Animal Behaviour. Mate sampling strategy in a field cricket: evidence for a fixed threshold strategy with last chance option

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1 Animal Behaviour 81 (11) 519e527 Contents lists available at ScienceDirect Animal Behaviour journal homepage: Articles Mate sampling strategy in a field cricket: evidence for a fixed threshold strategy with last chance option Oliver M. Beckers *, William E. Wagner, Jr School of Biological Sciences, University of Nebraska-Lincoln article info Article history: Received 13 July 1 Initial acceptance 12 August 1 Final acceptance 15 November 1 Available online 28 December 1 MS. number: A1-483 Keywords: acoustic communication female preference fixed threshold strategy Gryllus lineaticeps last chance option mate choice search strategy The strategy females use to sample potential mates can influence mate choice and thus sexual selection. We examined the mate sampling strategy of the cricket Gryllus lineaticeps. In our first set of experiments, we simultaneously presented three different chirp rates to females. The set consisted of three trials, each covering a different range of chirp rates. Independent of chirp rate range, female G. lineaticeps preferred rates that were above 3. chirps/s to rates that were below 3. chirps/s. Females did not discriminate among chirp rates that were below this threshold and did not discriminate among chirp rates that were above this threshold, suggesting that they express a fixed threshold sampling strategy. In our second experiment, females were presented sequentially with a fast chirp rate and then a slow chirp rate. When the interval between presentations was min, females showed significantly weaker responses to the slow rate than to the fast rate. However, when the interval between presentations was 24 h, female responses to the slow and fast rate did not significantly differ. The latter result suggests that females lower their threshold of acceptance when they have not recently experienced highly attractive song types. This lower acceptance threshold is probably adaptive, as it would allow females to avoid paying high search costs, and to reproduce, only when low-quality males are available. Our results are consistent with a rarely considered sampling strategy (fixed threshold with last chance option strategy) and highlights the importance of the timing of social experience for mate sampling. Ó 1 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. How females choose a mate is a central issue for understanding the causes and consequences of sexual selection. Most research on female mate choice has focused on female mating preferences, and we know that females often prefer to mate with males with particular types of traits (e.g. Basolo 199; Wilkinson & Reillo 1994; Gerhardt et al. ). Mate choice, however, is the result of interactions between environmental conditions (e.g. predation risk; Godin & Briggs 1995), mating preferences and sampling strategies (Wagner 1998). Much less is known about how females sample, assess and compare potential mates (i.e. female sampling or search strategies) than about female mating preferences. Understanding female sampling strategies is important because different strategies can lead to different mate choices. For example, the number of males sampled may differ depending upon the sampling strategy a female uses, and females that sample more males are likely to show a greater bias in their mate choices than females sampling fewer males (Janetos 198; Wagner 1998). The sampling strategies that females use can thus affect both how sexual selection acts on male traits and the benefits of mate choice itself. * Correspondence: O. M. Beckers, University of Nebraska, School of Biological Sciences, Lincoln, NE 68588, U.S.A. address: beckersom@unlserve.unl.edu (O.M. Beckers). Numerous sampling strategies have been proposed (e.g. Janetos 198; Wittenberger 1983; Real 199; Dombrovsky & Perrin 1994; Luttbeg 1996; Wiegmann et al. 1996), most of which involve comparing the traits of males that are sampled or comparing male traits to an internal standard (Janetos 198; Moore & Moore 1988). For all strategies, females are assumed to receive direct or indirect benefits from mating with males with preferred traits (preferred males are thus assumed to be of higher quality). Four of the more commonly proposed strategies are (1) the best-of-n strategy, (2) the sequential comparison strategy, (3) the fixed threshold strategy and (4) the variable threshold strategy. In the best-of-n strategy, a female samples a number (N) of males and then returns to the male with the highest quality (Janetos 198). In the sequential comparison strategy, a female continues searching as long as each new male encountered is of higher quality than the previous male. Once a female encounters a new male that is of lower quality than the previous male sampled, she chooses the previously encountered male (Wittenberger 1983). Finally, in the fixed threshold strategy (Janetos 198) and the variable (or adjustable) threshold strategy (Janetos 198; Reid & Stamps 1997), a female continues searching until she encounters a male whose quality exceeds an internal threshold value. The difference between these two strategies is how the threshold value is determined. In the fixed threshold strategy, females compare males against a fixed internal standard of acceptance (Janetos 198) /$38. Ó 1 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. doi:1.116/j.anbehav

