Long-term maintenance and eventual extinction of preference for a mate’s call in the female budgerigar

Animal Behaviour 82 (2011) 971e979

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Long-term maintenance and eventual extinction of preference for a mate’s call in the female budgerigar Hiroko Eda-Fujiwara a, b, *, Aya Kanesada a, Yasuharu Okamoto c, Ryohei Satoh d, Aiko Watanabe a, Takenori Miyamoto a a

Laboratory of Behavioral Neuroscience, Department of Chemical & Biological Sciences, Japan Women’s University Japan Society for the Promotion of Science Department of Psychology, Japan Women’s University d Department of Physiology, Kitasato University School of Medicine, Japan b c

a r t i c l e i n f o Article history: Received 12 January 2011 Initial acceptance 15 February 2011 Final acceptance 19 July 2011 Available online 24 August 2011 MS. number: 11-00040R Keywords: auditory memory budgerigar call Melopsittacus undulatus pair bond parrot vocal communication

Auditory memory is crucial for mate recognition in birds and for long-term maintenance of pair bonds in monogamous species. Parrots, unlike the majority of birds, can imitate sounds into adulthood. During pair bond formation, male budgerigars, Melopsittacus undulatus, which are monogamous parrots, imitate the calls of females. This imitation results in convergence of the two calls. We examined whether and how long the auditory memory of a mate’s call affects female behaviour in relation to pair bond maintenance in this vocally plastic species. Females were paired with a male and then separated, without auditory/visual stimulation from the mate. Females responded to the calls of their separated mates, which had been recorded prior to pair formation, significantly more than to a call from an unfamiliar male. These females continued to show a preference for their respective mates’ calls at 1 and 2 months after separation, demonstrating that the auditory memory of the mate’s call was retained for at least 1 month in the absence of stimulation from the mate. These results show that long-term auditory memory mediates the preferential response to a mate’s call in female budgerigars. However, females ceased to prefer their mates’ calls after 6 months of separation, indicating that the preference response of females is eventually extinguished. We discuss the possibility that the extinction of the preferential response may be explained by an underlying mechanism other than the decay of auditory memory. Ó 2011 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

In monogamous vertebrate species, it is common for each adult to mate repeatedly with the same partner and form a long-term pair bond (Mock & Fujioka 1990). In some species with long-term monogamous pair bonds, remaining with the same partner improves reproduction compared with mate switching (e.g. Adkins-Regan & Tomaszycki 2007). In other species, however, longterm mate retention may have nothing to do with reproductive advantages (Mock & Fujioka 1990). The maintenance of a pair bond often requires that animals are able to distinguish between conspecific individuals and to retain the memory of the mate’s characteristics for long periods of time. Monogamy is particularly common in birds, in which vocalization is important for mate recognition (Catchpole & Slater 2008). Previous studies have experimentally demonstrated auditory recognition of mates in colonial seabirds, which are often long lived and pair many times

* Correspondence: H. Eda-Fujiwara, Laboratory of Behavioral Neuroscience, Department of Chemical & Biological Sciences, Japan Women’s University, Bunkyoku, Tokyo 112-8681, Japan. E-mail address: [email protected] (H. Eda-Fujiwara).

with the same partner during their lives. It has been shown that seabirds respond selectively to playback of the distinctive vocalization of their respective mates (reviewed in Catchpole & Slater 2008). These studies suggest that adult birds acquire an auditory memory of their mates’ vocalizations and that they use these vocalizations to recognize their mates. Parrots, unlike the majority of birds (including songbirds), can learn to make new calls into adulthood (Farabaugh et al. 1994; Bradbury 2003; Vehrencamp et al. 2003; Balsby & Scarl 2008; Manabe et al. 2008; Balsby & Adams 2011). The budgerigar, Melopsittacus undulatus, is a monogamous parrot species. Both sexes in a budgerigar pair produce individually distinctive contact calls. At any given point in time, a bird has a repertoire of one to several different types of contact calls (Farabaugh et al. 1994; Hile et al. 2000). During pair bond formation, male budgerigars imitate the calls of females and thus create a new call type, but not vice versa (Hile et al. 2000). Previous studies have shown that call similarity is an important trait that female budgerigars use when choosing a mate. Upon meeting a strange male, the female budgerigar prefers a male whose call is similar to her own, although this similarity prior to pairing is not imitative (Moravec et al. 2006,

0003-3472/$38.00 Ó 2011 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.anbehav.2011.07.030

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2010). As courtship proceeds into pair formation, the male imitates his partner’s call and the female shows preference for a male with the ability to imitate her own call (Hile et al. 2005). Both sexes of a budgerigar pair produce contact calls more often when they are with their mates (Striedter et al. 2003), suggesting that these calls are associated with pair bond maintenance. Female budgerigars (and also males) respond preferentially to their mates’ calls, that is, they respond by calling to their mates’ calls more than to calls from other birds (Ali et al. 1993; Eda-Fujiwara et al. 2010). Females may show preferential responses towards their mates’ calls because (1) they may compare their mates’ calls to their own calls and estimate the similarity or (2) they may have auditory memories of their mates’ calls. In the budgerigar, the calls of mated birds diverge after the eggs are laid and the males begin to feed their mates (Moravec et al. 2006). Female budgerigars cannot rely on call similarity for recognizing their mates in the next breeding period, because the calls of mated birds would have diverged by that time. Pair members in the budgerigar resume their bonds after 70 days of separation in different aviaries (Trillmich 1976b). Thus, budgerigars can recognize their mates after 70 days of separation, although the relative importance of auditory and visual characteristics remains unknown. In the present study, we examined whether and how long the auditory memory of a mate’s call influenced female behaviour in relation to pair bond maintenance. To this end, female budgerigars were paired with a male and then separated, without auditory/visual stimulation from the mate. Upon and after separation from their mates, we examined responses of females in preference tests in which each female was exposed to the call of her mate and a call from an unfamiliar male. Preferential responses by females to their mates’ calls may be affected by auditory memory and also by call similarity. Since we aimed to minimize the effects of call similarity, we attended to three issues in our experiments. First, we assigned pairs; that is, we put a male and a female together in a breeding cage. This forced budgerigars to pair disassortatively with respect to their calls. Second, we used calls that were recorded before pairing as stimulus calls in the preference tests; therefore, they were not similar to the female’s own calls. Third, we prepared stimulus calls so that the call of the unfamiliar male might be as dissimilar to her call as was the mate’s call. We carried out two experiments. In experiment 1, we repeatedly conducted four preference tests for each female (at 0, 1, 2 and 6 months after separation from her mate). As the repeated stimulus exposure in experiment 1 may have led to learning between 0 and 6 months, we then conducted experiment 2 to test whether extinction of the preferential response was observed in the experimental paradigm by controlling for between-test learning. METHODS Subjects Budgerigars (24 males and 24 females) were obtained from a local supplier. All of the birds were judged to be adults based on forehead plumage (Wyndham 1980). Males and females were acquired from different breeders and were housed in separate rooms, with each bird in an individual wire-mesh cage (22
17 cm and 33 cm high), so that females were unfamiliar with males prior to the study. The individual cages were fitted with wooden perches, but not with nestboxes. Each bird could hear and see other birds of the same sex in the room. All birds were kept in a controlled environment suitable for breeding (23  3  C and a light cycle of 14:10 h light:dark) throughout the study. Food, which consisted of

