Sabethes Cyaneus

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I South, S.H., Arnqvist, G. (2009) Morphological variation of an ornament expressed in both sexes of the mosquito Sabethes cyaneus. Evolutionary Ecology Research, 11:1–21.

II South, S.H., Arnqvist, G. (in press) Male, but not female, preference for an ornament expressed in both sexes of the polygynous mosquito Sabethes cyaneus. Animal Behaviour (2011), doi:10.1016/j.anbehav.2010.12.014.

III South, S.H., Steiner, D., Arnqvist, G. (2009) Male mating costs in a polygynous mosquito with ornaments expressed in both sexes. Proceedings of the Royal Society B, 276: 3671-3678.

IV South, S.H.*, Arnqvist, G., Servedio, M.* Female preference for male courtship effort drives the evolution of male mate choice. (manuscript)

V South, S.H., Arnqvist, G. (2008) Evidence of monandry in a mosquito (Sabethes cyaneus) with elaborate ornaments in both sexes. Journal of Insect Behaviour, 21:451-459.

Reprints were made with permission from the respective publishers.

* These authors contributed equally to this work.

Cover photograph by Sandra South.

Contents

Introduction- Determining the intensity of sexual selection...... 7 My Study System- A theoretical challenge...... 10 General biology of mosquitoes...... 10 A unique group of mosquitoes: The sabethines ...... 11 The challenge: Elaborate ornaments in both sexes...... 12 My study species- Sabethes cyaneus...... 13 Aims of my thesis...... 14 1. Are female ornaments in S. cyaneus solely the result of an intersexual genetic correlation?...... 15 2. Is there selection via mutual mate choice on ornaments in S. cyaneus? ...19 3. How can male mate choice and male-male competition for mates occur simultaneously in S. cyaneus? Exploring the costs and benefits...... 21 Reducing the costs: does courtship constitute a significant reproductive investment by S. cyaneus males? ...... 22 Increasing the benefits: can female preferences for male courtship effort play a role? ...... 24 Interacting factors can affect the costs and benefits of male mate choice ...... 26 4. Why are females signalling? Exploring female mating rates and the opportunity for sexual selection in females...... 28 Conclusions and future directions...... 30 The relationship between costly female ornaments and the benefits of male mate choice...... 30 Female mating rates and the opportunity for sexual selection as assumed by the OSR ...... 31 Species recognition hypotheses- not an alternative to sexual selection ...32 The contribution of male mate choice to population fitness?...... 33 Sammanfattning på svenska...... 35 Acknowledgements...... 40 References...... 42

Abbreviations

OSR Operational sex ratio: the ratio of sexually active males to females PRR Potential rate of reproduction: the average of the maximum rate at which individuals can produce offspring BG Bateman gradient: the slope of the regression of offspring production on mating rate

Introduction- Determining the intensity of sexual selection

Elaborate male signals, such as the peacock's train, posed a conundrum for Darwin. He could not comprehend how signals such as these, that apparently reduced the survival of their bearer, could evolve (Darwin 1859). Darwin later explored the possibility that elaborate male signals may have evolved through sexual selection by female preferences for male traits during mate choice and/or by male-male contest competition for females (Darwin 1871). Sexual selection has since become one of the most active areas of study in behavioural ecology and evolution (Andersson 1994). Sexual selection theory has not only developed within the field of pre-copulatory sexual selection (i.e., mate choice and intrasexual competition), but also expanded to include the fields of both pre- and post-copulatory sexual conflict (Arnqvist and Rowe 2005) and post-copulatory sexual selection (e.g., sperm competition, Simmons 2001; Wedell et al. 2002) and cryptic female choice (Eberhard 1996). My thesis will focus largely on pre-copulatory sexual selection, in particular, sexual selection via mate choice. Selection on males which leads to the evolution of such elaborate traits can be broadly understood as being the result of females being a limiting resource which males need to compete for. The reason that females often appear to be a limiting resource to males is widely attributed to the greater investment per reproductive event of females than males; both in terms of investment in a limited number of large gametes (eggs) and other forms of parental investment such as pregnancy and postnatal care of offspring (Bateman 1948; Trivers 1972). Parental investment results in a longer 'time- out' of the mating pool for each reproductive event (i.e., a lower potential rate of reproduction [PRR]), such that sexually active members of the sex investing more in reproduction (often females) will be scarcer than those of the opposite sex at any given time (Clutton-Brock and Parker 1992). This ratio of sexually active males to sexually active females is known as the operational sex ratio (OSR) as first defined by Emlen (1976) and Emlen and Oring (1977). In their paper, Emlen and Oring (1977) emphasise that the operational sex ratio is not only affected by investment in parental care, but also by any number of other ecological factors such as sex differences in mortality, the distribution and availability of resources (e.g., nesting sites)

7 and the temporal distribution of mates. Thus, it is likely that the OSR of a population will be both temporally and spatially dynamic. However, the differences between the sexes in the intensity of sexual selection can not simply be understood in terms of the OSR. Fitness variation between individuals is necessary. The variation in reproductive fitness among members of each sex is known as the opportunity for sexual selection (Arnold and Wade 1984; Shuster and Wade 2003). This point can be portrayed by imagining a hypothetical population where the OSR is male biased such that, at any given time, there are more sexually active males than females. If these males were to father the same number of offspring, then there would be no opportunity for sexual selection regardless of the magnitude of the OSR as no particular male phenotype would confer a reproductive fitness advantage. However, this scenario is unlikely, especially considering the myriad of traits that could potentially increase a male's mating success. No- netheless, this fundamental proviso is useful to bear in mind, especially when considering the relationships between the PRR, OSR, and the opportunity for sexual selection. The opportunity for sexual selection (i.e., variance in reproductive success) was originally assumed to increase with an increasing PRR in males. This is because the fitness of males is thought to increase linearly with an increasing number of mates. Conversely, female fitness was expected to plateau after a low number of matings as females can gain more than enough sperm to fertilise all their eggs from a single mating and thus should invest in gametes and parental care. Thus, the fitness variation in males could potentially be much larger than in females, as individual male mating rates would scan a larger range of values and a correspondingly larger variation in reproductive fitness based on the number of matings obtained. On the other hand, all females are likely to mate and thus fitness variation in females would not be correlated to their mating rate but rather their intrinsic reproductive capacity. Therefore, males are expected to invest in traits which increase their mating rate, while females are expected to invest in traits which increase their fecundity. This episteme stemmed from the study by Bateman (1948), which showed that the offspring number of male Drosophila melanogaster increased linearly with an increasing number of matings, while that of females scarcely increased after their first mating. This gradient is now known as the Bateman gradient (BG), that is the slope of the regression of offspring production on mating rate, with a steep BG leading to strong directional selection on mating success (Arnold 1994; Arnold and Duvall 1994; Andersson and Iwasa 1996). However, an individual's PRR does not necessarily equate to the fitness benefits it stands to gain from mating multiply (i.e., the slope of the BG). Caution must therefore be taken when relating the relative PRRs of the sexes to the opportunity for sexual selection. Indeed, a less commonly cited aspect of Bateman's (1948) study is that in some cases, female fitness also increased with an increasing number of mates,

8 despite a lower PRR than males. Indeed, this phenonemon has since been seen in a wide number of species and is attributed to benefits to females from multiple mating such as increased egg production rate and fertility (e.g., Arnqvist and Nilsson 2000). Therefore, experimental measurement of the BG, in combination with the OSR (and hence the level of competition and mating rate able to be realised) is necessary to fully understand the factors contributing to the opportunity for sexual selection (Jennions and Kokko 2010). In one well-known study species, the pipefish Syngnathus typhle, information on both the relative levels of parental investment between the sexes and the BG are known. In this species, females deposit their eggs into a pouch on the male's ventral surface and the males then incubate the eggs for up to 40 days. Males are therefore limited in the amount of eggs they can fit in their pouch, whereas females are limited by the number of males they can mate with. This results in males becoming a limiting resource for females (Berglund and Rosenqvist 1990; Vincent et al. 1994). Accordingly, females had a steeper positive BG than that of males which was not significantly different from zero (Jones et al. 2005). Additionally, by altering the OSR, Jones et al. (2005) showed that the opportunity for sexual selection was highest in females with a female-biased OSR, and highest for males with a male-biased OSR. This species therefore represents the opposite scenario presented by Bateman (1948) in the female-limited Drosophila. Observa- tions of female-female competition, female ornaments and male mate choice led to this system becoming the archetype for 'sex-role reversal'. More recently, there have been increasing reports of mate choice even in the competitive sex. Indeed, in S. typhle there is now evidence of female mate choice coupled with female-female competition (Berglund et al. 2005). Correspondingly, in an increasing number of species with intense male-male competition and a male-biased OSR there is evidence of male mate choice (for reviews see Amundsen 2000; Bonduriansky 2001; Hooper and Miller 2008). Although originally seen as anomallies or cases where 'hidden' reproductive investments by males (leading to a female-biased OSR) had been overlooked, mate choice and competition are now accepted as often occurring simultaneously. This has lead to a reappraisal of what comprises a 'sex- role', with intrasexual competition appearing to be the only indicative phenotype resulting from strong sexual selection (Vincent et al. 1992). By return- ing to the pioneering sexual selection theory of Parker (1983), we can understand the evolution of mate choice coupled with intrasexual competition as a result of a trade-off between the costs and benefits. As the exploration of these costs and benefits comprises a significant proportion of this thesis, I will return to the costs and benefits of mate choice in more detail in Section 3 (Papers III-V).

