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1 Animal Behaviour 78 (2009) Contents lists available at ScienceDirect Animal Behaviour journal homepage: Sexual selection for genetic quality: disentangling the roles of male and female behaviour Nina Pekkala *, Mikael Puurtinen, Janne S. Kotiaho Centre of Excellence in Evolutionary Research, Department of Biological and Environmental Science, University of Jyväskylä article info Article history: Received 7 May 2009 Initial acceptance 9 July 2009 Final acceptance 24 August 2009 Available online 15 October 2009 MS. number: Keywords: condition dependence deleterious mutation Drosophila montana good genes ionizing radiation According to the good genes model of sexual selection, females choose males of good heritable genetic quality to obtain offspring with high fitness. However, better mating success of high-quality males can also be brought about by direct interference competition between males, or simply through elevated activity of high-quality males. We examined the roles of different processes leading to sexual selection for genetic quality in Drosophila montana. We manipulated genetic quality of male flies by inducing mutations with ionizing radiation. We then recorded the effects of inherited heterozygous mutations on several aspects of mating behaviour of males and females in two experiments. We found that mutations reduced the probability of courtship and extended the latency to courtship of the males, suggesting male activity plays a role in selection for genetic quality. However, the effects of mutations on mating success and mating behaviour of the flies were in general weak. No evidence for female mate choice or interference competition between males acting against heritable mutations was found. Ó 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. Sexual selection through mate choice, and in particular mate choice for indirect fitness benefits, is a major paradigm that today seems to enjoy almost unequivocal acceptance. A large body of theoretical work has been built to explain the evolution of mate choice in the absence of direct benefits (Andersson 1994; Andersson & Simmons 2006; Kokko et al. 2006), most of which critically depends on a few assumptions: male sexual signals are costly, and, because they are costly, they develop positive condition dependence (Andersson 1982, 1986; Rowe & Houle 1996; Kotiaho 2000, 2001; Hunt et al. 2004; Tomkins et al. 2004). Male condition depends on many underlying physiological and morphological traits and thus sums genetic variation over many loci (Rowe & Houle 1996; Kotiaho et al. 2001; Tomkins et al. 2004). This multitude of loci provides a large target for deleterious mutations. By choosing males with elaborate condition-dependent sexual signals, females can thus avoid males with deleterious mutations and have offspring with good genetic quality and high fitness. These so-called good genes models and related research emphasize the role of female mate choice in sexual selection for * Correspondence: N. Pekkala, Centre of Excellence in Evolutionary Research, Department of Biological and Environmental Science, P.O. Box 35, FIN University of Jyväskylä, Finland. address: nina.a.pekkala@jyu.fi (N. Pekkala). genetic quality of the male. However, as Whitlock & Agrawal (2009) pointed out, genetic quality can affect mating success by its effects on general activity and vigour of the male. Besides female choice, competition among males for matings can come in many forms, some involving direct communication or contact between males (interference competition) and others only indirect competition, for example via differences in the activity of males (Huntingford & Turner 1987; Andersson 1994). If the outcome of the mating competition among males depends on the condition of the male (Ligon et al. 1990; Kotiaho et al. 1999), and condition is determined by genetic quality, the winner of the competition should be of high genetic quality. Thus, competition among males, whether direct or indirect, can lead to the same outcome as female mate choice: apparent benefits in terms of good genes to the offspring. Clearly, the process of sexual selection can operate in many different ways, and special experimental designs are needed to unravel the mechanisms generating mating biases (see e.g. Wong & Candolin 2005; Kotiaho & Puurtinen 2007; Fitze et al. 2008). Theoretical models suggest that sexual selection could reduce mutational load from populations, but only a few empirical studies have evaluated the effectiveness of sexual selection against deleterious mutations and none of these studies have detailed the process of sexual selection for genetic quality (reviewed in Whitlock & Agrawal 2009). Our aim was to study experimentally the effectiveness of sexual selection against deleterious mutations, as /$38.00 Ó 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. doi: /j.anbehav