a commercial seed mixture, and water were provided ad libitum. Cuttlebone was provided during the period when birds were housed in pairs. All experimental procedures were approved by the Animal Experiments Committee of Japan Women’s University. After the study, the birds were housed as a mixed-sex flock of budgerigars in a large outdoor aviary that contained nestboxes and perches. Housing We used 12 males and 12 females in both experiments (experiments 1 and 2). Experiments 1 and 2 were conducted with different birds. Birds were initially housed singly and their vocalizations were recorded. After recording of all birds was completed, pairing was initiated. Each pair was housed in a wire-mesh cage (45
45 cm and 57 cm high) with a wooden nestbox (18.5
13.5 cm and 13 cm high) and a wooden perch for 46 days. Pairs were prevented from seeing other pairs in the same room by means of wooden barriers; however, they were in vocal contact with other pairs. After the pairing period, each female was isolated from her mate and kept in an individual cage without any subsequent auditory/visual stimulation from him. Because males and females were housed in separate rooms after the pairing period, each female received auditory/visual stimulation from other females, but not from any males. Budgerigar pair formation occurs over a span of days or weeks (Moravec et al. 2006). To assess the development of pair bonds, we performed continuous 120 min observations (scan sampling, Lehner 1996) between 0900 and 1100 hours on days 2 and 12 (in experiment 1) or 16 (in experiment 2) after the pairs were placed into breeding cages. Allopreening, in which one individual cleans the other’s feathers with its beak, is one of the behaviours associated with pair bonding in the budgerigar; it occurs more frequently with mates than with nonmates (Trillmich 1976a; Zocchi & Brauth 1991). Individual one-zero records of allopreening were taken every 3 min (i.e. 40 records per day per subject). The number of eggs in the nestboxes was checked in experiment 1 (on days 15, 21 and 24) and in experiment 2 (on days 15, 20 and 25). We assessed whether these data, which are known to be relevant to the pair bond, had any significant correlation with female preference. Recording and Analysis of Contact Calls Recording Contact calls are 100e300 ms in duration and strongly frequency modulated, mostly within the range of approximately 2e4 kHz (Fig. 1). The budgerigar contact call does not show marked sexual dimorphism in acoustic structure. We recorded and analysed the call repertoires of all birds prior to pair formation so that we could confirm that pair members did not have similar calls at this time, as determined by visual inspection of sonagrams (see Call classification below).

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The methods used to record contact calls were similar to those described by Hile et al. (2000). Briefly, during recording sessions prior to pairing, each subject (male or female) was placed with one of three males, which were only used for the recording sessions and were socially unfamiliar to all of the subjects used for pairing. Two birds at a time (a female or a male with a male only used for the recording) were placed in a soundproof chamber to encourage the birds to vocalize. The soundproof chamber contained two cages separated by a transparent Plexiglas divider 10 mm thick. One microphone (ECM-T150; Sony Corp., Tokyo, Japan) was placed on each side of the divider and connected to a digital audio tape recorder (TCD-D8; Sony Corp., Tokyo, Japan). Sounds from both microphones were stereorecorded onto a tape so that sound intensities could be compared between the two channels and thereby caller identity could be determined. Recording continued for 2 (in most birds) to 5 days (in one bird) so that at least 80 contact calls per bird were recorded. Call classification The vocalizations recorded during the present study were filtered (band-pass filter: 1.5e5.5 kHz) and displayed as sonagrams (fast Fourier transformation, FFT, size: 256, temporal resolution 3 ms) using the sound analysis software Avisoft SASLab Pro (Avisoft Bioacoustics, Berlin, Germany). Farabaugh et al. (1994) classified contact calls on the basis of their frequency modulation patterns on sonagrams. This procedure proved reliable for classifying contact calls within individuals; average interobserver agreement for four independent observers was 93% in a pilot analysis conducted using 880 calls (80 calls recorded from each of 11 males). Contact calls of each bird before pairing were classified into call types by an observer (one of four observers in our pilot analysis mentioned above) naïve to the identity of the birds. Of all call types for each bird, one call type occurs most frequently and is therefore termed ‘the dominant contact call’. We determined this call for each bird. After classifying contact calls into call types, we paired birds disassortatively with respect to calls. All the call types of each male were compared with those of his mate, by visual inspection of sonagrams. The observer, who classified calls, rated the degree of similarity as 1 (no similarity), 2 (fair similarity) or 3 (good similarity). All comparisons were rated as 1. Stimulus call Each recorded mate’s call used in the preference tests was randomly chosen from recordings of the male’s dominant call type made before pairing. To avoid pseudoreplication (Kroodsma 1990), for each experiment, we prepared 12 calls from 12 males that were unfamiliar to the females; these recordings had been made before pairing in previous pairing experiments using different birds from those used in the present study. The 12 unfamiliar calls were also chosen from the 12 unfamiliar males’ dominant call types. Then, by visual inspection of sonagrams, we assigned to each female two stimulus calls: a mate’s (familiar male’s) call and an unfamiliar male’s call, under the condition that both the familiar and unfamiliar calls were scored ‘1’ (no similarity) on our ordinal scale of similarity with all of the call types in each female (Fig. 1). Sonagram cross-correlation analyses We conducted sonagram cross-correlation analyses using Avisoft Correlator version 2.2 and verified that both calls in each stimulus call pair used in experiment 1 were dissimilar to the call types of each female. Each female had one to five contact call types. All of the female’s call types prior to pairing were compared with each of the two stimulus calls. For example, in the case of a female with three call types, we randomly selected five exemplar calls for each of the female’s call types and

