Mate preferences by male guppies (Poecilia reticulata) in relation to the risk of sperm competition

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1 Behav Ecol Sociobiol (2004) 55: DOI /s ORIGINAL ARTICLE Laura D. Dosen Robert Montgomerie Mate preferences by male guppies (Poecilia reticulata) in relation to the risk of sperm competition Received: 6 May 2003 / Revised: 10 September 2003 / Accepted: 10 October 2003 / Published online: 13 November 2003 Springer-Verlag 2003 Abstract We investigated male mate preferences in relation to the perceived risk of sperm competition in the guppy (Poecilia reticulata), a freshwater fish with a promiscuous mating system. Our laboratory experiments revealed that male mate choice behaviour is not influenced by the presence of rival males that are merely in close proximity to a potential mate, as there was no significant difference in the amount of time that males spent with females that were recently either alone or in close proximity to four rival males. Male mate choice behaviour was, however, strongly influenced by the presence of rival males in a second experiment, where those rivals were permitted to copulate with one of the females. In that situation, males spent significantly more time with, and directed significantly more sigmoid courtship displays toward, females that they had recently seen alone compared to females they had seen receiving forced copulations from up to four rival males. Our results therefore indicate that male guppies are sensitive to the risk of sperm competition and alter their mate choice behaviour in an adaptive fashion. Keywords Guppies Mate choice behaviour Mate preferences Sperm competition Introduction Male mate choice is predicted to occur whenever (1) males have more than one female to choose from, (2) females differ noticeably in fecundity, (3) mating with any one female diminishes sperm reserves, thereby reducing a male s chance of fertilizing other females in Communicated by K. Lindström L. D. Dosen ()) R. Montgomerie Department of Biology, Queen s University, Kingston, Ontario, K7L 3N6, Canada dosenl@biology.queensu.ca Tel.: Fax: the short term, or (4) males are at risk of sperm competition (Andersson 1994). Most studies examining male mate choice have focused on male mating preferences when females vary in fecundity, showing that males do indeed prefer to mate with females that are more fecund (Rowland 1982, 1989; Sargent et al. 1986; CôtØ and Hunte 1989; Nuttall and Keenleyside 1993; Grant et al. 1995; Ptacek and Travis 1997; Kraak and Bakker 1998; Dosen and Montgomerie, unpublished data). Only recently, however, has attention been focused on male mating tactics with respect to sperm competition risk and intensity (Wedell et al. 2002). Males can gain information about the probability (or risk) of sperm competition at a particular mating by using various cues such as the operational sex ratio (Evans and Magurran 1999), presence of rival males, and information about female mating status (i.e., evidence of recent matings; Wedell et al. 2002). If mating with any one female reduces a male s future reproductive success by diminishing his sperm reserves, then selection should favor males who pay particular attention to socio-sexual cues that provide information about the risk of sperm competition at any given mating. Although recent sperm competition models (Parker et al. 1996, 1997) make independent predictions about whether a male should reduce or increase his ejaculate expenditure in the face of varying levels of sperm competition risk versus intensity, the optimal mate choice strategy will also depend on other mating opportunities (Dewsbury 1982; Wedell et al. 2002). If, for example, a male observes a particular female in close proximity to, or engaging in copulation with, another male and an unmated (or virgin) female is also available, he should preferentially mate with the unmated female. By mating with females that are unmated, males can potentially achieve greater reproductive success. Indeed, males of many species have been shown to adjust their mate preferences with respect to both female mating status (i.e., evidence of recent matings) and the presence of rival males, as would be predicted by sperm competition theory (Parker et al. 1996, 1997). In male

2 Drosophila melanogaster, for example, it has been shown that males discriminate mated from virgin females and preferentially court virgins, and for longer periods of time (Cook 1975). Similarly, Schwagmeyer and Parker (1990) found that male thirteen-lined ground squirrels (Spermophilus tridecemlineatus) avoided copulating with previously-mated females when the gain in fitness fell below the average gain from mating with other females in the population. Male flour beetles (Tribolium castaneum) also preferentially approached and copulated with virgins rather than previously mated females (Lewis and Iannini 1995). Recent work on a variety of fish species also illustrates how males can alter their reproductive decisions in response to sperm competition as predicted by theory (Parker et al. 1996, 1997). Male rainbow darters (Etheostoma caeruleum), for example, were more likely to abstain from spawning when sperm competition intensity was high (Fuller 1998). Similarly, male European bitterling (Rhodeus sericeus) attempt to avoid sperm competition by leading females to mussels (where they spawn) that had no rival males in their vicinity (Smith et al. 2003). In this study, we examined the mating