Introduction

Animals often communicate in network environments, in which multiple unintended receivers may be present [1]. The ability to direct signals to specific individuals (addressing) can therefore provide an advantage by affecting only the behavior of the intended receiver and not the whole network.

Mechanisms for addressing individuals include vocally matching aspects of the addressee’s vocalisation, timing vocalisations relative to those of the addressee, and orientating towards the intended receiver [2], [3]. Addressing by vocal matching can be accomplished in two different ways: (1) by using a vocalisation type that resembles the one used by the addressee and which is already part of the repertoire of the addressor, or (2) through vocal imitation in which the addressor alters its vocalization type to resemble the variant of the addressee. Several territorial species perform vocal matching using song types from their repertoires, e.g. great tits (Parus major) [4], [5], western medowlarks (Sturnella neglecta) [6] and song sparrows (Melospiza melodia) [7]. In these species, song type matching by a simulated intruder elicits strong responses from territory owners and thus indicates that matching addresses the matched individual [7], [8].

Whereas call or song type matching is limited by the number of vocalisation types in the repertoire, imitation provides a more flexible mechanism for matching. Vocal imitations of individuals’ distinct vocalisations have the potential to address specific individuals within a communication network; however, such addressing has not been shown experimentally for any species. Vocal imitation of conspecifics’ individually specific vocalisations during interactions in non-territorial contexts has been observed among bottlenose dolphins (Tursiops truncatus) [9], [10], [11], galahs (Eolophus roseicapillus) [12] and orange-fronted conures (Aratinga canicularis) [13], [14]. The flexibility of vocal imitation will enable individuals to uniquely address all conspecifics in a communication network, without requiring prior knowledge of or familiarity with the interacting individuals. Spectacled parrotlets seem to address specific individuals in small stable flocks, which was interpreted as vocal labelling [15]. It is, however, possible that the contact calls of spectacled parrotlets could involve imitations, but that hypothesis has not yet been tested.

In this experiment, we simulate vocal imitation by using variants of contact calls similar to an intended receiver’s contact call to determine if specific receivers can be addressed in a non-territorial communication network. Previous experiments on orange-fronted conures have demonstrated their ability to rapidly imitate contact calls of other individuals [13], [14], and that imitations of contact calls have signal value for receivers [16]. The functional significance of contact call imitation for receivers has not yet been explicitly tested, although this is crucial for understanding the evolution of vocal imitation among conspecifics. Our playback experiment tests the potential of addressing a specific individual in a communication network by vocal imitation. We monitor the response of both intended and non-intended receivers in the communication network. To our knowledge, no experiments have monitored how non-intended receivers in such networks respond, although these individuals may also respond to the stimuli and indirectly affect the response of the focal bird.

Our study species, the orange-fronted conure, communicates in large and dynamic communication networks. Outside the breeding season they are non-territorial and live in flocks with fission-fusion dynamics with frequent changes in flock composition [17]. Flock fusions are always preceded by exchanges of contact calls [13]. The contact calls of the orange-fronted conure show individual and sexual distinctiveness [18], [19], and are uttered both when individuals are alone (solo contact calls) and during vocal interactions. Solo contact calls show very little variation, whereas contact calls given during interactions are more variable (unpublished data). During natural contact exchange and during vocal interactions with playback [13], [14], orange-fronted conures often imitate contact calls by modifying the call’s fine scale structure. The imitations gradually increase the similarity between individuals’ calls, resulting in a convergent interaction [13], [14]. In other interactions, however, orange-fronted conures can decrease the similarity between the contact calls, resulting a divergent interaction. Imitations of contact calls have signal value for orange-fronted conures as imitation elicits high contact calls rates in the receiver [16].

Previous experiments have only tested the signal function of imitation within dyadic interactions with playback to a single subject at a time [16]. In this experiment, we create a simple communication network by joining two wild-caught orange-fronted conures from different flocks in the same aviary and presenting playback stimuli to them simultaneously. Our aim was to determine if specific receivers can be addressed in a non-territorial communication network. To do so, we simulated vocal imitation through playback of variants of contact calls similar to that of an intended receiver. If vocal imitation addresses specific individuals, we expected the imitated individual to be the primary respondent to the playback. Orange-fronted conures respond with most calls in interactions with the opposite sex and where female test-birds generally gave more calls than male test birds. Given the strong influence of sex in other experiments [16], we expected an overall stronger response of female than male test birds. Most experiments on vocal matching have only monitored the vocal response of the focal bird [4], [5], [8], [20] although other individuals of the local network respond after eavesdropping on the experiment [21],[22],[23],[24]. Our experimental setup, however, enabled us to monitor the vocal response of the whole communication network rather than just the focal bird.

