Male Mating Success and Survival in the Field with Respect to Size and Courtship Song Characters in

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1 Journal of Insect Behavior, Vol. 8, No. 1, 1995 Male Mating Success and Survival in the Field with Respect to Size and Courtship Song Characters in Drosophila littoralis and D. montana (Diptera: Drosophilidae) Jouni Aspi 1'2 and Anneli Hoikkala t Accepted May 26, 1994; revised June 30, 1994 We investigated the importance of male song and morphological characters to the male mating success in a two-year field study in natural populations of D. littoralis and D. montana, We compared the properties of mating flies with those of a random male sample taken at the same time and place. In D. littoralis the male's size had no effect on his mating success, while in D. montana small males had a mating advantage in the field during the first study year. Females preferred males with short sound pulses in both species. We also examined the relationship between male morphological and song characters and viability by collecting male flies in late summer and comparing the means of male characters to those of overwintered flies the next spring. In D. littoralis male size had no effect on overwinter survival. In D. montana large flies survived better than small flies. In both species the shifts in song characters during the winter dormancy were opposite to those caused by sexual selection. Our results, accordingly, imply a possible balance between the forces of sexual and natural selection, which act in opposing directions on attractive male traits. KEY WORDS: love songs; morphology; mating success; sexual selection; natural selection. INTRODUCTION Males of most Drosophila species produce songs during courtship by means of wing vibrations (Bennet-Clark and Ewing, 1968). Several laboratory experi- Department of Genetics, University of Oulu, FIN-90570, Oulu 57, Finland. 2To whom correspondence should be addressed, /95/ / Plenum Publishing Corporation

2 b8 Aspi and Hoikkala ments have suggested that male song has a substantial effect on female mate choice. These experiments have shown that wingless mutants or wing-amputated males have reduced mating success (Bennet-Clark and Ewing, 1969; von Schilcher, t976; Kyriacou and Hall, 1982; Hoikkala, 1988; Liimatainen et al., 1992) and that the success of wingless males can be restored by subjecting females to playback songs (von Schilcher, 1976; Kyriacou and Hall, 1982). Among the boreal D. virilis group species, the male song seems to be an especially important courtship stimulus for the female. Absence of song totally blocks courtship of some species of this species group (Hoikkala, 1988; Liimatainen et al., 1992). The structure of male songs in the D. virilis group has been described (Hoikkala et al., 1982), but it is not known whether intraspecific variation in song characters has any effect on female mate choice in these species. Body size may also be an important determinant of male mating success. The influence of male body size for mating success has been shown in several species in the genus Drosophila. Generally, large males tend to have an advantage in obtaining copulations (Partridge et al., 1987a; Wilkinson, 1987; Hoffmann, 1987; Taylor and Keki6, 1988; Santos et al., 1988; James and Jaenike, 1992), although not always (Boake, 1989). In insects there may be interaction between male size, songs and mating success because the songs of large males differ from those of small males in various ways (see Searcy and Andersson, 1986; Partridge et al., 1987b, Tuckerman et al., 1993). Two sexual selection models have different predictions about the observable changes in attractive male traits due to sexual and natural selection. In the "Fisherian" or "arbitrary trait" models of sexual selection (Fisher, 1958; Lande, 1981; Kirkpatrick, 1982; Pomiankowski et al., 1991), the sexually selected male character is costly, and the models predict that in populations assumed to be in evolutionary equilibrium, the forces of sexual and natural selection should act in opposing directions on attractive male traits (Lande, 1981; Bradbury and Anderson, 1987; Arnold, 1983; Heisler and Curtsinger, 1990). In the "viability indicator" models (Zahavi, 1975, 1977) the male character is also initially costly, but the cost of the male character cannot necessarily be observed in the field. In the original (Zahavi, 1975), "pure epistasis" model (see Iwasa et al., 1991) natural selection tends to weed out males with low viability and full expression of the male character. In another major variant, the "conditional" viability indicator model (Zahavi, 1977; Andersson, 1986; Pomiankowski, 1987; Tomlinson, 1988; Iwasa et al., 1991), the sexually selected trait is plastic and reflects male phenotypic quality. Males in good condition may both express the sexually selected character more fully and show higher survival than inferior phenotypes. According to these models there may be thus even a positive correlation between the male character value and survival in field tests (e.g., Zeh and Zeh, 1988).