preferences of male guppies (Poecilia reticulata) in response to sociosexual cues that could predict the risk of sperm competition. Specifically, we wanted to determine whether male guppies would adjust their behavioral response to (1) the presence of rival males, and (2) information on female mating status (recently copulated or not). We predicted that the guppies would adjust their behaviour in accordance with sperm competition theory, such that males would prefer to associate with females that have not been observed in the presence of, or recently copulating with, rival males. Copulation in guppies can be achieved via two alternative mating tactics: (1) cooperative mating (usually only achieved with receptive or virgin females), and (2) coercive or forced mating (gonopodial thrusts, most often directed at unreceptive females; Liley 1966). Because females are only receptive when they are virgins or for a short period following parturition, females receive forced copulations at a high frequency (Houde 1997). Farr (1975), for example, has reported that male guppies from some Trinidadian streams perform gonopodial thrusts at a rate of up to five per 10 min. Although it was previously thought that forced copulations were not successful in transferring sperm, recent studies have shown that such copulations can indeed result in the transfer of large amounts of sperm into the gonoduct of unreceptive females (Pilastro and Bisazza 1999; Matthews and Magurran 2000; Evans et al. 2003). In addition, females can store sperm for up to 6 months from just a single insemination (Winge 1937; Kadow 1954). Thus, the promiscuous and highly sexual behaviour exhibited by male guppies, in combination with the ability of females to store viable sperm for many months, likely results in high sperm competition risk. Consequently, male mating behaviour is expected to be sensitive to information on female mating status. Methods 267 We studied guppies that were descendants of fish collected from a population in Venezuela in Fish were maintained on a 12:12 light:dark photoperiod at 24 25C and fed commercial flake food (Hartz Wardley Total Colour) twice a day. Test males were housed separately from females. All females were housed with non-test males and were thus gravid and sexually unreceptive (female guppies are most responsive to male courtship when they are not gravid; Liley 1966). Following Ptacek and Travis (1997), we used gravid females to ensure that male choice would not be confounded by female choice. In addition, because gravid female guppies show increased abdominal distension as their brood develops, we matched both females used in a given trial with respect to both standard length (SL) and abdominal distension. Thus, we controlled for any variation in male preferences that might have been caused by variation in female fecundity and reproductive status (stage of reproductive cycle or brood development). After females were used in a trial, they were kept separate from unused females. Our experimental apparatus was a cm glass aquarium (Fig. 1), divided into five compartments by solid plexiglas partitions sealed with silicone; compartments at either end of the tank measured cm and the middle compartment measured cm. The partitions prevented water flow between compartments ensuring that males could neither identify females or detect their reproductive status by pheromonal cues (Crow and Liley 1979). We drew vertical lines on the glass of the middle compartment to divide it into three 6.7-cm wide zones so that we could record the location of the male. When a male was in one of the zones adjacent to a female s compartment, we considered that he had made a choice whereas the central zone was considered no choice. We used beige-colored gravel and beige paper covering both ends and the back of the test tank to provide a uniform background color for male assessment of females. The aquarium was illuminated from 30 cm above with a 15-W full spectrum Sun-Glo fluorescent tube (Hagen, Montreal); there was no other lighting in the room. The test tank was maintained at the same temperature as Fig. 1 Experimental tank in which the central compartment contained the test male guppy (Poecilia reticulata) held within a square plexiglas tube, and the front compartments at each end contained females. In experiment 1, rival males were randomly placed in one of the end compartments separated from the female s compartment by a plexiglas partition. In experiment 2, rival males were randomly placed in one of the females compartments. Solid lines represent glass; dashed lines are plexiglas; shaded lines are opaque partitions that were removed during the observation and test periods of each trial