2 5 O.M. Beckers, W.E. Wagner Jr / Animal Behaviour 81 (11) 519e527 This means that the female uses the same threshold acceptance criteria regardless of the mating opportunities that are available to her (Reid & Stamps 1997). In the variable threshold strategy, the standard changes in response to the information gathered during sampling, such as in response to information about the costs of searching and the distribution of male quality. We consider the onestep decision strategy (Janetos 198), sequential search strategy (Real 199) and optimal stopping rule (Dombrovsky & Perrin 1994) as variations of the variable threshold strategy. Mate sampling strategies have to balance the benefits obtained by finding a preferred mate with the costs associated with increased sampling effort (Janetos 198; Real 199). These costs include the time and energy used for sampling, the risk of predation during sampling and opportunity costs, such as reduced time available for foraging, the loss of previously sampled mates due to their death, emigration, or selection by other females (Daly 1978; Parker 1983; Real 199). In addition, the ability of a female to remember the characteristics and locations of previously sampled males may constrain the sampling strategy that a female uses (Janetos 198). In fact, all of the above-described strategies, with the exception of the fixed threshold strategy, require some degree of memory, a trait long assumed to be limited to vertebrates (but see Dukas 8). While the fixed threshold strategy does not require that females remember previously sampled males, it has a major drawback: a female that uses this strategy may risk not mating if the threshold is set too high, or, alternatively, risk accepting a low-quality male if the threshold is set too low (Jennions & Petrie 1997). However, it has been suggested that there could be a time-dependent decrease in the threshold value, which would improve the success of this strategy by granting a last chance option for the female if no high-quality males are available (Janetos 198; Real 199; Jennions & Petrie 1997). To our knowledge, the last chance option has not been demonstrated in any mating system. We studied female mate sampling behaviour in the variable field cricket, Gryllus lineaticeps. Male crickets produce songs to attract silent females for mating. Previous studies using single-speaker or two-speaker testing paradigms have shown that female G. lineaticeps prefer fast chirp rates over intermediate rates, and intermediate chirp rates over slow rates (Wagner 1996; Wagner & Reiser ; Wagner & Basolo 7a). Female G. lineaticeps directly benefit from mating with males that produce fast chirp rates under some environmental conditions because these males transfer seminal fluid products that increase female fecundity (Wagner & Harper 3; Tolle & Wagner, in press). We examined how female G. lineaticeps sample male chirp rates. In our first set of experiments, we tested female preferences using three sets of chirp rates. Each set consisted of three simultaneously presented chirp rates (slower, intermediate, and faster), but the sets differed in whether the chirp rates were centred at the slow, intermediate, or high end of the range. This acoustic environment is more complex than those used in the previous studies with G. lineaticeps, but it more closely simulates natural choruses in which many males sing simultaneously. We hypothesized that if females use the sequential comparison strategy, one of the variable threshold strategies, or the best-of-n strategy, they should consistently discriminate against the slower chirp rate(s) within each set of chirp rates (unless the threshold is set very low, in which case they will not discriminate among any of the chirp rates). As a result, whether a given chirp rate is more or less attractive should depend on the other chirp rates presented at the same time. In contrast, if females use a fixed threshold strategy, whether a given chirp rate is more or less attractive should be independent of the other chirp rates presented at the same time. Additionally, females should not discriminate among chirp rates that are above the threshold value or among chirp rates that are below the threshold value. In a second set of experiments, we tested whether female G. lineaticeps use a last chance option when males are not readily available. Previous results have shown that females show weaker responses to a slow chirp rate if they have experienced a fast chirp rate within the previous min (Wagner et al. 1). We tested whether this effect of acoustic experience weakens over time, which would be consistent with a last chance option as proposed by Janetos (198). GENERAL METHODS Animals We used females from a laboratory population of G. lineaticeps maintained at the University of Nebraska-Lincoln. We collected adult females from Academy, California, U.S.A. (Wagner & Basolo 7a) to establish the laboratory population. Most field-collected females had mated before capture in the field and laid fertile eggs in the laboratory. Individuals hatching from those eggs constituted the first laboratory generation. We actively managed the matings of subsequent laboratory generations by pairing males and females from unrelated families to reduce inbreeding and maintain genetic diversity. We tested females of the second and older laboratory generations for our experiments and tested no more than three females from a given family in a treatment group. The number of full-sibling families represented in each treatment ranged from 16 to 28. Juvenile crickets were reared in family containers until the penultimate instar, at which time we transferred them to individual containers. Once separated into individual containers, the nymphs were acoustically isolated from singing adult males. We checked the individual