9 My Study System- A theoretical challenge

General biology of mosquitoes The biology of mosquitoes (Diptera: Culicidae) has been extensively studied due to their medical importance as disease vectors during adult female blood feeding. In the large majority of species females are unautogenous, meaning that they require a blood meal in order to begin egg development (Becker et al. 2003). Only females develop the mouthparts needed to pierce the skin and blood feed (Clements 1992). Both male and female adults can also ob- tain energetic requirements from water and nectar (Magnarelli 1979). Aquatic environments are required for egg, larval and pupal development. Females of some species can lay eggs on damp surfaces which are regularly flooded, with the eggs entering a stage of dormancy until submersion. The larval stage is the only aquatic stage which feeds, with a diet of microorgan- isms, algae, protozoa, invertebrates and detritus. Many species are capable of hibernation during cold and dry periods, although different species enter hibernation at different stages: eggs- tribe Aedini; larvae- tribes Aedini and Culicini and Subfamily Anophelinae; adults- tribes Culisetini, Uranotaeniini and Subfamily Anophelinae (Becker et al. 2003). The typical life-cycle of a mosquito is shown in Figure 1.

Figure 1. The mosquito life-cycle. Diagram by S. South.

10 It was originally considered that there was a general lack of sexual selection in mosquitoes (Hancock et al. 1990a). This was primarily considered to be so because of limited sexual dimorphism and because males typically swarm at feeding or emergence sites, as in many insect species (Nielsen and Haeger 1960; Haddow and Corbet 1961; Anderson 1974). Within these swarms, scramble competition polygyny was thought to predominate, i.e., there is little overt competition or contact among males and pairings are assumed to be random (Thornhill and Alcock 1983). In other species of mosquitoes active pre-copulatory female choice is constrained because males monopolise females. In Deinocerites cancer, males hold on to female pupae in order to mate with them immediately after emergence (Downes 1966; Provost and Haeger 1967). Alternatively, in some species pre-copulatory female choice is prevented as females are receptive to mating only after a blood meal and males may pursue females, mating with them immediately after feeding (Gillett 1971). Post-copulatory female choice is also prevented in the majority of species as females are monandrous (i.e., mate once only) (e.g., Gillies 1956; Goma 1963; Jones 1973; Mahmood and Reisen 1980; Reisen et al. 1984; Baimai and Green 1987). In a subset of species, female monandry is known to be the result of substances transferred in the male ejaculate (Klowden 2001; Klowden 2006). However, evidence that sexual selection may indeed be a strong force driving mosquito evolution is provided by recent experiments showing size- dependent mating success in mosquito swarms (Ng'habi et al. 2008) and that males and females interact before mating by matching their flight tones (Cator et al. 2009; Robert 2009; Pennetier et al. 2010; Ritchie and Immonen 2010). As mosquitoes are generally nocturnal, males of the majority of species use their plumose antennae to locate females by their specific wing-beat frequency (Belton 1974). Thus, there is scope for both male- and female mate choice based on wing beat frequencies.

A unique group of mosquitoes: The sabethines The mosquito tribe Sabethini has taken a unique evolutionary path with diurnal activity and a greater reliance on vision (Shannon 1931; Haddow and Corbet 1961). The structure of sabethine eyes has adaptations similar to members of the higher Diptera (Land et al. 1999). The tribe Sabethini have a distribution 30 degrees North and South of the equator and are active all year round, with activity peaks at high temperatures and humidity. They oviposit in plant-held waters and females are thought to blood feed primarily on arbo- real primates, although they occasionally feed on humans at ground level (Galindo et al. 1951). The sabethines are particularly unique within the mosquitoes because they have moved from mating in swarms to mating on a substrate; males patrol

11 for females perched on branches and align with them to mate (Hancock et al. 1990a). The improved visual perception of sabethine mosquitoes appears to allow males to locate conspecific females despite the fact that they are often stationary and their wing beat frequencies can not be used in mate location. In support of an argument for increased reliance on visual cues, colourful signals are common in the tribe Sabethini, in contrast to the dull colours possessed by nocturnal mosquitoes (Shannon 1931; Haddow and Corbet 1961). Members of this tribe are covered by brilliant metallic coloured flat scales, with large variation in the patterns and colours between closely related sympatric species (Judd 1996). One genus of sabethine mosquitoes - Sabethes- is particularly striking and was described by Harbach and Peyton (1990) as ‘a paragon of beauty among the genera of mosquitoes’. Members of the genus Sabethes not only have iridescent scales covering their scutum but also possess striking structures on their legs which appear to be ornaments (i.e., sexually selected traits). Spe- cies of the subgenera sabethes, sabethoides and davismyia have white scales on their tarsi which contrast with their iridescent colouration. Three species of the sabethes subgenus also have strikingly elongated scales on their legs which form paddle-like structures (henceforth referred to as 'paddles'). These species express varying degrees of white scaling on the leg paddles and expression of paddles on the legs; S. (sabethes) tarsopus has paddles on all three legs, S. (sabethes) gymnothorax has paddles on the fore- and mid-leg, and S. (sabethes) cyaneus has paddles only on the mid-legs (Harbach 1991).

The challenge: Elaborate ornaments in both sexes Within the three Sabethes sabethes species with the most elaborate paddles, the size of the paddles in males and females appears to be equal. There are also reports of other sabethine species which are yet to be classified in which relatively small paddles occur in males and are absent or reduced in females (R. Harbach, personal communication). The large variation in paddle size, number and colour among closely related species suggests that these traits may be sexually selected, as rapid divergence of traits is a hallmark of sexual selection (Coyne and Orr 2004). In support of a role of sexual selection in the evolution of these paddles, male courtship displays have been documented in four species of mosquitoes in which the ornamented legs bearing the paddles are conspicuously dis- played in front of the females while the pairs are aligned on a branch (Okazawa et al. 1986; Hancock et al. 1990a; Philips et al. 1996; Zsemlye et al. 2005). Finally, a preliminary study conducted in one of these species, Sabethes cyaneus, showed that removal of the paddles affected mating success of males and females (Hancock et al. 1990b), yet not flying ability, sug-

12 gesting that these paddles do indeed play a role in sexual selection and are true ornaments. If we assume, based on the mating system of the majority of mosquitoes, that Sabethes females are monandrous and males are polygynous (i.e., mate multiply) and that neither sex invests in offspring other than in the form of gametes, then it is difficult to explain the occurrence of ornaments in both male and female sabethine mosquitoes. This is because this situation will create a heavily male-biased OSR, with many males competing to mate with a small number of sexually active females. Furthermore, as females mate once only, the opportunity for sexual selection as affected by the BG and opportunity for sexual selection is expected to be lower in females than males. Thus, we would not expect females to be under selection to employ ornaments to increase their mating rate.

My study species- Sabethes cyaneus During my doctoral studies I have explored the evolution of ornaments in the sabethine mosquito Sabethes cyaneus. I chose this species because not only because both males and females possess elaborate iridescent mid-leg paddle ornaments (Figure 2, see also cover photograph), but also because males display these ornaments to the females in an elaborate courtship display (the courtship display is described in detail by (Hancock et al. 1990a) (Figure 2, see also online video supplement to Paper II). The courtship display involves a highly stereotyped sequence of behaviours which culminate in the female lowering her abdomen so enabling genital coupling. It is interesting to note that in this species a significant portion of the courtship involves a ‘superfi- cial coupling’ stage in which the male and female are joined at the genitals yet sperm is unable to be transferred. During this stage the male continues to display to the female, and there is also the opportunity for tactile courtship using the male genitalia (see Eberhard 1996). Another important feature of this courtship display is that females are capable of disrupting courtship at any stage by pushing the male off the branch using her hind legs or by simply not lowering their abdomen and thus preventing males from copulating. In all the experiments I conducted using S. cyaneus, the terraria used were large enough to allow males and females to engage freely in feeding, resting, egg-laying and courtship. I did not observe any behavioural divergences from those observed in the wild (Hancock et al. 1990a,b). An ad libitum supply of honey-soaked sponges and deionised water wicks were also provided. Ample perching sites allowing all individuals to perch were provided by sticks that ran the length of the terraria, reflecting the natural abundance of perching sites in nature (Hancock et al. 1990a).