3 Determinants of Male Mating Success in Drosophila 69 In this paper we have studied the association of morphological characters and male song structure with male mating success in the field among the two most abundant boreal D. virilis-group species (D. littoralis Meigen and D. montana Stone, Griffen & Patterson). We also studied the association between male characters and overwinter survival to examine whether the preferred characters are deleterious and whether there is a balance between natural and sexual selection in the field. Boreal species of the D. virilis group offer a number of advantages in studying sexual selection fulfilling most of the structural assumptions premised in sexual selection models (see Andersson, I987). The species are polygynous and have no parental care. They are almost or completely univoltine, the successive generations being reproductively isolated. The main viability selection occurs before the flies mate, i.e., during the larval stage and winter diapause. Because the flies overwinter as adults, and the mating season in early spring is very short (Lumme et al., 1978; Aspi et al., 1993) the varying ages of flies cannot affect male mating success as in other Drosophila species (see Long et al., 1980; Santos et al., 1988; Hoffman, 1990). MATERIALS AND METHODS Male Mating Success in the Field We investigated the importance of male morphological and song characters to mating success by comparing the properties of mating flies with those of a random sample taken at the same time and place in natural populations of D. littoralis and D. montana. Collections were made on 7 days between May 5 and June 1 in spring of 1988 and on 6 days between April 28 and May 24 in spring of 1989 in northern Finland (65 40'N, 23 35'E). These sampling periods cover the whole mating season of the species (see Aspi et al., 1993). The flies were trapped by exposing plastic jars baited with fermenting malt to flies for several hours. Jars were covered frequently by a net and the mating pairs as well as solitary flies were gently removed with an aspirator. Samples of flies were caught also on birch (Betula pubescens) sap fluxes in spring of Since no differences in the studied characters (see below) between flies collected on malt baits and on sap fluxes were found, the samples were pooled. The collection methods are provided in detail by Aspi et al. (1993). Male Overwinter Survival in the Field Since the winter diapause is the longest and probably the most demanding life stage in the adult life, we examined the relationship between male characters and viability by collecting the newly emerged male flies in late summer 1988

4 70 Aspi and Hoikkala and comparing the means of male characters among young flies to those of overwintered flies belonging to the same cohort in the spring of Morphological Measurements Three linear measurements were made on each male: the length of the thorax, the length of the wing, and the width of the wing. The left wing and the body were mounted on a glass microscope slide covered with a thin layer of glycerol, and the measurements were made using a dissecting microscope with an ocular micrometer. Wing width was measured from the intersection of the second longitudinal vein and the distal wing margin to the intersection of the fifth longitudinal vein with the distal wing margin. Wing length was measured along longitudinal vein 3 from the anterior cross vein to the intersection with the distal wing margin. For statistical analysis each measurement was transformed to natural logarithms. Courtship Songs The songs of the wild-caught males were recorded in the laboratory, when the males were courting conspecific laboratory-reared females in a plastic netcovered petridish (diameter, 50 mm; height, 12 mm). Song recordings were made with a JVC condenser microphone and a Sony TC-FX 33 cassette recorder. Oscillograms of the songs were analyzed by a Gold 1425 digital oscilloscope. The song of D. littoralis consists of discrete sound pulses (Fig. 1). For this species we analyzed five sound pulses per male. We measured the length of each sound pulse (henceforth PL) and the distance from the beginning of the pulse to the beginning of the next one (IPI) and counted the number of cycles in each pulse (CN). The song of D. montana consists of trains of sound pulses (Fig. 1). For this species we analyzed the fourth sound pulse from five pulse trains as in D. littoralis and counted the number of pulses (PN) and measured the length of the pulse train (PTL) in each pulse train. Means were then calculated for each character over these five sound pulses (in D. littoralis) or five pulse trains (in D. montana) to decrease within-male variation in song characters. For statistical analysis PTL, IPI, and PL were transformed to natural logarithms in both species. The repeatabilities of all these song characters are generally rather high (Aspi and Hoikkala, 1993). Statistical Analysis The statistical analysis of male characters involved estimation of selection differentials and selection gradients. Directional selection differentials are the differences between the mean value of a trait before and after selection and represent the total effect of selection (Falconer, 1981; Lande and Arnold, 1983;