containers daily and recorded the date of the moult to adulthood. Females and males used in our experiments were tested between 7 and 12 days after adult moult. Family and individual containers had a paper towel substrate and cardboard shelters. Crickets were provided with water and Purina cat chow ad libitum (Wagner et al. 1). Crickets were reared on a 14:1 h light:dark cycle. Humidity ranged between 3% and 75%, and temperatures ranged between 21. C and 31 C. Our research adhered to the ASAB/ABS guidelines for the use of animals in research, the legal requirements of the U.S.A., and all guidelines of the University of Nebraska. Stimuli We generated acoustic stimuli that varied in chirp rate, while other song parameters were kept constant. We used one pulse of a natural chirp (pulse duration ¼ 11 ms, dominant frequency ¼ 5.17 khz; Wagner & Basolo 7a) and copied it eight times while holding the interpulse interval constant at 4 ms to construct a chirp with a duration of 116 ms. This chirp was copied, and the interchirp interval was varied by 1551 ms, 439 ms, 217 ms, 122 ms and 69 ms to construct stimuli with chirp rates of.6, 1.8, 3., 4.2 and 5.4 chirps/s, respectively. Stimuli were burned on CD and broadcast using Sony D-NF43 CD players (experiment 1, mate sampling experiment), or broadcast using a Macintosh Quadra 84AV computer (experiment 2, experience experiment), TEAC A-H3 amplifiers and KLH 97 loudspeakers. The stimuli were calibrated at peak amplitudes of 7 1 db SPL (re: mpa) at a distance of 3.5 cm from the loudspeaker, using a CEL-254 sound level meter. This amplitude corresponds to the amplitude of male songs measured at 3.5 cm distance (W.E.W., unpublished data). Experimental Set-up Females were tested in a square chamber (Fig. 1) measuring m (width length height). The chamber was

3 O.M. Beckers, W.E. Wagner Jr / Animal Behaviour 81 (11) 519e Experiment 1: Mate Sampling Release circle Empty lined with corrugated foam to reduce echoes. It was also equipped with dim red light mounted on the ceiling, which allowed females to be observed with a video camera mounted on the ceiling (Lorex SG4915R) and a Sylvania SRC134AC TV/VCR system outside the chamber. In each of the four corners of the chamber, a speaker circle with a radius of 26 cm was drawn on the floor. Between each speaker circle and the arena wall was a space of 3 cm to allow crickets to walk along the arena wall freely without entering a circle. Loudspeakers broadcasting acoustic stimuli were placed in the centre of the circles. Note that we used only three locations to place the loudspeakers in the first experiment (i.e. one location always remained empty). In the second experiment, the loudspeaker remained at the same location in all trials and the other three locations remained empty. Experiments were conducted at ambient temperatures of 23 1 C. Statistical Analyses Speaker circle Loudspeaker Male circle Figure 1. Schematic diagram of the test arena (top view). See Methods for the dimensions of release circle, male circle and speaker circle. Note that for the experience experiment (experiment 2), we used only one loudspeaker and loudspeaker circle. For our statistical analysis, we used procedures outlined in Zar (1999) or JMP statistical software (version 7..1, SAS Institute, Inc., Cary, NC, U.S.A.). In the mate sampling experiment (experiment 1), we compared within each trial the time that females spent near each of the three speakers using repeated measure Friedman tests. Post hoc comparisons (Wilcoxon signed-ranks tests) were used to compare the time that females spent near pairs of speakers. We also compared female choices between the three loudspeakers (i.e. the first speaker approached) using chi-square tests. Post hoc comparisons (binomial tests) were used to compare female choices between pairs of speakers. The significance levels of the multiple post hoc tests were adjusted using sequential HolmeBonferroni corrections (Holm 1979). In the experience experiment (experiment 2), we compared female responses between trials within treatments using Wilcoxon signed-ranks tests and compared female responses between treatments using ManneWhitney tests. All statistical comparisons used two-tailed tests. Methods We first tested whether females adjust their responses to a given chirp rate based on the alternatives that are simultaneously available. We used three chirp rate treatments, each consisting of a different range of chirp rates. In the first treatment, we broadcast songs with.6, 1.8 and 3. chirps/s (slow treatment). In the second treatment, we broadcast songs with 1.8, 3. and 4.2 chirps/s (intermediate treatment). And in the last treatment we broadcast songs with 3., 4.2 and 5.4 chirps/s (fast treatment). Chirp rates from 1 chirp/s to 4.5 chirps/s reflect approximately the natural range of G. lineaticeps song (Wagner & Reiser ). Thus, chirp rates of.6 chirps/s and 5.4 chirps/s were unusually slow and fast, respectively. We simultaneously broadcast the three different chirp rates within a treatment from the three different loudspeakers. We switched the stimuli between the loudspeakers pseudorandomly after each trial, using all nine possible combinations throughout each treatment. This resulted in the different chirp rates being presented approximately the same number of times from each speaker in each treatment. Each female was tested only once. We placed one male in front of each of the three loudspeakers. Males were muted by sealing their forewings with beeswax (Wagner et al. 7). Each male was tied to a weight (28 g; Dipsey Swivel sinker, Water Gremlin Co., MN, U.S.A.) with a string (w4 cm) to confine the male to the