2 1358 N. Pekkala et al. / Animal Behaviour 78 (2009) well as to disentangle the relative importance of female mate choice, interference among males, and male activity for sexual selection for genetic quality. We manipulated genetic quality of Drosophila montana by exposing the fathers of the experimental males to ionizing radiation known to produce a wide range of mutations in DNA (reviewed in Evans & DeMarini 1999). To mimic the situation in natural populations where novel mutations are likely to occur mainly as heterozygotes, we studied the effects of mutations from males heterozygous for the new heritable mutations. METHODS Study Species Drosophila montana is a boreal species belonging to the D. virilis group of Drosophila flies. In nature, D. montana flies gather on food patches to court and mate (Aspi et al. 1993). Courting consists of the male chasing the female, touching the female with its legs, head and mouthparts, and producing a courtship song by vibrating its wing (Hoikkala & Lumme 1987; Liimatainen et al. 1992; personal observation). Courtship song is a species-specific signal that females require before allowing the male to mate (Liimatainen et al. 1992). Both female choice and male male competition are made possible at the gatherings of the flies, and the species has become a model species for sexual selection studies (Hoikkala & Aspi 1993; Aspi & Hoikkala 1995; Hoikkala et al. 1998; Ritchie et al. 1998; Hoikkala & Suvanto 1999; Klappert et al. 2007). Maintenance and Manipulation of Flies The flies for the study came from a laboratory stock originating from Kawasaki, Japan. The stock had been inbred for 20 generations with full sib mating to create a (nearly) homozygous line, and then reared in bottles containing malt medium in constant light at 19 1 C. For the experiments, the flies were sexed under CO 2 anaesthesia 1 4 days after their emergence and maintained in single-sex groups in plastic vials (diameter 23.5 mm, height 75.0 mm) containing malt medium. We manipulated genetic quality of randomly selected 4 7-dayold male flies by inducing new mutations with ionizing gamma radiation. An inbred line was used to minimize the initial variation in genetic quality so that the effects of induced mutations would be easier to detect. Nine doses of radiation were used: 0, 5, 10, 15, 20, 25, 30, 35 and 40 Grays (Gy). The number of mutations induced is expected to increase with increasing dose of radiation (Edington 1957; reviewed in Evans & DeMarini 1999). For the 0 Gy level (control) the flies were treated otherwise the same but were not given any radiation. The maximum amount of radiation was set to 40 Gy because of the substantial decrease in fertility of the males at high doses of radiation (Fig. 1). We treated males for each radiation dose. Once the irradiated males were sexually mature (14 17 days old) they were each mated to a nonirradiated stock female. Male offspring of these matings, heterozygous for the new heritable mutations, were used in the experiments. These males are called experimental males from here on. For the last 7 days before the experiments, the experimental males were each reared individually in a plastic vial containing malt medium. For mating experiment 2 (see Experimental set-up), male flies from the laboratory stock were anaesthetized with CO 2 1 week before the experiments and marked for identification by cutting off a small piece from the tip of their wing. After that they were reared in plastic vials containing malt medium in groups of four until the experiments. Number of offspring (95% CI) All flies used in the experiments were virgin. The flies were days old during the experiments, the age distribution between the radiation treatments and the two mating experiments being the same. Experimental Set-up Figure 1. Relationship between radiation dose and fertility of the irradiated males. Symbols represent mean number of offspring and error bars the 95% confidence interval. Offspring were counted from tubes where a stock female and an irradiated male (or a control male in the case of 0 Gy) were allowed to mate and lay eggs for 4 days (linear regression: F 1,435 ¼ , P < 0.001). We aimed to determine the roles of different processes of sexual selection for genetic quality by examining the effects of inherited heterozygous mutations of the experimental males on several aspects of mating behaviour of the flies. We conducted two mating experiments. In mating experiment 1 (ME1), an experimental male was introduced to a stock female. In mating experiment 2 (ME2), an experimental male and a stock male were simultaneously introduced to a stock female. In ME1, effects of inherited mutations on mating behaviour of the flies are thus an outcome of female choice and/or activity of the male, whereas in ME2 there is also a possibility for interference among males and an opportunity for the female to compare the two males and thus choose between them. By comparing the effects of inherited mutations on different aspects of mating behaviour in the two experiments we can evaluate the relative importance of female choice, interference among males, and male activity on sexual selection for genetic quality. In both mating experiments we recorded the