3 268 the holding tanks. Experiments were conducted from hours EDST. All fish were fed to satiation before experiments commenced. Experiment 1: presence of rival males This experiment was conducted to determine if a male guppy would prefer to associate with females that were not in the presence of rival males, as predicted by sperm competition theory. We used a dichotomous choice design in which males could choose between a lone female and a female that was in the presence of four rival males. Previous experiments with guppies have revealed that the amount of time a female spends near a particular male in a dichotomous choice experiment like this is a good indicator of her preference for that male because copulation usually follows during a subsequent mating trial (Kodric-Brown 1989). To conduct an experimental trial, we chose two females of approximately the same body size (<2 mm difference in SL) and abdominal distension, and randomly placed one of them in each of the end compartments of the test tank. At the same time, we chose a male (haphazardly) and placed him in a rectangular plexiglas tube ( cm) within the central compartment (Fig. 1). We also placed opaque partitions in front of both females compartments to prevent the male from observing them. All three fish were allowed to acclimate for 10 min, then four rival males were chosen haphazardly from their holding tank and placed in one of the end compartments (chosen at random) next to a female s compartment (Fig. 1). Once the rival males began to take notice of the female in the compartment next to them (30 60 s), the opaque partitions were removed, and the test male was allowed to observe both females (one alone, one in close proximity to four rival males), from within his tube, for 20 min. By restraining the test male in a tube, we were able to prevent him from getting close to and interacting with the other fish during the 20-min observation period during each trial; such interaction could have possibly have altered the behaviour of the rival males. After this 20-min period when the test male could observe both females, we reinstalled the opaque partitions and removed the four rival males from the tank. Once the females began to exhibit normal swimming behaviour (30 60 s after the rival males were removed), and did not show any visible signs of stress (hiding in the corner of the tank or remaining stationary), we again removed the opaque partitions and released the test male by gently lifting the plexiglas tube out of the test tank. We then observed a 10-min test period during which we recorded the amount of time the male spent in each of the three zones of his compartment and the number of sigmoid displays he directed at each female. We ran 18 trials, testing each male once. Because we had available only 16 healthy females that could be matched in dyads for both SL and abdominal distension, some females were used in more than one trial, but never on the same day. Thus for 8 trials we used female dyads that had not previously been used in this experiment and for the remaining 10 trials we chose females haphazardly from the pool of previously used females. After males were tested, they were used only as rival males in subsequent trials, but they were never used as a test male and rival male on the same day. The average standard length of males used in this experiment was 15.8 mm ( 0.31, mm, 18) (mean SE, range, n), whereas females averaged 20.7 mm ( 0.50, mm, 16). There was no significant difference between the standard lengths of the two females used together in each trial (Wilcoxon paired sample test, T + =64.5, P=0.36, n=18 female dyads). Experiment 2: copulations by rival males This experiment was conducted to determine if a male guppy would prefer to associate with females that had not been observed receiving copulations from other males. Thus, this experiment was identical to Experiment 1 except that there were no partitions in the females compartments to separate them from the rival males. In addition, during the 20-min period when the test male was allowed to observe the females, we recorded from videotapes: (1) the total amount of time the test male spent observing (oriented toward) the female that was housed with four rival males, and (2) the total number of forced copulations (gonopodal contact with a female while she was swimming away from or not paying attention to a male; Kodric-Brown 1993) that the female received from rival males while the test male was watching her. Although male guppies also engage in cooperative copulations with females, males only performed forced copulations during our experiments, probably because males are most likely to engage in coercive matings with females that are not sexually receptive (Houde 1997). We ran 16 trials for this experiment, using the same males from experiment 1 and testing each male once. Because experiment 2 was conducted >50 days after experiment 1, there is no reason to believe that the results of these experiments were not independent. Because we had available only 24 healthy females that could be matched in dyads for both SL and abdominal distension, 8 females were used in more than one trial, but never on the same day; 10 trials were performed using female dyads that had not participated in previous trials. After males were tested, they were used only as rival males in subsequent trials, but never as a test male and rival male on the same day. To control for male courtship and copulation activity, no rival males were used in more than one trial per day. It has previously been shown that male sexual activity in guppies decreases after both cooperative and forced copulations due to diminished sperm reserves (Pilastro and Bisazza 1999). Thus, in our experiment, rival males were given at least 24 h to replenish their sperm reserves before being used in a subsequent trial. The average standard length of males was 16.5 mm ( 0.35, mm, 16), whereas females averaged 21.0 mm ( 0.38, mm, 24). There was no significant difference between the standard lengths of the two females that were used together in each trial (paired t=1.06, P=0.31, n=16 female dyads). Statistical analyses Because we had to re-use some individuals during our experiments, there was a possibility that male and female