front of the loudspeaker. The weight was placed in the centre of a small male circle (radius ¼ 5cm), and males could move around in this small circle (Fig. 1). We included the males to reduce the probability that females would prematurely leave a loudspeaker because they could not find a male. The males were never related to the females tested in same trial. The males were pseudorandomly chosen and thus probably varied in size and other characteristics. We reused males in multiple trials, but we never reused males that were touched by a female during a trial. At the beginning of a trial, the female was placed under a cup (radius ¼ 2 cm) in the centre of the arena for 1 min for acclimation. This location was 1.14 m from each loudspeaker. After the acclimation period, we began broadcasting the stimuli and lifted the cup to release the female. The trials began when we lifted the cup and lasted for 15 min. For each trial, we measured the total time the female spent in each of the three loudspeaker circles. Preferences measured in this manner are correlated with female mate choices in other animals (e.g. White & Galef 1999; Morris et al. 1; this study, see Results). We also determined which chirp rate the female chose by noting which small male circle the female entered first, or which male the female touched first, accounting for the cases when the male was at the edge of the male circle, preventing the female from entering. We used both the time spent in each speaker circle and initial choice as measures of female preference. Females that did not enter a male circle, or that did not touch a male during the 15 min trial were excluded from analyses. Results In the treatment with the lowest range of chirp rates (Fig. 2a), there was significant variation in the time that females spent in the three circles associated with loudspeakers (Friedman test: c 2 2 ¼ 26., P <.1). The post hoc tests revealed that females showed significantly weaker responses to the two slowest chirp rates (.6 and 1.8 chirps/s) than to the fastest rate (Wilcoxon signedranks tests:.6 versus 3. chirps/s: T ¼ 224., N ¼ 33, P <.1, corrected a ¼.17; 1.8 versus 3. chirps/s: T ¼ 167., N ¼ 33, P <.2, corrected a ¼.25). However, female responses to the two slowest rates did not significantly differ (Wilcoxon signed-ranks test: T ¼ 24.5, N ¼ 33, P ¼.9, corrected a ¼.5). The same pattern of preference was reflected in the initial choice of females

4 522 O.M. Beckers, W.E. Wagner Jr / Animal Behaviour 81 (11) 519e (a) (b) (c) Time in association (s) (d) 3 (e) 3 (f) No. of females approached first Chirp rate (chirps/s) Figure 2. Preferences of female G. lineaticeps in a complex acoustic environment. Three different chirp rates were broadcast simultaneously in each trial. (aec) Mean SE time that females spent in proximity to each loudspeaker broadcasting a different chirp rate. (def) Number of the same females choosing each chirp rate. Note that the range of highly attractive chirp rates was independent of the alternative chirp rates presented in each trial (aec or def). We tested 33, 48 and 47 females in the slow (a, d), intermediate (b, e) and fast (c, f) trials, respectively. Asterisks and grey shading of bars indicate significant differences (P.4). (Fig. 2d). There was significant variation in the initial choice among the three loudspeakers (chi-square test: c 2 2 ¼ 31.9, P <.1). Significantly more females chose the speaker broadcasting the fastest rate (binomial tests:.6 versus 3. chirps/s, P <.1, corrected a ¼.17; 1.8 versus 3. chirps/s, P <.1 at corrected a ¼.25). The number of females that chose the speakers broadcasting the two slowest chirp rates did not differ significantly (binomial test: P ¼.16, corrected a ¼.5). In the intermediate chirp rate treatment (Fig. 2b), there was significant variation in the time that females spent in each of the three circles (Friedman test: c 2 2 ¼ 14.45, P <.1). The post hoc tests showed that female responses to the two faster chirp rates, 3. and 4.2 chirps/s, were significantly stronger than those to the slowest chirp rate of 1.8 chirps/s (Wilcoxon signed-ranks tests: 1.8 versus 3. chirps/s: T ¼ 313., N ¼ 48, P <.1, corrected a ¼.17; 1.8 versus 4.2 chirps/s: T ¼ 233., N ¼ 48, P ¼.1, corrected a.25). However, female responses to the two fastest chirp rates did not significantly differ (Wilcoxon signed-ranks test: T ¼ 78., N ¼ 48, P ¼.43, corrected a ¼.5). The same pattern of preference was reflected in the initial choice of females (Fig. 2e). The number of choices showed significant variation among chirp rates (chi-square test: c 2 2 ¼ , P <.1). Significantly more females chose the two fastest rates (1.8 chirps/ s; binomial tests: 1.8 versus 3. chirps/s: P <.1, corrected a ¼.17; 1.8 versus 4.2 chirps/s: P ¼.4, corrected a ¼.25). The number of females choosing the two fastest chirp rates did not differ significantly (binomial test: P ¼.11, corrected a ¼.5). In the treatment with the fastest chirp rates (3., 4.2 and 5.4 chirps/s; Fig. 2c), female responses did not differ between the three rates (Friedman test: c 2 2 ¼ 2.76, P >.1). There was also no significant difference in the initial choices of females among loudspeakers (chi-square test: c 2 2 ¼.298, P >.75; Fig. 2f). Together, these results suggest that in a complex acoustic environment containing more than two sound sources, females have a fixed threshold of preference based on chirp rate: they preferred chirp rates equal to or greater than 3. chirps/s to all lower chirp rates, but they did not discriminate among chirp rates below this threshold or among chirp rates equal to or above this threshold, regardless of the alternatives that were presented.