following aspects of behaviour of the experimental male or the female. (1) Male courtship (yes/no). Male courtship was recorded as a discrete variable: male courted or did not court the female. Any form of male courtship behaviour (chasing, touching or producing a courtship song) was recognized as courtship. In ME1, male courtship was considered to indicate activity of the male. In ME2, male courtship could also be influenced by interference among males. We expected mutations to reduce courtship of the males. (2) Latency to courtship (s). Latency to courtship is a measure of how long it takes the male to start courtship (i.e. time between beginning of the experiment and beginning of courtship). In ME1, latency to courtship was considered to indicate activity of the male. In ME2, latency to courtship could be further influenced by interference among males. We expected mutations to extend latency to courtship. (3) Amount of courtship required by female (s). Courtship of D. montana males typically consists of periods of courting and periods of not courting. In D. montana, forced copulations are extremely rare. Summing the periods of active courtship before mating thus gives the amount of active courtship required by the female before mating. The

3 N. Pekkala et al. / Animal Behaviour 78 (2009) amount of courtship required by the female was considered to be influenced by female choice. We expected females to require longer courtship from males with more mutations. (4) Mating success (yes/no). Mating success was recorded as a discrete variable: the male mated or did not mate. In ME1, mating success could be influenced by female choice and activity of the male. In ME2, interference among males and relative female mate choice could have an additional effect. We expected mutations to reduce mating success of the males. (5) Latency to mating (s). Latency to mating measures how long it takes the male to mate with the female (i.e. time between beginning of the experiment and beginning of copulation). In ME1, latency to mating could be influenced by male activity and female choice. In ME2, interference among males and relative female mate choice could also play a role. We expected mutations to extend latency to mating. (6) Duration of mating (s). If females mate with several males, extended copulation may be beneficial to the male because of different aspects of sperm competition (Simmons 2001). Multiple mating with several males is common among D. montana females, although why this occurs is not known (Aspi 1992; Aspi & Lankinen 1992; see also Arnqvist & Nilsson 2000). Drosophila montana females terminate the copulation by vigorously kicking with their hindlegs to dislodge the male. The males try to resist this behaviour, and prefer longer copula durations than females (Mazzi et al. 2009). Duration of mating could thus depend on both female choice and persistence (activity) of the male. We expected mutations to shorten mating duration of the males. (7) Order of courtship (first/second). Order of courtship was recorded as a discrete variable (in ME2 only): the experimental male started to court first or second. If only one of the males courted the female, the courting male was considered to be the first to court and the noncourting one to be the second. Order of courtship could be affected by the activity of the male and interference among the males. We expected mutations to reduce the probability of being the first to court. The experiments were conducted in petri dishes (diameter 5.0 cm, height 0.7 cm) covered with nylon net. The floor of the dish was covered with a moistened filter paper to create optimal conditions for the flies. The paper was allowed to dry before use in a new trial. First the male (in ME1) or males (in ME2) and then the female were placed into the petri dish. Timing began from introduction of the female. We observed the behaviour of the flies until copulation occurred, or until 30 min had elapsed. Both mating experiments were performed each day in randomized order for 25 days, ME1 twice a day and ME2 four times a day. For each experiment, all treatments of radiation were observed simultaneously. Thus, the environmental conditions (temperature, time of day, air moisture, etc.) did not differ between the radiation treatments. In ME1, all nine doses of radiation were included. In ME2, because of the more demanding simultaneous observation of two males, only doses of 0, 5, 10, 20, 30 and 40 Gy were included. Statistical Analyses For all statistical analyses we used SPSS (SPSS Inc., Chicago, IL, U.S.A.). All the P values are from two-tailed tests. The discrete variables were tested with logistic regression. Thus, courtship and mating success of the experimental males were tested as the regression of the proportion of males that courted or mated the female, respectively, on the radiation dose of the parental males. The relationship between radiation dose and order of courtship was tested similarly in ME2. The latency measures, amount of courtship required by the female and duration of mating were tested with