Figure 2. A male (left) Sabethes cyaneus courting a female (middle) and a resting male perched on the stick (right). Note that both males and females possess the elaborate paddle-like ornaments on their mid-legs. The sexes can easily be distinguished by the naked human eye as the male genitalia cause the males abdomen to flare at the tip, while the female abdomen tapers to a point.

Aims of my thesis

In my thesis I investigate sexual selection in Sabethes cyaneus by exploring the following topics:

1. Are female ornaments in S. cyaneus solely the result of an intersexual genetic correlation? (Paper I)

2. Is there selection via mutual mate choice on ornaments in S. cyaneus? (Paper II)

3. How can male mate choice and male-male competition for mates occur simultaneously in S. cyaneus? Exploring the costs and benefits. Reducing the costs: does courtship constitute a significant reproductive investment by S. cyaneus males? (Paper III) Increasing the benefits: can female preferences for male courtship effort play a role? (Paper IV)

4. Why are females signalling? Exploring female mating rates and the opportunity for sexual selection in females. (Paper V)

14 1. Are female ornaments in S. cyaneus solely the result of an intersexual genetic correlation?

I began my doctoral studies by exploring the possibility that the ornamental 'paddles' of female S. cyaneus may purely reflect an intersexual genetic correlation, with direct selection on the ornament in males only (Darwin 1871; Lande 1980; Lande and Arnold 1985; Lande 1987). This hypothesis is plausible because, as discussed above, the mating system of S. cyaneus makes it unlikely that females would benefit from signalling in order to compete for access to matings. In order to explore this possibility I used the powerful tool of geometric morphometrics to explore the pattern of ornament expression in relation to body size. The details and results of this study are presented in Paper I. I found that sexual dimorphism in the size (Figure 3) and shape (Figure 4) of the ornaments was slight. In both sexes, paddle size increased with body size. The tibial portion of the paddle did not differ significantly between the sexes (Figure 3a). However, the tarsal portion of the paddle did differ significantly between the sexes, with the relationship between paddle size and body size being steeper in females than males (Figure 3b). This revealed that the paddles were proportionately larger in females. The relationship between paddle shape and size was different between the sexes, such that male paddles changed shape more rapidly with paddle size than female paddles (Fig- ure 4a). For the first principle component of paddle shape, male paddles tend to be slightly broader at the widest point than do female paddles and the breadth of the paddle increases with paddle area (Figure 4b). This lack of sizeable sexual dimorphism suggests that it is unlikely that the female paddles are solely due to a genetic correlation. This is because it is probable that expression of the ornamental paddles is associated with costs such as increased conspicuousness to predators (Godin and McDonough 2003), impaired ability and energetic costs during activity (Evans 2004; Allen and Levinton 2007) and production costs during ontogeny (Munoz et al. 2008). Hence, there is expected to be strong selection against the paddles in females which would quickly result in a disruption of the genetic correlation between the sexes and a subsequent reduction in paddle size in females (Fisher 1930; Lande 1980; Lande 1987; Bonduriansky and Rowe 2005).

Figure 3. The relationship between body size and (a) tibial paddle area and (b) tarsal paddle area. Lines (solid: females; dotted: males) represent ordinary linear regressions (see Paper I for details). Please note, that for ease of measurement, I divided the paddle into two sections for these analyses. The first section being the portion of the paddle that occurs on the first and second tarsi, and the second section being the portion of the paddle that occurs on the tibia.

I also explored a series of the classic hallmarks of condition-dependent traits that are under directional sexual selection. Handicap models of sexual selection predict that traits should positively covary with the bearer's condition (Andersson 1986; Pomiankowski 1987; Grafen 1990a; Iwasa and Po- miankowski 1994; Andersson and Iwasa 1996) and thus act as honest indicators of quality (Johnstone 1995; Maynard Smith and Harper 2003; Lailvaux and Irschick 2006). Condition is narrowly defined as the pool of resources available to an individual for allocation to fitness enhancing traits (Rowe and Houle 1996; Lorch et al. 2003; Hunt et al. 2004; Tomkins et al. 2004) and

16 will therefore reflect both an individuals genetic quality and their environment.

Figure 4. a) The relationship between tarsal paddle area and shape, represented by the first principle component (PC1) of variation in tarsal paddle shape. Lines represent ordinary linear regressions. b) The upper and lower extremes of the first principle component describing tarsal paddle shape. See Paper I for details.

First, I found that phenotypic variation in ornamental paddle size was much greater than for any other trait (Figure 5). This pattern is expected for condition-dependent ornaments because as the traits become increasingly linked to condition due to strong directional sexual selection they will also reflect the large magnitude of genetic and environmental variation (Rowe and Houle 1996; Cuervo and Møller 1999; Badyaev 2004). Second, I found evidence that for one component of tarsal paddle shape there was a negative relationship between fluctuating asymmetry and body size. This has been suggested to be a hallmark of condition-dependent sexual traits as the degree of asymmetry may be highest in low-quality individuals who are less able to buffer

17 stress during development (Swaddle 2003). Third, I showed that the size of the tibial component of the paddle showed positive allometry (i.e., a slope greater than one) when regressed against body size. In other words, large individuals had proportionately larger traits than small individuals. Positive allometry has been suggested to be a trademark of condition-dependent sexual traits as high-quality individuals in better condition are expected to have more resources available to allocation to sexually selected traits without experiencing a significant total fitness cost of re-distributing these resources from viability-selected traits (Grafen 1990a; Grafen 1990b; Green 1992; Petrie 1992; Emlen and Nijhout 2000; Kodric-Brown et al. 2006, but see Bonduriansky 2007).

Figure 5. Phenotypic variation of morphological traits, shown both as proportional variances and coefficients of variation (see Paper I for details).

These multiple lines of evidence suggested that both male and female paddle ornaments are under strong directional sexual selection. Nonetheless, it remained plausible that the costs of ornament expression are low enough in females to permit to non-adaptive expression, that we are observing the early stages of the evolution of sexual dimorphism and/or that the more pro- nounced expression of the ornaments in females is due to a link with other developmental pathways not under sexual selection. I therefore interpreted these results with caution and used this study as motivation to explore direct selection on both male and female paddles.

18 2. Is there selection via mutual mate choice on ornaments in S. cyaneus?

Upon consideration of the hallmarks of directional sexual selection on female paddles of S. cyaneus (Section 1 above, details in Paper I), I decided to further explore direct selection on both male and female paddles via mutual mate choice (for details see Paper II). Mutual mate choice for ornaments occurring in both sexes of socially monogamous species, such as the crested auklet (Jones and Hunter 1999), are easily understood in terms of an equal male and female investment in parental care leading to an approximately equal OSR (Johnstone et al. 1996; Johnstone 1997; Kokko and Johnstone 2002). However, as discussed above, in species with intense male-male competition and a male-biased OSR it was traditionally thought to be unlikely that female ornaments were the result of male mate choice, being attributed instead to a non-adaptive genetic correlation. My study (Paper II) is therefore one of the few that investigates mutual mate choice for sexually homologous ornaments in a species with male-male competition (for reviews see Amundsen 2000; Kraaijeveld et al. 2007) I explored mutual mate choice on paddle size by using phenotypic engineering (Sinervo and Basolo 1996), i.e., experimental manipulation of ornament size. This approach provides a powerful tool, as it allows the testing of the fitness effects of single traits in isolation (Sinervo and Basolo 1996; Travis and Reznick 1998). Phenotypic engineering increases the statistical power, allowing the detection of even weak selection, although results may be biased if the underlying fitness function is non-linear (Sinervo and Basolo 1996; Travis and Reznick 1998). Regardless of any potential bias, this approach remains informative as the complete removal of traits (such as the paddles) allows approximation of the fitness of a mutant phenotype lacking the trait which is relevant for understanding the origin and maintenance of the trait in question (Sinervo and Basolo 1996; Travis and Reznick 1998; Maklakov and Arnqvist 2009). The experimental design involved placing together virgin sexually mature males and females whose paddles were ei- ther intact, partially-removed or completely-removed. All individuals were given a unique marking on their thorax allowing them to be distinguished during observations. I then recorded which females males chose to align with on the perching stick and court and which pairs mated.