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compared these 15 (five exemplars multiplied by three types) exemplars with each of the two stimulus calls, resulting in 30 pairwise comparisons. We then averaged five cross-correlation values for each of the female’s call types and obtained three averages for each of the two stimulus calls. The maximum value among the three averages for the mate’s or unfamiliar call was defined as the ‘Similarity’ value for each of the two stimulus calls of each female. Then, we tested the hypothesis that there was no significant difference in mean ‘Similarity’ values between the mate’s calls and unfamiliar calls. In previous studies of male budgerigars, calls that were more than 75% similar (cross-correlation values greater than 0.75) to the mate’s call were defined as imitation calls (Hile et al. 2000; Striedter et al. 2003). In cross-correlations between two calls from different call types, values greater than 0.75 are very unlikely to occur by chance (Hile et al. 2000). Using the threshold of 0.75, we assessed whether each Similarity value was low enough so that male and female calls could be classified into different call types. Preference Test Experiment 1 For each female, we conducted four preference tests at days 5  4, 32  3, 68  3 and 182  5 after separation, which were designated ‘0 month (m0)’, ‘1 month (m1)’, ‘2 months (m2)’ and ‘6 months (m6)’ (Fig. 2a). Each stimulus pair was used repeatedly in the four tests (i.e. m0, m1, m2 and m6) for one female subject. One female died on day 104 after the m0 test. Therefore, the sample size was 12 females in three tests (i.e. m0, m1 and m2) and 11 females in the last test (i.e. m6). Each female was separated from her mate and placed in an individual cage in a sound-attenuating chamber for at least 1 h before the start of stimulus presentation at m0. The soundattenuating chamber was equipped with two speakers (AS-5; Kenwood Corp., Tokyo, Japan) placed at either end of the cage and connected to a DAT recorder (TCD-D8; Sony Corp., Tokyo, Japan). Each speaker was assigned one of the two stimulus calls. In a 2 h trial, tested females were presented with two call stimuli: that is, a series of the mate’s call and a series of an unfamiliar male’s call were broadcast alternately (series duration ¼ 10 s, with one call every 2 s; 2 s interval between series). Calls were broadcast at a peak value of 80e85 dB SPL (NA-14 Sound Level Meter; Rion Corp., Tokyo, Japan; A-weighting, slow response), which was measured at the end of the cage. The two calls (a mate’s and an unfamiliar call) in a stimulus pair were adjusted to have the same peak amplitude. The vocal activity of the female was recorded with a DAT recorder, and we counted the calls produced during the broadcast of the stimuli. After the 2 h trial, the bird was kept in the sound-attenuating chamber and presented with the same stimuli in a 2 h trial on the next day. In the m0 test, individual females were subjected to two 2 h trials, with each call being broadcast from the alternate speaker in the two trials to control for possible side preferences. Data from the two trials for each bird were pooled for the test at m0. We used the same procedure for the other three tests (i.e. at m1, m2 and m6). Experiment 2 For each female, we conducted preference tests at days 5  4 and 150  5 after separation, which are designated ‘0 month (m0)’ and ‘5 months (m5)’ (Fig. 3a). The procedure was identical to that in experiment 1. We started with 12 females, but could only use six females in the m0 test because of logistical problems. One female died on day 71 after the m0 test, and then 11 birds were tested in the m5 test. The data from one female were lost owing to technical

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Figure 2. (a) The time course of experiment 1. After pairs of adult male and female budgerigars were housed together for 46 days, each female was isolated from her mate and then given repeated preference tests with the mate’s call versus the call of an unfamiliar male: T1(m0), T1(m1), T1(m2) and T1(m6). (b) Mean  SE preference ratios (number of calls by each female for her mate divided by the total number of calls). The dashed line indicates chance level. *P < 0.05.

problems in the m5 test. There were no vocal responses from two of the six females in the m0 test or from one of the 10 females in the m5 test. Therefore, the sample size was four females in the m0 test and nine females in the m5 test. Statistical Analysis We conducted repeated measures analyses of variance (ANOVAs) with individual subject as a repeated factor, one-sample t tests (two tailed) and two-sample t tests (two tailed) after the raw data were log transformed for the total number of calls and arcsine transformed for the preference ratio and the Similarity value to satisfy the assumptions of the parametric tests (Zar 2009). We used two-tailed Wilcoxon signed-ranks tests for the pairwise comparison of allopreening between day 2 and day 12 (in experiment 1) or 16 (in experiment 2) after the start of pairing. The relationship between the preference ratio and data associated with pair bonds was examined using a Spearman rank correlation coefficient. Levels of significance were set at P < 0.05. Data were analysed using StatView version 5 (SAS Institute, Inc., Carey, NC, U.S.A.). Means  SE are given.

T2(m0)

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Figure 3. (a) The time course of experiment 2. Each female was isolated from her mate and immediately given a preference test, T2(m0), with her mate’s call versus the call of an unfamiliar male, as in experiment 1. Females were given another test, T2(m5), at day 150 after separation. (b) Each open circle represents a preference ratio (number of calls by each female for her mate divided by the total number of calls) for one female, and the filled circles represent means for T2(m0) and T2(m5). The dashed line indicates chance level.

