiology 64, 617–625. Holwell GI & Herberstein ME. 2010. Chirally dimorphic male genitalia in praying mantids (Ciulfina: Liturgusidae). Journal of Morphology 271, 1176–1184. Hosken DJ, Minder AM & Ward PI. 2005. Male genital allometry in Scathophagidae (Diptera). Evolutionary Ecology 19, 501–515. Hosken DJ & Stockley P. 2004. Sexual selection and genital evolution. Trends in Ecology and Evolution 19, 87–93. Hotzy C, Polak M, Rönn JL & Arnqvist G. 2012. Phenotypic engineering unveils the function of genital morphology. Current Biology 22, 2258–2261. House CM, Lewis Z, Hodgson DJ et al. 2013. Sexual and natural selection both influence male genital evolution. PLoS ONE 8, e63807. House CM & Simmons LW. 2003. Genital morphology and fertilization success in the dung beetle Onthophagus taurus: an example of sexually selected male genitalia. Proceedings of the Royal Society of London B 270, 447–455. House CM & Simmons LW. 2007. No evidence for condition-dependent expression of male genitalia in the dung beetle Onthophagus taurus. Journal of Evolutionary Biology 20, 1322–1332. Ilango K & Lane RP. 2000. Coadaptation of male aedeagal filaments and female spermathecal ducts of the old world phlebotomine sand flies. Journal of Medical Entomology 37, 653–659. Jagadeeshan S & Singh RS. 2006. A time-sequence functional analysis of mating behaviour and genital coupling in Drosophila: role of cryptic female choice and male sex-drive in the evolution of male genitalia. Journal of Evolutionary Biology 19, 1058–1070. Kamimura Y. 2012. Correlated evolutionary changes in Drosophila female genitalia reduce the possible infection risk caused by male copulatory wounding. Behavioral Ecology and Sociobiology 66, 1107–1114. Kamimura Y & Polak M. 2011. Does surgical manipulation of Drosophila intromittent organs affect insemination success? Proceedings of the Royal Society of London B 278, 815–816. Kawano K. 2002. Character displacement in giant rhinoceros beetles. American Naturalist 159, 255–271. © 2013 Australian Entomological Society 16 L W Simmons Kawano K. 2003. Character displacement in stag beetles (Coleoptera: Lucanidae). Annals of the Entomological Society of America 96, 503– 511. Kawecki TJ, Lenski RE, Ebert D, Hollis B, Olivieri I & Whitlock M. 2012. Experimental evolution. Trends in Ecology and Evolution 27, 547– 560. Koshio C, Muraji M, Tatsuta H & Kudo S. 2007. Sexual selection in a moth: effect of symmetry on male mating success in the wild. Behavioral Ecology 18, 571–578. Kuntner M, Coddington JA & Schneider JM. 2009. Intersexual arms race? Genital coevolution in Nephilid spiders (Araneae, Nephilidae). Evolution 63, 1451–1463. Lamy J-B, Plomion C, Kremer A & Delzon S. 2012. QST < FST as a signature of canalization. Molecular Ecology 21, 5646–5655. Lande R & Arnold SJ. 1983. The measurement of selection on correlated characters. Evolution 37, 1210–1226. van Lieshout E. 2011. Male genital length and mating status differentially affect mating behaviour in an earwig. Behavioral Ecology and Sociobiology 65, 149–156. van Lieshout E & Elgar MA. 2011. Longer exaggerated male genitalia confer defensive sperm-competitive benefits in an earwig. Evolutionary Ecology 25, 351–362. Lloyd JE. 1979. Mating behavior and natural selection. Florida Entomologist 62, 17–34. Losos JB. 1999. Uncertainty in the reconstruction of ancestral character states and limitations on the use of phylogenetic comparative methods. Animal Behaviour 58, 1319–1324. Macagno ALM, Pizzo A, Parzer HF, Palestrini C, Rolando A & Moczek AP. 2011. Shape – but not size – codivergence between male and female copulatory structures in Onthophagus Beetles. PLoS ONE 6, e2889. Márquez EJ & Knowles LL. 2007. Correlated evolution of multivariate traits: detecting co-divergence across multiple dimensions. Journal of Evolutionary Biology 20, 2334–2348. Mayr E. 1963. Animal Species and Evolution. Harvard University Press, Cambridge, Massachusetts, USA. McKay JK & Latta