Animal Behaviour 81 (2011) 519e527

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Animal Behaviour

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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 The strategy females use to sample potential mates can influence mate choice and thus sexual selection. fi Article history: We examined the mate sampling strategy of the cricket Gryllus lineaticeps. In our rst set of experiments, Received 13 July 2010 we simultaneously presented three different chirp rates to females. The set consisted of three trials, each Initial acceptance 12 August 2010 covering a different range of chirp rates. Independent of chirp rate range, female G. lineaticeps preferred Final acceptance 15 November 2010 rates that were above 3.0 chirps/s to rates that were below 3.0 chirps/s. Females did not discriminate Available online 28 December 2010 among chirp rates that were below this threshold and did not discriminate among chirp rates that were MS. number: A10-00483 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 Keywords: the interval between presentations was 20 min, females showed significantly weaker responses to the acoustic communication slow rate than to the fast rate. However, when the interval between presentations was 24 h, female female preference responses to the slow and fast rate did not significantly differ. The latter result suggests that females fixed threshold strategy Gryllus lineaticeps lower their threshold of acceptance when they have not recently experienced highly attractive song last chance option types. This lower acceptance threshold is probably adaptive, as it would allow females to avoid paying mate choice high search costs, and to reproduce, only when low-quality males are available. Our results are consistent search strategy 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 2010 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 Numerous sampling strategies have been proposed (e.g. Janetos the causes and consequences of sexual selection. Most research on 1980; Wittenberger 1983; Real 1990; Dombrovsky & Perrin 1994; female mate choice has focused on female mating preferences, and Luttbeg 1996; Wiegmann et al. 1996), most of which involve we know that females often prefer to mate with males with comparing the traits of males that are sampled or comparing male particular types of traits (e.g. Basolo 1990; Wilkinson & Reillo 1994; traits to an internal standard (Janetos 1980; Moore & Moore 1988). Gerhardt et al. 2000). Mate choice, however, is the result of inter- For all strategies, females are assumed to receive direct or indirect actions between environmental conditions (e.g. predation risk; benefits from mating with males with preferred traits (preferred Godin & Briggs 1995), mating preferences and sampling strategies males are thus assumed to be of higher quality). Four of the more (Wagner 1998). Much less is known about how females sample, commonly proposed strategies are (1) the best-of-N strategy, (2) the assess and compare potential mates (i.e. female sampling or search sequential comparison strategy, (3) the fixed threshold strategy and strategies) than about female mating preferences. Understanding (4) the variable threshold strategy. In the best-of-N strategy, a female female sampling strategies is important because different strategies samples a number (N) of males and then returns to the male with the can lead to different mate choices. For example, the number of highest quality (Janetos 1980). In the sequential comparison strategy, males sampled may differ depending upon the sampling strategy a female continues searching as long as each new male encountered a female uses, and females that sample more males are likely to is of higher quality than the previous male. Once a female encounters show a greater bias in their mate choices than females sampling a new male that is of lower quality than the previous male sampled, fewer males (Janetos 1980; Wagner 1998). The sampling strategies she chooses the previously encountered male (Wittenberger 1983). that females use can thus affect both how sexual selection acts on Finally, in the fixed threshold strategy (Janetos 1980) and the variable male traits and the benefits of mate choice itself. (or adjustable) threshold strategy (Janetos 1980; 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 * Correspondence: O. M. Beckers, University of Nebraska, School of Biological fi Sciences, Lincoln, NE 68588, U.S.A. determined. In the xed threshold strategy, females compare males E-mail address: [email protected] (O.M. Beckers). against a fixed internal standard of acceptance (Janetos 1980).