5 Determinants of Male Mating Success in Drosophila 71 A - - SOUND CYCLE SOUNO PULSE INTERPULSE INTERVAL (tpi) B -- SOUND CYCLE SOUND PULSE INTERPULSE INTERVAL (IPI) PULSE TRAIN I I ms Fig. 1. Oscillogmms of cou~ship songs of wild-caught males in D. littoralis (A)and in D. montana (B). Endler, 1986). The directional sexual selection differentials were estimated as the difference between the mean character value for copulating males and mean pooled character value for all the males (Lande and Arnold, 1983). To examine whether the selection differentials in the 2 years were different, we tested whether the deviations from the mean of the two classes differed between the years. This procedure is analogous to Levene's test (e.g., Snedecor and Cochran, t980). The directional natural selection differentials for different characters were estimated as the difference in means of the fall and spring population. Following Endler (1986) we use term variance selection (stabilizing or disruptive) for selection affecting the variance of phenotypic traits. Variance selection differentials were estimated only when the fitness maximum (or minimum) occurred at some intermediate point of the phenotype distribution (see Mitchell-Olds and Shaw, 1987). Variance selection differentials for these characters were calculated as the difference between the variance for the two groups of males, subtracting the changes due to directional selection (Lande and Arnold, 1983). All morphological characters were significantly correlated within both species. The correlations between song characters varied, the highest being between PL and CN in both species and, also, between PTL and PN in D. montana (see Hoikkala and Lumme, 1987). All correlations between morphology and song characters were low (< 0.3). Because the studied characters within both variable sets were correlated, the observed changes in phenotypic means could be due

6 72 Aspi and Hoikkala to indirect selection affecting a correlated character. Thus we also estimated selection gradients for each trait to measure the direct effects of selection in each trait independent of any indirect effects caused by selection on other characters (Lande and Arnold, 1983; Arnold and Wade, 1984a,b). Because our study approach to both selection episodes was cross-sectional rather than longitudinal (Arnold and Wade, 1984b), the normally used multiple partial regression was not appropriate. Instead directional selection gradients were calculated by multiplying the standardized selection-differential vector by the inverse of the phenotypic variance-covariance matrix (Lande and Arnold, 1983). Significance levels for selection gradients could not be estimated but these gradients are still valuable when considering possible contrasts between selection differentials and gradients. Sexual selection differentials and gradients were estimated for both years and also for pooled data if the selection differentials were not different between years. Because the numbers of males in different variable sets were not equal, and due to low correlations between character sets, the morphology and songs were analyzed separately to maximize the sample sizes and to simplify interpretation of results of statistical analysis. The methods described by Lande and Arnold (1983) can sometimes give misleading results depending on the form of the fitness function in the range of the character (MitchelI-Olds and Shaw, 1987; Schtuter 1988). Although only selection coefficients matter under the model of Lande and Arnold (1983), Turelli and Barton (1990) have shown that the entire shape of the distribution of the fitness function can influence evolutionary dynamics. These reasons provide motivation for nonparametric descriptions of fitness surfaces, as proposed by Schluter (1988). Fitness functions were estimated using the nonparametric cubicspline technique (Schluter 1988), which provides a univariate nonparametric estimate of fitness probabilities across the range of the considered character. The estimation of selection gradients involves also other than statistical assumptions (Lande and Arnold, 1983; Endler 1986; Mitchell-Olds and Shaw, 1987; Rausher, 1992). In our study they may involve unmeasured male characters influencing mating success and environmental correlations between male characters and success. Inferring causal relationships regarding the effects of phenotypic characters on fitness on the basis of selection gradients should thus be made cautiously (e.g., Mitchell-Olds and Shaw, 1987). Estimation of Variance in Copulatory Success Among Males Selection coefficients are normally expressed in units of standard deviation (selection intensity sensu Falconer, 1981). Estimation of standardized sexual selection coefficients requires an estimate of the variance in male mating success (Amold and Wade, 1984a,b). In insects variance can be estimated in the field only rarely (e.g., McLain, 1987, 1991; several authors in Clutton-Brock, 1988).