Jonckheere Terpstra analysis. This is a powerful nonparametric test developed for situations where the groups to be compared have a natural order (here radiation doses of the fathers of the experimental males; Sheskin 2004). The trials inwhich courtship or mating did not happen within the 30 min observation were excluded from the analysis of the respective latency measures. The correlation between latency to courtship and latency to mating was analysed with Spearman nonparametric correlation. Estimates for effect size (r) of mutations on all variables were calculated from the test statistics and sample sizes following Rosenthal (1991). RESULTS In ME1 with one experimental male, 95.0% of the males courted the female and 85.1% of these mated. Mutations reduced the probability of courtship of the males (Table 1, Fig. 2). In addition, mutations extended the latency to courtship of the males that did court (Table 1, Fig. 3). Total time to mating was longer for males that started their courtship later, as latency to courtship and latency to mating correlated positively (r S ¼ 0.273, N ¼ 337, P < 0.001). Mutations had no statistically significant effects on the other variables measured (Table 1). In ME2 with one experimental male and one stock male, 85.2% of the experimental males courted the female and 69.1% of these mated. In 59.4% of the trials one of the males and in 38.8% of the trials both males courted the female. None of the measured variables was significantly affected by mutations of the experimental males (Table 2). As in ME1, latency to courtship and latency to mating were positively correlated (r S ¼ 0.445, N ¼ 326, P < 0.001). In the vast majority of the ME2 trials, the males had some physical contact with each other (wing vibration and touching). In some cases the males tried to court simultaneously and interrupt each other s courtship. When we compared the probability of courtship and the latency to courtship of the experimental males in the control (0 Gy) treatment between ME1 and ME2, it was clear that there was interference competition among males in ME2. The presence of the competing stock male reduced the courtship probability (Pearson c 1 2 ¼ 7.822, P ¼ 0.005) and increased the courtship latency of the experimental males (Mann Whitney U test: U ¼ , N 1 ¼ 48, N 2 ¼ 83, P < 0.001). However, interference among males did not make sexual selection against deleterious mutations stronger, as no significant effects of mutations were detected on any of the recorded variables, and the effect size estimates were not inflated compared to ME1 (Tables 1, 2). In both mating experiments, statistically nonsignificant effects were not due to low power of tests, as even very weak effects of radiation (r > 0.11) would be significant at the a ¼ 0.05 level, given the amount of data in the study (see Tables 1, 2). See the Appendix for descriptive statistics for all the variables measured in ME1 and ME2. Table 1 Effect of induced mutations on variables recorded in mating experiment 1 Variable N Test statistic df P r Male courtship 424 c 2 ¼ Latency to courtship 401 J T¼ Amount of courtship required 334 J T¼ by female Mating success (all males) 417 c 2 ¼ Mating success (males that courted) 396 c 2 ¼ Latency to mating 339 J T¼ Duration of mating 309 J T¼ N denotes the number of mating trials and r is the effect size estimate, calculated from the test statistic (J T is the standardized Jonckheere Terpstra statistic). Significant results are indicated in bold.

4 1360 N. Pekkala et al. / Animal Behaviour 78 (2009) Proportion of males that performed courtship (95% CI) DISCUSSION Figure 2. Proportion of experimental males that courted the female in mating experiment 1 as a function of radiation dose of the parental males (confidence intervals are calculated following Zar 1999). Overall, we found inherited heterozygous mutations of D. montana males to have relatively weak effects on mating behaviour of the flies. Most strikingly, mating success of the males was not affected by mutations in either of the mating experiments. However, in the experiment with only one male (ME1), we found mutations reduced the probability of courtship of the males. In addition, in the cases where males did court, the more mutated males started their courtship later. These results suggest a role for male activity in sexual selection for genetic quality in D. montana.in nature, lower probability of courtship would inevitably lead to lower mating success of the males: if you don t court you don t get to mate. Also, latency to courtship could be crucial for mating success of the males. In laboratory experiments, D. montana females often accept the male that courts first (here in 76% of the cases in the mating experiment with two males). If the same is true in nature, beginning the courtship before other males will result in a major benefit in competition over mates. Furthermore, longer latency to mating is likely to decrease the overall mating success of the males, that is, reduce the number of matings the male gets in his entire lifetime (Shackleton et al. 