0003-3472/$38.00 Ó 2010 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.anbehav.2010.11.022 520 O.M. Beckers, W.E. Wagner Jr / Animal Behaviour 81 (2011) 519e527

This means that the female uses the same threshold acceptance In a second set of experiments, we tested whether female G. lin- criteria regardless of the mating opportunities that are available to eaticeps use a last chance option when males are not readily available. her (Reid & Stamps 1997). In the variable threshold strategy, the Previous results have shown that females show weaker responses to standard changes in response to the information gathered during a slow chirp rate if they have experienced a fast chirp rate within the sampling, such as in response to information about the costs of previous 20 min (Wagner et al. 2001). We tested whether this effect searching and the distribution of male quality. We consider the one- of acoustic experience weakens over time, which would be consistent step decision strategy (Janetos 1980), sequential search strategy (Real with a last chance option as proposed by Janetos (1980). 1990) and optimal stopping rule (Dombrovsky & Perrin 1994)as variations of the variable threshold strategy. GENERAL METHODS Mate sampling strategies have to balance the benefits obtained by finding a preferred mate with the costs associated with increased Animals sampling effort (Janetos 1980; Real 1990). These costs include the time and energy used for sampling, the risk of predation during We used females from a laboratory population of G. lineaticeps sampling and opportunity costs, such as reduced time available for maintained at the University of Nebraska-Lincoln. We collected adult foraging, the loss of previously sampled mates due to their death, females from Academy, California, U.S.A. (Wagner & Basolo 2007a)to emigration, or selection by other females (Daly 1978; Parker 1983; establish the laboratory population. Most field-collected females had Real 1990). In addition, the ability of a female to remember the mated before capture in the field and laid fertile eggs in the labora- characteristics and locations of previously sampled males may tory. Individuals hatching from those eggs constituted the first constrain the sampling strategy that a female uses (Janetos 1980). In laboratory generation. We actively managed the matings of subse- fact, all of the above-described strategies, with the exception of the quent laboratory generations by pairing males and females from fixed threshold strategy, require some degree of memory, a trait long unrelated families to reduce inbreeding and maintain genetic diver- assumed to be limited to vertebrates (but see Dukas 2008). While the sity. We tested females of the second and older laboratory genera- fixed threshold strategy does not require that females remember tions for our experiments and tested no more than three females previously sampled males, it has a major drawback: a female that from a given family in a treatment group. The number of full-sibling uses this strategy may risk not mating if the threshold is set too high, families represented in each treatment ranged from 16 to 28. or, alternatively, risk accepting a low-quality male if the threshold is Juvenile crickets were reared in family containers until the set too low (Jennions & Petrie 1997). However, it has been suggested penultimate instar, at which time we transferred them to individual that there could be a time-dependent decrease in the threshold value, containers. Once separated into individual containers, the nymphs which would improve the success of this strategy by granting a ‘last were acoustically isolated from singing adult males. We checked chance option’ for the female if no high-quality males are available the individual containers daily and recorded the date of the moult (Janetos 1980; Real 1990; Jennions & Petrie 1997). To our knowledge, to adulthood. Females and males used in our experiments were the ‘last chance option’ has not been demonstrated in any mating tested between 7 and 12 days after adult moult. system. Family and individual containers had a paper towel substrate We studied female mate sampling behaviour in the variable field and cardboard shelters. Crickets were provided with water and cricket, Gryllus lineaticeps. Male crickets produce songs to attract Purina cat chow ad libitum (Wagner et al. 2001). Crickets were silent females for mating. Previous studies using single-speaker or reared on a 14:10 h light:dark cycle. Humidity ranged between 30% two-speaker testing paradigms have shown that female G. line- and 75%, and temperatures ranged between 21.0 C and 31 C. Our aticeps prefer fast chirp rates over intermediate rates, and inter- research adhered to the ASAB/ABS guidelines for the use of animals mediate chirp rates over slow rates (Wagner 1996; Wagner & Reiser in research, the legal requirements of the U.S.A., and all guidelines 2000; Wagner & Basolo 2007a). Female G. lineaticeps directly benefit of the University of Nebraska. from mating with males that produce fast chirp rates under some environmental conditions because these males transfer seminal Stimuli fluid products that increase female fecundity (Wagner & Harper 2003; Tolle & Wagner, in press). We generated acoustic stimuli that varied in chirp rate, while We examined how female G. lineaticeps sample male chirp rates. other song parameters