19 I found that males prefer mates with large paddles (Figure 6), while I did not detect a preference for male paddle size in females. This result came as a surprise for four reasons. First, we would predict female mate choice for male paddles based on my confirmation of male polygyny (Paper III) which provides further evidence that the opportunity for sexual selection in males (as affected by the BG) is high in male S. cyaneus. This is a prediction supported by the fact that the males predominately display the paddles to females during courtship. A previous study also did not find a female preference for male courtship or paddles in S. cyaneus (Hancock et al. 1990b). I think it is likely that our and previous experiments failed to find female preferences for male traits because none has independently manipulated these traits, and it is thus possible that a combination of paddle size and courtship determines male attractiveness to females.

Figure 6. The relationship between female paddle manipulation and female attractiveness (number of times courted by males). Mean and standard error of raw data are given. An illustration of a representative paddle from each treatment group is shown above the corresponding bar. See Paper II for details.

The second call for surprise is that male mate choice is expected to increase the costs of competition to males and I have observed male-male competition for females (unpublished data) as would be predicted by the mating system (see Section 3). Third, as investment in ornaments by females is expected to detract from the benefits of choice, the evolution of male preferences for elaborate female ornaments is unlikely (see Future directions). Finally, if we assume that S. cyaneus females are monandrous, all females should mate and therefore should not be under selection to signal to attract mates (see Section 4).

20 3. How can male mate choice and male-male competition for mates occur simultaneously in S. cyaneus? Exploring the costs and benefits.

My observations of male mate choice in S. cyaneus led me to reconsider my understanding of male mate choice theory. I find it useful to conceptualise the factors which can contribute to the evolution of male mate choice using a cost-benefit analysis approach. Figure 7 presents a simplified flow chart visualising the two most commonly explored factors affecting the evolution of male mate choice: i) the 'Triver's path' (light grey) and, ii) the 'Female quality path' (dark grey). In the following two sections I elaborate on these two pathways while integrating my own research on the possible costs and benefits of male mate choice in S. cyaneus.

Figure 7. Flow chart illustrating the relationships between female quality variance, male reproductive investment (and the resulting OSR) and the cost/benefit relationship determining the direction of selection for male mate choice. See text for details.

21 Reducing the costs: does courtship constitute a significant reproductive investment by S. cyaneus males? As depicted by the positive sign of the arrow between the OSR and the costs of male mate choice in Figure 7, the costs of male mate choice increase with an increasing OSR. The costs of male mate choice include lost future mating opportunities through, for example, reducing the time available for future mate location (Deutsch and Reynolds 1995; Johnstone 1997). The likelihood of finding a suitable future mate will be lower the higher the OSR, for example, if females mated by other males have left the mating pool. Males may also suffer costs of male mate choice through risks associated with assessment itself, such as increased conspicuousness to predators (Crowley et al. 1991; Rowe 1994). This may also positively correlate with the OSR, for example if large groups of males competing for preferred females increases their conspicuousness to predators. Finally, when all males express the same preference for a female trait, male choice will also increase competition as all males will compete for a subset of the females (Servedio and Lande 2006). The costs of male mate choice can therefore be reduced through a reduction in the OSR. The OSR can be affected by a variety of factors such as sex differences in mortality, the distribution and availability of resources (e.g., nesting sites) and the temporal distribution of mates (Emlen and Oring 1977). However, here I focus on one particular body of factors, namely male reproductive investment. This relationship is depicted by the path I have named the 'Triver's path' (light grey highlights in Figure 7) due to the pioneering theory on this topic proposed by Trivers (1972). As portrayed by the pipefish example in the Introduction, male reproductive investment can increase male 'time-out' from the mating pool and lead to a female-biased OSR. The Triver's path has become one of the most well-studied aspects of male mate choice theory (e.g., Burley 1977; Kokko and Monaghan 2001; Kokko and Johnstone 2002; Hooper and Miller 2008; Kokko and Jennions 2008). Empirical studies have explored male mate choice correlated with male reproductive investment in the form of, for example; large spermatophores (Rutowski 1982; Sakaluk 1985; Gwynne and Simmons 1990; Hyashi 1993; Savalli and Fox 1998; Fedorka et al. 2004; Paukku and Kotiaho 2005; Morse 2010) and male care of offspring (Berglund and Rosenqvist 1990; (Vincent et al. 1994; Wong and Svensson 2009). Male S. cyaneus do not provide any form of parental care, and there is no evidence that a substantial ejaculate or spermatophore is transferred to females during copulation (W. Foster, personal communication). Reports of male mate choice coinciding with an apparent lack of male reproductive investment and a high OSR have recently been increasing in a wide range of

22 taxa (for reviews see Amundsen 2000; Bonduriansky 2001; Hooper and Miller 2008). In Paper III, I suggest that male courtship costs may constitute a 'hidden' investment which many of these species (with male-male competition resulting from the male-biased OSR) will experience. I explore this possibility by disentangling the costs of i) male courtship, ii) copulation and iii) the combined effects of courtship and copulation by comparing three treat- ments with males housed alone, with females who were rendered unable to mate or with receptive females. Males remained in their assigned treatment groups from sexual maturity until death. Females were replaced regularly ensuring males were given ample opportunity to court and mate virgin females of a wide variety of phenotypes. I found that males showed additive longevity costs of coursthip and copulation, with males facing up to a 25% reduction in lifespan (Figure 8).

Figure 8. Survival across the three treatment groups; 1- Males housed alone, 2- Males only able to court females as they were housed with females rendered unable to mate, and 3- Males who were able to court and copulate as they were housed with receptive females. See Paper III for details.

My study, presented in detail in Paper III, comprises one of the few which have considered the importance of male courtship costs in determining the direction of selection on male mate choice (for another exception see Bonduriansky 2001). This may in part be due to the important role the costs of male signals play in maintaining the honesty of signals of quality (Zahavi 1975; Zahavi 1977; Grafen 1990a; Grafen 1990b; Kotiaho 2000; Getty 2006) through trade-offs with other fitness-related traits (Williams 1966; Cordts and Partridge 1996; Kotiaho and Simmons 2003; Paukku and Kotiaho 2005; Simmons and Kotiaho 2007). Alternatively, the lack of atten- tion given to courtship costs in mutual mate choice theory may be attribut- able to the fact that this concept is confounded by its implications for the

23 constraints on male choosiness (Parker 1983; Watson et al. 1998). If male mate choice is realised in part as increased courtship intensity or duration with preferred females (e.g., as in lizards, Weiss 2002; Ruiz et al. 2010: wolf spiders, Roberts and Uetz 2005; Taylor et al. 2006: cockroaches, Lihoreau et al. 2008: guppies, Herdman et al. 2004; Ojanguren and Magurran 2004: and fiddler crabs, Reading and Backwell 2007), then the cost of exhibiting a preference for a high quality female will increase not only due to the costs associated with male mate choice (see references above) but also as an intrinsic property of expressing the preference. However, males may not increase their total courtship output but rather redistribute their courtship with a bias towards preferred females (see also Increasing the benefits section below). In the next section I will explore how this intrinsic cost of choice may be offset by the benefits of male mate choice.

Increasing the benefits: can female preferences for male courtship effort play a role? A less explored option is that a reduction in the costs associated with male mate choice is not necessary to see the spread of male preferences, and that the evolution of male mate choice is instead driven by large benefits which offset the costs of choice. The benefits of male mate choice will depend on the variance in female quality, and thus the direct and indirect benefits of male mate choice, as I have depicted by the 'Female quality path' highlighted in dark grey in Figure 7. The majority of studies exploring the benefits of male mate choice have focussed on female quality variance in the form of fecundity variance (Parker 1983; Owens and Thompson 1994; Servedio and Lande 2006; Servedio 2007; Nakahashi 2008), as this is expected to constitute a large component of female fitness and therefore also their male mating partner's fitness (Bateman 1948). Empirical studies have confirmed that male preferences tend to be for large, virgin and therefore often more fecund females in variety of species including newts (Verrell 1986), spiders (Hack et al. 1997; Bel-Venner et al. 2008) and moths (Xu and Wang 2010). It is likely that the body size of female S. cyaneus is correlated with their fecundity, as has been shown in numerous studies of mosquitoes (Packer and Corbet 1989; Briegel 1990a; Briegel 1990b; Lounibos et al. 1990; Livdahl and Wiley 1991; Blackmore and Lord 2000; Briegel and Timmermann 2001; Armbruster and Hutchinson 2002). Since paddle size and body size are positively correlated in S. cyaneus (Paper I) and the paddles wear with age (personal observation), it is possible that the paddles act as honest signals of fecundity. Whether the benefits of choosing a more fecund female outweigh the increased competition costs associated with courting preferred females is

24 difficult to quantify, however, and will of course also depend on the variance in female fecundity. I began to consider if there were other potential benefits of male mate choice that had been overlooked. Following my observation in the mutual mate choice experiment (Paper II) that males were more likely to court preferred females, I began to consider how this would affect male mating success. I predicted that males with a preference may gain more mates in species where males express their preference for females through increased courtship and females prefer, and thus are more likely to mate with, males who court more. I explore this question in Paper IV using a series of haploid population genetic models which assume male polygyny, female monandry and thus intense male-male competition and high costs of male mate choice (based on the model of Servedio and Lande 2006). I found that, when males court preferred females more, a female preference for male courtship effort can lead to a spread of a male preference allele (Figure 9). This effect was dependent on the starting frequencies of the female trait and the male preference allele, which is intuitive as the higher the frequency of males with the preference relative to females with the trait, the higher the costs of competition for males.