We used a Poisson regression model (Kutner et al. 2005) to examine whether the strength of the female’s response (i.e. the number of calls in response to call stimulation) was determined by each of three variables: (1) separation time (m0, m1, m2 or m6); (2) call familiarity (mate’s call or an unfamiliar call); and (3) call similarity (Similarity values for the stimulus call versus female call types; see Appendix). For each regression coefficient, we calculated the 95% highest posterior density (HPD) interval, which is a Bayesian estimate, corresponding to the confidence interval in the standard statistical method. Bayesian analysis was employed because maximum likelihood methods require large data sets. If the HPD interval of a regression coefficient (e.g. the coefficient showing the effect of call similarity) does not include zero (the value in our null hypotheses), it indicates that the variable (call similarity in this example) has a significant effect on the strength of the female’s response. RESULTS Experiment 1 We compared the Similarity values for ‘the mate’s call versus female call types’ with those for ‘the unfamiliar call versus female call types’. The Similarity values did not differ significantly between

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the two (two-sample t test: t11 ¼ 0.60, P ¼ 0.56; mate’s call: mean  SE ¼ 0.48  0.03; unfamiliar call: 0.50  0.03). Thus, matched calls in stimulus call pairs were not similar to those of the experimental females. The Similarity values for the mate’s call ranged from 0.34 to 0.65, and those for the unfamiliar call from 0.29 to 0.60. Furthermore, the Similarity values for ‘the mate’s versus the unfamiliar calls’ ranged from 0.30 to 0.66 (mean  SE ¼ 0.47  0.03). All of the Similarity values were less than 0.75, the threshold mentioned above (‘Sonagram cross-correlation analyses’). The preference ratio (the number of calls in response to the mate’s call divided by the grand total) changed between months (i.e. 0, 1, 2 and 6 months after pair separation, repeated measures ANOVA: F3,30 ¼ 2.94, P ¼ 0.048; Fig. 2b). Post hoc Bonferroni tests revealed a significant difference in preference ratios between m0 and m6 (P < 0.05). Females significantly preferred their respective mates’ calls, that is, their preference ratio was significantly different from chance (0.5) at m0 (one-sample t test: t11 ¼ 5.35, P ¼ 0.0002), m1 (t11 ¼ 3.07, P ¼ 0.011), and m2 (t11 ¼ 5.17, P ¼ 0.0003). However, at m6, the preference ratio was not significantly different from 0.5 (t10 ¼ 1.15, P ¼ 0.28). Based on this result, that neither of the stimuli presented to each female was preferred at m6, one might expect that the total number of calls during playback at m6 would be lower than those at m0, m1 and m2. We compared means of the total number of calls between months (m0: 514  155 calls; m1: 373  126; m2: 352  105; m6: 204  61, N ¼ 11) with a directional heterogeneity test (Rice & Gaines 1994). There was a significant decrease with time in the total number of calls during playback (the ordered heterogeneity test: rsPc ¼ 0.71, N ¼ 11, P < 0.05). We performed analyses with a Poisson regression model, using the data from 12 females, to evaluate the effect of call similarity on the female’s response and to separate the effect of call familiarity from that of call similarity (Table 1). We found a significant positive effect of call similarity on the strength of the female’s response to her mate’s call and a significant positive effect on the female’s response to the unfamiliar call, as the HPD intervals for the regression coefficients for call similarity did not include zero. Regarding call familiarity, there was a significantly stronger response to the mate’s call than to the unfamiliar call at m0, m1 and m2, as the HPD intervals for the coefficients for call familiarity did not include zero. However, at m6, there was a significantly stronger response to the unfamiliar call than to the mate’s call, as the HPD interval of the coefficient for call familiarity at m6 did not include zero, and the limits of the interval were less than zero. These results indicate that the preferential response to a mate’s call was extinguished at m6. The effect of call similarity was significantly greater during the playback of the mate’s call than that of the unfamiliar call, as the HPD interval of the coefficient did not include zero, and thus there was an interaction between call similarity and call familiarity.

Table 1 Results of the analyses with a Poisson regression model 95% HPD interval of the regression coefficient Effect of call similarity on female’s response To the mate’s call To the unfamiliar call Effect of call familiarity At m0 At m1 At m2 At m6 Similarity*Familiarity

[0.692, 1.486] [0.116, 0.787] [0.841, 1.052] [0.776, 0.996] [0.805, 1.019] [0.651, 0.247] [0.314, 1.064]

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Experiment 2 On separation from the mate in experiment 2 (m0), each of the females tested (N ¼ 4) showed a preference ratio that was significantly different from 0.5 (chi-square test for each individual female: c21 ¼ 10.1, 145.4, 8.9 and 19.3, respectively, P < 0.05 for all females tested; Fig. 3b), indicating a preferential response to the mate’s call by each female. The sample size was too small for a group-level statistical analysis, but at a group level, a preferential response to the mates’ calls by female budgerigars on separation was demonstrated in experiment 1 and in other studies that used the same protocol (Eda-Fujiwara et al. 2010; H. Eda-Fujiwara, A. Kanesada & T. Miyamoto, unpublished data). Therefore, it is highly probable that, at a group level, females formed memories of their mates’ calls in experiment 2, as they did in experiment 1. At 5 months after separation (m5), the preference ratio was not significantly different from 0.5 (one-sample t test: t8 ¼ 0.87, P ¼ 0.41; Fig. 3b), indicating extinction of the preferential response to mates’ calls by female budgerigars at m5. Two of the females showed a preference ratio that was significantly different from 0.5 (chi-square test for each individual female: c21 ¼ 21.63 and 16.2, respectively, P < 0.05 for both of the females), but the other seven females did not show significant preferences. Two females at m5 showed preference ratios that revealed greater differences from 0.5 than did that of the female with the smallest preference ratio at m0 (Fig. 3b). Nevertheless, these females at m5 did not show preference ratios that were significantly different from 0.5. This lack of significance is related to the total number of calls produced by each female during playback. The total number of calls during playback is small in each of the two females at m5 (28 calls and 86 calls) compared with the two females at m0, which had preference ratios lower than the mean value but large numbers of calls (151 calls and 599 calls). The total number of calls during playback did not differ significantly between months (two-sample t test: t11 ¼ 1.49, P ¼ 0.16). Pair Bonding Both sexes engaged in allopreening with their mates during the paired-housing period. Birds showed significantly more allopreening on day 12 than on day 2 in experiment 1 (Wilcoxon signedranks test: allopreening by males towards females: z ¼ 2.58, N ¼ 12, P ¼ 0.01; allopreening by females towards males: z ¼ 2.67, N ¼ 12, P ¼ 0.008). Similarly, birds showed significantly more allopreening on day 16 than on day 2 in experiment 2 (allopreening by males towards females: z ¼ 2.38, N ¼ 12, P ¼ 0.018; allopreening by females towards males: z ¼ 2.55, N ¼ 12, P ¼ 0.011). These results suggest that behaviours associated with pair bonding increased during the period of pairing. Because allopreening increased during the period of pairing in males and females, we pooled the data for members of each pair. There was no significant correlation between the preference ratio at m0 and allopreening on day 12 after the start of pairing in experiment 1 (Spearman rank correlation: rS ¼ 0.078, N ¼ 12, P ¼ 0.797). There was a significant correlation between the preference ratio at m6 and allopreening on day 12 in experiment 1 (rS ¼ 0.768, N ¼ 11, P ¼ 0.015), but there was no such correlation between the preference ratio at m5 and allopreening on day 16 after the start of pairing in experiment 2 (rS ¼ 0.565, N ¼ 9, P ¼ 0.110). Six of 12 females laid eggs by day 24 after the start of pairing in experiment 1 and by day 25 in experiment 2. In experiment 1, there was no significant correlation between the preference ratio at m0 and the number of eggs on day 24 (rS ¼ 0.305, N ¼ 12, P ¼ 0.31) or between the preference ratio at m6 and the number of eggs on day 24 (rS ¼ 0.353, N ¼ 11, P ¼ 0.264). In experiment 2, there was no