were kept constant. We used one pulse of In our first set of experiments, we tested female preferences using a natural chirp (pulse duration ¼ 11 ms, dominant frequency ¼ 5.17 three sets of chirp rates. Each set consisted of three simultaneously kHz; Wagner & Basolo 2007a) and copied it eight times while presented chirp rates (slower, intermediate, and faster), but the sets holding the interpulse interval constant at 4 ms to construct a chirp differed in whether the chirp rates were centred at the slow, inter- with a duration of 116 ms. This chirp was copied, and the interchirp mediate, or high end of the range. This acoustic environment is more interval was varied by 1551 ms, 439 ms, 217 ms, 122 ms and 69 ms complex than those used in the previous studies with G. lineaticeps, to construct stimuli with chirp rates of 0.6, 1.8, 3.0, 4.2 and but it more closely simulates natural choruses in which many males 5.4 chirps/s, respectively. Stimuli were burned on CD and broadcast sing simultaneously. We hypothesized that if females use the using Sony D-NF430 CD players (experiment 1, mate sampling sequential comparison strategy, one of the variable threshold experiment), or broadcast using a Macintosh Quadra 840AV strategies, or the best-of-N strategy, they should consistently computer (experiment 2, experience experiment), TEAC A-H300 discriminate against the slower chirp rate(s) within each set of chirp amplifiers and KLH 970 loudspeakers. The stimuli were calibrated rates (unless the threshold is set very low, in which case they will at peak amplitudes of 70 1 dB SPL (re: 20 mPa) at a distance of not discriminate among any of the chirp rates). As a result, whether 30.5 cm from the loudspeaker, using a CEL-254 sound level meter. a given chirp rate is more or less attractive should depend on the This amplitude corresponds to the amplitude of male songs other chirp rates presented at the same time. In contrast, if females measured at 30.5 cm distance (W.E.W., unpublished data). use a fixed threshold strategy, whether a given chirp rate is more or less attractive should be independent of the other chirp rates pre- Experimental Set-up sented at the same time. Additionally, females should not discrim- inate among chirp rates that are above the threshold value or among Females were tested in a square chamber (Fig. 1) measuring chirp rates that are below the threshold value. 2.2 2.2 2.7 m (width length height). The chamber was O.M. Beckers, W.E. Wagner Jr / Animal Behaviour 81 (2011) 519e527 521

Experiment 1: Mate Sampling

Speaker circle Methods We first tested whether females adjust their responses to a given chirp rate based on the alternatives that are simultaneously avail- able. We used three chirp rate treatments, each consisting of a different range of chirp rates. In the first treatment, we broadcast Male circle songs with 0.6, 1.8 and 3.0 chirps/s (slow treatment). In the second treatment, we broadcast songs with 1.8, 3.0 and 4.2 chirps/s (intermediate treatment). And in the last treatment we broadcast songs with 3.0, 4.2 and 5.4 chirps/s (fast treatment). Chirp rates from fl Release circle 1chirp/sto4.5chirps/sre ect approximately the natural range of G. lineaticeps song (Wagner & Reiser 2000). Thus, chirp rates of 0.6 chirps/s and 5.4 chirps/s were unusually slow and fast, respec- tively. 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. Empty We placed one male in front of each of the three loudspeakers. Loudspeaker Males were muted by sealing their forewings with beeswax (Wagner et al. 2007). Each male was tied to a weight (28 g; Dipsey Swivel w Figure 1. Schematic diagram of the test arena (top view). See Methods for the sinker, Water Gremlin Co., MN, U.S.A.) with a string ( 4cm)to dimensions of release circle, male circle and speaker circle. Note that for the experi- confine the male to the front of the loudspeaker. The weight was ence experiment (experiment 2), we used only one loudspeaker and loudspeaker placed in the centre of a small ‘male circle’ (radius ¼ 5cm),and circle. males could move around in this small circle (Fig. 1). We included the males to reduce the probability that females would prematurely lined with corrugated foam to reduce echoes. It was also equipped leave a loudspeaker because they could not find a male. The males with dim red light mounted on the ceiling, which allowed females were never related to the females tested in same trial. The males to be observed with a video camera mounted on the ceiling (Lorex were pseudorandomly chosen and thus probably varied in size and SG4915R) and a Sylvania SRC20134AC TV/VCR system outside the other characteristics. We reused males in multiple trials, but we chamber. In each of the four corners of the chamber, a ‘speaker never reused males that were touched by a female during a trial. circle’ with a radius of 26 cm was drawn on the floor. Between each At the beginning of a trial, the female was placed under a cup speaker circle and the arena wall was a space of 3 cm to allow (radius ¼ 2 cm) in the centre of the arena for 10 min for acclimation. crickets to walk along the arena wall freely without entering This location was 1.14 m from each loudspeaker. After the acclima- a circle. Loudspeakers broadcasting acoustic stimuli were placed in tion period, we began broadcasting the stimuli and lifted the cup to the centre of the circles. Note that we used