behaviors between trials would not be independent, even though we adopted several protocols to minimize bias, as described above. Regardless, we reanalyzed all of the results reported below using only trials in which females had not appeared in previous trials during each experiment. Although this reanalysis reduced sample sizes to 8 and 10 trials in experiments 1 and 2, respectively, our results remained significant and effect sizes were similar. We tested all distributions for normality (Shapiro-Wilks test) and performed nonparametric analyses when the assumption of normality was violated and could not be satisfied with an appropriate data transformation. Descriptive statistics are reported as mean ( SE, range, n). Results Experiment 1: presence of rival males During the 10-min test period, males spent only 21% ( s, s, 18) of their time in the no choice zone, and performed only 6% of their sigmoid displays there. Thus test males spent 79% of their time in one of the two choice zones during the 10-min test period, but there was no significant difference in the amount of time they spent associating with the female that was alone versus the female that was previously in close proximity to four rival males (paired t=0.24, P=0.81,

4 269 with one of the females (i.e., when there was no plexiglas partition separating the rival males from the female), he spent significantly more time during this 10-min test period associating with the female that was previously alone (paired t=2.42, P=0.03, n=16 males; Fig. 2a). In addition, males directed significantly more sigmoid displays toward females that were previously alone than to females previously housed with and receiving copulations from four rival males (paired t=3.29, P=0.005, n=16; Fig. 2b). During this 10-min test period, there was no significant correlation between the amount of time that test males spent with the female that was previously alone and the number of forced copulations that he had seen the other female receive during the preceding 20-min observation period of each trial (Spearman rank correlation, r S = 0.34, P=0.21, n=15). Discussion Fig. 2a, b Behavior of test male guppies toward females during the 10-min test period in the experimental trials, comparing females previously alone to those either in close proximity to (Experiment 1, n=18 trials), or the recipient of copulations from (Experiment 2, n=16 trials), rival males. a Amount of time that the male spent with each female. b Number of sigmoid displays that the male directed toward each female. Box plots show 10th, 25th, 50th (median), 75th and 90th percentiles with horizontal lines, and all data points outside this range n=18 males; Fig. 2a). Nor was there a significant difference in the number of sigmoid displays the test male directed toward the two females (T + =55.5, P=0.80, n=18 males; Fig. 2b). Experiment 2: copulations by rival males On average, test males spent 57% ( s, 94 1,066 s, 15) of the 20-min observation period in this experiment observing the female that was housed with four rival males. Test males also observed these females receiving an average of 4.8 forced copulations ( 1.1, 0 16, 15) during this 20-min observation period. Note that sample size is reduced for these two measures due to failure of one of the videotapes. During the 10-min test period, males spent only 17% ( s, s, 16) of their time in the no choice zone, and performed 19% of their sigmoid displays there. When the test male had observed other males copulate In this study, we showed that male guppies are indeed sensitive to the socio-sexual cues that might predict the risk of sperm competition when choosing a female to mate with. Thus, like females, the degree of choosiness a male guppy exhibits when selecting a mate depends on the costs of engaging in sexual behaviour. When there was an obvious cost to choosing a mate due to perceived sperm competition risk (experiment 2), males responded adaptively by reducing the amount of time they spent with the female that had previously been the recipient of forced copulations from multiple males, and by increasing the amount of time they spent with the female that did not present an immediate sperm competition risk. Our findings are thus similar to those of White and Galef (1999) who found that male Japanese quail (Coturnix japonica) decreased their tendency to associate with particular females after seeing them mate with another male. Although the presence of rival males has been shown, in other species, to be an important cue that males use to fine-tune their mating behaviour in response to the perceived risk and intensity of sperm competition (Schwagmeyer and Parker 1990; Fuller 1998; Pilastro et al. 2002; Schaus and Sakaluk 2003; Smith et al. 2003), the presence of male rivals near a female in experiment 1 did not apparently influence a test male s perceived sperm competition risk. This finding is likely a result of the test male not observing any copulations or direct contact between the fish (due to the presence of the plexiglas barrier) during the 20-min observation period of each trial. Thus our results show that males can assess, even at some distance, the difference between females actually receiving copulations from, versus those simply in the presence of, rival males. Therefore, it appears that observing males actually forcing copulations on a female serves as a more reliable cue of sperm competition risk than observing four males that are merely in close proximity to a female.