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correlation between the preference ratio at m5 and the number of eggs on day 25 (rS ¼ 0.110, N ¼ 9, P ¼ 0.756).

Previous studies have demonstrated that female behaviours related to pairing are affected by call similarity in the budgerigar (Hile et al. 2005; Moravec et al. 2006, 2010). Our results show that the auditory memory of a mate’s call as well as call similarity affects the preference response for a long period of time in the female budgerigar. The relative importance of the auditory memory and call similarity needs to be considered to improve our understanding of female behaviours related to pairing. We used calls recorded prior to pairing as stimuli to examine the effect of the auditory memory on the female’s response. Male budgerigars use imitations of their respective mates’ calls more when females are nearby (the audience effect, Striedter et al. 2003). Females might have responded more strongly to calls that males had vocalized towards their mates at the end of the pairing period. However, it is possible that the mate call type selected for the preference tests played a role in mate recognition at the end of the pairing period. Male budgerigars in the study by Plummer & Striedter (2002) retained at least some of their initial call types at 5 weeks after the start of pairing. Furthermore, when males are with their mates, they also produce other call types in addition to the imitation of their mates’ calls (18.8% imitation of all calls; Striedter et al. 2003).Thus, the call type used as stimuli in our study was likely to be vocalized by the male in front of his mate at the end of the pairing period, although the mate’s call used as a stimulus was not necessarily representative of that male anymore. In the present study we assumed that a female’s preference for the mate’s call used in the preference tests was absent before pairing. Of the two male calls that were used as stimuli in the preference tests, neither was similar to the mate’s calls prior to the pairing. We can speculate that, if the females had been tested with these two calls before pairing, they would have shown no preferences related to the call similarity. The unfamiliar calls in the present study were used as mates’ calls in previous experiments and were preferred by females in those experiments (Eda-Fujiwara et al. 2010; H. Eda-Fujiwara, A. Kanesada & T. Miyamoto, unpublished data), suggesting that the unfamiliar calls were potent enough to elicit responses from the female. Variation in the strength of the pair bond may affect the strength of the auditory memory of the mate’s call, and then account for variation in the female’s responses. We found no significant relationship between the preference ratio upon separation from their mates and the strength of the pair bond. This could be because we had not assessed the strength of the pair bond near the end of the pairing period, when the assessment would have been more appropriate. Meanwhile, there was a significant correlation between the preference ratio after a long separation and allopreening in experiment 1, but not in experiment 2. It is possible that the strength of the pair bond affects how quickly the preferential response to a mate’s call is extinguished in the female, but this needs further consideration.

(Morris & Erickson 1971). However, the relative importance of auditory and visual memories in mate recognition was not determined in previous studies. The present results demonstrate that female budgerigars retain auditory memories of mates’ calls for at least 1 month. Female budgerigars maintained a preference for the mate’s call at both 1 and 2 months after separation (m1 and m2 tests in experiment 1). The present findings do not conclusively show that the auditory memory of a mate’s call is retained for 2 months without auditory stimuli from the mate. It may be that re-exposure to the mate’s call at m1 affected the auditory memory of the mate’s call. Recent neuroscience studies in rodents have shown that reactivation of a memory through re-exposure to salient training stimuli results in a process known as reconsolidation, which mediates strengthening of the memory (e.g. a contextual fear memory in rats, Rattus norvegicus; Lee 2008). If the results of those general studies hold for the auditory memory of a mate’s call in the budgerigar, the reactivation of the memory of the mate’s call through re-exposure at m1 would strengthen the auditory memory. Then, the reactivated auditory memory might be retained at m2. Young male songbirds form auditory memories of the songs of adult conspecifics (tutor songs), after which they progressively form their own songs through a sensorimotor process of matching their own vocal output with the stored memory of the tutor song (reviewed in Bolhuis & Gahr 2006). The tutor song memory is retained for a long time: for at least 1 year in male nightingales, Luscinia megarhynchos (Geberzahn & Hultsch 2003) and or at least 165 days in male zebra finches, Taeniopygia guttata (Funabiki & Konishi 2003). Although females do not sing in many songbird species, several studies have shown that the auditory memory of the father’s song, which is acquired by young females, is retained for a period exceeding 2 months (Riebel et al. 2002; Riebel 2003). In the zebra finch, young females acquire auditory memories of tutor songs and show a preference for tutor songs over unfamiliar songs at 115 (Riebel 2000), 95 (Clayton 1988) and 65 days (Miller 1979) after separation from the tutor. In songbirds, there is a phase early in life when vocal imitation is most easily accomplished, called the ‘sensitive period’. In contrast, vocal imitation in parrots is prevailingly seen in adulthood (Farabaugh et al. 1994; Bartlett & Slater 1999; Hile & Striedter 2000; Bradbury 2003; Vehrencamp et al. 2003; Balsby & Scarl 2008; Manabe et al. 2008; Balsby & Adams 2011). Very few studies have assessed the retention period of auditory memories acquired in adulthood, as opposed to memories formed by young birds. Park & Dooling (1985) trained three budgerigars (one female and two males) with operant techniques to discriminate between contact calls. They showed that these budgerigars could retain learned discriminations for a period of 180 days without intervening experience with the stimulus calls. However, female budgerigars in the present study ceased to prefer their mates’ call after separation for comparably long periods. There must be a memory when there is a preference, whereas the disappearance of a preference does not necessarily mean that the memory has disappeared. Therefore, we cannot conclude whether the auditory memory of a mate’s call was retained at m6 (experiment 1) or m5 (experiment 2) in the present study (but see next section).