only three locations to release the female. The trials began when we lifted the cup and place the loudspeakers in the first experiment (i.e. one location lasted for 15 min. For each trial, we measured the total time the always remained empty). In the second experiment, the loud- female spent in each of the three loudspeaker circles. Preferences speaker remained at the same location in all trials and the other measured in this manner are correlated with female mate choices in three locations remained empty. Experiments were conducted at other animals (e.g. White & Galef 1999; Morris et al. 2010; this study, ambient temperatures of 23 1 C. see Results). We also determined which chirp rate the female chose by noting which small ‘male circle’ the female entered first, or which Statistical Analyses male the female touched first, accounting for the cases when the male was at the edge of the male circle, preventing the female from For our statistical analysis, we used procedures outlined in Zar entering. We used both the time spent in each speaker circle and (1999) or JMP statistical software (version 7.0.1, SAS Institute, Inc., initial choice as measures of female preference. Females that did not Cary, NC, U.S.A.). In the mate sampling experiment (experiment 1), enter a male circle, or that did not touch a male during the 15 min we compared within each trial the time that females spent near trial were excluded from analyses. each of the three speakers using repeated measure Friedman tests. Post hoc comparisons (Wilcoxon signed-ranks tests) were used to Results compare the time that females spent near pairs of speakers. We In the treatment with the lowest range of chirp rates (Fig. 2a), also compared female choices between the three loudspeakers there was significant variation in the time that females spent in the (i.e. the first speaker approached) using chi-square tests. Post hoc three circles associated with loudspeakers (Friedman test: 2 comparisons (binomial tests) were used to compare female c2 ¼ 26.20, P < 0.001). The post hoc tests revealed that females choices between pairs of speakers. The significance levels of the showed significantly weaker responses to the two slowest chirp multiple post hoc tests were adjusted using sequential rates (0.6 and 1.8 chirps/s) than to the fastest rate (Wilcoxon signed- HolmeBonferroni corrections (Holm 1979). In the experience ranks tests: 0.6 versus 3.0 chirps/s: T ¼224.00, N ¼ 33, P < 0.0001, experiment (experiment 2), we compared female responses corrected a ¼ 0.017; 1.8 versus 3.0 chirps/s: T ¼167.00, N ¼ 33, between trials within treatments using Wilcoxon signed-ranks P < 0.0002, corrected a ¼ 0.025). However, female responses to the tests and compared female responses between treatments using two slowest rates did not significantly differ (Wilcoxon signed-ranks ManneWhitney tests. All statistical comparisons used two-tailed test: T ¼24.5, N ¼ 33, P ¼ 0.09, corrected a ¼ 0.05). The same tests. pattern of preference was reflected in the initial choice of females 522 O.M. Beckers, W.E. Wagner Jr / Animal Behaviour 81 (2011) 519e527

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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 (aecordef). 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 0.0004).

(Fig. 2d). There was significant variation in the initial choice among (Fig. 2e). The number of choices showed significant variation 2 2 the three loudspeakers (chi-square test: c2 ¼ 31.09, P < 0.001). among chirp rates (chi-square test: c2 ¼ 13.625, P < 0.001). Significantly more females chose the speaker broadcasting the Significantly more females chose the two fastest rates (1.8 chirps/ fastest rate (binomial tests: 0.6 versus 3.0 chirps/s, P < 0.0001, cor- s; binomial tests: 1.8 versus 3.0 chirps/s: P < 0.0001, corrected rected a ¼ 0.017; 1.8 versus 3.0 chirps/s, P < 0.0001 at corrected a ¼ 0.017; 1.8 versus 4.2 chirps/s: P ¼ 0.0004, corrected a ¼ 0.025). The number of females that chose the speakers broad- a ¼ 0.025). The number of females choosing the two fastest chirp casting the two slowest chirp rates did not differ significantly rates did not differ significantly (binomial test: P ¼ 0.11, corrected (binomial test: P ¼ 0.16, corrected a ¼ 0.05). a ¼ 0.05). In the intermediate chirp rate treatment (Fig. 2b), there was In the treatment with the fastest chirp rates (3.0, 4.2 and significant variation in the time that females spent in each of the 5.4 chirps/s; Fig. 2c), female responses did not differ between the 2 2 three circles (Friedman test: c2 ¼ 14.045, P < 0.001). The post hoc three rates (Friedman test: c2 ¼ 2.76, P > 0.1). There was also no tests showed that female responses to the two faster chirp rates, significant difference in the initial choices of females among 2 3.0 and 4.2 chirps/s, were significantly stronger than those to the loudspeakers (chi-square test: c2 ¼ 0.298, P > 0.75; Fig. 2f). slowest chirp rate of 1.8 chirps/s (Wilcoxon signed-ranks tests: 1.8 Together, these results suggest that in a complex acoustic envi- versus 3.0 chirps/s: T ¼313.00, N ¼ 48, P < 0.0001, corrected ronment containing more than two sound sources, females have a ¼ 0.017; 1.8 versus 4.2 chirps/s: T ¼233.00, N ¼ 48, P ¼ 0.0001, a fixed threshold of preference based on chirp rate: they preferred corrected a 0.025). However, female responses to the two fast- chirp rates equal to or greater than 3.0 chirps/s to all lower chirp est chirp rates did not significantly differ (Wilcoxon signed-ranks rates, but they did not discriminate among chirp rates below this test: T ¼ 78.00, N ¼ 48, P ¼ 0.43, corrected a ¼ 0.05). The same threshold or among chirp rates equal to or above this threshold, pattern of preference was reflected in the initial choice of females regardless of the alternatives that were presented. O.M. Beckers, W.E. Wagner Jr / Animal Behaviour 81 (2011) 519e527 523