5 O.M. Beckers, W.E. Wagner Jr / Animal Behaviour 81 (11) 519e Experiment 2: Effect of Experience Methods Our first experiment indicated that female G. lineaticeps use a fixed threshold strategy for mate sampling. However, it has been shown that previous acoustic experience with a fast chirp rate reduces female responses to a slow chirp rate (Wagner et al. 1). In a second experiment, we tested whether this effect of acoustic experience weakens over time. If females show stronger responses to a less attractive (slow) song after a longer period of silence, it would indicate a lowering of the threshold as result of a lack of a recent exposure to attractive song types, which would be consistent with the last chance option proposed by Janetos (198). Using a repeated measures design, we measured female responses in two treatments, each consisting of two trials separated by different durations of silence. In the first treatment, the first and second trials were separated by e3 min. This was designed to represent a reasonable time interval separating female encounters with different males on the same night. In the second treatment, the first and second trials were separated by 24e26 h. This was designed to represent a reasonable time interval separating female encounters with different males on sequential nights. For simplicity, we refer to the time periods used as min and 24 h. In both treatments, we tested female responses to a chirp rate of 4.2 chirps/s in the first trial (a more preferred chirp rate in experiment 1) and responses to 1.8 chirps/s chirp rate in the second trial (a less preferred chirp rate in experiment 1). In each trial we measured the time the female spent in the circle with the loudspeaker. A female was tested in only one of the treatments. We compared female responses to the high and low chirp rates within each treatment. The last chance option predicts that females will show a stronger response to the high chirp rate when the low chirp rate is presented min later. In contrast, females should show similar responses to the two chirp rates when the low chirp rate is presented 24 h later. We also compared the relative responses of females to the low chirp rates in the two treatments (time in association with the low chirp rate/time in association with the high chirp rate). Because some females spent s in association with a speaker in one of the trials, we added 1 s to all of the female responses. The last chance option predicts that females will show stronger relative responses to the low chirp rate when it is presented 24 h after the high chirp rate than when it is presented min after the high chirp rate. In two control treatments, we measured female attraction to a chirp rate of 4.2 chirps/s, with the trials being separated by min or 24 h, respectively. These control treatments tested for sequential testing effects and age effects. For example, females may show weak responses when retested after min, regardless of the chirp rate with which they are tested, but their responses may recover after 24 h. Each female was tested in only one of the treatments. We compared female responses within each treatment and between treatments, as described above. A loudspeaker was placed in the centre of a circle (radius ¼ 26 cm) in one corner of the arena (Fig. 1). At the beginning of the trial, we placed the female under a cup (radius ¼ 4 cm) inside a small circle in the centre of the arena ( release circle ; radius ¼ 5 cm). The distance between the female in the centre of the arena and the loudspeaker was 1.14 m. During the acclimation period, we kept the female under the cup for 5 min, while broadcasting the test or control stimulus. At the end of the acclimation period we released the female by lifting the cup. The trial started when the female left the release circle in centre of the arena and lasted for 1 min. In each trial, we measured the total time the female spent inside the circle associated with the broadcasting loudspeaker. If a female left the release circle within 1 min, but did not enter the circle around the loudspeaker, the female was assigned a zero for the total time spent inside the circle. Females that did not leave the release circle within 1 min after lifting the cup or that failed to enter the circle in both trials of a series were excluded from analyses. These females were considered unresponsive to male song. However, females that failed to enter the circle in only one of the trials were included. Results Females showed significantly stronger responses to the initial fast chirp rate than to the slow chirp rate presented min later (Wilcoxon signed-ranks test: T ¼ 112.5, N ¼ 32, P ¼.33; Fig. 3a). However, there was no significant difference in female responses to the initial fast chirp rate and the slow chirp rate presented 24 h later (Wilcoxon signed-ranks test: T ¼ 76., N ¼ 35, P ¼.218; Fig. 3b). The relative responses of females to the slow chirp rate, however, did not differ significantly between the two treatments (ManneWhitney test: U ¼ 165.5, N 1 ¼ 32, N 2 ¼ 35, P ¼.782). In two