2005; see also McGhee et al. 2007). In our study, mutations did not affect mating latency of the males directly, but latency to courtship did correlate positively with latency to mating. Other, indirect evidence for the effect of genetic quality on male activity in D. montana can be found from the study Latency to courtship (median, s) Figure 3. Latency to courtship (median,s) of the experimental males in mating experiment 1 as a function of radiation dose of the parental males. Table 2 Effect of induced mutations on variables recorded in mating experiment 2 Variable N Test statistic df P r Male courtship 562 c 2 ¼ Latency to courtship 477 J T¼ Amount of courtship required 325 J T¼ by female Mating success (all males) 561 c 2 ¼ Mating success (males that courted) 478 c 2 ¼ Latency to mating 329 J T¼ Duration of mating 288 J T¼ Order of courtship 553 c 2 ¼ N denotes the number of mating trials and r is the effect size estimate, calculated from the test statistic (J T is the standardized Jonckheere Terpstra statistic). of Hoikkala & Suvanto (1999), where they reported D. montana males performing high-frequency courtship song to be more active in their courtship behaviour. Courtship song frequency has been suggested to indicate genetic quality in D. montana (Hoikkala et al. 1998). In contrast to the experiment with one male (ME1), in the mating experiment with two males (ME2), no significant effects of mutations on courtship behaviour of the males were detected. Observations of contact between the males in most of the mating trials and reduced courting of the experimental males in the presence of the other male indicate interference between the males. However, no evidence for selection against mutations via interference competition was found. Rather than amplifying the effects of mutations seen in ME1, the presence of the other male seemed to mask these effects in ME2. Most likely the small effects of mutations detected in ME1 were concealed by the strong effects of the competing male on courtship probability and courtship latency of the males in ME2. It is difficult to relate these findings to natural settings where the effects of male activity may be greatly intensified compared to the confined space of a petri dish, but interference between males may also be frequent. Previous studies have suggested that D. montana females prefer genetically superior males, based on evidence of relationships between courtship song frequency and genetic quality of the males (Hoikkala et al. 1998) and between courtship song frequency and female preference (Ritchie et al. 1998; Hoikkala & Suvanto 1999; but see Klappert et al. 2007). However, we found no evidence for female mate choice against males with more mutations. In ME1, this might be an artefact of the experimental design, females accepting any male when they did not have better males to choose from (Hoikkala & Aspi 1993). However, in ME2, females did have two males to choose from, but still no significant effect of mutations on the mating success of the males, amount of courtship required by the female, latency to mating or duration of mating was detected. Only a few empirical studies have examined the role of sexual selection in removing deleterious mutations, despite theoretical models suggesting that this may affect the reproductive output of individuals and thus the viability of populations, and even the maintenance of sexual reproduction (reviewed in Whitlock & Agrawal 2009). Whitlock & Bourguet (2000) as well as Sharp & Agrawal (2008) documented how mutations that reduce female fecundity and egg-to-adult survivorship may also reduce male mating success in Drosophila melanogaster. MacLellan et al. (in press) showed, again in D. melanogaster, that deleterious mutations may harm males ability to find mates. However, all of these studies used single or a few mutations with clearly visible phenotypic effects, either homozygous recessives (Whitlock & Bourguet 2000; MacLellan et al., in press) or heterozygous mutations with phenotypically dominant effects (Sharp & Agrawal 2008). In nature, mutations can, however, occur in many different loci and have any

5 N. Pekkala et al. / Animal Behaviour 78 (2009) degree of dominance. In a more realistic setting with bulb mites, Rhizoglyphus robini, Radwan (2004) found that sexual selection eliminated randomly induced heterozygous mutations from populations, but he did not identify the process by which less mutated males were favoured. We found that random mutations decreased the courting probability and increased the latency to courtship in D. montana males. To influence population fitness, sexual selection would have to remove mutations that reduce the reproductive output of the population. With the current data set we are unable to determine whether the induced mutations only affected male mating behaviour, or if they also affected other fitness traits. However, given the presumed multitude of loci affecting condition (Rowe & Houle 1996; Kotiaho et al. 2001; Tomkins et al. 2004), it would be surprising if randomly induced mutations only