General experimental design

This study consisted of two playback experiments. Experiment 1 was designed to test whether Japanese great tits discriminate between calls with different note types (ABC and D calls) and, if so, whether they also extract a compound meaning from combined calls (ABC–D). If the combination of ABC and D calls obeys compositional syntax, tits are expected to show different responses to the two different note units and a compound response to the combined calls. We examined the response of Japanese great tits to playbacks of ABC calls, D calls, ABC–D calls and the background noise (control).

Experiment 2 was designed to test whether tits respond to the combination of ABC and D calls through the recognition of the note-ordering rule. If they perceive the combined calls (ABC–D calls) as a single meaningful unit but not as separated and independent calls (ABC and D calls), they are expected to respond differently to the natural (ABC–D) and reversed (D–ABC) sequences. We tested the response of tits to playbacks of ABC–D and D–ABC calls.

Study population and call recordings

Experiments were conducted in a colour-ringed population of Japanese great tits in a mixed deciduous–coniferous forest near Karuizawa, Nagano Prefecture, Japan (36°19′–22′N, 138°32′–37′E). For all playbacks, we used ‘chicka’ mobbing calls that were previously recorded from Japanese great tits (ten males and seven females) from the study population in 2009 and 2010 (refs 25, 30). The ‘chicka’ calls were elicited by exposure to either a taxidermic model of a crow or a marten near the nest boxes. Calls were recorded using an LS370 parabolic microphone (Fuji Planning Corporation, Tokyo, Japan) connected to an R-09HR digital audio recorder (sampling rate, 48 kHz; sample size, 16 bits; Roland Corporation, Shizuoka, Japan). Detailed information on call recordings has been provided elsewhere25,30.

Playback stimuli

Adobe Audition 3.0 software and Raven Pro 1.3 software40 were used to construct the playback stimuli. We chose four types of notes (A, B, C and D) from recordings of every source individual on the basis of the sound quality (for example, the bird was close to the microphone when it called and the background noise was low). Although A, B and C notes were typically produced as a single note in a call, D notes always occurred as a string of multiple notes. Therefore, we used a single A, B and C note and a string of seven to ten D notes to construct the playback calls. These four note types were combined into an ABC–D call with natural intervals between the notes (50–150 ms, measured for each individual of the recording source). We thus obtained a total of 21 ABC–D calls from the recording files (11 calls from the recordings of 10 males and 10 calls from the recordings of 7 females).

In Experiment 1, we prepared three call treatments (ABC, D and ABC–D calls; Fig. 1a–c) and a control treatment (background noise). ABC and D call types were constructed by eliminating either D or ABC note units from each of the 21 ABC–D calls. Calls were repeated in a sound file at a rate of 30 calls per minute (one call every 2 s, total duration 90 s). This calling rate is within the range of the natural repetition rates for ‘chicka’ calls during the nestling period25,30. Low-frequency noise (<1 kHz) was filtered out and the calls were amplified on a computer. The background noise files were created in the same way as the call files, using the parts where no birds were calling in the same recordings as call treatments. Thus, we constructed 21 unique sets of playback stimuli (ABC, D and ABC–D calls, and background noise). To avoid pseudoreplication41, we played back each exemplar only once to each focal individual (n=21). To each focal individual, we played back three call types that originated from the same calling individual (matched-pairs design), ensuring that any acoustic features other than the note combinations (for example, the intervals between different notes) were constant over these three call treatments. All of the sound files were saved in WAV format (16-bit accuracy, 48.0-kHz sampling rate) onto an SD memory card.

In Experiment 2, we prepared two types of calls: ABC–D (natural sequence) and D–ABC (artificially reversed sequence) calls (Fig. 1d). We chose 17 different ABC–D calls that originated from different individuals (10 male calls and 7 female calls). D–ABC calls (n=17) were constructed by using these ABC–D calls and re-ordering the sequence by moving D notes before A notes. The intervals between D and A notes within D–ABC calls were set at the same durations as those between C and D notes in their original ABC–D calls, ensuring that any acoustic features other than note orderings did not differ between ABC–D and D–ABC calls (balanced design). These calls were recorded in a sound file at a rate of 20 calls per minute (one call every 3 s, total duration 90 s), which was saved in WAV format (16-bit accuracy, 48.0-kHz sampling rate) onto an SD memory card. This calling rate is within the natural range25,30 and ensures that each call is separated by at least 1.6 s from any preceding calls, reducing the chances that receivers could perceive ABC–D sequences from adjacent D–ABC calls. As with Experiment 1, unique exemplars were used for each focal individual to avoid pseudoreplication41.