Materials and Methods

We conducted the experiment during the non-breeding season from June 16 to August 6, 2007, at Santa Rosa National Park, Area de Conservación Guanacaste, Costa Rica. We captured orange-fronted conures in mist nests, using playback of contact calls to lure them in. In this experiment we used 36 orange-fronted conures from 27 different flocks. Four aviaries (dimensions: 3.5×1.8×1.8 m) housed the test birds. Aviaries were located in the natural habitat of dense shrubbery at least 300 m apart and were thus considered acoustically isolated from each other. All aviaries provided rain cover and several branches for perching. In the aviary, test birds had unlimited access to water and food (nancite fruits, Byrsonima crassifolia). Test birds spent 2–7 days in the aviary. For the first 2–4 days, each test bird was housed individually and their individually distinctive solo contact calls were recorded, which we used to specifically tailor the playback stimuli to the individuals in each trial (see below). Once the solo contact calls for both test birds had been obtained, we moved one of the test birds to the aviary of the other individual for the playback experiment. Because flock mates of some species converge their contact calls [25], [26], [27], we always used birds from different flocks to form our new flocks to ensure that contact calls differed. Although imitation in orange-fronted conures occurs rapidly during an interaction, sustained convergence of individually-specific contact calls is unlikely in the timescale of this experiment.

Orange-fronted conures are sexually monomorphic [28]. Sex was therefore determined molecularly from blood samples extracted from the wing vein after the experiment (primer description [29], [30]). The molecular sexing showed that we had 16 female and 20 male test birds. Of the 18 pairs, three pairs consisted of females, 5 pairs consisted of males and 10 pairs consisted of a male and a female. To enable individual identification, we gave each test bird distinctive marks on breast and/or head using felt pens.

Playback Stimuli and Solo Contact Calls of Test Birds

The contact calls used for playback had been recorded between mid-June and early-August 2005 and 2006 from 14 male and 10 female orange-fronted conures from the local area that were temporarily held individually in the aviaries. The stimuli calls were recorded using a Marantz PMD 670 or 690 solid state recorder and a Sennheiser 816T MKH microphone placed 0.5 metres from the corner of the aviary, so the distance between the microphone and the test birds never exceeded 4.5 m.

We recorded contact calls of the test birds between 05∶30–08∶30 am and 03∶00–05∶00 pm using a Marantz PMD 670 solid state recorder and a Sennheiser ME67 K6 supercardioid microphone positioned as described above. We extracted solo contact calls from the recordings using Syrinx version 2.5 s (John Burt http://www.Syrinxpc.com). Acoustic characteristics of the extracted contact calls were measured on spectrograms using a transform size of 512 and a fixed time line of 0.5 s/line. Contact calls can be divided into three segments (Figure 1): a rising harmonic series ascending about 1 kHz over the duration of the segment (P1), a middle section with deep (up to 3 kHz) step-like frequency modulations (P2) where the main part of the energy is between 3 kHz to 6 kHz (P2), and a decreasing harmonics series in which the frequency descends 1 to 3 kHz over the duration of the segment (P3) [17], [18]. For each contact call we measured the length of the entire call and the length of each of the three segments, P1, P2 and P3.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 1. Spectrograms (A–C) of orange-fronted conure contact calls from three different playback trials to different flocks.

In each spectrogram the first call is the playback call, the second call is a solo contact call of the target bird (imitated bird) and the third call is a solo call of the non-imitated bird. The three playback calls came from three different flocks. The numbers above the solo contact calls are their cross-correlation similarity relative to the playback call. In A and B the playback call came from a male and in C a female. The test birds in A and C were all females and in B the test birds were both males. The three parts of the contact call are marked below the first call in spectrogram C. The spectrogram has been prepared in Avisoft (FFT = 256, Overlap = 75%, Blackman window).

https://doi.org/10.1371/journal.pone.0049747.g001

To identify a representative set of individually-distinct solo contact calls for each individual, we chose the 12 most similar solo contact calls given by each test bird, except for three birds from which we only obtained 10 or 11 solo contact calls. The evaluation of similarity was based on the length of the different segments (P1–P3) and the frequency contour of the contact call, in particular P2. Once the 12 most similar solo calls of a test bird were identified, we calculated the mean total call length and mean length of each of the three segments, P1, P2, and P3.