control treatments (Fig. 3c, d), we tested female responses to a fast chirp rate (4.2 chirps/s) in both the initial and the second trial using the same time intervals as in the previous tests. Females showed no significant difference in their responses to an initial fast chirp rate and the same chirp rate presented min later (Wilcoxon signed-ranks test: T ¼ 31.5, N ¼ 27, P ¼.46; Fig. 3c). In addition, females showed no significant difference in their responses to an initial fast chirp rate and the same chirp rate presented 24 h later (Wilcoxon signed-ranks test: T ¼ 15., N ¼ 28, P ¼.74; Fig. 3d). The relative responses of females to the second fast chirp rate did not significantly differ between the two treatments (ManneWhitney test: U ¼ 728.5, N 1 ¼ 27, N 2 ¼ 28, P ¼.649). Together, these results provide mixed support for the last chance option hypothesis. Females showed significantly stronger responses to a high chirp rate than to a low chirp rate presented min later, but they did not show significantly stronger responses to a high chirp rate than to a low chirp presented 24 h later. The control experiment suggests that the weak response to the low chirp rate presented min after a high chirp rate was not simply a consequence of females showing weak responses to all calls types presented min after an initial test. These results thus suggest that females discriminate against a slow chirp rate if they have recently heard a fast chirp rate, but that they will respond strongly to a slow chirp rate if they have not recently heard male song. We were, however, unable to show that the relative responses of females to the low chirp rate song were significantly different between the two treatments. DISCUSSION We examined female mate sampling strategies in G. lineaticeps. Our three-stimulus experiment suggested that females use a fixed threshold strategy in complex acoustic environments: females responded strongly in each trial to chirp rates that were equal to or greater than 3. chirps/s, regardless of the other song types that were simultaneously present. Similarly, females responded only weakly to chirp rates below 3. chirps/s, regardless of the other song types that were simultaneously present. These results suggest a threshold-like preference pattern. In contrast to our current results, previous experiments using single-stimulus and two-stimulus testing paradigms indicated that female G. lineaticeps show finer levels of discrimination among chirp rates. In single-stimulus experiments, there was a linear and stabilizing component to female responses: females tended to respond more strongly to higher chirp rates across a range of 1.8e4.2 chirps/s, but responded more strongly to intermediate and high chirp rates than to very high chirp rates (Wagner & Basolo 7a). Similarly, in most two-speaker choice tests, female G. lineaticeps preferred higher

6 524 O.M. Beckers, W.E. Wagner Jr / Animal Behaviour 81 (11) 519e527 min 24 h 8 (a) 8 (b) Time in association (s) 1 8 (c) (d) Chirp rate (chirps/s) Figure 3. Preferences of female G. lineaticeps in sequential single-stimulus presentations. Mean SE time that females spent in proximity to the loudspeaker. (a) Attraction in the initial trial was tested to a fast chirp rate (4.2 chirps/s) and in the second trial to a slow chirp rate (1.8 chirps/s; N ¼ 32). Trials were separated by e3 min. (b) Female responses to the same stimuli used in (a), but trials were separated by 24e26 h (N ¼ 35). (c, d) Control treatments. Female attraction to the fast chirp rate (4.2 chirps/s) was tested in the initial and the subsequent trial. (c) Trials separated by e3 min (N ¼ 26). (d) Trials separated by 24e26 h (N ¼ 28). Asterisk and grey shading of bar indicate significant difference (P ¼.33). to lower chirp rates, both at the low and high ends of the testing range of 1.75 to 4. chirps/s (Wagner & Reiser ). The contrasting results between the previous studies and the current study may be explained by the different experimental designs. In the current study, we used a more complex acoustic environment with three simultaneously presented songs to measure female responses. Our simulated acoustic environment approximates a more natural setting in a highdensity chorus of G. lineaticeps (O.M.B., personal observation). The crickets tested in the previous studies, however, were from a different population than those tested in the current study. It is also possible that females from different populations use different sampling strategies. In some frogs, the addition of multiple sound sources or background noise has been shown to reduce female discrimination among call traits (e.g. Gerhardt 1982, 1987; Telford et al. 1989; Marquez & Bosch 1997; Schwartz et al. 1) comparedtosimpler two-stimulus environments (e.g. Gerhardt 1982; Marquez 1995; Gerhardt et al. 1996, ; Schwartz et al. 1). Acoustic interference and masking of overlapping calls (e.g. Schwartz 1987; Gerhardt & Klump 1988; Schwartz & Gerhardt 1989) under complex acoustic