Experiment 2: Effect of Experience circle around the loudspeaker, the female was assigned a zero for the total time spent inside the circle. Females that did not leave the Methods release circle within 10 min after lifting the cup or that failed to Our first experiment indicated that female G. lineaticeps use enter the circle in both trials of a series were excluded from anal- a fixed threshold strategy for mate sampling. However, it has been yses. These females were considered unresponsive to male song. shown that previous acoustic experience with a fast chirp rate However, females that failed to enter the circle in only one of the reduces female responses to a slow chirp rate (Wagner et al. 2001). trials were included. In a second experiment, we tested whether this effect of acoustic experience weakens over time. If females show stronger responses Results to a less attractive (slow) song after a longer period of silence, it Females showed significantly stronger responses to the initial fast would indicate a lowering of the threshold as result of a lack of chirp rate than to the slow chirp rate presented 20 min later a recent exposure to attractive song types, which would be (Wilcoxon signed-ranks test: T ¼112.50, N ¼ 32, P ¼ 0.033; Fig. 3a). consistent with the last chance option proposed by Janetos (1980). However, there was no significant difference in female responses to Using a repeated measures design, we measured female the initial fast chirp rate and the slow chirp rate presented 24 h later responses in two treatments, each consisting of two trials separated (Wilcoxon signed-ranks test: T ¼76.00, N ¼ 35, P ¼ 0.218; Fig. 3b). by different durations of silence. In the first treatment, the first and The relative responses of females to the slow chirp rate, however, did second trials were separated by 20e30 min. This was designed to not differ significantly between the two treatments (ManneWhitney represent a reasonable time interval separating female encounters test: U ¼ 1065.5, N1 ¼ 32, N2 ¼ 35, P ¼ 0.782). with different males on the same night. In the second treatment, In two control treatments (Fig. 3c, d), we tested female responses the first and second trials were separated by 24e26 h. This was to a fast chirp rate (4.2 chirps/s) in both the initial and the second designed to represent a reasonable time interval separating female trial using the same time intervals as in the previous tests. Females encounters with different males on sequential nights. For showed no significant difference in their responses to an initial fast simplicity, we refer to the time periods used as 20 min and 24 h. In chirp rate and the same chirp rate presented 20 min later (Wilcoxon both treatments, we tested female responses to a chirp rate of signed-ranks test: T ¼31.5, N ¼ 27, P ¼ 0.46; Fig. 3c). In addition, 4.2 chirps/s in the first trial (a more preferred chirp rate in exper- females showed no significant difference in their responses to an iment 1) and responses to 1.8 chirps/s chirp rate in the second trial initial fast chirp rate and the same chirp rate presented 24 h later (a less preferred chirp rate in experiment 1). In each trial we (Wilcoxon signed-ranks test: T ¼15.00, N ¼ 28, P ¼ 0.74; Fig. 3d). measured the time the female spent in the circle with the loud- The relative responses of females to the second fast chirp rate did not speaker. A female was tested in only one of the treatments. significantly differ between the two treatments (ManneWhitney We compared female responses to the high and low chirp rates test: U ¼ 728.50, N1 ¼ 27, N2 ¼ 28, P ¼ 0.649). within each treatment. The last chance option predicts that females Together, these results provide mixed support for the last chance will show a stronger response to the high chirp rate when the low option hypothesis. Females showed significantly stronger responses chirp rate is presented 20 min later. In contrast, females should to a high chirp rate than to a low chirp rate presented 20 min later, but show similar responses to the two chirp rates when the low chirp they did not show significantly stronger responses to a high chirp rate rate is presented 24 h later. We also compared the relative than to a low chirp presented 24 h later. The control experiment responses of females to the low chirp rates in the two treatments suggests that the weak response to the low chirp rate presented (time in association with the low chirp rate/time in association with 20 min after a high chirp rate was not simply a consequence of the high chirp rate). Because some females spent 0 s in association females showing weak responses to all calls types presented 20 min with a speaker in one of the trials, we added 1 s to all of the female after an initial test. These results thus suggest that females discrim- responses. The last chance option predicts that females will show inate against