Each trial consisted of a loop with the same stimulus call played back 10 times with 10 second intervals between calls. A stimulus call was chosen from our library of contact calls on basis of its similarity to the contact calls of the test bird, with similarity assessed on the basis of frequency contour and the averaged measures of the 12 solo contact calls described above. Selection was based primarily on the total lengths, secondarily on the lengths and contours of P2, and finally on the length and contours of P1 and P3. We chose these criteria becuase the time parameters of the contact calls contribute most to individual differences among orange-fronted conures [19].

Our aim during playback was to imitate one of the two test birds. Because the two birds inevitably differed in the length of their solo contact calls, we selectively imitated the test bird with the shortest contact call by choosing stimulus calls that were slightly shorter (approximately 5–10 msec) than the average length of the solo contact calls of the test bird. Thus it ultimately made the stimuli even less similar to the non-imitated bird. Similarly, the test bird with the longest solo contact call was imitated by stimulus calls that were longer than the average length of its solo contact calls. The selected stimulus calls were filtered (0.5–11 kHz) and the amplitude standardised with respect to peak amplitude.

Playback experiments have demonstrated that the sex of test birds and stimulus birds influences the response. Female test birds are generally more responsive than male test birds, and heterosexual interactions elicit stronger responses than interactions with the same sex [16]. We therefore included the sex of both test- and stimulus-birds in the design. To account for any effect of sex of the stimulus birds, we imitated each test bird with calls from both a female and a male stimulus bird. Each pair of test birds hence received a total of 4 trials and the playback imitated each of the test birds twice. Within a pair of test birds 4 different stimulus birds were used for the 4 trials.

Results

Treatment Success

Overall, we obtained a relatively high cross-correlation similarity between playback calls and solo contact calls for both test birds. Solo contact calls from imitated test birds showed significantly higher similarity to the playback calls than those from non-imitated test birds (Figure 1 & 2, Mixed model F_1,39 = 4.56, p = 0.039). Neither test birds’ sex nor stimuli birds’ sex (mixed model test bird sex F_1,39 = 0.07, p = 0.79; stimuli birds sex F_1,39 = 0.04, p = 0.85) or any of the second order interactions involving these factors affected the average similarity values significantly (mixed models all F_1,39≤0.50, p≥0.48). These results show that the playbacks successfully imitated the targeted test birds. Furthermore, no bias with regard to sex of the test bird or stimulus-bird could be detected.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 2. Playbacks successfully imitated the targeted birds.

Playback calls (LS mean ± SE) had a higher similarity to the solo calls of imitated than non-imitated test birds. The similarity between the individuals solo contact call and the playback was quantified by spectrographic cross-correlation.

https://doi.org/10.1371/journal.pone.0049747.g002

Imitation×Test Birds’ Sex

The significant interaction between imitation and test bird sex for the call rate during playback indicated that male and female test birds responded differently when imitated by the playback (Figure 4, Table 1). Post hoc pairwise comparisons showed that males had a significantly higher call rate when imitated by the playback compared to non-imitating playback (Figure 4, least square means t = 3.78, p = 0.0005), whereas the response of imitated and non-imitated females did not differ significantly (Figure 4, least square means t = 0.56, p = 0.58). Although not significant, imitated male test birds tended to respond more than imitated female test birds (Figure 4, least square means t = 1.76, p = 0.09), and non-imitated female test birds tended to call more than non-imitated male test birds (Figure 4, least square means t = 2.01, p = 0.052). The interactions between imitation and test bird sex for latency and post-playback call rate were not significant (Table 1). Likewise the interactions between imitation and stimulus-bird sex showed no significance for any of the response variables (Table 1).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 1. Responses of test birds.

https://doi.org/10.1371/journal.pone.0049747.t001

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 3. Imitation affected the vocal response.

Imitated birds responded with higher call rates and shorter response latency than non-imitated birds. Contact call rates (a) and response latency (b) (LS mean ± SE) of imitated and non-imitated test birds during playback.

https://doi.org/10.1371/journal.pone.0049747.g003

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 4. Sex-specific responses to imitation.