Retention of Auditory Memory of Mate’s Call

Extinction of Preferential Response to Mate’s Call

Adult birds of several species are capable of retaining recognition memories of mates for a long time. For example, whitethroated sparrows, Zonotrichia albicollis (a songbird) recognize each other after 17 days of separation (Wessel & Leigh 1941), and ring doves, Streptopelia risoria, recognize mates even after 7 months

The preferential response to a mate’s call, which was learned during the pairing period, was eventually extinguished after a long period of separation from the mate in female budgerigars. Is the extinction of the preferential response to a mate’s call explained by auditory memory? Conceivably, the extinction of preferential

DISCUSSION Effect of Auditory Memory of Mate’s Call on Female Responses

H. Eda-Fujiwara et al. / Animal Behaviour 82 (2011) 971e979

responses could be caused by either of two possibilities: (1) that the auditory memory of the mate’s call decays over time and (2) that the auditory memory is retained, but the preference is extinguished. The former possibility is not likely, as the previous study involving operant conditioning (Park & Dooling 1985) showed that budgerigars could retain auditory memories of contact calls for a period of 180 days without intervening experience with the stimulus calls. This suggests that the females retained the auditory memory of their mate’s call for at least 6 months after separation. The extinction of the preferential response to a mate’s call may not be explained by the decay of auditory memory. The analysis with our Poisson regression model revealed that there was a significantly stronger response to the unfamiliar call than to the mate’s call at m6. This result supports the possibility that after long-term separation the auditory memory is retained, but the preference is extinguished. It is well known that learned behavioural responses to stimuli in rodents decrease when those stimuli become irrelevant to the animal. This phenomenon is called extinction of the learned behaviour. In the auditory fear-conditioning paradigm in rodents, freezing to a tone-conditioned stimulus is rapidly extinguished when tones are given without a foot shock (Quirk 2002). However, learned preference in female birds for auditory signals (e.g. song) is not a conditioned response that is extinguished when no further reinforcement occurs. As far as we know, the present result is the first report that a nonconditioned preferential response is extinguished in adult birds. In female songbirds, preferential learning of a tutor’s song early in life has a stable influence on a preferential response to the song in adulthood (Clayton 1988; Riebel 2000). In fact, female zebra finches showed a preference for the tutor’s song over unfamiliar songs at 155 days after separation from the tutor, after having one intervening experience with the tutor’s song at 95 days (Clayton 1988). In the present study, female budgerigars did not show a preferential response to their mates’ calls after a period of separation exceeding 150 days. In female budgerigars, a preferential response acquired in adulthood may be extinguished without difficulty compared with that in female zebra finches, which is acquired early in life. Separation of maleefemale pairs in a monogamous species, such as the zebra finch, elicits behavioural changes that would promote locating the partner or strengthening an existing pair bond through the regulation of the hypothalamicepituitaryeadrenal axis (Remage-Healey et al. 2003). Similarly, monogamous budgerigars might have physiological mechanisms that help maintain pair bonds after a short separation from the mate. However, the present study shows that a long separation of pairs causes behavioural changes that would weaken an existing pair bond. The underlying mechanisms of call preference extinction remain to be investigated. The preferential response by females can be viewed as a suite of processes, including perceptual processes and those involved in decision making. Investigating the mechanisms for call preference extinction will require experimental designs in which processes involved in decision making are taken into consideration. Acknowledgments We thank Katharina Riebel, Shoji Hamao, Sergio Castellano and two anonymous referees for constructive comments on the manuscript, and Akiko Kubota and Yumi Yoshihara for data analyses. H.E.-F. and A.K. contributed equally to this work. This work was partly supported by a Multi-Career Path Support Model for Female Researchers project of JWU to H.E.-F., and by JSPS Grant-inAid for JSPS Fellows (22-40174) to H.E.-F.