7 Determinants of Male Mating Success in Drosophila 73 It has been suggested that the proportions of copulating and solitary males can be used to estimate the variance in male mating success (e.g., Arnold and Wade, 1984b). However, in our case the males were scanned only once. Since even the most attractive can not be in copula constantly, this approach will give overly large variance estimates for male mating success. Accordingly, to get a more reliable estimates we used an approach similar to Zuk (1988; see also Markow and Sawka, 1992), where variance in male mating success was estimated in separate mate choice experiments. For the estimates of variance in male mating success random samples of flies were caught on May 1, 4, and 13 in Males were identified, but because it is not possible to identify the species of female flies on the basis of external traits, the female samples consisted of a mixture of D. virilis group species. Two experiments were conducted. In the first all females and D. montana males caught on May 1 were used; in the second females and D. littoralis males caught on May 4 and 13 were used. The sex ratios and alien female species composition were thus similar to those faced by the males in the field (see Aspi et al., 1993). Observations were made using a mating chamber, which was large enough (16 cm in height, 17.5 cm in breadth, and 24.5 cm in length) for a female to terminate the courtship. To make the external circumstances as natural as possible the experiments were performed in a climate room, where the temperature was 12 C. The relative humidity within the chamber was 70%. Prior to the experiment, the females were aspirated into the chamber and allowed to habituate for 5 min. After the habituation period, all the males were aspirated into the chamber simultaneously. The copulating pairs were caught with an aspirator, examined without anesthesia with a dissecting microscope, and marked with a small dot of acrylic paint on the ventral surface of the thorax. If the individual was already marked, we used a different color to make the next mark. After marking, the flies were released back into the chamber. The treatment lasted no longer than the copulation duration of these flies [4 to 6 min (Aspi, 1992)]. The experiment was continued until as many copulations were observed as there were males present. The frequency of mated and nonmated individuals was used to obtain a weighted variance for calculating standardized selection differentials and gradients using the formula provided by Zuk (1988). Standardized variances of copulation success were calculated as the variance of the matings divided by the square of the mean number of matings (Wade and Arnold, 1980). RESULTS Variance in Male Copulation Success in the Laboratory In the experiment to estimate the variance in male copulation success the number of copulations per male ranged from zero to four in both species. The distribution of copulations among males is listed in Table I, together with the

8 74 Aspi and Hoikkala Table I. The Distribution of Numbers of Male Matings Observed and Expected in Mating Chamber Experiments in Two Drosophila Species" No. of matings / N D D. littoralis Observed Expected D. montana Observed Expected IIII III " * 0.7 "N is the number of males used in experiment, t is the standardized variance of the copulatory success, and D is the test value of the Kolmogorov-Smirnov test. *Significant with Kolmogorov-Smimov test at level P < Poisson distribution, which is expected if the mating time is short compared to total time of the experiment (Sutherland, 1985). The duration of the experiment was 9 h 20 min in D. littoralis and 10 h 15 min in D. montana. Since the copulations in both species last only for 4 to 6 min, each courtship occupied only a negligible fraction of the total experimental time. The goodness of fit of observed versus expected Poisson distribution was tested using the Kolmogorov- Smimov test (Table I) and also a dispersion test suggested by Sutherland (1985). The distribution of copulations deviated significantly from random in both tests. The observed frequencies were greater than expected in the tails and less than expected in the center of the distribution data in both species, i.e., some males were more successful and some less successful than expected. The estimated standardized variances in copulatory success may be even conservative as the capturing and painting process probably disturbed the treated males. Male Morphology and Mating Success in the Field Male size had no effect on mating success in D. littoralis. The means of the morphological characters of the males found in copula did not differ from the mean of solitary males in either of the years (Table II). The fitness function presented for the first principal component (general size; Fig. 2A) explains 85 % of the total variation in morphological characters. It decreases monotonically (as well as fitness functions for separate characters), suggesting that variance selection is not present. In D. montana the smaller males were more successful in achieving copulation than larger males in 1988 (Table II), but in 1989 there were no significant differences between the males found in copula and solitary males. The selection differential estimates for thorax length were significantly different between years,