Experiment 1

We tested the responses of Japanese great tits to playbacks of ABC, D and ABC–D calls. We conducted this experiment on 21 adult great tits (10 males and 11 females from 21 different pairs) during their first breeding attempt of the season. All experimental birds bred in nest boxes that were attached to tree trunks 1.8 m above the ground. The average brood size of these pairs was 7.8±1.5 (mean±s.d., n=21). The experimental trials were carried out from 3 June to 15 June 2012 when the nestlings were 10–17 (12.4±1.7) days old.

An AT-SPG50 loudspeaker (Audio-Technica Corporation, Tokyo, Japan) was hung from a tree and fixed 1.8±0.2 m from the ground and 5.3±1.0 m from the nest (mean±s.d., n=21). The loudspeaker was connected to an R-09 HR digital audio recorder with EXC-12A extension cords (JVC Kenwood Corporation, Kanagawa, Japan), which enabled the control of playbacks from an observation position 15 m away from the nest. Playbacks commenced when a focal individual was within 5 m of the nest and their mate was absent. Calls were played back at a standardized volume (75 dB re 20 μPa at 1 m from the loudspeaker measured using an SM-325 sound level meter; AS ONE Corporation, Osaka, Japan) and background noise was played back at the same amplitude as the background noise level of the call playbacks (50 dB re 20 μPa at 1 m). Focal birds received playbacks of calls that were constructed from unfamiliar individuals (that is, not their mates or neighbours), to eliminate any influence of familiarity. No more than two trials were conducted at the same nest in a single day and playbacks at the same nest were separated by at least 2 h to reduce habituation. The order of the playbacks was randomized. We used the same position for setting the loudspeaker in all treatments at each site to control for its possible effect on the behavioural response. Trials were conducted in calm and dry weather between 08:30 and 16:00 h (Japan Standard Time).

To determine the tits’ responses to different treatments, we recorded the following behavioural variables during 90 s of playbacks: (1) number of horizontal scans: we counted the number of movements that birds made with their heads from left to right or right to left (approximately a 180° turn) and (2) approaching the loudspeaker: we recorded whether birds approached within 2 m of the loudspeaker during the playback. These behavioural variables were commented onto an R-09HR digital audio recorder. We also recorded the latency to feed nestlings by using a GZ-MG880 digital video camera (JVC Kenwood Corporation) set ca. 10 m from the nest. Behavioural observations were continued until each playback had ended and the adults entered the nest box to feed the chicks.

Experiment 2

We tested the responses of Japanese great tits to naturally combined ABC–D calls and artificially reversed D–ABC calls. We conducted this experiment with 34 individual great tits (ABC–D calls: 11 males and 6 females; D–ABC calls: 12 males and 5 females). The minimum distance between experimental sites was 400 m, to ensure the collection of data from different individual tits21. Trials were carried out between 6 November and 19 November 2015, during the non-breeding season, when tits, such as other members of the Paridae, are threatened by a variety of predators and produce a corresponding variety of alarm calls22,23,24.

First, we searched for a flock of Japanese great tits. On finding a flock, we hung an AT-SPG50 loudspeaker from a tree at 1.8±0.1 m from the ground (mean±s.d., n=34). The loudspeaker was connected to an R-09 HR digital audio recorder with EXC-12A extension cords, which enabled the control of playbacks from an observation position ca. 10 m away from the loudspeaker. Then, we commenced the playback when a tit came within 15 m of the loudspeaker. We defined the individual that was closest to the loudspeaker as the focal individual and focussed on this individual during the playback. Trials were carried out under calm and dry weather between 08:45 and 15:30 h (Japan Standard Time). ABC–D and D–ABC treatments were alternated with each other on successive trials so that responses to both treatments were observed under largely similar conditions.

As with Experiment 1, we measured two behavioural variables: (1) number of horizontal scans and (2) the probability of approaching within 2 m of the loudspeaker. These variables were commented onto an R-09HR digital audio recorder.