Figure 9. Polymorphic equilibria in the two locus model (male preference allele P2 with frequency p2, and female trait allele T2 with frequency t2), for different levels of the strength of the female preference (a). The evolutionary trajectories to reach these equilibria are almost horizontal (very little evolution occurs in t2). For starting values above each line the equilibria generally lie along the line p2 = 1 (not shown). Throughout the figure the strength of the male preference (expressed as increased courtship) is equal. Increasing a leads to higher equilibrium frequencies of P2. See Paper IV for details.

25 In my model, males with a preference biased their courtship towards preferred females and correspondingly courted non-preferred females less, thus, males with a preference did not increase their total courtship output as in Servedio and Lande (2006). This assumption is important as it is improbable that males with a preference would have a higher total courtship output than males without a preference due to selection via female mate choice for opti- mal courtship output through fitness trade-offs (Kokko et al. 2002) and thus courtship as an honest signal of male quality (Pomiankowski 1987; Johns- tone 1995; Lailvaux and Irschick 2006; Seymour and Sozou 2009). Further- more, male mate choice expressed as increased courtship of preferred females will not result in a greater total cost of courtship under the biased courtship scenario and thus the costs of male mate choice will also be mini- mised (see Reducing the costs section above).

Interacting factors can affect the costs and benefits of male mate choice There are other interacting effects of factors on the costs and benefits of male mate choice in addition to the effects of males with a preference bias- ing their courtship towards preferred females. I have portrayed these effects by the finer diagonal dark and light grey lines in Figure 7 and the accompa- nying signs of the relationships they depict. Female quality variance may decrease the costs of male mate choice by reducing the costs of assessment as quality differences between females may be easier to detect. Male reproductive investment could both increase and decrease the benefits of male mate choice. For example, if males invest a much larger amount in care of the offspring than females and offspring survival is therefore largely dependent on the male's genotype, then the direct benefits to males of choosing a particular female could be reduced. Alternatively, a high male reproductive investment may increase the benefits of male mate choice as a male's PRR will be reduced and their reproductive fitness will become increasingly dependent on their partner's fecundity rather than their own mating rate (e.g., Jones et al. 2005). The evolution of male mate choice in itself may also feed back into the benefits of male mate choice, and will definitely affect the costs of male mate choice (as indicated by the signs of the curved thin black arrows in Figure 7). The evolution of male mate choice may increase the costs of male mate choice as males may face increasing competition from other males with a preference as the preference spreads (Servedio and Lande 2006). Alterna- tively, males may decrease competition costs through corresponding male and female preferences for mates of similar quality resulting in assortative mating (Fawcett and Johnstone 2003; Härdling and Kokko 2005; Venner et

26 al. 2010). The spread of a male preference may increase the benefits of male mate choice by enhancing the strong directional selection on high quality females (Nakahashi 2008). Alternatively, male preferences may reduce the benefits of male mate choice. An interesting example of how this may occur comes from a study in Drosophila which found that females preferred by males actually suffered decreased fecundity due to the costs associated with male harassment (Long et al. 2009). Finally, the evolution of male mate choice may have no, or little, effect on the benefits. This is especially likely if male preferences are based on indices of female quality which are under strong natural selection (e.g., body size) and the male preference function does not have the opportunity to act at the extremes due to strong viability selection removing the individuals from the population before male mate choice can occur (Lande 1981; Hall et al. 2000; Nakahashi 2008, see also Future directions section).

27 4. Why are females signalling? Exploring female mating rates and the opportunity for sexual selection in females.

I find the most exciting implication of my doctoral studies is the evidence of a female ornament under sexual selection by male mate choice in a species with a male-biased OSR and the resulting male-male competition. It is difficult to understand why females should invest in an ornament that appears costly in terms of production costs during ontogeny (Munoz et al. 2008), increased conspicuousness to predators (Godin and McDonough 2003) and impaired ability and energetic costs during flight (Evans 2004; Allen and Levinton 2007). In order for this female signal to evolve due to direct selection via male mate choice, there must be benefits to females of signalling that outweigh these costs (Maynard Smith and Harper 2003). Female mating rates may affect the opportunity of sexual selection on females through increased variation in components of female fitness that are positively correlated with their mating rate (i.e., a steeper Bateman gradient) (see Introduc- tion and references therein). Due to the apparently intense sexual selection resulting in the elaborate ornaments expressed in both sexes of this species, I predicted that S. cyaneus females are polyandrous (i.e., mate multiply). This would likely reflect a shift from monandry as the majority of mosquito species are monandrous (see My study system- General biology of mosquitoes and references therein). In Paper V I explored female mating rate by first mating females with sterilised males (by irradiation) and then providing them with the opportunity to mate with novel, virgin, fertile males for the remainder of their life- time and multiple gonotrophic cycles (achieved though repeated blood meals). Thus, an increase in the number of viable eggs after the introduction of fertile males would provide conclusive evidence of female remating. I found that females did not remate, as the slope of the proportion of eggs hatched with time did not differ from zero for the majority of females, and was negative for the remainder (Figure 10). This confirmed that, as in other species of mosquitoes, female S. cyaneus are monandrous and male S. cyaneus are polygynous (Paper III). Therefore, increased female mating rates (and a possibly steeper related BG) can not explain the apparently intense sexual selection on female S. cyaneus, as females cannot be under selection

28 to secure benefits of additional matings (Arnqvist and Nilsson 2000; Wedell et al. 2002).

Figure 10. The overall proportion of eggs hatched (pooled over all females) over time (per 7-10 day egg count interval increments). Line represents overall regression line and is included only to visualise the main trend in data. See Paper V for details.

Even if S. cyaneus females are monandrous they may benefit from attracting a large number of courting males that they can then choose between (Bradbury 1981). As female monandry precludes female post-copulatory choice (Eberhard 1996; Simmons 2001), a female's pre-copulatory choice of male may be even more consequential for her fitness under monandry than under polyandry (see Shuster and Wade 2003). The confirmation that female S. cyaneus are monandrous also affects the evolution of male preferences for female traits (Section 3). The mating rate of females may affect the benefits of choice to males (Section 3- Increasing the benefits, see also Bonduriansky 2001 for a review). For example, if females mate once only and reject all future mates, then there would be strong selection on males to effectively distinguish between virgin and mated females. As the paddles wear with age (personal observation), males may benefit from choosing females with large paddles who are also more likely to be unmated and therefore receptive. Opposingly, as discussed above, sperm competition is absent under female monandry and males may therefore not be under as strong selection to distinguish the mating status of females (Simmons 2001). Alternatively, if female fecundity is determined by their intrinsic quality rather than their mating rate and females are no longer able to be courted by males after copulation, there may be no effect of female mating rate on the benefits of male mate choice. However, this does not appear to be an option in S. cyaneus as mated females also perch on sticks and males court them eagerly (unpublished data).

29 Conclusions and future directions

My studies exploring selection on the sexually homologous ornaments of S. cyaneus provide evidence that both the male and female ornaments are under sexual selection via mate choice (Papers I and II). I found a significant longevity cost of male courtship in S. cyaneus which may result in a lower OSR (Paper III). This may facilitate the evolution of male mate choice by reducing the costs associated with choice. Furthermore, I show that, when females have a preference for male courtship effort, the costs of male mate choice may be offset by the increased female acceptance rate of males who express their preference by courting preferred females more (Paper IV). Somewhat surprisingly, I found that female S. cyaneus are monandrous and are therefore unlikely to be under selection to signal their quality to males in order to increase their mating rate (Paper V). Although females may benefit from signalling by attracting many males which they can then choose between, I feel that further factors remain to be elucidated in order to explain the evolution of female ornaments in S. cyaneus. I will briefly consider this topic further in this section by asking why males would show a preference for a trait which is likely to detract from the benefits likely to be of interest (i.e., female fecundity) and whether the assumption that all females are likely to mate solely due to a male-biased OSR is justified. I will also discuss why I believe species recognition hypotheses do not provide a unique explanation for the evolution of female ornaments. Finally, I will introduce some possible wider implications of male mate choice on individual and population fitness.