influenced courtship behaviour of the males. The good genes models of sexual selection and the great amount of related research emphasize the role of females choosing goodquality males to mate with (Zahavi 1975, 1977; Pomiankowski 1988; Maynard Smith 1991; Andersson 1994; Kokko et al. 2006). However, it is often forgotten that the mechanisms generating mating bias cannot be inferred from possible indirect fitness benefits from mating with genetically superior males; if genetic quality and male competitiveness or male activity are correlated, genetically superior males will be more successful even without active female choice (Kokko et al. 2003; Kotiaho & Puurtinen 2007). We found no evidence for active female mate choice for genetically superior males; nor did we find evidence for direct male male competition acting against inherited mutations of the males. Instead, the results suggest that sexual selection for genetic quality may operate simply via the effects of male activity. Acknowledgments We thank Professor Anneli Hoikkala for practical advice with the mating experiments. We also thank the staff of the Radiology Hospital of Central Finland Health Care District in Jyväskylä, who kindly gave their equipment and expertise for our use, in particular physicist Pekka Sjöholm. The study was funded by Academy of Finland s Centre of Excellence in Evolutionary Research. References Andersson, M Sexual selection, natural selection and quality advertisement. Biological Journal of the Linnean Society, 17, Andersson, M Evolution of condition-dependent sex ornaments and mating preferences: sexual selection based on viability differences. Evolution, 40, Andersson, M Sexual Selection. Princeton, New Jersey: Princeton University Press. Andersson, M. & Simmons, L. W Sexual selection and mate choice. Trends in Ecology & Evolution, 6, Arnqvist, G. & Nilsson, T The evolution of polyandry: multiple mating and female fitness in insects. Animal Behaviour, 60, Aspi, J Incidence and adaptive significance of multiple mating in females of two boreal Drosophila virilis -group species. Annales Zoologici Fennica, 29, Aspi, J. & Hoikkala, A Male mating success and survival in the field with respect to size and courtship song characters in Drosophila littoralis and D. montana (Diptera, Drosophilidae). Journal of Insect Behavior, 8, Aspi, J. & Lankinen, P Frequency of multiple insemination in a natural population of Drosophila montana. Hereditas, 117, Aspi, J., Lumme, J., Hoikkala, A. & Heikkinen, E Reproductive ecology of the boreal riparian guild of Drosophila. Ecography, 16, Edington, C. W The induction of recessive lethals in Drosophila melanogaster by radiations of different ion density. Genetics, 41, Evans, H. H. & DeMarini, D. M Ionizing radiation-induced mutagenesis: radiation studies in Neurospora predictive for results in mammalian cells. Mutation Research, 437, Fitze, P. S., Cote, J., Martinez-Rica, J. P. & Clobert, J Determinants of male fitness: disentangling intra- and inter-sexual selection. Journal of Evolutionary Biology, 21, Hoikkala, A. & Aspi, J Criteria of female mate choice in Drosophila littoralis, D. montana, and D. ezoana. Evolution, 47, Hoikkala, A. & Lumme, J The genetic basis of evolution of the male courtship sounds in the Drosophila virilis group. Evolution, 41, Hoikkala, A. & Suvanto, L Male courtship song frequency as an indicator of male mating success in Drosophila montana. Journal of Insect Behavior, 12, Hoikkala, A., Aspi, J. & Suvanto, L Male courtship song frequency as an indicator of male genetic quality in an insect species, Drosophila montana. Proceedings of the Royal Society B, 265, Hunt, J., Bussiere, L. F., Jennions, M. D. & Brooks, R What is genetic quality? Trends in Ecology & Evolution, 19, Huntingford, F. & Turner, A Animal Conflict. New York: Chapman & Hall. Klappert, K., Mazzi, D., Hoikkala, A. & Ritchie, M. G Male courtship song and female preference variation between phylogeographically distinct populations of Drosophila montana. Evolution, 61, Kokko, H., Brooks, R., Jennions, M. D. & Morley, J The evolution of mate choice and mating biases. Proceedings of the Royal Society B, 270, Kokko, H., Jennions, M. D. & Brooks, R Unifying and testing models of sexual selection. Annual Review of Ecology, Evolution, and Systematics, 37, Kotiaho, J. S Testing the assumptions of conditional handicap theory: costs and condition dependence of a sexually selected trait. Behavioral Ecology and Sociobiology, 48, Kotiaho, J. S Costs of sexual traits: a mismatch between theoretical considerations and empirical evidence. Biological Reviews, 76, Kotiaho, J. S. & Puurtinen, M Mate choice for indirect genetic benefits: scrutiny of the current paradigm. Functional Ecology, 21, Kotiaho, J. S., Alatalo, R. V., Mappes, J. & Parri, S Honesty of agonistic signalling and effects of size and motivation asymmetry in contests. Acta Ethologica, 2, Kotiaho, J. S., Simmons, L. W. & Tomkins, J. L Towards a resolution of the lek paradox. Nature, 410, Ligon, J. D., Thornhill, R., Zuk, M. & Johnson, K Male male competition, ornamentation