9 Table il, Male Morphology and Mating Success in D. littoralis and D. montana in 1988 and 1989" i i Character Copulating Solitary s' /3' Copulating Solitary s' [3' D. littoralis (N = 38) (N = 80) (N = 32) (N = 97) Thorax length : : Wing length : Wing width D. montana (N = 28) (N = 126) (N = 50) (N = 147) Thorax length : : " : : Wing length : : : : Wing width : : : : "Means (+SE) for males found in copula and solitary males are provided, and in parentheses are the number of males studied. Directional sexual selection differentials (s') and gradients (,6") are given in units of standard deviation. *P < 0.05; t test between the means of copulating and solitary males ,i

10 76 Aspi and Hoikkala A 1.o 1~1988 B t u ,8 - ~ f 11 0,6- I E ~ ~ / ' ~ I /~'~" / ~" ~" ~'/ / / 0,0 0.0,~ t5 Generol size (PC1) Generol size (PC1) Fig. 2. Nonparametric fitness functions for male mating success in relation to general size in D. littoralis (A) and in D. montana (B). and thus the means and selection coefficients are not given for pooled data. In D. montana the first principal component also explained most (90%) of the multivariate variation in morphological characters. The fitness function for the first principal component shows bimodality in male size in relation to fitness in both years (Fig. 2B). Accordingly, the relationship between male relative fitness and morphological characters may be more complicated than that of the parametric model of Lande and Arnold (1983). The selection gradients (Table II) may be misleading and the importance of separate characters to male mating success cannot be evaluated. Existence of disruptive selection with respect to male size in D. montana in 1988 was supported by the fact that the variances of all morphological characters were larger among the mated males than among solitary ones. Variance selection differentials in 1988 for thorax length, wing length and wing width were 0.95 (F = 2.01, P < 0.05), 0.89 (F = 2.22, P < 0.01), and 1.16 (F = 2.65, P < 0.001). In 1989 there were no differences in variances between different groups. Male Courtship Songs and Mating Success in the Field The fitness functions of all song characters in D. littoralis were monotonically increasing or decreasing indicating no variance selection. The means of song characters differed significantly (t test, P < for each character) between the study years, and thus the means for pooled data are not given. In the second year the males had shorter IPIs and sound pulses (PL) and lower number of cycles in a pulse (CN) compared with the first year (see Table III). In D. littoralis there was also some inconsistency in selection differentials between the years (Table IH), although the differences were not significant. In 1988 there were no significant differences between the means of mating and

11 Table 111. Male Song Characters and Mating Success in D. littoralis and D. montana in Years 1988 and 1989" Character Copulating Solitary s' /~' Copulating Solitary s' f3' s' ~' D. littoralis (N = 38) (N =* 80) (N = 23) (N = 73) PL : : : :i: ** * IPI : :4.5 0.t CN : ± : : ** D. montana (N = 28) (N = 113) (N = 47) (N = 83) PTL ± ± : : PN 8.6 5: ± : * PL : : * : t 5: ** "* IPI : ± : : CN 4.7 5: : ± _ "Means (+SE) for males found in copula and solitary males are provided, and in parentheses are the number of males studied. Directional sexual selection differentials (s') and gradients (/~') are given in units of standard deviation. *P < 0.05; t test between the means of copulating and solitary males. **P < 0.01; t test between the means of copulating and solitary males. ga m ~a gl IIQ iii