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References Adkins-Regan, E. & Tomaszycki, M. 2007. Monogamy on the fast track. Biology Letters, 3, 617e619. Ali, N. J., Farabaugh, F. & Dooling, R. 1993. Recognition of contact calls by the budgerigar (Melopsittacus undulatus). Bulletin of the Psychonomic Society, 31, 468e470. Balsby, T. J. S. & Adams, D. M. 2011. Vocal similarity and familiarity determine response to potential flockmates in orange-fronted conures (Psittacidae). Animal Behaviour, 81, 983e991. Balsby, T. J. S. & Scarl, J. C. 2008. Sex-specific responses to vocal convergence and divergence of contact calls in orange-fronted conures (Aratinga canicularis). Proceedings of the Royal Society B, 275, 2147e2154. Bartlett, P. & Slater, P. J. B. 1999. The effect of new recruits on the flock specific call of budgerigars (Melopsittacus undulatus). Ethology, Ecology and Evolution, 11, 139e147. Bolhuis, J. J. & Gahr, M. 2006. Neural mechanisms of birdsong memory. Nature Reviews Neuroscience, 7, 347e357. Bradbury, J. W. 2003. Vocal communication in wild parrots. In: Animal Social Complexity: Intelligence, Culture, and Individualized Societies (Ed. by F. B. M. DeWaal & P. L. Tyack), pp. 293e316. Cambridge, Massachusetts: Harvard University Press. Catchpole, C. K. & Slater, P. J. B. 2008. Bird Song: Biological Themes and Variations. 2nd edn. Cambridge: Cambridge University Press. Clayton, N. S. 1988. Song discrimination learning in zebra finches. Animal Behaviour, 36, 1016e1024. Eda-Fujiwara, H., Hata, Y., Inanuma, M., Watanabe, A., Satoh, R. & Miyamoto, T. 2010. Sex differences in vocal behavior associated with pair bonding in the budgerigar, a monogamous parrot. Society for Neuroscience Abstracts 815.1. Farabaugh, S. M., Linzenbold, A. & Dooling, R. J. 1994. Vocal plasticity in budgerigars (Melopsittacus undulatus): evidence for social factors in the learning of contact calls. Journal of Comparative Psychology, 108, 81e92. Funabiki, Y. & Konishi, M. 2003. Long memory in song learning by zebra finches. Journal of Neuroscience, 23, 6928e6935. Gamerman, D. & Lopes, H. F. 2006. Markov Chain Monte Carlo. 2nd edn. : Chapman & Hall. Geberzahn, N. & Hultsch, H. 2003. Long-time storage of song types in birds: evidence from interactive playbacks. Proceedings of the Royal Society B, 270, 1085e1090. Hile, A. G. & Striedter, G. F. 2000. Call convergence within groups of female budgerigars (Melopsittacus undulatus). Ethology, 106, 1105e1114. Hile, A. G., Plummer, T. K. & Striedter, G. F. 2000. Male vocal imitation produces call convergence during pair bonding in budgerigars, Melopsittacus undulatus. Animal Behaviour, 59, 1209e1218. Hile, A. G., Burley, N. T., Coopersmith, C. B., Foster, V. S. & Striedter, G. F. 2005. Effects of male vocal learning on female behavior in the budgerigar, Melopsittacus undulatus. Ethology, 111, 901e923. Kroodsma, D. E. 1990. Using appropriate experimental designs for intended hypotheses in ‘song’ playbacks, with examples for testing effects of song repertoire sizes. Animal Behaviour, 40, 1138e1150. Kutner, M. H., Nachtsheim, C. J., Neter, J. & Li, W. 2005. Applied Linear Statistical Models. 5th edn. McGraw-Hill. Lee, J. L. C. 2008. Memory reconsolidation mediates the strengthening of memories by additional learning. Nature Neuroscience, 11, 1264e1266. Lehner, P. N. 1996. Handbook of Ethological Methods. 2nd edn. Cambridge: Cambridge University Press. Manabe, K., Dooling, R. J. & Brittan-Powell, E. F. 2008. Vocal learning in budgerigars (Melopsittacus undulatus): effects of an acoustic reference on vocal matching. Journal of the Acoustical Society of America, 123, 1729e1736. Miller, D. B. 1979. Long-term recognition of father’s song by female zebra finches. Nature, 280, 389e391. Mock, D. W. & Fujioka, M. 1990. Monogamy and long-term pair bonding in vertebrates. Trends in Ecology & Evolution, 5, 39e43. Moravec, M. L., Streidter, G. F. & Burley, N. T. 2006. Assortative pairing based on contact call similarity in budgerigars, Melopsittacus undulatus. Ethology, 112, 1108e1116. Moravec, M. L., Striedter, G. F. & Burley, N. T. 2010. ‘Virtual parrots’ confirm mating preferences of female budgerigars. Ethology, 116, 961e971. Morris, R. L. & Erickson, C. J. 1971. Pair bonding maintenance in the ring dove (Streptopelia risoria). Animal Behaviour, 19, 398e406. Park, T. J. & Dooling, R. J. 1985. Perception of species-specific contact calls by budgerigars (Melopsittacus undulatus). Journal of Comparative Psychology, 99, 391e402. Plummer, T. K. & Striedter, G. F. 2002. Brain legions that impair vocal imitation in adult budgerigars. Journal of Neurobiology, 53, 413e428. Quirk, G. J. 2002. Memory for extinction of conditioned fear is long-lasting and persists following spontaneous recovery. Learning & Memory, 9, 402e407. Remage-Healey, L., Adkins-Regan, E. & Romero, L. 2003. Behavioral and adrenocortical responses to mate separation and reunion in the zebra finch. Hormones and Behavior, 43, 108e114. Rice, W. R. & Gaines, S. D. 1994. Extending nondirectional heterogeneity tests to evaluate simply ordered alternative hypotheses. Proceedings of the National Academy of Sciences, U.S.A., 91, 225e226.

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Riebel, K. 2000. Early exposure leads to repeatable preferences for mate song in female zebra finches. Proceedings of the Royal Society B, 267, 2553e2558. Riebel, K. 2003. The ‘mute’ sex revisited: vocal production and perception learning in female songbirds. Advances in the Study of Behavior, 33, 49e86. Riebel, K., Smallegange, I. M., Terpstra, N. J. & Bolhuis, J. J. 2002. Sexual equality in zebra finch song preference: evidence for a dissociation between song recognition and production learning. Proceedings of the Royal Society B, 269, 729e733. Striedter, G. F., Freibott, L., Hile, A. G. & Burley, N. T. 2003. For whom the male calls: an effect of audience on contact call rate and repertoire in budgerigars, Melopsittacus undulatus. Animal Behaviour, 65, 875e882. Trillmich, F. 1976a. Spatial proximity and mate-specific behaviour in a flock of budgerigars (Melopsittacus undulatus; Aves, Psittacidae). Zeitschrift fur Tierpsychologie, 41, 307e331. Trillmich, F. 1976b. The influence of separation on the pair bond in budgerigars (Melopsittacus undulatus; Aves, Psittacidae). Zeitschrift fur Tierpsychologie, 41, 396e408. Vehrencamp, S. L., Ritter, A. F., Keever, M. & Bradbury, J. W. 2003. Responses to playback of local versus distant contact calls in the orange-fronted conures, Aratinga canicularis. Ethology, 109, 37e54. Wessel, J. P. & Leigh, W. H. 1941. Studies in the flock organization of the whitethroated sparrows, Zonotrichia albicollis. Wilson Bulletin, 53, 222e230. Wyndham, E. 1980. Diurnal cycle, behaviour and social organization of the budgerigar Melopsittacus undulatus. Emu, 80, 25e33. Zar, J. H. 2009. Biostatistical Analysis. 5th edn. London: Prentice Hall. Zocchi, D. C. & Brauth, S. E. 1991. An experimental study of mate directed behaviour in the budgerigar Melopsittacus undulatus. Behaviour, 9, 49e57.

Appendix Analyses with a Poisson Regression Model We introduced five variables (including four dummy variables) into our Poisson regression model.