and the role of testosterone in sexual selection in red jungle fowl. Animal Behaviour, 40, Liimatainen, J., Hoikkala, A., Aspi, J. & Welbergen, P Courtship in Drosophila montana: the effects of male auditory signals on the behaviour of flies. Animal Behaviour, 43, McGhee, K. E., Fuller, R. C. & Travis, J Male competition and female choice interact to determine mating success in the bluefin killifish. Behavioral Ecology, 18, MacLellan, K., Whitlock, M. C. & Rundle, H. D. In press. Sexual selection against deleterious mutations via variable male search success. Biology Letters, doi: /rsbl Maynard Smith, J Theories of sexual selection. Trends in Ecology & Evolution, 6, Mazzi, D., Kesäniemi, J., Hoikkala, A. & Klappert, K Sexual conflict over the duration of copulation in Drosophila montana: why is longer better? BMC Evolutionary Biology, 9, 132. Pomiankowski, A. N The evolution of female mate preferences for male genetic quality. Oxford Surveys in Evolutionary Biology, 5, Radwan, J Effectiveness of sexual selection in removing mutations induced with ionizing radiation. Ecology Letters, 7, Ritchie, M. G., Townhill, R. M. & Hoikkala, A Female preference for fly song: playback experiments confirm the targets of sexual selection. Animal Behaviour, 56, Rosenthal, R Meta-analytic Procedures for Social Research, Revised edn. Newbury Park, California: Sage. Rowe, L. & Houle, D The lek paradox and the capture of genetic variance by condition dependent traits. Proceedings of the Royal Society B, 263, Shackleton, M. A., Jennions, M. D. & Hunt, J Fighting success and attractiveness as predictors of male mating success in the black field cricket, Teleogryllus commodus: the effectiveness of no-choice tests. Behavioral Ecology and Sociobiology, 58, 1 8. Sharp, N. P. & Agrawal, A. F Mating density and the strength of sexual selection against deleterious alleles in Drosophila melanogaster. Evolution, 62, Sheskin, D Handbook of Parametric and Nonparametric Statistical Procedures. Boca Raton, Florida: Chapman & Hall/CRC. Simmons, L. W Sperm Competition and its Evolutionary Consequences in the Insects. Princeton, New Jersey: Princeton University Press. Tomkins, J. L., Radwan, J., Kotiaho, J. S. & Tregenza, T Genic capture and resolving the lek paradox. Trends in Ecology & Evolution, 19, Whitlock, M. C. & Agrawal, A. F Purging the genome with sexual selection: reducing mutation load through selection on males. Evolution, 63, Whitlock, M. C. & Bourguet, D Factors affecting the genetic load in Drosophila: synergistic epistasis and correlations among fitness components. Evolution, 54, Wong, B. B. M. & Candolin, U How is female mate choice affected by male competition? Biological Reviews, 80, Zahavi, A Mate selection: selection for a handicap. Journal of Theoretical Biology, 53, Zahavi, A Cost of honesty (further remarks on handicap principle). Journal of Theoretical Biology, 67, Zar, J. H Biostatistical Analysis. Upper Saddle River, New Jersey: Prentice Hall.

6 1362 N. Pekkala et al. / Animal Behaviour 78 (2009) APPENDIX Table A1 Number of mating trials (N total) and number and proportion of males that courted and mated in ME1 N total N courted % Courted N mated % Mated (from total) % Mated (from courted) Total Radiation dose expresses the radiation dose of the fathers of the experimental males. Table A2 Latency to courtship and mating in ME1 Latency to courtship (s) Latency to mating (s) N Mean SE Median N Mean SE Median Total N denotes number of mating trials. Radiation dose expresses the radiation dose of the fathers of the experimental males. Table A3 Amount of courtship reqiured by female and duration of mating in ME1 Amount of courtship required by female (s) Duration of mating (s) N Mean SE Median N Mean SE Median Total N denotes number of mating trials. Radiation dose expresses the radiation dose of the fathers of the experimental males. Table A4 Number of mating trials (N total) and number and proportion of experimental males that courted and mated in ME2 N total N courted % Courted N mated % Mated (from total) % Mated (from courted) Total Radiation dose expresses the radiation dose of the fathers of the experimental males.

7 N. Pekkala et al. / Animal Behaviour 78 (2009) Table A5 Latency to courtship and mating for experimental males in ME2 Latency to courtship (s) Latency to mating (s) N Mean SE Median N Mean SE Median Total N denotes the number of mating trials. Radiation dose expresses the radiation dose of the fathers of the experimental males. Table A6 Amount of courtship required by female and duration of mating in ME2 Total amount of courtship required by female (s) Amount of courtship required by female (s) Duration of mating (s) N Mean SE Median N Mean SE Median N Mean SE Median Total Total amount of courtship is the amount of courtship of the two males (experimental male and stock male) together. Amount of courtship required by female and duration of mating are expressed for experimental males. N denotes the number of mating trials. Radiation dose expresses the radiation dose of the fathers of the experimental males.