Variation, but no covariance, in female preference functions and male song in a natural population of Drosophila montana

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1 ANIMAL BEHAVIOUR, 2005, 70, doi: /j.anbehav Variation, but no covariance, in female preference functions and male song in a natural population of Drosophila montana MICHAEL G. RITCHIE*, MARI SAARIKETTU &ANNELIHOIKKALA *Environmental & Evolutionary Biology, University of St Andrews ydepartment of Biology, University of Oulu (Received 6 September 2004; initial acceptance 18 October 2004; final acceptance 23 January 2005; published online 29 August 2005; MS. number: 8265) Studies of the variance and covariance between female mating preferences and sexually selected male traits in natural populations are rare. In D. montana, male courtship song is an important target of sexual selection. We analysed the variance in components of song and preferences among F 1 families from a natural population. All song traits varied substantially, with among-family variance components ranging from 30 to 65%. The greatest variation was in carrier frequency, which is the most important predictor of mating success. This is compatible with the trait capturing mutational and other components of genetic variance in condition because of condition-dependent expression. There was also variation for some components of preference variation, with significant variation among sisters within families, and among families. Females varied in overall responsiveness, but not in the slope of the linear female preference function for male song carrier frequency. Such variation might be expected to generate assortative mating, with more choosy females mating with higher quality males, but there was no covariance across families between female responsiveness and male carrier frequency. Substantial variation in the level of responsiveness might allow low-quality males to achieve some mating success and counteract the buildup of a strong genetic covariance between preferences and traits. Ó 2005 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. Studies of variation in courtship signals and preferences are very important to many areas of evolutionary biology, because the variance and covariance between these will determine the extent and nature of sexual selection and may influence population divergence and speciation (Lande 1982; Ryan & Rand 1993; Andersson 1994; Bakker & Pomiankowski 1995). In particular, few studies have accurately quantified the variance in female preferences within populations, or examined the components or sources of any such variation (Brooks & Endler 2001). Significant variability in preferences has been detected in some systems (Jennions & Petrie 1997; Hedrick & Weber 1998), but the nature and genetic basis of such variability is not well explored (Gray & Cade 1999; Brooks & Endler 2001). Female preferences are complex with different Correspondence: M. G. Ritchie, Environmental & Evolutionary Biology, Dyers Brae House, University of St Andrews, St Andrews, Fife KY16 9TS, U.K. ( mgr@st-and.ac.uk). M. Saarikettu is at the Department of Biology, University of Oulu, PO Box 3000, Oulu, Finland. A. Hoikkala is now at the Department of Biological and Environmental Sciences, University of Jyväskylä, PO Box 35, Jyväskylä, Finland. components of preference contributing to variation in the outcome of mate choice (Wagner et al. 1995; Reinhold et al. 2002) and may be particularly difficult to analyse if females vary because of complex, multitrait preferences (Blows et al. 2003). Females may vary in responsiveness (how likely they are to mate overall), discrimination (how choosy they are among males) and in the nature of the female preference function, the relation between a trait and the probability of mating, which may have linear, quadratic or correlational terms, or unorthodox shapes (Ritchie 1996; Blows et al. 2003). Highly influential models of Fisher s runaway process have identified the extent of genetic covariance in traits and preferences as a key determinant of whether this process could lead to runaway evolution and exaggeration of these behaviours (Lande 1981; Arnold 1983). As a result, many studies have attempted either to detect indirectly or to quantify directly such a genetic covariance, and several have identified significant covariance in a range of organisms (Bakker 1993; Houde 1994; Wilkinson & Reillo 1994; Blows 1999; Gray & Cade 1999). However, covariances have not always been detected (Gilburn & Day 1994; Day & Gilburn 1997). Genetic covariance may be found if there is an intrinsic genetic association between /05/$30.00/0 Ó 2005 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

2 850 ANIMAL BEHAVIOUR, 70, 4 genes caused by pleiotropy or tight genetic linkage, although there is little direct evidence for the very strong pleiotropy predicted by the genetic coupling hypothesis (Butlin & Ritchie 1989; Boake 1991). Nevertheless, if there is substantial genetic variation for traits and preferences, assortative mating would be expected to generate covariance within populations because of gametic phase linkage disequilibrium. Some studies have indicated that covariances may be unstable in the laboratory as might be expected if assortative mating generates and maintains such disequilibria (Houde 1994; Pomiankowski & Sheridan 1994; Blows 1999; Gray & Cade 1999). Although much of the research in this area has taken place in the context of Fisher s runaway process, other forms of sexual selection such as good genes processes would also generate covariance. However, it has increasingly come