a slow chirp rate if they have recently heard a fast chirp stronger relative responses to the low chirp rate when it is rate, but that they will respond strongly to a slow chirp rate if they presented 24 h after the high chirp rate than when it is presented have not recently heard male song. We were, however, unable to 20 min after the high chirp rate. show that the relative responses of females to the low chirp rate song In two control treatments, we measured female attraction to were significantly different between the two treatments. a chirp rate of 4.2 chirps/s, with the trials being separated by 20 min or 24 h, respectively. These control treatments tested for DISCUSSION sequential testing effects and age effects. For example, females may show weak responses when retested after 20 min, regardless of the We examined female mate sampling strategies in G. lineaticeps. chirp rate with which they are tested, but their responses may Our three-stimulus experiment suggested that females use a fixed recover after 24 h. Each female was tested in only one of the threshold strategy in complex acoustic environments: females treatments. We compared female responses within each treatment responded strongly in each trial to chirp rates that were equal to or and between treatments, as described above. greater than 3.0 chirps/s, regardless of the other song types that A loudspeaker was placed in the centre of a circle (radius ¼ were simultaneously present. Similarly, females responded only 26 cm) in one corner of the arena (Fig. 1). At the beginning of the weakly to chirp rates below 3.0 chirps/s, regardless of the other trial, we placed the female under a cup (radius ¼ 4 cm) inside song types that were simultaneously present. These results suggest a small circle in the centre of the arena (‘release circle’; radius ¼ a threshold-like preference pattern. 5 cm). The distance between the female in the centre of the arena In contrast to our current results, previous experiments using and the loudspeaker was 1.14 m. During the acclimation period, we single-stimulus and two-stimulus testing paradigms indicated that kept the female under the cup for 5 min, while broadcasting the female G. lineaticeps show finer levels of discrimination among chirp test or control stimulus. At the end of the acclimation period we rates. In single-stimulus experiments, there was a linear and stabi- released the female by lifting the cup. The trial started when the lizing component to female responses: females tended to respond female left the release circle in centre of the arena and lasted for more strongly to higher chirp rates across a range of 1.8e4.2 chirps/s, 10 min. In each trial, we measured the total time the female spent but responded more strongly to intermediate and high chirp rates inside the circle associated with the broadcasting loudspeaker. If than to very high chirp rates (Wagner & Basolo 2007a). Similarly, in a female left the release circle within 10 min, but did not enter the most two-speaker choice tests, female G. lineaticeps preferred higher 524 O.M. Beckers, W.E. Wagner Jr / Animal Behaviour 81 (2011) 519e527

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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 20e30 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 20e30 min (N ¼ 26). (d) Trials separated by 24e26 h (N ¼ 28). Asterisk and grey shading of bar indicate significant difference (P ¼ 0.033).

to lower chirp rates, both at the low and high ends of the testing range that females from different populations use different sampling of 1.75 to 4.0 chirps/s (Wagner & Reiser 2000). The contrasting results strategies. between the previous studies and the current study may be In some frogs, the addition of multiple sound sources or back- explained by the different experimental designs. In the current study, ground noise has been shown to reduce female discrimination we used a more complex acoustic environment with three simulta- among call traits (e.g. Gerhardt 1982, 1987; Telford et al. 1989; neously presented songs to measure female responses. Our simulated Marquez & Bosch 1997; Schwartz et al. 2001)comparedtosimpler acoustic environment approximates a more natural setting in a high- two-stimulus environments (e.g. Gerhardt 1982; Marquez 1995; density chorus of G. lineaticeps (O.M.B., personal observation). The Gerhardt et al. 1996, 2000; Schwartz et al. 2001). Acoustic interfer- crickets tested in the previous studies, however, were from a different ence and masking of overlapping calls (e.g. Schwartz 1987; Gerhardt population than those tested in the current study. It is also possible & Klump 1988; Schwartz & Gerhardt 1989)undercomplexacoustic O.M. Beckers, W.E. Wagner Jr / Animal Behaviour 81 (2011) 519e527 525 conditions probably make it more difficult for females to distinguish including another field cricket (Bailey & Zuk 2008, 2009). In the (small) differences among the calls of individuals (Marquez & Bosch current study, we sequentially presented females with a highly 1997). Simple acoustic conditions may thus exaggerate female attractive fast chirp rate followed by a less attractive slow chirp rate. selectivity (Schwartz et al. 2001). Furthermore, our results suggest Females showed weaker responses to the slow chirp rate presented that females may switch from a comparison strategy in a simple 20 min after the fast