7 O.M. Beckers, W.E. Wagner Jr / Animal Behaviour 81 (11) 519e conditions probably make it more difficult for females to distinguish (small) differences among the calls of individuals (Marquez & Bosch 1997). Simple acoustic conditions may thus exaggerate female selectivity (Schwartz et al. 1). Furthermore, our results suggest that females may switch from a comparison strategy in a simple acoustic environment (i.e. from evaluating the relative chirp rates of the available songs and then responding more strongly to the higher chirp rate song) to a threshold strategy in a complex acoustic environment (i.e. to evaluating whether a given chirp rate is low or intermediate/high and then responding strongly to any intermediate/high chirp rates they detect). The results of some studies of other animals have suggested that females categorically classify the calls of conspecifics and heterospecifics (Baugh et al. 8) and the calls of conspecifics and predators (Wyttenbach et al. 1996). Our results suggest that categorical perception may also play a role in the context of mate assessment. According to the predictions of a fixed threshold strategy, a searching female should mate with the first male that exceeds the threshold value, and this value is set before females begin to search for potential mates (Janetos 198; Real 199; Reid & Stamps 1997). Because of the static quality of the fixed threshold strategy, it has been considered inferior to other strategies under most conditions (Janetos 198; Janetos & Cole 1981). For example, females using a fixed threshold strategy discriminate against low-quality males, independent of the costs involved in searching for a better male that would exceed the threshold. This can be especially costly if, in some generations or environments, the quality of most males is below the threshold value. In this case, females would incur high costs due to extended search and may even forgo mating (Jennions & Petrie 1997). Also, females using this search strategy would not distinguish among high-quality males, resulting in lost opportunities to mate with males that provide the highest quality mating benefits. However, it has been suggested that a fixed threshold strategy may be advantageous if the environment is constant (Real 199; Collins et al. 6) and/or search costs are high and females have to make quick mating decisions (Mazalov et al. 1996; Reid & Stamps 1997). Evidence for fixed threshold strategies have been found for the cockroach Nauphoeta cinerea (Moore & Moore 1988), the red junglefowl Gallus gallus (Zuk et al. 199) and the cricket Gryllus sigillatus (Ivy & Sakaluk 7). Janetos (198) suggested another, but rarely considered version of the fixed threshold strategy: the fixed threshold with last chance option. Here, females use a fixed threshold strategy as long as male quality is high. However, if the females have not encountered a sufficiently attractive male after some period of time, they will mate with the next available male regardless of the male s attractiveness (Janetos 198). Thus, a female expressing a fixed threshold strategy with a last chance option would behave differently in different social environments (i.e. her threshold would be higher if some available males have traits that exceed the initial threshold, but the threshold would decline over time if no males with traits above this threshold are encountered). In a theoretical comparison between the fixed threshold strategy with last chance option, bestof-n strategy and one-step decision strategy, the latter two strategies only resulted in greater mating benefits if the number of the sampled males was large (Janetos & Cole 1981). It was further suggested that the selective advantage of the best-of-n and the one-step decision strategies over the fixed threshold strategy with last chance option can be fairly small (Janetos & Cole 1981). Previous work on G. lineaticeps suggested that females change their responses to slow chirp rate song depending upon whether they recently encountered high chirp rate song (Wagner et al. 1). Social experience is likewise known to affect female mating preferences in a limited number of other invertebrate systems (e.g. Hebets 3; Dukas 5; Hebets & Vink 7; Fincke et al. 7), including another field cricket (Bailey & Zuk 8, 9). In the current study, we sequentially presented females with a highly attractive fast chirp rate followed by a less attractive slow chirp rate. Females showed weaker responses to the slow chirp rate presented min after the fast chirp rate (Fig. 3a), but showed stronger responses to the slow chirp rate presented 24 h after the fast chirp rate (Fig. 3b). Thus, females accepted slower rates if no faster chirp rates had recently been experienced. However, we were unable to show a significant difference in female response strength to the slow rate in the two treatments, which may partially be due to a lack of statistical power (i.e. there was substantial variation in female responses to the slow rate after 24 h; see Fig. 3b). Our results thus provide mixed support for the last option hypothesis. Previous studies have shown age-related effects on preferences (Moore & Moore 1; Lynch et al. 5) and age-related effects on mate assessment (Baugh & Ryan 9). Less is known about time-related experience effects on preferences. As outlined above, female preferences can change with the acoustic environment in which they are tested (e.g. two-speaker versus three-speaker experiments). An alternative to the onespeaker experimental design we used to test time-dependent changes in female preferences would be a three-speaker design in which three above-threshold chirp rates are presented in the first trial and, after min or 24 h, three below-threshold rates are presented in a second trial. The fixed threshold strategy with last chance option predicts that females under these conditions would show weak responses to all three slow chirp rates after min but strong responses after 24 h. Deviations from these predictions would suggest that females switch to a comparative strategy when only slow chirp rates are present (e.g. if they responded more strongly to the higher of the slow chirp rates after min), or that females use a pure fixed threshold strategy in complex acoustic environments (e.g. if they continued to show weak responses to all three slow chirp rates after 24 h). For G. lineaticeps females, plasticity in their responses to slow chirp rate songs would probably be beneficial, considering that population density can fluctuate remarkably throughout the breeding season. Male singing activity is usually low at the beginning of the season and increases over the summer and early autumn (W.E.W., personal observation). In some populations, the acoustically orienting fly Ormia ochracea parasitizes G. lineaticeps in the late summer and early autumn (Wagner 1996), resulting in high mortality for singing males (59% of males can be parasitized and thus near death at a given time; Martin & Wagner 1), whereas silent females are less often parasitized (w6%; Martin & Wagner 1). As a result, few males but many receptive females are present after a few weeks of fly parasitism (W.E.W., personal observation). Additionally, the flies have the same preferences for faster chirp rates as G. lineaticeps females (Wagner 1996; Wagner & Basolo 7b), which should selectively remove the most attractive males from the population. Females expressing a pure fixed threshold strategy are thus likely to incur high search costs, and perhaps never mate, under some conditions. Mate sampling has been investigated in various vertebrate and invertebrate systems (reviewed in Jennions & Petrie 1997). As a general pattern, it has been proposed that a best-of-n strategy is most likely to be found in mating systems with low costs for sampling multiple males (Reynolds & Gross 199; Gibson & Bachman 1992). These conditions are most often realized in species that form leks, where females can quickly sample many males within brief time periods. Accordingly, females of at least some lekking species show sampling behaviour that supports a best-of-n strategy (Trail & Adams 1989; Fiske & Kålås 1995; Rintamäki et al. 1995). Most studies investigating mate sampling behaviour, however, have found considerable variability among individuals (e.g. sequence and

8 526 O.M. Beckers, W.E. Wagner Jr / Animal Behaviour 81 (11) 519e527 number of males visited prior to choice) and have often supported more than one strategy for a given species (e.g. Bensch & Hasselquist 1992; Choudhurry & Black 1994; Dale & Slagsvold 1996). It is possible that females of a given species use more than one mate sampling strategy. Strategies differ in costliness (e.g. time costs, energy costs, predation risk), and some individuals may not be able to afford to use a more costly strategy and instead use a different strategy (Jennions & Petrie 1997). Most studies of female strategies have been based on field observations of female behaviour (e.g. Fiske & Kålås 1995; Dale & Slagsvold 1996; Fagundes et al. 7). However, this approach has been criticized for two reasons. First, the same sampling sequence may support more than one strategy (Gibson & Langen 1996). Second, repeated sampling of a given male may be due to memory errors (Wiegmann et al. 1996) and/or due to imperfect information gained about male quality (Luttbeg 1996), rather than due to the use of a best-of-n sampling strategy. Thus, it may be necessary to use more than one approach (e.g. observations and controlled experiments) to clearly identify the sampling strategy used. In addition, as our results suggest, female sampling strategies may vary among social conditions. In G. lineaticeps, for example, females may use a comparison strategy when male densities are low but use a fixed threshold strategy with last chance option when male densities are higher. Acknowledgments We thank two anonymous referees and J. Podos, A. L. Basolo, L. Sullivan-Beckers and E. A. Hebets for their helpful criticism and editing of the manuscript. This research was supported by a National Science Foundation research grant awarded to W.E.W. (NSF ). References Bailey, N. W. & Zuk, M. 8. 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