to be realized that male traits and female preferences may often be out of step during evolutionary change (Ryan & Rand 1995). Species have been described where females show preferences for heterospecific males (e.g. Basolo 1995), and this can come about either because males of their species have lost a preferred trait despite the female preference, or because females have arbitrary preferences which males of their own species have not yet exploited (Shaw 1995). Sexual conflict in the outcome of mating generates selection on males to overcome female reluctance to mate, which gives rise to a dynamic interaction between traits and preferences (Gavrilets 2000; Gavrilets et al. 2001) during which covariances might be expected to change rapidly and unpredictably (there is one intriguing case of a negative covariance, Morris et al. 1996). Acoustic communication systems offer a good opportunity to analyse variation in male traits, because these are relatively easily identified and measured, and in preferences, because playback of synthetic signals allows quantification of variation in the components of female preferences. In Drosophila montana, male song is an obligatory component of mating success (Liimatainen et al. 1992); females show a receptive gesture to males whose song they like by spreading their wings (Vuoristo et al. 1996), and they will do this to synthetic song in the laboratory (Ritchie et al. 1998). This allows researchers to carry out repeated playback experiments to females, and hence to analyse their consistency and variation. Previously we have analysed the shape of female preference functions for acoustic signals in inbred laboratory strains of D. montana. Females prefer males with high-frequency song, which has probably evolved under sexual selection as an indicator of male quality, because the offspring of males with high-frequency song have higher viability (Hoikkala et al. 1998). Previous field studies have implicated song frequency as a predictor of mating success (Aspi & Hoikkala 1995). In this study, we quantified variation in preference functions, male songs and their covariance in a natural population. METHODS Drosophila montana flies were collected in the summer of 2000 from Oulu, Finland and 27 lines were established from females mated in the wild. All the individuals studied here are F 1 progeny of these females. The flies were maintained in malt medium bottles in continuous light at 19 C. Males and females were sexed when they were 2 days old and kept in separate malt medium vials. The flies were used when they were sexually mature (21 28 days old). Males used in the playback experiments had their wings removed under carbon dioxide anaesthesia (to silence them) 1 day before the experiments. For song recording, males were confined in chambers made of a petri dish (diameter 55 mm, height 13 mm) with sexually mature virgin females. Floors of the chambers were covered with moistened filter paper and ceilings were made of nylon net. Male songs were recorded with a JVC condenser microphone and Sony TC-FX33 cassette recorder at 20 G 1 C. To analyse the song parameters we used the Signal Sound Analysis System (Engineering Design, Berkeley, CA, U.S.A.). Songs were digitized at a sampling rate of 2.5 khz after sound above 500 Hz was filtered. We measured the length of pulse trains and the number of pulses in these trains from oscillograms of the recorded songs. Interpulse interval, pulse length and the number of cycles were obtained from the fourth pulse in every pulse train. Carrier frequency was measured from Fourier spectra of the pulse trains. We measured three pulse trains for each of three males per family and used the mean per male in the analysis. The synthesized songs were made with the Signal Sound Analysis System (Engineering Design). Three songs were produced with carrier frequencies of 200, 260 and 320 Hz, while other parameters were kept constant (a pulse train consisted of nine pulses with a total intertrain interval of 1400 ms, interpulse interval of 31 ms and pulse length of 20 ms). These parameters were typical of the study population (Suvanto et al. 2000) and the preference function is known to be linear over this range (Ritchie et al. 2001) The songs were played via a subwoofer (Boston Acoustics, Inc., Peabody, MA, U.S.A. Micromedia PC Sound System) with a sound pressure level of 110 db (measured with a Tecpel sound level meter DSL-330). For playback, females were individually placed in round chambers (diameter 25 mm, height 10 mm) with a wingless male from another isofemale strain (Ritchie et al. 1998). The chambers had plastic walls and both floors and ceilings were made of acoustically transparent nylon net. They were held approximately 2 cm above the subwoofer. We observed each female for 10 min during playback and scored the incidence of wing spreading. The playback experiments were conducted between 0800 and 1200 hours at 20 G 1 C. Three females from each strain were studied and these triplets were observed simultaneously. For each triplet only one song was played on any one day. Playback sequence was randomized. All songs were used twice so every triplet was observed six times. In a control experiment we observed 50 D. montana fly pairs in silence and saw no wing lifting. All male song traits conformed to normality and their variation was analysed with a one-way ANOVA (males within families). Preference data were binomial and were analysed with a generalized linear model with a logit link