RG. 2002. Adaptive population divergence: markers, QTL and traits. Trends in Ecology and Evolution 17, 285–291. McPeek MA, Shen L & Farid H. 2009. The correlated evolution of threedimensional reproductive structures between male and female damselflies. Evolution 63, 73–83. McPeek MA, Shen L, Torrey JZ & Farid H. 2008. The tempo and mode of three-dimensional morphological evolution in male reproductive structures. American Naturalist 171, E158–E178. Merilä J & Crnokrak P. 2001. Comparison of genetic differentiation at marker loci and quantitative traits. Journal of Evolutionary Biology 14, 892–903. Moreno-Garcia M & Cordero C. 2008. On the function of male genital claspers in Stenomacra marginella (Heteroptera: Largidae). Journal of Ethology 26, 255–260. Nagata N, Kubota K, Takami Y & Sota T. 2009. Historical divergence of mechanical isolation agents in the ground beetle Carabus arrowianus as revealed by phylogeographical analyses. Molecular Ecology 18, 1408–1421. Otronen M. 1998. Male asymmetry and postcopulatory sexual selection in the fly Dryomyza anilis. Behavioral Ecology and Sociobiology 42, 185–192. Parker GA. 1979. Sexual selection and sexual conflict. In: Sexual Selection and Reproductive Competition in Insects (eds MS Blum & NA Blum), pp. 123–166. Academic Press, London, UK. Patterson BD & Thaeler CS. 1982. The mammalian baculum: hypotheses on the nature of bacular variability. Journal of Mammalogy 63, 1–15. Perry JC & Rowe L. 2012. Sexual conflict and antagonistic coevolution across water strider populations. Evolution 66, 544–557. Petrie M. 1988. Intraspecific variation in structures that display competitive ability: large animals invest relatively more. Animal Behaviour 36, 1174–1180. Phillips PC & Arnold SJ. 1989. Visualizing multivariate selection. Evolution 43, 1209–1222. Pizzo A, Roggero A, Palestrini C, Moczek AP & Rolando A. 2008. Rapid shape divergences between natural and introduced populations of a © 2013 Australian Entomological Society horned beetle partly mirror divergences between species. Evolution and Development 10, 166–175. Polak M & Rashed A. 2010. Microscale laser surgery reveals adaptive function of male intromittent genitalia. Proceedings of the Royal Society of London B 277, 1371–1376. Polihronakis M. 2006. Morphometric analysis of intraspecific shape variation in male and female genitalia of Phyllophaga hirticula (Coleoptera: Scarabaeidae: Melolonthinae). Annals of the Entomological Society of America 99, 144–150. Polihronakis M. 2009. Hierarchical comparative analysis of genetic and genitalic geographical structure: testing patterns of male and female genital evolution in the scarab beetle Phyllophaga hirticula (Coleoptera: Scarabaeidae). Biological Journal of the Linnean Society 96, 135–149. Preziosi RF & Fairbairn DJ. 2000. Lifetime selection on adult body size and components of body size in a waterstrider: opposing selection and maintenance of sexual size dimorphism. Evolution 54, 558–566. Puniamoorthy N, Kotrba M & Meier R. 2010. Unlocking the ‘Black box’: internal female genitalia in Sepsidae (Diptera) evolve fast and are species-specific. BMC Evolutionary Biology 10, 275. Richards OW. 1927a. Sexual selection and related problems in the insects. Biological Reviews 2, 298–364. Richards OW. 1927b. The specific characters of British bumblebees (Hymenoptera). Transactions of the Royal Entomological Society of London 75, 233–265. Richmond MP, Johnson S & Markow TA. 2012. Evolution of reproductive morphology among recently diverged taxa in the Drosophila mojavensis species cluster. Ecology and Evolution 2, 397–408. Ritchie MG. 2007. Sexual selection and speciation. Annual Review of Ecology, Evolution, and Systematics 38, 79–102. Robinson JV & Novak KL. 1997. The relationship between mating system and penis morphology in ischnuran damselflies (Odonata: Coenagrionidae). Biological Journal of the Linnean Society 60, 187– 200. Rodriguez V. 1995. Relation of flagellum