The relationship between costly female ornaments and the benefits of male mate choice As there needs to be significant benefits of male mate choice in order to offset the costs (see Section 3), it has been predicted that female ornaments which detract from female fecundity (and thus the benefits to males of choice) are unlikely to evolve (Fitzpatrick et al. 1995). Nonetheless, my mate choice experiment (Paper II) found that male S. cyaneus prefer large female paddles, a result supported by the morphometric study (Paper I) showing positive allometry.

30 Chenoweth et al. (2006) suggested that stabilising male preference functions for female ornaments may be one explanation for observations of male preferences for costly female traits. Unfortunately, we were unable to test for male preferences for female ornaments that were enlarged beyond the natural range, due to experimental constraints (i.e., the brittle structure of the paddles). Thus, it remains a possibility that the true male preference function in S. cyaneus is stabilising yet viability selection has removed individuals with large trait values from the population before mate choice can occur (Lande 1981; Hall et al. 2000; Nakahashi 2008). An alternative explanation is that the viability optimum for trait size may be higher than the fecundity optimum (Nakahashi 2008). Thus, if one estimates the absolute fitness of individuals bearing the trait allele as the average fecundity multiplied by the proportion surviving (Hunt and Hodgson 2010); females with the trait can have the highest absolute fitness despite a small viability fitness component. Alternatively, it may be that the underlying male preference function for female paddles is indeed directional, and that there are other conditions that can lead to the evolution of directional and open-ended male preferences. For example, Chenoweth et al. (2006) assumed an exponentially increasing cost function. Other cost functions, such as a linear cost function, can result in signals of direct benefits becoming correlated with condition (Price et al. 1993). The proposal that further exploration of the theory explaining the evolution of directional male preferences for apparently costly female signals of direct benefits is supported not only by this study, but also an increasing number of other empirical studies (e.g., LeBas et al. 2003; Siefferman and Hill 2005; Bitton et al. 2008; Doutrelant et al. 2008; Simmons and Em- len 2008; Wright et al. 2008).

Female mating rates and the opportunity for sexual selection as assumed by the OSR In a recent review, Rhainds (2010) noted the pervasive paradigm in the entomological literature that all females are likely to mate in mating systems characterised by a larger female than male reproductive investment, a male- biased OSR and the resulting intense male-male competition. This is a paradigm which is reflected in the prediction that female fitness is largely determined by their intrinsic fecundity rather than their mating rate (Bateman 1948). Rhainds (2010) explored whether this episteme is supported in empirical studies by comparing mating failures in females (i.e., that females do not mate) in 100 species of arachnid and insect species. This review revealed that female mating failures are common. High female mating failures are associated with, for example, low population density (i.e., the Allee effect, Allee 1932), a female-biased OSR (e.g., Unnithan and Saxena 1991; Rhainds

31 et al. 1999) and phenotypic variation in body size with large females most likely to mate (e.g., Rhainds et al. 1999; del Castillo and Núñez-Farfán 2002). The relationship between female body size and likelihood to mate may be the result of male mate preferences for large females (see Bonduriansky 2001 and Rhainds 2010, and references therein). Thus, it is possible that in S. cyaneus, factors such as a strong male mating preference for large females and/or a low population density may lead to a large proportion of females who fail to mate. This in turn may lead to selection on females to develop traits (such as the ornaments) which increase their attractiveness to males and therefore likelihood to mate. However, surely if the OSR was male-biased and males were therefore competing intensely for females, selection for mate attraction or location mechanisms would be on the males and not females. This prediction ties in with the prediction that males of species with a male-biased OSR should not be choosy when their mating opportunities are limited due to the costs of choice (see Section 3). Thus, the evolution of male mate choice driven by the fecundity benefits of choice (rather than by a reduction in the costs) may play an important role in determining female fitness components resulting from their mating success and/or rate.

Species recognition hypotheses- not an alternative to sexual selection It has been suggested that female traits have evolved as a species recognition mechanism, thereby facilitating matings between individuals with similar recognition systems (Paterson 1978). Paterson (1985) argued that differences between species could evolve between sympatric (or parapatric) populations with the sole purpose of reproductive isolation. This episteme has its base in a body of theories widely accepted during the 1960s and 1970s which predicted reproductive isolation would be beneficial during interspecific competition (for a review see Cardé and Baker 1984). I believe that there is no direct selection for species recognition because the maintenance of species boundaries is not necessarily adaptive. Nonetheless, hybrid offspring are often inferior to offspring produced from conspecific matings, and there is abundant evidence that individuals prefer conspecifics (for a review see Coyne and Orr 2004). In my opinion, preferences for conspecifics most likely reflect the rapid divergence of intraspecific mating preferences and sexually selected traits rather than hybrid avoidance in itself. These traits may then play a secondary role in the avoidance of hybrid production when the post-zygotic barriers to reproduction are present (e.g., hybrid inviability or sterility) (West-Eberhard 1984) and may sometimes result in further character displacement in reproductive traits due to reinforcement (see below).

32 However, when traits and preferences have diverged rapidly as predicted by sexual selection theory (Coyne and Orr 2004), individuals will often show preferences for traits which only conspecifics possess even in the absence of selection against hybrids. When closely related species have not sufficiently diverged in their mating preferences and traits for complete pre-zygotic reproductive isolation, and inferior hybrids are thus often produced, there may be selection on individuals to avoid mating with members of other species. However, this does not form a unique body of theory with predictions unique to any other mating preference which has evolved due to the indirect fitness benefits (e.g., female preferences for MHC diversity, Tregenza and Wedell 2000; Milinski 2006). Löfstedt (1993) argued that female moth pheromones were the result of natural selection acting against inferior hybrids. However, if one rejects the episteme that the maintenance of species boundaries is adaptive in itself, one quickly can see that male mating preferences for female moth pheromones must be the result of sexual, not natural, selection via male mate choice. Imagine a situation where hybrids have a fitness 2% that of offspring from a conspecific mating. Now consider the relative cost of hybrid production for males and females in a population where males invest less in reproduction and have a higher PRR and a steeper BG than females. This will result in a large opportunity for sexual selection in males. Many of the males will therefore be likely not to mate at all, and therefore have a fitness of zero. Thus, a heterospecific mating may still result in a fitness benefit for these males. Additionally, given that males have a higher PRR than their realised rate of reproduction (due to a lower female PRR), the time lost on a heterospecific mating will not pose a large cost to future mating success. A female on the other hand will pay large costs of mating with a heterospecific male. This will result in selection primarily acting on female preferences for conspecifics rather than male preferences, unless the costs of male mate choice are reduced due to a lower OSR. However, if the benefits of choosing a conspecific are large enough relative to that of mating with a heterospecific to outweigh the costs of male mate choice, one may see the evolution of male preferences for conspecifics. Thus, it is clear that predictions determining the strength of selection on male and female mating preferences for conspecifics do not differ in any way from those generated by extant sexual selection theory.

The contribution of male mate choice to population fitness? My study joins a growing number of empirical reports of male mate choice in species with an apparently male-biased OSR and resulting male-male

33 competition (for reviews see Amundsen 2000; Bonduriansky 2001; Hooper and Miller 2008). In many of these species males are reported to show preferences for more fecund females. Thus, male mate choice coincides with viability selection on females and was therefore predicted not to significantly affect population fitness (Bonduriansky 2009). This prediction was chal- lenged by a recent study in Drosophila melanogaster by Long et al. (2009) who found that male preferences for large, more fecund females actually led to a decrease in the mean female fecundity of a population. This is likely because large females suffered from the costs of male harassment (Partridge and Fowler 1990) and possibly from receiving more harmful ejaculates (Chapman et al. 1995). Another possibility is that, when there is a mismatch between the fecundity and viability optimum for a preferred female trait, and the male preference follows the fecundity optimum (Nakahashi 2008), the effects of the male preference on population fitness will vary with a chang- ing environment and the resulting selective pressures. My study also presents another exciting avenue for the possible population fitness effects of male preferences, namely the evolution of costly female ornaments. Although individual female fitness may be increased by expressing a costly trait and thereby attracting more males (or by mating at all), the mean female fitness of a population may be decreased as females may suffer a fecundity cost of expressing the trait. The population fitness effects of male mate choice and female ornaments remains largely unexplored. An obvious place to begin is experiments exploring effects known to result from female mate choice, such as artificial selection experiments conducted both with and without the opportunity for male mate choice. Another exciting possibility is that female gametes may compete in externally fertilising species or species with male incubation; plausibly leading to selection acting on female gametes which is analogous to post-copulatory sexual selection acting on male gametes (e.g., sperm competition, Simmons 2001; Wedell et al. 2002). I believe that empirical studies exploring the population fitness effects of male mate choice and female ornaments will elucidate exciting new mechanisms and provide valu- able insight into the intriguing traits possessed by many species.