3 RITCHIE ET AL.: SONG PREFERENCE COVARIANCE IN FLIES 851 function. Terms are described in the Results. For statistical analyses, we used either Minitab12 or Genstat6 (Genstat Committee 1993). In an experiment such as this, variance among families can be regarded as a measure of broadsense heritability. Nonadditive sources of variance, maternal and common rearing effects will inflate this, so it is not a reliable measure of narrow-sense (additive genetic) heritability (Falconer & Mackay 1996). It is safe to consider the variance components as a general measure of genetic variability, but not a specific component of genetic architecture. RESULTS Table 1 shows the components of variance for male song traits among families. Families differed in all traits, including song carrier frequency. Variance components among families ranged from 30 to 65%. Table 2 summarizes the variation in female preferences. Terms used in the model were song carrier frequency (as a linear covariate), family, females (nested within family), family! song interaction and the three-way interaction. The frequency term tests whether females discriminate between the three songs, the family term tests whether families differ in overall responsiveness, and the female term whether sisters within families differ in responsiveness. The family! song interaction term tests whether families differ in preference function (i.e. the slope of the relation between song and preference) and the three-way interaction term whether the sisters vary in preference function. The first three terms were significant, the latter two not (and the model did not converge satisfactorily when we attempted to fit the latter two). Overall, higher song frequencies were preferred. Families differed in the level of overall responsiveness, with some families responding more readily to all song types. There were also significant differences in responsiveness between sisters within families. However, the slopes of the preference functions, at the level of families or sisters, did not differ significantly. Hence, in this population preference functions differed significantly in height, but not slope. Figure 1 shows the fitted preference functions (from a binomial model including only the significant terms). There were few females of high responsiveness. One would expect that variation in female responsiveness would generate assortative mating with the least responsive females mating with the better males (in this species characterized by those with higher song frequency). Figure 2 shows the covariance between family mean carrier frequency and responsiveness, estimated from a correlation analysis between family means (the different natures of the statistical models used to analyse male and female data precluded a more formal ANCOVA approach to estimating the genetic covariance). There was no suggestion of a significant covariance between these two traits (Pearson correlation: r 25 Z 0.148, P Z 0.47). Female preference variation was unrelated to any song trait. DISCUSSION The amount of genetic variability in sexually selected traits, and the covariance between selected traits and preferences, remains a topic of considerable interest in evolutionary biology. In particular, few studies have quantified variance in female preferences and their covariance with male traits (Bakker & Pomiankowski 1995; Jennions & Petrie 1997; Brooks & Endler 2001). Here, we have characterized the variability in courtship song and components of female preferences in a natural population of D. montana. All song traits showed considerable variation among families. The most important trait for mating success, song frequency, showed the greatest evidence of genetic variation with 65% of the variance being among families (average of the other traits Z 39%). Pomiankowski & Møller (1995) found that, in general, targets of sexual selection have a higher additive genetic variance than other traits, perhaps because nonlinear preferences would favour modifier genes. Preference functions for song frequency in D. montana do have a quadratic component, but this is stabilizing and the preference function is effectively linear over the normal range of male song variation (Ritchie et al. 2001). A more likely explanation for higher variance in the target of sexual selection here is condition dependence of the trait. Carrier frequency in D. montana is condition dependent because it correlates with overwinter survival (Hoikkala & Isoherranen 1997) and offspring quality (Hoikkala et al. 1998). Such traits are expected to capture genetic variance, as mutational input to their heritability will be greater than for other traits not influenced by genes affecting condition (Rowe & Houle 1996; Kotiaho et al. 2001). Female preference variation was also detected in this population, but not in all components of preferences. Overall, females preferred song of higher carrier frequency, as expected. There was substantial variability both among families and among females within families in the responsiveness, that is, the probability of accepting song of a given carrier frequency. However, the shape of the preference function (the extent of discrimination against males with low carrier frequency) did not vary. Hence, Table 1. Mean square (and variance component, as a percentage) for song trait variation among families from a one-way ANOVA Component Pulse number Pulse train length Pulse length Cycle number Interpulse interval Carrier frequency Families (NZ27) 1.87 (35) (51) 11.0 (30) 0.56 (31) 21.2 (48) (65) Males (NZ3) 0.71 (65) (49) 4.8 (70) 0.24 (69) 5.6 (52) (35) P 0.001! !0.001!0.001 The variance components among families are an indication of the broad-sense heritabilities of the traits.