length to reproductive success in male Chelymorpha alternans Boheman (Coleoptera: Chrysomelidae: Cassidinae). Coleopterists’ Bulletin 49, 201–205. Rodriguez V, Windsor D & Eberhard WG. 2004. Tortoise beetle genitalia and demonstrations of a sexually selected advantage for flagellum length in Chelymorpha alternans (Chrysomelidae, Cassidini, Stolaini. In: New Developments in the Biology of Chrysomelidae (eds P Jolivet, JA Santiago-Blay & M Schmitt), pp. 739–748. SPB Academic Publishing, The Hague, Netherlands. Rodríguez RL & Al-Wathiqui N. 2011. Genotype x environment interaction is weaker in genitalia than in mating signals and body traits in Enchenopa treehoppers (Hemiptera: Membracidae). Genetica 139, 871–884. Rodríguez RL & Al-Wathiqui N. 2012. Genotype × environment interaction in the allometry of body, genitalia and signal traits in Enchenopa treehoppers (Hemiptera: Membracidae). Biological Journal of the Linnean Society 105, 187–196. Roig-Alsina A. 1993. The evolution of the apoid endophallus, its phylogenetic implications, and functional significance of the genital capsule (Hymenoptera, Apoidea). Bollettino di Zoologia 60, 169–183. Rönn J, Katvala M & Arnqvist G. 2007. Coevolution between harmful male genitalia and female resistance in seed beetles. Proceedings of the National Academy of Sciences of the United States of America 104, 10921–10925. Rowe L. 1994. The costs of mating and mate choice in water striders. Animal Behaviour 48, 1049–1056. Rowe L & Arnqvist G. 2012. Sexual selection and the evolution of genital shape and complexity in water striders. Evolution 66, 40–54. Ryan MJ & Rand AS. 1993. Species recognition and sexual selection as a unitary problem in animal communication. Evolution 47, 647–657. Sakurai G, Himuro C & Kasuya E. 2012. Intra-specific variation in the morphology and the benefit of large genital sclerites of males in the adzuki bean beetle (Callosobruchus chinensis). Journal of Evolutionary Biology 25, 1291–1297. Sánchez V, Hernández-Baños BE & Cordero C. 2011. The evolution of a female genital trait widely distributed in the lepidoptera: comparative evidence for an effect of sexual coevolution. PLoS ONE 6, e22642. Sexual selection and genital evolution Sasabe M, Takami Y & Sota T. 2010. QTL for the species-specific male and female genital morphologies in Ohomopterus ground beetles. Molecular Ecology 19, 5231–5239. Shapiro AM & Porter AH. 1989. The lock-and-key hypothesis: evolutionary and biosystematic interpretation of insect genitalia. Annual Review of Entomology 34, 231–245. Simmons LW. 2001. Sperm Competition and Its Evolutionary Consequences in the Insects. Princeton University Press, Princeton, New Jersey, USA. Simmons LW, Beveridge M & Krauss S. 2004. Genetic analysis of parentage within experimental populations of a male dimorphic beetle, Onthophagus taurus, using amplified fragment length polymorphism. Behavioral Ecology and Sociobiology 57, 164–173. Simmons LW & Fitzpatrick JL. 2012. Sperm wars and the evolution of male fertility. Reproduction 144, 519–534. Simmons LW & García-González F. 2008. Evolutionary reduction in testes size and competitive fertilization success in response to the experimental removal of sexual selection in dung beetles. Evolution 62, 2580–2591. Simmons LW & Garcia-Gonzalez F. 2011. Experimental coevolution of male and female genital morphology. Nature Communications 2, 374. doi:10.1038/ncomms1379 Simmons LW & Holley R. 2011. Offspring viability benefits but no apparent costs of mating with high quality males. Biology Letters 7, 419– 421. Simmons LW, House CM, Hunt J & García-González F. 2009. Evolutionary response to sexual selection in male genital morphology. Current Biology 19, 1442–1446. Simmons LW & Tomkins JL. 1996. Sexual selection and the allometry of earwig forceps. Evolutionary Ecology 10, 97–104. Song H & Bucheli SR. 2010. Comparison of phylogenetic signal between male genitalia and non-genital characters in insect