12 78 Aspi and Hoikkala solitary males, but in 1989 copulating males had significantly shorter sound pulses (PL) and fewer cycles per pulse (CN) than solitary males. The selection gradients for PL were rather consistent, and also the largest ones in both years and in pooled data, suggesting that PL was the target of female choice (Table HI). Obviously females seem to prefer shorter pulses in this species. Selection gradients for the other characters were not similar in different years, probably because they are sensitive to small perturbations in data containing closely correlated characters (e.g., Endler, 1986). Accordingly, the interpretation of selection gradients was based on pooled data, since it would give the most reliable results. These gradients suggest that strong direct female preference for short pulses produced an indirect selection to decrease CN in 1989, although it was not itself a target of female choice. In D. montana there were no significant differences in the means of song characters between the study years. The length of a pulse (PL) was shorter among copulating males than among solitary males in both study years, and in 1989 also the number of pulses in a pulse train (PN) was larger among copulating males than among solitary males (Table HI). In this species, there were no either significant differences in selection differentials between years. The magnitude of selection gradients for PL and CN in the pooled data were at least twofold larger than the values for other characters, suggesting that these characters were the main targets of female preference. The significant increase in PN in 1989 was probably only an indirect product of direct preference for short PL, which is negatively correlated with PN. The second largest selection gradient for CN was positive suggesting direct preference for more sound energy per pulse, although there was no significant difference in the mean of this character between the two groups in either years or in pooled data. Male Morphology and Overwinter Survival in the Field In both species all the fitness functions for principal components (Fig. 3) as well as separate characters were monotonically increasing. In D. littoralis the means of the males of the late summer and the spring populations did not differ for any morphological characters studied, whereas in D. montana there were significant differences in the means of all characters (Table IV). Generally, larger males seem to have survived better during the winter dormancy. According to the selection gradients the main target of selection was the width of the wing. Since the smaller males were more successful in obtaining copulations during the mating season and the magnitudes of sexual and natural selection differentials were quite similar, there seems to be a balance between sexual and natural selection in D. montana.

13 Determinants of Male Mating Success in Drosophila 79 A,0 0.8 ~1.0 [ 0.8 I o.>.= 0,6 0,6 _o 0.4 / J i i i i i Generol size (PC1) 0.0 I O 2 "4 Generol size (PC1) Fig. 3. Nonparametric fitness functions for male overwinter survival in relation to general size in D. littoralis (A) and in D. montana (B). Male Courtship Songs and Overwinter Survival in the Field The fitness functions of the male winter survival for song characters did not have any modes or dips in either species. In D. littoralis all song characters were significantly different in autumn and in spring populations (Table V). The net result of viability selection in PL and CN was opposite to sexual selection, i.e., the overwintered males had longer sound pulses including more sound cycles than the males of the late summer population. According to the selection gradients PL was the probable target of viability selection, and during the winter dormancy males with long pulses were favored. In D. montana the selection differentials for most song characters were opposite in sign to the selection differentials of sexual selection (Table V). Only the selection differentials for PNs and IPIs were significantly different in the late summer and spring populations. PN and PL had the largest selection gradients indicating that they were the main targets of selection. The direct selection was opposite to sexual selection in PN but not in PL. The net changes in these characters were, however, opposite in sign compared to the changes due to sexual selection. Thus it seems that also in this species the changes in songs during the winter dormancy were opposite to those caused by sexual selection, although the targets of sexual and natural selection may not have been the same. DISCUSSION The results of the present study suggest that the magnitude of sexual selection on male characters may vary between years. In D. littoralis the size of the courting male appeared not to be critical to male mating success, whereas in D.