Usage of D calls in a non-predatory context

Japanese great tits produce D calls not only in predatory contexts but also in non-predatory contexts such as when visiting their nests. We investigated the usage and function of D calls in a non-predatory context, testing the hypothesis that D calls serve to recruit conspecifics. If this hypothesis is true, then we predict that (1) tits produce D calls more often when they visit the nest alone than when their mated partner is also present and (2) a caller’s mate is more likely to visit the nest when the caller produces D calls than when it does not. We therefore investigated the effect of social context on the usage of D calls and whether the production of D calls increases the visitation of their mate to the nest.

We observed n=187 nest visitations of 40 adults (19 males and 21 females) at 22 nests from 3 June to 15 June 2012, when nestlings were 10–17 days old. When a parent visited within 5 m of the nest box with a food item, we noted (1) the sex of the parent, (2) whether it gave D calls and (3) whether its mate was present within 5 m of the nest box. In the case in which a parent visited the nest alone (n=136), we also noted (4) whether the mate visited within 5 m of the nest before the first bird entered the nest box. Observations were made at 15 m from the nest box, a distance from which the tits’ behaviour was not disturbed.

Statistical analysis

All the statistical analyses were performed using R for Mac OS X version 3.1.1 (ref. 42). In the analysis of Experiment 1, we used generalized linear mixed models for primary analyses, which include the treatment as a fixed term and individual identity of focal birds as a random term. Trial order and sex were also entered as covariates. We used a negative binomial error distribution and log-link function (glmer.nb in the package lme4 (ref. 43)) for the analysis of the number of horizontal scans and a binomial error distribution and logit-link function (glmer in the package lme4 (ref. 43)) for the analysis of the probability of approaching behaviour (yes or no). In some trials, tits visited the nest boxes and flew out of sight immediately after feeding chicks. Therefore, we determined the time duration in which we could observe the behaviour of the tits as the observation time and included this term in the analysis of horizontal scans as a log-transformed offset. For the analysis of approaching behaviour, it was not possible to run the model because of the absence of variance in background noise control treatment (no birds approached to the loudspeaker during this treatment). Therefore, we combined background noise and ABC calls in this analysis, as there was no significant difference between these two treatments (sign test, P=0.5). We used likelihood ratio tests to calculate P-values of each term. In the event of a significant effect of treatment, we further conducted pair-wise comparisons by using non-parametric statistics: Wilcoxon signed-ranks tests (wilcox.paired.multcomp in the package RVAideMemoire44) for the number of horizontal scans (standardized by observation time) and sign tests for approaching to the loudspeaker (cochran.qtest in the package RVAideMemoire44). When making these multiple comparisons, sequential Bonferroni corrections were applied for the adjustments of P-values. To investigate the correlation between scanning and approaching behaviours, we used Spearman’s rank-order correlations (cor.test in the default package stats).

In the analysis of Experiment 2, we ran generalized linear models including treatment as a fixed term and sex as a covariate. We used a negative binomial error distribution and log-link function (glm.nb in the package MASS45) for the analysis of horizontal scans and a binomial error distribution and logit-link function (glm in the package stats) for the analysis of approaching behaviour. We standardized the number of scans by observation time, as in some cases the focal individuals flew away from the sight during the trials.

In the analysis of the usage of D calls, we ran generalized linear mixed models with a binomial error distribution and a logit-link function (glmer in the package lme4 (ref. 43)). To test the effect of social context on the production of D calls, we fitted social context (mate present or absent) as a fixed term and the probability of D calling (yes or no) as a dependent variable. To test the effect of D calling on the recruitment of a mate to the nest, we fitted the production of D calls (yes or no) as a fixed term and the probability of recruitment (yes or no) as a dependent variable. In both models, we also included sex of focal birds as a covariate and individual identity of focal birds and individual nest as random terms. All tests were two-tailed and the significance level was set at α=0.05.

Ethical statement

All experiments were performed in accordance with relevant guidelines and regulations. All experimental protocols were approved by the Animal Care and Use Committees at the Rikkyo University and SOKENDAI (The Graduate University for Advanced Studies), and adhered to the Guidelines for the Use of Animals in Research of the Animal Behavior Society/Association for the Study of Animal Behaviour. This research was performed under permission from the Ministry of the Environment and the Forestry Agency of Japan.