systematics. Cladistics 26, 23–35. Sota T & Kubota K. 1998. Genital lock-and-key as a selective agent against hybridization. Evolution 52, 1507–1513. Soulier-Perkins A. 2001. The phylogeny of the Lophopidae and the impact of sexual selection and coevolutionary sexual conflict. Cladistics 17, 56–78. Tadler A. 1999. Selection of a conspicuous male genitalic trait in seedbug Lygaeus simulans. Proceedings of the Royal Society of London B 266, 1773–1777. Tadler A & Nemeschkal HL. 1999. Selection of male traits during and after copulation in the seedbug Lygaeus simulans (Heteroptera, Lygaeidae). Biological Journal of the Linnean Society 68, 471–483. Takami Y. 2003. Experimental analysis of the effect of genital morphology on insemination success in the ground beetle Carabus insulicola. Ethology Ecology and Evolution 15, 51–61. Takami Y & Soto T. 2007. Rapid diversification of male genitalia and mating strategies in Ohomopterus ground beetles. Journal of Evolutionary Biology 20, 1385–1395. Tarasov SI & Solodovnikov AY. 2011. Phylogenetic analyses reveal reliable morphological markers to classify mega-diversity in 17 Onthophagini dung beetles (Coleoptera: Scarabaeidae: Scarabaeinae). Cladistics 27, 1–39. Tatarnic NJ & Cassis G. 2010. Sexual coevolution in the traumatically inseminating plant bug genus Coridromius. Journal of Evolutionary Biology 23, 1321–1326. Thornhill R & Alcock J. 1983. The Evolution of Insect Mating Systems. Harvard University Press, Cambridge, Massachusetts, USA. Tsuchiya K & Hayashi F. 2008. Surgical examination of male genital function of calopterygid damselflies (Odonata). Behavioral Ecology and Sociobiology 62, 1417–1425. Tuxen SL. 1970. Taxonomists Glossary of Genitalia in Insects. Scandinavian University Press, Copenhagen, Denmark. Vahed K, Lehmann AW, Gilbert JDJ & Lehmann GUC. 2011. Increased copulation duration before ejaculate transfer is associated with larger spermatophores, and male genital titillators, across bushcricket taxa. Journal of Evolutionary Biology 24, 1960–1968. Waage JK. 1979. Dual function of the damselfly penis: sperm removal and transfer. Science 203, 916–918. Wenninger EJ & Averill AL. 2006. Influence of body and genital morphology on relative male fertilization success in oriental beetle. Behavioral Ecology 17, 656–663. Werner M & Simmons LW. 2008. The evolution of male genitalia: functional integration of genital sclerites in the dung beetle Onthophagus taurus. Biological Journal of the Linnean Society 93, 257–266. Whitlock MC. 2008. Evolutionary inference from QST. Molecular Ecology 17, 1885–1896. Wilkinson GS. 1993. Artificial sexual selection alters allometry in the stalk-eyed fly Cyrtodiopsis dalmanni (Diptera: Diopsidae). Genetical Research 62, 213–222. Wilkinson GS & Taper M. 1999. Evolution of genetic variation for condition-dependent traits in stalk-eyed flies. Proceedings of the Royal Society of London B 266, 1685–1690. Wojcieszek JM, Austin P, Harvey MS & Simmons LW. 2012. Micro-CT scanning provides insight into the functional morphology of millipede genitalia. Journal of Zoology 287, 91–95. Wojcieszek JM & Simmons LW. 2012. Evidence for stabilizing selection and slow divergent evolution of male genitalia in a millipede (Antichiropus variabilis). Evolution 66, 1138–1153. Wojcieszek JM & Simmons LW. 2013. Divergence in genital morphology may contribute to mechanical reproductive isolation in a millipede. Ecology and Evolution 3, 334–343. Xu J & Wang Q. 2010. Form and nature of precopulatory sexual selection in both sexes of a moth. Naturwissenschaften 97, 617–625. Yassin A & Orgogozo V. 2013. Coevolution between male and female genitalia in the Drosophila melanogaster species subgroup. PLoS ONE 8, e57158. Zunino M. 1979. Gruppi artificiali e gruppi naturali negli Onthophagus (Coleoptera, Scarabaeoidea). Bollettino dei Musei di Zoologia e Anatomia comparata, Torino 1, 1–18. Accepted for publication 10 July 2013. © 2013 Australian Entomological Society