chirp rate (Fig. 3a), but showed stronger acoustic environment (i.e. from evaluating the relative chirp rates of responses to the slow chirp rate presented 24 h after the fast chirp the available songs and then responding more strongly to the higher rate (Fig. 3b). Thus, females accepted slower rates if no faster chirp chirp rate song) to a threshold strategy in a complex acoustic envi- rates had recently been experienced. However, we were unable to ronment (i.e. to evaluating whether a given chirp rate is low or show a significant difference in female response strength to the slow intermediate/high and then responding strongly to any interme- rate in the two treatments, which may partially be due to a lack of diate/high chirp rates they detect). The results of some studies of statistical power (i.e. there was substantial variation in female other animals have suggested that females categorically classify the responses to the slow rate after 24 h; see Fig. 3b). Our results thus calls of conspecifics and heterospecifics (Baugh et al. 2008) and the provide mixed support for the last option hypothesis. Previous calls of conspecifics and predators (Wyttenbach et al. 1996). Our studies have shown age-related effects on preferences (Moore & results suggest that categorical perception may also play a role in the Moore 2001; Lynch et al. 2005) and age-related effects on mate context of mate assessment. assessment (Baugh & Ryan 2009). Less is known about time-related According to the predictions of a fixed threshold strategy, experience effects on preferences. a searching female should mate with the first male that exceeds the As outlined above, female preferences can change with the threshold value, and this value is set before females begin to search acoustic environment in which they are tested (e.g. two-speaker for potential mates (Janetos 1980; Real 1990; Reid & Stamps 1997). versus three-speaker experiments). An alternative to the one- Because of the static quality of the fixed threshold strategy, it has speaker experimental design we used to test time-dependent been considered inferior to other strategies under most conditions changes in female preferences would be a three-speaker design in (Janetos 1980; Janetos & Cole 1981). For example, females using which three above-threshold chirp rates are presented in the first a fixed threshold strategy discriminate against low-quality males, trial and, after 20 min or 24 h, three below-threshold rates are pre- independent of the costs involved in searching for a better male sented in a second trial. The fixed threshold strategy with last chance that would exceed the threshold. This can be especially costly if, in option predicts that females under these conditions would show some generations or environments, the quality of most males is weak responses to all three slow chirp rates after 20 min but strong below the threshold value. In this case, females would incur high responses after 24 h. Deviations from these predictions would costs due to extended search and may even forgo mating (Jennions suggest that females switch to a comparative strategy when only & Petrie 1997). Also, females using this search strategy would not slow chirp rates are present (e.g. if they responded more strongly to distinguish among high-quality males, resulting in lost opportu- the higher of the slow chirp rates after 20 min), or that females use nities to mate with males that provide the highest quality mating a ‘pure’ fixed threshold strategy in complex acoustic environments benefits. However, it has been suggested that a fixed threshold (e.g. if they continued to show weak responses to all three slow chirp strategy may be advantageous if the environment is constant rates after 24 h). (Real 1990; Collins et al. 2006) and/or search costs are high and For G. lineaticeps females, plasticity in their responses to slow females have to make quick mating decisions (Mazalov et al. 1996; chirp rate songs would probably be beneficial, considering that Reid & Stamps 1997). Evidence for fixed threshold strategies have population density can fluctuate remarkably throughout the been found for the cockroach Nauphoeta cinerea (Moore & Moore breeding season. Male singing activity is usually low at the beginning 1988), the red junglefowl Gallus gallus (Zuk et al. 1990) and the of the season and increases over the summer and early autumn cricket Gryllus sigillatus (Ivy & Sakaluk 2007). (W.E.W., personal observation). In some populations, the acoustically Janetos (1980) suggested another, but rarely considered version orienting fly Ormia ochracea parasitizes G. lineaticeps in the late of the fixed threshold strategy: the fixed threshold with last chance summer and early autumn (Wagner 1996), resulting in high mortality option. Here, females use a fixed threshold strategy as long as male for singing males (59% of males can be parasitized and thus near quality is high. However, if the females have not encountered death at a given time; Martin & Wagner 2010), whereas silent females a sufficiently attractive male after some period of time, they will are less often parasitized (w6%; Martin & Wagner 2010). As a result, mate with the next available male regardless of the male’s attrac- few males but many receptive females are present after a few weeks tiveness (Janetos 1980). Thus, a female expressing a fixed threshold of fly