14 Table IV. Male Morphology and Overwinter Survival in D. littoralis and D. montana" Autumn Spring Autumn Spring Character (N = 296) (N = 113) s' /~' (N = 215) (N = 183) s' ~' Thorax length : : : : * Wing length 2, : : * -0,639 Wing width :0.01 1,33 5: , : * 1,099 "Means (+SE) for autumn and spring populations are provided. Directional sexual selection differentials (s') and gradients (/~') are given in units of standard deviation. *P < 0.001; t test between the means of autumn and spring populations. t~ an P. II D. littoralis D. montana

15 Table V. Male Song Characters and Overwinter Survival in D, littoralis and D. montana" IIIIIIIIIII I I IIIIIIIII I IIIIIII IIIII I III IIIIIIII I D. littoralis D. montana Autumn Spring Autumn Spring Character (N = 49) (N = 96) s' /3' (N = 27) (N = t30) s' /3' PTL I- 2, ,448 PN , ** PL 42.4 _ *** : _ IPI , * _ "* 0,303 CN : ** -0,053 4, , ,412 "Means (+SE) for autumn and spring populations are provided. Directional sexual selection differentials (s') and gradients (/3') are given in units of standard deviation. *P < 0.05; t test between the means of autumn and spring populations. **P < 0.01; t test between the means of autumn and spring populations. ***P < 0.00[; t test between the means of autumn and spring populations. m I= ran Bo,,,I llll,i i iiiiiiiiilllll INIIIIII

16 82 Aspi and Hoikkala montana small males had a mating advantage, but only during one of the study years. Both D. littoralis and D. montana females appeared to prefer the males with short sound pulses. In D. montana the preference was significant during both study years, but in D. littoralis only during the second year. These results indicate that the mating success of certain types of males is not so definite as laboratory experiments often lead us to think. The mating success of different types of males depends largely on the environmental conditions and on the phenotypic variation among the available males in field, being not solely due to sexual selection exercised by the females. Due to the masking effects of these factors, the real targets of sexual selection may be difficult to find out in the field. Male mating success with respect to size has been studied in several natural populations of Drosophila (Partridge et al., 1987a; Taylor and Keki6, 1988; Santos et al., 1988; James and Jaenike, 1992), and in all of these studies large males have appeared to be more successful in obtaining matings than small males. In D. montana the small males, if any, appeared to have a mating advantage. Because of the better maneuverability of a small body compared with a large one, the small males may have a mating advantage in circumstances where agility is an important component of mating success (McLachlan and Allen, 1987; Steele and Partridge, 1988). However, the possible reason for the difference between our results and the previous field studies may lie also in the possibility that the better mating success of large males in some Drosophila species is due not to female choice but to direct contests or scramble competition between males (Partridge et al., 1987b; Wilkinson, 1987) and that the importance of male contests on mating success may vary in different kinds of mating systems or ecological conditions (see Markow and Ricker, 1992). Direct contests favoring male-male competition ability and large size will be important determinants of male mating success in circumstances where small ephemeral resource patches are available (see Hoffmann and Cacoyianni, 1989). Scramble competition is mostly adaptive in crowded conditions which probably prevail in natural populations of D. melanogaster (Crossley and Wallace, 1987). In D. littoralis and D. montana direct fights between males are rare (Liimatainen et al., 1992; Aspi et al., 1993; Hoikkala and Aspi, 1993), and small resource patches seem to be absent during the mating season of these species (Aspi et al., 1993). Because the boreal populations of Drosophila are rather sparse, scramble competition between males may not be as important component of male mating success as among Drosophila in other ecological contexts. The mating success of small D. montana males was better than that of the large males during only one of our study years, suggesting that it was affected by other factors than active female choice. The sizes of the available males may also depend on environmental factors. Stalker (1980) has suggested a sizedependent relationship between temperature and flying ability of Drosophila.