5 270 An alternative explanation for our findings is that, in experiment 2, a test male may have associated more with the lone female simply because he thought she might be sexually receptive, whereas the female in the presence of four rival males clearly was not (because she did not cooperate in copulations). This explanation is unlikely, however, because females who are sexually receptive respond to a male s courtship displays by gliding toward him (Liley 1966). No female was observed to do this in either experiment despite numerous sigmoid displays by both the test male (Fig. 2) and rival males. Thus, in experiment 2 each test male should have perceived both females as unreceptive even during the 20-min observation period of the trials as neither female responded to his (or the rival males ) displays. While this alternative explanation seems unlikely, it is certainly worth addressing in future studies. We expected, but did not find, a significant correlation between the number of forced copulations a female received and the amount of time the test male spent with the female that was previously alone. We can suggest two potential explanations for the absence of such a relationship. First, because test males did not spend the entire 20- min observation period of the trial watching all the interactions that occurred between the four rival males and the female, they could not have had information on the total number of copulations a female received, nor the number of males that actually copulated with the female. Second, keeping track of and remembering the total number of interactions that occurred between the female and all four rivals may have been a cognitively difficult task. Thus, observing just a few copulations in combination with the presence of multiple rival males seems to be enough for a male to perceive a sperm competition risk. It is particularly interesting that males have the ability to make adaptive mate choice decisions based on the male-female interactions that they had observed previously, rather than on their immediate perception of risk. The test males mate choice decisions were not made when they could see rival males in the presence of, or mating with a female, but rather up to 10 min later (when the sperm competition risk is nevertheless unchanged). Similarly, in mate choice copying experiments, female sailfin mollies (Poecilia latipinna) have been shown to remember and copy the mate choices of other females up to 1 day after observing a model female and male interact (Witte and Massmann 2003). It would be interesting to look at the duration of a male guppy s ability to respond to sperm competition previously observed, and to determine whether males can recognize individual females with respect to sperm competition risk even if the females are in a different location (e.g., different ends of the experimental tank). In conclusion, our study provides behavioral support for models of sperm competition (Parker et al. 1996, 1997). Male guppies adjusted their behavioral response to the risk of sperm competition by preferring to associate with females that did not receive copulations from multiple males. Whether male guppies, like many other fish species examined to date (Candolin and Reynolds 2002; Pilastro et al. 2002; Smith et al. 2003), have the ability to fine tune their ejaculate expenditure in response to sperm competition as predicted by theory, awaits further investigation. Acknowledgements We thank Helen Rodd for advice on caring for guppies and answering the many questions we had about guppy behaviour and Felix Breden for providing the guppies. This study was conducted under Queen s University Animal care permit R1 and was funded by the Natural Sciences and Engineering Research Council of Canada in the form of research and equipment grants to R.M. and a postgraduate scholarship to L.D.D. R.M. was supported by a Canada Council Killam Research Fellowship. References Andersson M (1994) Sexual selection. Princeton University Press, Princeton, N.J. Candolin U, Reynolds JD (2002) Adjustments of ejaculation rates in response to risk of sperm competition in a fish, the bitterling. Proc R Soc Lond B 269: Cook, R (1975) Courtship of Drosophila melanogaster: rejection without extrusion. Behaviour 53: CôtØ IM, Hunte W (1989) Male and female mate choice in the redlip blenny: why bigger is better. 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