X1 ¼ 1ðmateÞ; 0ðunfamiliarÞ X2 ¼ 1ðm0Þ; 0ðother than m0Þ X3 ¼ 1ðm1Þ; 0ðother than m1Þ X4 ¼ 1ðm2Þ; 0ðother than m2Þ X5 ¼ the Similarity value ðthe stimulus call versus the female call typesÞ We fitted the model

Vij ¼ mi þ a1 X1 þ a2 X2 þ a3 X3 þ a4 X4 þ a21 X1 X2 þ a31 X1 X3 þa41 X1 X4 þ a5 X5 þ a51 X1 X5

(A1)

where Vij is the strength of response that female i shows under condition j.

j ¼ ðX1 ; X2 ; X3 ; X4 ; X5 Þ Table A1 shows the summary of equation (A1) with each dummy variable set to 0 or 1. lij is determined by Vij, as shown below, and there exists a Poisson distribution of lij values. Yij is the number of calls made by female i under condition j, which is a value sampled from the population.

Yij wPoisson lij




When Y is sampled from the population with a Poisson distribution (l),

PðYÞ ¼

lY expðlÞ Y!

The simplest form of the Poisson regression model (Kutner et al. 2005) is the linear function, where lij is determined by Vij. Adjusting units of the coefficients in equation (A1), we can find

lij ¼ Vij Model L However, in our data, when a female produced a greater total number of calls during playback, there was an increase in the difference between conditions within the female. Thus,

lij ¼ exp Vij




Model E

would be a better model for our data. We performed Bayesian analyses with Model L and Model E for our data set in experiment 1. Then, we calculated deviance information criterion (DIC; Gamerman & Lopes 2006) values for each model as a measure of the ‘goodness of fit’ of a given regression model.

DICðModel LÞz8080:5 DICðModel EÞz5796:4 Model E had a smaller DIC value and thus was more appropriate for our data set than Model L. Therefore, we performed the following analyses with Model E. Table A2 shows means and 95% highest posterior density (HPD) intervals for parameters in posterior distributions estimated by the Markov chain Monte Carlo (MCMC) method. The 95% HPD interval is a Bayesian estimate, corresponding to the confidence interval in the standard statistical method. If the HPD interval of a parameter does not include zero (the value in our null hypotheses), it indicates that the factor related to the parameter has a significant effect on the strength of the female response. At m6, the effect of the mate’s call was expressed as a1. The 95% HPD interval of a1 did not include zero, and the limits of the interval were less than zero. Thus, at m6, there was a significantly greater response to the unfamiliar call than to the mate’s call. The effects of the unfamiliar call at m0, m1 and m2 were expressed as a2, a3 and a4, respectively. The 95% HPD intervals of a2, a3, and a4 did not overlap, and thus the effects of the unfamiliar call decreased significantly over time. However, the effect at m2 was not significantly different from that at m6, as the 95% HPD interval of a4 included zero. The interactions between the mate’s call and each of the separation periods (m0, m1 and m2) were expressed as a21, a31 and a41, respectively. These parameters (a21, a31 and a41) indicated the effects of the mate’s call compared with the unfamiliar call at m0, m1 and m2, respectively. The 95% HPD intervals of a21, a31 and a41 did not include zero, and thus there was a significantly stronger response to the mate’s call than to the unfamiliar call at m0, m1 and m2. The effects of separation periods (m0, m1 and m2) on the response to the mate’s call were expressed by a2 þ a21, a3 þ a31 and a4 þ a41, respectively. The 95% HPD intervals of these parameters did not include zero; therefore, the strength of the response to the mate’s call at m6 was significantly decreased compared with the strength at m0, m1 and m2. Furthermore, the 95% HPD intervals of a2 þ a21, a3 þ a31 and a4 þ a41 did not overlap; therefore, the strength of the response to the mate’s call decreased significantly over time. The parameters a5 and a5 þ a51 indicated the effects of similarity on the strength of the response to the unfamiliar call and to the mate’s call, respectively. There was a significant positive effect of call similarity on the strength of the response to the mate’s call. Furthermore, there was also a significant positive effect on the response to the unfamiliar call, as the 95% HPD intervals of a5 and a5 þ a51 did not include zero. The interaction between call similarity and call familiarity was expressed as a51. The effect of call similarity was significantly greater during playback of the mate’s call than during that of the unfamiliar call, as the 95% HPD interval of a51 did not include zero.

H. Eda-Fujiwara et al. / Animal Behaviour 82 (2011) 971e979 Table A1 The strength of response (Vij) by female i under condition j, j ¼ (X1, X2, X3, X4, X5)

At At At At

m0; m1; m2; m6;

X2¼1 X3¼1 X4¼1 X2¼X3¼X4¼0

Unfamiliar; X1¼0

Mate; X1¼1

miþa2þa5X5 miþa3þa5X5 miþa4þa5X5 miþa5X5

miþa1þ(a2þa21)þ(a5þa51)X5 miþa1þ(a3þa31)þ(a5þa51)X5 miþa1þ(a4þa41)þ(a5þa51)X5 miþa1þ(a5þa51)X5

Table A2 Means and 95% highest posterior density (HPD) intervals for parameters in posterior distributions estimated by the Markov chain Monte Carlo (MCMC) method Parameter

Mean

95% HPD interval

m1 m2 m3 m4 m5 m6 m7 m8 m9 m10 m11 m12 a1 a2 a3 a4 a21 a31 a41 a5 a51 a2þa21 a3þa31 a4þa41 a5þa51

5.07 4.41 2.52 5.33 2.53 3.82 4.32 2.18 4.59 5.13 3.50 4.48 0.429 0.345 0.144 0.030 0.953 0.883 0.916 0.413 0.675 1.297 1.027 0.886 1.089

[4.90, 5.22] [4.24, 4.57] [2.32, 2.74] [5.13, 5.49] [2.32, 2.74] [3.58, 4.00] [4.13, 4.48] [1.96, 2.40] [4.43, 4.72] [4.95, 5.28] [3.31, 3.67] [4.64, 5.00] [0.651, 0.247] [0.268, 0.422] [0.064, 0.232] [0.124, 0.045] [0.841, 1.052] [0.776, 0.996] [0.805, 1.019] [0.116, 0.787] [0.314, 1.064] [1.229, 1.365] [0.959, 1.100] [0.811, 0.955] [0.692, 1.486]

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