17 Determinants of Male Mating Success in Drosophila 83 He found that small flies have relatively large wing-load indices (ratio between thorax volume and wing area), and they can fly in colder temperatures than large flies. When calculating the wing load indices of flies in our data, we found significant differences between collecting days in this ratio in D. montana in spring 1988 (F = 4.05, P < 0.01, df = 4,147), but not in spring 1989 (F = 1.86, P > 0.1, df = 5,156). In spring 1988 the wing-load index was significantly correlated with mean daily temperature (r = , P < 0.05). Because small flies have relatively large wing-load indices also in our data (correlation coefficient between wind load index and general size, r = , P < 0.001), they could probably fly in colder temperatures than large flies. The mean daily temperature of collecting days in 1988 was only 2.7 C, while in spring 1989 it was 7.7 C. This might explain why the smaller males were selectively favored in the cold spring of 1988 but not in the warmer spring of Thus although there was mate choice with respect to size (as defined by Halliday, 1983), there was not necessarily female preference or "active" female choice (see Halliday, 1983; Parker, 1983). In some other animal groups structural song characters can give cues about male size (Tuckerman et al., 1993). However, we found only low correlations between morphological and song characters. This is consistent with other Drosophila studies, in which no significant correlations between structural song characters and size have been found (Partridge et al., 1987). Several laboratory experiments have shown that the presence or absence of song affects female choice in Drosophila (Bennet-Clark and Ewing, 1969; yon Schilcher, 1976; Kyriacou and Hall, 1982; Hoikkala, 1988; Liimatainen et al., 1992). The only attempts, so far, to study the importance of variation within the species in song characters on female choice has been carried out by Cowling (1980) and Greenacre et al. (1993). In Cowling's study the range of IPIs in male songs was equally acceptable to the females in D. melanogaster. Cowling (1980) also tried to examine the past selective events which might have affected the songs in their history using a diatlel analysis. He found that IPI and sine song frequency were characterized by a high degree of additivity and no significant dominance, indicating that no directional or stabilizing selection had altered these characters during their past history. Greenacre et al. (1993) have studied preferences of females homozygous for mutant per allele, which alter rhythmic components of male song. In this study the females carrying the per mutant also preferred wild-type over mutant songs. In our study both D. littoralis and D. montana females appeared to exercise selection on male courtship songs preferring males with short sound pulses and in D. montana probably also males with lot of sound energy per pulse. Because the songs are not used in male contests, we believe that the association between male song characters and mating success is due to female preference (cf. Searcy and Andersson, 1986). The selection for short PL in D. littoralis was evident

18 84 Aspi and Hoikkala only in spring This could be because all males had longer sound pulses on average in 1988 than in 1989, and the females may not have had favorable males to choose from. We have previously shown (Hoikkala and Aspi, 1993) that the number of courting males may affect female choice with respect to male song. A female can accept a male producing less favorable songs, if there are no other males available. As mentioned above the spring of 1988 was cold and the number of males courting the female may have been lower than in the warmer spring of 1989, and thus the differences in selection differentials may reflect relative mate choice. The only attempt so far to examine the balance between sexual and viability selection in Drosophila has been made by Wilkinson (1987). He estimated the intensity of sexual and viability selection on male wing length under laboratory conditions in a recently captured D. melanogaster population. After estimating the heritability of the character, he found that the expected standardized response due to sexual selection was opposite in sign and similar in magnitude compared to the standardized response due to viability selection. The results of the present study suggest a balance between sexual and natural selection in some male characters in both species. The large males survived better during the winter, but the small males may have had an advantage in obtaining copulations in D. montana. The changes in the song characters during the winter were opposite to those caused by sexual selection in both species. In D. littoralis the targets of both sexual and natural selection in song characters were the same. In D. montana the shifts in song characters during the winter dormancy were also opposite to those caused by sexual selection, even though the targets of sexual and natural selection were different. However, as Heisler (1985) has shown, the balance between natural and sexual selection can be obtained even if the targets of different selection episodes would not be the same. The results of the present study thus fulfil the predictions of the "arbitrary trait" model of the balance between two selection episodes, given that the observed changes in male songs during diapause are due to selection. We cannot, however, reject the possibility that the difference between the means of the males of the summer and spring populations were due to other factors than selection. Our attempts to maintain these flies overwintering in outdoor chambers in winters and to estimate survival rates and assign individual fitness values to overwintering flies were not successful. Changes in means of song characters may thus be due to changes in the songs of individual males during the winter dormancy, males may have migrated selectively from the area, or our traps may not have been equally attractive to different phenotypes in different seasons. Assuming that the changes in the means of song characters are due to changes within individual males, this observation could also be accommodated by the viability indicator model. If the altered songs reflect the phenotypic condition of the males and the songs of

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