parasitism (W.E.W., personal observation). Additionally, the strategy with a last chance option would behave differently in flies have the same preferences for faster chirp rates as G. lineaticeps different social environments (i.e. her threshold would be higher if females (Wagner 1996; Wagner & Basolo 2007b), which should some available males have traits that exceed the initial threshold, selectively remove the most attractive males from the population. but the threshold would decline over time if no males with traits Females expressing a ‘pure’ fixed threshold strategy are thus likely to above this threshold are encountered). In a theoretical comparison incur high search costs, and perhaps never mate, under some between the fixed threshold strategy with last chance option, best- conditions. of-N strategy and one-step decision strategy, the latter two strat- Mate sampling has been investigated in various vertebrate and egies only resulted in greater mating benefits if the number of the invertebrate systems (reviewed in Jennions & Petrie 1997). As sampled males was large (Janetos & Cole 1981). It was further a general pattern, it has been proposed that a best-of-N strategy is suggested that the selective advantage of the best-of-N and the most likely to be found in mating systems with low costs for sampling one-step decision strategies over the fixed threshold strategy with multiple males (Reynolds & Gross 1990; Gibson & Bachman 1992). last chance option can be fairly small (Janetos & Cole 1981). These conditions are most often realized in species that form leks, Previous work on G. lineaticeps suggested that females change where females can quickly sample many males within brief time their responses to slow chirp rate song depending upon whether periods. Accordingly, females of at least some lekking species show they recently encountered high chirp rate song (Wagner et al. 2001). sampling behaviour that supports a best-of-N strategy (Trail & Adams Social experience is likewise known to affect female mating prefer- 1989; Fiske & Kålås 1995; Rintamäki et al. 1995). Most studies ences in a limited number of other invertebrate systems (e.g. Hebets investigating mate sampling behaviour, however, have found 2003; Dukas 2005; Hebets & Vink 2007; Fincke et al. 2007), considerable variability among individuals (e.g. sequence and 526 O.M. Beckers, W.E. Wagner Jr / Animal Behaviour 81 (2011) 519e527 number of males visited prior to choice) and have often supported Gerhardt, H. C. & Klump, G. M. 1988. Masking of acoustic signals by the chorus more than one strategy for a given species (e.g. Bensch & Hasselquist background noise in the green treefrog: a limitation of mate choice. Animal Behaviour, 36, 1247e1249. 1992; Choudhurry & Black 1994; Dale & Slagsvold 1996). It is possible Gerhardt, H. C., Dyson, M. L. & Tanner, S. D. 1996. Dynamic acoustic properties of that females of a given species use more than one mate sampling the advertisement calls of gray treefrogs: patterns of variability and female e strategy. Strategies differ in costliness (e.g. time costs, energy costs, choice. Behavioral Ecology, 7,7 18. Gerhardt, H. C., Tanner, S. D., Corrigan, C. M. & Walton, H. C. 2000. Female predation risk), and some individuals may not be able to afford to use preference functions based on call duration in the gray tree frog (Hyla a more costly strategy and instead use a different strategy (Jennions & versicolor). Behavioral Ecology, 11, 663e669. Petrie 1997). Most studies of female strategies have been based on Gibson, R. M. & Bachman, G. C. 1992. The costs of female choice in a lekking bird. e fi Behavioral Ecology and Sociobiology, 3,300 309. eld observations of female behaviour (e.g. Fiske & Kålås 1995; Dale & Gibson, R. M. & Langen, T. A. 1996. How do animals choose their mates? Trends Slagsvold 1996; Fagundes et al. 2007). However, this approach has in Ecology & Evolution, 11,468e470. been criticized for two reasons. First, the same sampling sequence Godin, J.-G. & Briggs, S. E. 1995. Female mate choice under predation risk in the guppy. Animal Behaviour, 51,117e130. may support more than one strategy (Gibson & Langen 1996). Second, Hebets, E. A. 2003. Subadult experience influences adult mate choice in an repeated sampling of a given male may be due to memory errors arthropod: exposed female wolf spiders prefer males of a familiar phenotype. (Wiegmann et al. 1996) and/or due to imperfect information gained Proceedings of the National Academy of Sciences, U.S.A., 100, 13390e13395. about male quality (Luttbeg 1996), rather than due to the use of Hebets, E. A. & Vink, C. J. 2007. Experience leads to preference: experienced females prefer brush-legged males in a population of syntopic wolf spiders. a best-of-N sampling strategy. Thus, it may be necessary to use more Behavioral Ecology, 18,1010e1020. than one approach (e.g. observations and controlled experiments) to Holm, S. 1979. A simple sequentially rejective multiple test procedure. Scandinavian e clearly identify the sampling strategy used. In addition, as our results Journal of Statistics, 6,65 70. Ivy, T. M. & Sakaluk, S. K. 2007. Sequential mate choice in decorated crickets: suggest, female sampling strategies may vary among social condi- females use a fixed internal threshold in pre- and postcopulatory choice. Animal tions. 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