34 Sammanfattning på svenska

Evolutionen av homologa ornament hos hanar och honor: hanligt partnerval under hanlig konkurrens hos en neotropisk mygga Extraordinära hanliga signaler, såsom påfågelhanens stjärtfjädrar, utgjorde en gång i tiden en utmaning för Charles Darwin. Han sökte förklara hur signaler såsom dessa, vilka uppenbarligen minskade överlevnaden hos deras bärare, kunde utvecklas. Darwin föreslog att sådana hanliga signaler har utvecklats via sexuell selektion i form av honliga preferenser för vissa hanliga karaktärer under partnerval och/eller via konkurrens mellan hanar över tillgången på honor. Studiet av sexuell selektion har under de senaste decen- nierna växt till att bli ett av evolutionsbiologins mest aktiva forskningsfält. Vi vet idag att sexuella selektion bland hanar generellt sett är starkare ju fler sexuellt aktiva hanar det finns i förhållande till honor i en population och ju starkare relationen är mellan antalet parningar och fitness hos hanar jämfört med hos honor. Således finner vi stark sexuell selektion hos hanar när hanar investerar mindre i reproduktionen än honor och därför har potential att para sig med fler partners, vilket är vanligt hos många djurarter. Då hanar är rela- tivt talrika favoriseras egenskaper vilka ökar hanars förmåga att konkurrera om parningar, antingen genom direkt konkurrens med andra hanar eller genom att konkurrera om honornas gunst. Hos en del djur investerar hanar mer i reproduktionen än vad honorna gör, såsom hos kantnålsfiskar som uppvisar hanlig vård av ägg, och där honorna istället konkurrerar om hanarnas gunst. I mitt avhandlingsarbete har jag fokuserat på evolutionen av karaktärer som har utvecklats som en direkt följd av parningspreferenser hos reproduktiva partners, så kallade ornament. Då vi jämför hanar med honor har vi av tradition utgått ifrån att styrkan av parningspreferenser hos ett kön är negativt korrelerad med intensiteten i konkurrensen inom detta kön. Detta har följt från resonemanget att individer av det kön som konkurerar mest med varandra inte ’har råd att vara kräsna’. På senare tid har det emellertid visat sig att hanar ofta föredrar vissa typer av honor, trots intensiv konkurrens hanar emellan. Detta beror sannolikt på att hanar kan dra nytta av preferenser, till exempel genom att välja att para sig med mer fruktsamma honor, och därigenom kan uppväga de kostnader som är förknippade med partnerval (t.ex. en ökad konkurrens om de mest fruktsamma honorna). I motsats till honligt partnerval förväntar vi oss dock inte

35 att hanligt partnerval normalt sett ska vara baserat på rent dekorativa egenskaper hos honor. Detta beror, enkelt uttryckt, på att honliga ornament (vilka är kostsamma för honor) minskar de fördelar som hanar kan uppnå via hanligt partnerval. Dessutom är det svårt att förstå varför honor skulle dra nytta av att signallera för att attrahera många hanar i situationer då det finns ett överflöd av hanar i populationen. Trots detta finns det ett ökande antal rapporter om honliga ornament hos arter med reproduktiv konkurrens hanar emellan. Honliga ornament som har sin direkta motsvarighet hos hanar (dvs är homologa) antogs tidigare ofta vara icke-adaptiva hos honor och ett resultat av en genetisk korrelation mellan könen. På senare tid har man emellertid börjat beakta möjligheten att sådana honliga ornament även kan vara ett resultat av hanligt partnerval, även hos arter med konkurrens hanar emellan. Min studie-art: Sabethes cyaneus Mitt avhandlingsarbete har rört evolutionen av hanliga och honliga ornament hos arter med påtaglig konkurrens hanar emellan. Jag har utforskat detta fält genom att utveckla teoretiska modeller och genom experimentella studier av en neotropisk insekt – myggan Sabethes cyaneus (Diptera: Culicidae). Både hanar och honor av S. cyaneus uppvisar spektakulära, regnbågsskimrande paddel-liknande ornament på deras mellanben, något som gör denna grupp av myggor (Sabethini) unik då andra myggor saknar liknande utsmyckning- ar. Denna art är på många sätt ett perfekt system för att utröna evolutionen av honliga ornament, då hanar vid ett ytligt betraktande inte förefaller investera mer än spermier i reproduktionen medan honor investera mer både i form av produktionen av ägg och genom kostnader i samband med att honor söker värdar för att suga blod. Tidigare forskning har visat att, som förväntat, hanar förefaller konkurrera över honor och hanar uppvaktar honor via ett komplext uppvaktningsbeteende. Förekomsten av ornament hos honor hos S. cyaneus är därför en utmaning för gällande sexuell selektionsteori.

36 Mål I min avhandling undersöker jag evolutionen av hanliga och honliga ornament hos S. cyaneus genom att söka besvara följande frågor: 1. Är honliga ornament hos S. cyaneus enbart ett resultat av en genetisk korrelation mellan könen? (Arbete I) 2. Förekommer hanligt partnerval av honliga ornament hos S. cyaneus? (Arbete II) 3. Hur kan hanligt partnerval och konkurrens hanar emellan uppträda samtidigt hos S. cyaneus? Här har jag utforskat flera distinkta kostnader och fördelar. Reducering av kostnader: utgör uppvaktning av honor en betydande reproduktiv investering för S. cyaneus hanar? (Arbete III) Ökning av fördelar: kan honliga preferenser för hanligt uppvaktningsbeteende spela en roll? (Arbete IV) 4. Varför signalerar honor? Här har jag undersökt potentiella fördelar och kostnader för honor. (Arbete V) Är honliga ornament hos S. cyaneus enbart ett resultat av en genetisk korrelation mellan könen? Jag har utforskat denna fråga i arbete I, genom att noga undersöka den feno- typiska variationen i form och storlek av hanliga och honliga ornament (hä- danefter ’paddlar’) med hjälp av geometrisk morfometri. Jag fann mycket ringa könsdimorfism i paddelns form och storlek. Detta tyder på att det är osannolikt att honliga paddlar enbart är ett resultat av en genetisk korrelation, eftersom kostnader i samband med paddeltillväxt skulle resultera i ett starkt selektionstryck mot paddlar hos honor och därigenom en minskning av deras storlek. Jag fann dessutom flera ytterligare bevis för att paddlar hos honor inte bara är ett resultat av en genetisk korrelation utan under direkt selektion. Faktum är att dessa honliga ornament uppvisade många egenskaper som är klassiska kännetecknen för sexuellt selekterade karaktärer. Likväl är det inte omöjligt att kostnaderna för ornamentens uttryck är tillräckligt lågt hos honor för att resultera i ett icke-adaptivt uttryck. Jag tolkade därför dessa resultat med viss försiktighet och använde denna studie primärt som en motivation för att undersöka direkt partnerval av dessa ornament hos både hanar och honor. Är honliga ornament hos S. cyaneus under sexuell selektion via hanligt partnerval? Efter de något överraskande bevisen för sexuell selektion på honliga paddlar hos S. cyaneus som presenterades i arbete I, bestämde jag mig för att vidare utforska direkt val av både hanlig och honlig paddelmorfologi via ömsesidigt partnerval. Jag gjorde detta i arbete II. Paddelns storlek ändrades i dessa