4 852 ANIMAL BEHAVIOUR, 70, 4 Table 2. Analysis of deviance for female preferences Component df Mean deviance Deviance ratio P Song frequency !0.001 Family !0.001 Females within families Song*family Song*family*female Residual Preference sexual selection will constantly favour males with high carrier frequency, but some females will overall be choosier than others. We would still expect this to generate a positive covariance because choosy females are likely to mate only with high-quality males, but we did not detect a correlation between preference and song. Our design looked for this rather indirectly and may not have detected a small correlation. The family mean approach is relatively crude, for example multiple mating will mean each family may consist of half-sibs. However, the approach has detected such a covariance in other systems (e.g. Bakker 1993). Less choosy females would have a greater probability of accepting a low-quality male, so selection generating a covariance would be less strong than in situations where, for example, there was substantial variation in the slopes of preference functions. Effectively, all females here preferred similar (high song frequency) males, but the presence of more responsive females will ensure that low-quality males achieve some mating success, and counteract the build-up of a positive genetic covariance. In Lande s (1981) model, the choosiness of the females was important under some models of female preference. However, this influenced the line of Preference Song frequency (Hz) Figure 1. Fitted preference functions. In this model, one line is fitted per family, but several are overlapping Song frequency (Hz) Figure 2. The covariance between song carrier frequency and female preference among families. equilibrium more than the establishment or size of the covariance. Why such females should persist in a population is an interesting question. Sexual selection would be expected to act against them, at least in stable populations. At low density, such females may have an advantage if mating opportunities are rare (Kaneshiro & Boake 1987). These females may also obtain other advantages if there is a viability cost associated with choosiness. Brooks & Endler (2001) found very similar results with a study of preference variation in a natural guppy, Poecilia reticulata, population. Females varied in responsiveness but to a much lesser extent in discrimination. Only responsiveness showed heritable variation (and artificial selection on mate choice was unsuccessful, Hall et al. 2004; but see Houde 1994). The guppies showed complex variation in discrimination among females, with females differing in which trait they assessed as well as responsiveness. In D. montana, the communication system seems more simple, with song carrier frequency being consistently found to be the most important trait for mating success. This could mean that selection can more effectively diminish genetic variation in important components of preference variation than in systems such as the guppies. In addition, perhaps female preferences are more likely to go to fixation than male traits because they are less condition dependent, which would lead to a decline in covariance. An interesting contrast with these results is provided by Gryllus integer in which females of one population had stabilizing preferences for the number of pulses in a chirp and showed significant variance in peak preference, and there was a significant covariance between male song and peak preference in an F 1 analysis. However, in this population females did not vary in choosiness (defined as the width of the stabilizing function; Gray & Cade 1999). Other populations of this species have different patterns of variation in preferences (Hedrick & Weber 1998), so the functions must vary genetically at some level. Ecological factors may counteract the build-up of strong covariance between preferences and traits, despite variance in both traits. In seaweed flies, Coelopa frigida, positive covariance is related to ecological stability. It is

5 RITCHIE ET AL.: SONG PREFERENCE COVARIANCE IN FLIES 853 thought that stable resources (seaweed in low tidal populations) allow large population sizes to be stable, allowing the build-up of covariance. In less stable (tidal) conditions good genes mate choice predominates, which in this species leads to dissassortative mating for much of the genome. Hence, the strength of a genetic covariance between preference and trait depends on the tidal range to which the population is exposed (Day & Gilburn 1997). Nichols & Butlin (1989) showed how genetic drift in modest population sizes can counteract the build-up of covariance between traits and preferences by a Fisher process. Also, sexual selection may be inconsistent across generations. In a neighbouring population of D. montana, the strength of sexual selection on song and morphology varied between years (Aspi & Hoikkala 1995). Genotype by environment interactions, affecting the reliability of traits and or preferences, also have the potential to disturb a simple build-up of covariance between the traits and preferences. Jia & Greenfield (1997) found that attractive signals of wax moths, Achroia grisella, correlated with fitness only under some environmental conditions. In conclusion, we found significant variation in some but not all components of mate choice variation in a natural population of D. montana, but not an expected genetic covariance between preferences and male traits. The fact that all females preferred males with high values of the trait, and varied only in overall responsiveness, will probably have mitigated against a build-up of a genetic covariance between trait and preference. Hence, this study reiterates the need to distinguish different components of preference variation to understand the nature and strength of sexual selection in natural populations (Brooks & Endler 2001). 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