37 experiment, genom direkt fenotypisk manipulation. Den experimentella de- signen inbegrep studiet av replikerade grupper av oparade hanar och honor vars paddlar antingen var (i) intakta, (ii) minskade till sin storlek eller (iii) helt avlägsnade. Alla individer märktes och jag registrerade sedan alla reproduktiva interaktioner mellan individer. Jag fann något överraskande att hanar, men inte honor, baserar sitt partnerval på partnerns paddelmorfologi. Som diskuterats ovan är detta förvånande, då hanar förväntas erfara kostnader av partnerval och främst förmodas uppvisa preferenser för honliga egenskaper som indikerar hög fekunditet hos honor (t.ex. kroppsstorlek). Dessut- om förväntas inte honor vara under selektion för att signalera, då de sannolikt kommer att attrahera fler hanar än vad de behöver för att para sig vid ett optimalt antal tillfällen. Jag fortsatte därför att ytterligare utforska de faktorer som kan påverka denna dynamik. Hur kan hanligt partnerval och konkurrens hanar emellan upp- träda samtidigt hos S. cyaneus - kostnader och fördelar? Reducering av kostnader: utgör uppvaktning av honor en betydande reproduktiv investering för S. cyaneus hanar? I arbete III undersökte jag möjligheten att hanar kan ha "dolda" reproduktiva kostnader vilka minskar deras parningsfrekvens och vilka därmed kan ändra förhållandet mellan sexuellt aktiva hanar och honor. Jag undersökte om hanlig investering i uppvaktningsbeteende skulle kunna utgöra en sådan kost- nad. Jag åstadkom detta genom att experimentellt separera kostnaderna för (i) hanlig uppvaktning, (ii) parning och (iii) den kombinerade effekterna av uppvaktning och parning, i en serie försök där jag jämförde tre behandlingar: (i) hanar hållna ensamma, (ii) hanar hållna med honor som inte kunde para sig och (iii) hanar hållna med normala och mottagliga honor. Jag kunde här dokumentera betydande kostnader (ca 25% minskning av livslängden) för hanar med såväl uppvaktning av honor som av parningar i sig. Det är dock osannolikt att denna investering i reproduktion hos hanar överstiger de inve- steringar som görs av honor. Jag drar därför slutsatsen att det är troligt att hanligt partnerval är behäftat med väsentliga fördelar för hanar. Ökning av fördelar: kan honliga preferenser för hanligt uppvaktningsbeteende spela en roll? I arbete IV undersökte jag möjligheten att hanliga parningspreferenser kan spridas genom en honlig preferens för hanligt uppvaktningsbeteende. Om hanar uttrycker preferens genom att företrädesvis uppvakta attraktiva honor, och därmed ökar chansen att bli accepterade av dessa honor, finns det en möjlighet att de därigenom kan övervinna den konkurrensnackdel som de upplever genom att uppvakta honor som har många potentiella partners. Jag undersökte detta scenario i en serie haploida populationsgenetiska modeller, vilka antar hanlig polygyni, honlig monandri och därmed en stark konkur-

38 rens mellan hanar och höga potentiella kostnader för hanligt partnerval. Jag fann att när hanar uppvaktade attraktiva honor mer, kan en honlig preferens för hanligt uppvaktningsbeteende leda till en spridning av gener för hanlig partnerpreferens. Detta är en unik fördel av hanligt partnerval vilken, så vitt jag vet, inte har tidigare har undersökts. Varför signalerar honor? I arbete III och IV fann jag belägg för faktorer som främjar hanligt partnerval hos S. cyaneus. Men varför honor har evolverat ornament när de sannolikt blir uppvaktade av mer än tillräckligt många hanar förblev något av ett mysterium. Jag beslutade därför att undersöka hur ofta S. cyaneus honor parar sig. Hos de flesta myggor parar sig honor endast en enda gång i sitt liv. Om honor parar sig flera gånger kan detta emellertid leda till att variansen i honlig fitness ökar, som ett direkt resultat av skillnader i parningsfrekvens. Detta kan i sin tur leda till naturlig selektion hos honor för att attrahera flera hanar genom signalering. Jag förutsade därför att honor av S. cyaneus skiljer sig från honor av andra myggor genom att para sig med flera hanar. Jag undersökte denna möjlighet i arbete V, där jag först lät honor para sig med en steril hane (steriliserad genom bestrålning) och sedan ge honor möj- lighet att para sig med nya, oparade normala hanar under återstoden av sin livstid och genom flera gonotrofiska cykler. En ökning av antalet fertila ägg efter införandet av fertila hanar skulle här utgöra bevis på att honor parar sig fler än en gång. I motsats till min förutsägelse fann jag att S. cyaneus honor endast parar sig en enda gång under sitt liv. Även om detta sannolikt reduce- rar selektionen för ornament hos honor finns fortfarande en möjlighet att honor kan dra nytta av att attrahera många hanar för att därigenom utöka antalet hanar som de sedan kan välja mellan. Slutsatser Min avhandling är ett sällsynt men tydligt exempel på förekomsten av hanligt partnerval av ett honligt ornament hos en djurart där konkurrensen hanar emellan förefaller stark. Resultaten har viktiga konsekvenser för sexuell selektionsteori och indikerar att förhållandena för evolutionen av honliga ornament inte är så restriktiv som man tidigare trott. Jag tror också att fram- tida undersökningar av effekterna av sexuell selektion hos honor på popula- tionsfitness kan komma att generera flera viktiga nya insikter.

39 Acknowledgements

My deepest thanks to my primary supervisor Göran. I am really grateful to you for introducing me to this unique system and gave me the opportunity to work with you on them. I think that I am in a rather unique position as I have not once during my doctoral studies felt uninspired. This is partly due to the mosquitoes themselves, everything that I have learnt about these peculiar species has only made understanding them in terms of current sexual selection theory even more of a challenge. Nevertheless, my passion for the theoretical implications of my work could not have developed without your guidance, wealth of knowledge on sexual selection and statistical methods, lively discussions and opposition. I always found our discussions challeng- ing and inspiring and I hope that I can look forward to many more in the future! To my secondary supervisor, Niclas, I am extremely grateful that you agreed to take me on. I particularly appreciate all the support you gave me during the planning and execution of my experiments, not only for encourag- ing me not to give up when everything seemed to be going wrong but also for coming up with practical solutions. I also appreciate your ability to laugh at any situation, and make me laugh, no matter how bad it seems! To Woody and Robin, this project would have been impossible without your kind donation of the Sabethes to my laboratory and your advice on rearing and conducting experiments on mosquitoes. Thank you for always quickly responding to my many queries since Woody, the depth of your knowledge of and experience working with mosquitoes awes me. Spending the second month of my doctoral studies with you both in Ohio remains one of my fondest memories, I hope that I can soon return to go out for some good Ethiopian food with you both! To Maria, thank you so much for bothering to reply to my first inquisitive mail and then for walking me through how exactly we could explore the question I was asking. You never once made me feel stupid or like you didn't have time for my confused emails (which there have been plenty of), I couldn't have had a better introduction to modelling! Thank you also for letting me come to you in person for a couple of weeks. To Ray, thank you for taking the time to show me around the forests of North-Western Trinidad and welcoming me into your home. Thank you also to my field assistants Anil, David and Sylvester (aka Johnny or Bud). A special extra thanks to Anil and his family for their hospitality at Christmas.

40 I would also like to extend a special thanks to Ingrid. Your positive comments and kind words really have helped me to deal with my fear of public speaking and many other moments of insecurity during my studies. I am also grateful to both yourself and Anders for being kind enough to share with me a little of your wealth of knowledge on 'sex roles' and all that is good and bad about the term itself. I am also honoured to have had the opportunity to work with Mats, Anna, Ted and Lars, at multiple points during my doctoral studies your insightful comments have made me realise that there is so much I want to learn! Thank you to Reija and Ingela for helping me with all sorts of annoying little- and not-so-little problems. A special thanks also to Bo Stenerlöw for the help with the caesium source for irradiating the mosquitoes. I would also like to acknowledge the Swedish Research Council grant awarded to Göran Arnqvist and the grants awarded to me which funded my attendance at various conferences throughout my PhD, the purchase of literature and laboratory running costs; ASAB conference travel grant, Stiftelsen för Zoologisk Forskning, Rektors Wallenbergmedel and EBC Graduate School on Genomes and Phenotypes research travel grant to UNC. I would also like to thank John for hosting me during my six month sabbatical at the University of Exeter in Cornwall where I got to take a break while working with the simple concept of 'condition'. I am really happy that I got to meet you; I have learnt a lot from you (and have much much more to learn), you have always been there for a chat and advice when I need it, and your work and personal ethics are inspirational! I was grateful to receive funding for this stay from Stiftelsen för Zoologisk Forskning, KVA, Nilsson-Ehle Dona- tionerna and Anna Marie Lundins stipendiefond from Smålands Nation. I owe extra thanks to Mirjam who not only repeatedly helped me by taking care of my delicate mosquitoes while I was away, but who was also a much-appreciated office mate for the past few years. Thank you also to Dianna: I am so glad you decided to come and do a Masters with me- my most enjoyable lab memories are those when we were working down there together! Thank you also for continuing to show an interest in the mosquitoes and my work. Thanks also to Katherine, Sara and Johan for taking care of my mosquitoes while I was away. Thank you especially to all the people who were kind enough to get involved in my work by proof-reading, listen- ing to me practice talks and/or giving me advice on grant applications and papers: Alex M., Björn R., Claudia F., Dad, Damian D., Jessica A., Johanna R., John H., Judith M., Kate St O., Locke R., Mark E., Martin E., Mia O., Mum, Nina W., Peter H.-R., Simone I., Ted M., Trine B. and Urban F.. I would also like to thank all the people at the EBC whose friendship, inspiring and/or relaxing conversations and advice have always made me look forward to coming into work. A special final thanks to Mum, Dad, Michael, Richard, Peter and my friends both here and overseas who have provided me with unconditional love and support even during my absence.

41 References

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