Males gave higher call rates (LS means ± SE) when imitated than when not imitated, whereas females did not differ in their response to imitation versus non-imitation.

https://doi.org/10.1371/journal.pone.0049747.g004

Discussion

When multiple orange-fronted conures were simultaneously exposed to the same contact call stimulus, the individual whose call had the highest similarity to the playback call responded the strongest. This suggests that an orange-fronted conure may address a specific individual within a flock by imitating its contact calls. Imitations between calling individuals are common during interactions between free-ranging birds in separate flocks (unpublished data) as well as during individual birds’ responses to playback [13], [14]. Given that orange-fronted conures frequently communicate within large communication networks with many potential receivers, which may be from several different flocks, the ability to selectively address specific individuals may be of particular importance.

Our experiment monitored the response of both intended and non-intended receivers to a sender that did not vary its calls during the interaction. In some natural interactions, the degree of call modification is asymmetric among the interactants; immitation often results from one individual modifying its call more the other individual, rather than a mutual convergence. The playback therefore laid within the natural range of behaviours in orange-fronted conures. Natural interactions between members of flocks would not allow us to discern who addressed who as both flocks (i.e. communication networks) may attempt to address each other. Only an experiment would allow such conclusions. The receiver responses, therefore, are essential for testing whether imitations of contact calls can address other individuals and, ultimately, for understanding the function and evolution of vocal imitation.

Conures can imitate contact calls almost immediately upon hearing them [14], therefore, addressing individuals within a network by imitation does not require any long-term prior experience with specific individuals. This rapid imitation ability is essential, given the fission-fusion flock dynamics that result in a large social network with frequent turnovers in flock composition. In comparison, turnovers in the communication networks of territorial species are less frequent and involve fewer individuals [33]. The relatively small and stable network of territorial species may explain why several of them use song type matching with discrete, existing song types for addressing birds in the neighbourhood [8], [34], [35], [36], [37]. Addressing of specific individuals in a communication network can also be achieved by vocal labeling of individuals, where a specific vocalisation is linked to a specific individual [15], [38], [39]. However, vocal labelling only works in small and/or stable social networks, as it requires prior knowledge of and a learned internal representation of the interacting individuals [40]. Vocal labelling is, therefore, unlikely in large networks with high turnover involving many individuals. In contrast, the plasticity that vocal imitation provides, allows for the addressing of specific individuals with which the addressor has only a limited knowledge. Many species of parrots live part of their lives in social flocks [28] and vocal imitation in parrots may, therefore, have evolved, to enable addressing of specific individuals in communication networks with high turnovers involving many different individuals.

A previous study [16] suggests that convergent contact calls with similarities about 0.6–well below the imitations of the present study (Figure 1 & 2)–are affiliative signals for orange-fronted conures, as male orange-fronted conures call more in response to convergent than to divergent series of calls in a non-agonistic contest [16]. The current experiment provides further insight by revealing that imitations, can be used as a way of addressing individuals. Imitations of the contact calls can thus address and initiate an interaction with a specific individual. Interestingly, males and females responded differently to imitation of their solo contact calls. Males primarily responded more when addressed, whereas females responded both when addressed and when not addressed. Therefore, being addressed seems to be more important to males than to females. In European siskins (Carduelis spinus), which live in flocks part of the year, high ranking males showed mutual hostility towards each other and never made calls that imitated each other. In contrast low-ranking males were less hostile and imitated their flock mates [26]. If imitation of contact calls signals willingness to take a subordinate position in a flock fusion, then the contact call exchanges and imitations after establishing contact by addressing may potentially be used for negotiating dominance position after a flock fusion [16]. Imitation may thus serve two functions: first, it may function in addressing individuals, as demonstrated here. Second, imitation in prolonged interactions may negotiate dominance position after a flock fusion. The strong difference in male and female responses may provide insight to the social organization of flocks in orange-fronted conures. This interpretation suggests that dominance hierarchy/leadership is mainly important to males. Previous experiments support this view [16], since females will respond and attempt to imitate calls with both low and high similarity to their own solo calls, whereas males mainly respond when the playback imitates them. Alternatively, males may respond to imitation because being addressed by imitation is perceived as a challenge. This scenario would be similar to song type matching interactions in passerines [41]. It follows from this interpretation that females do not experience a challenge in the same way by being addressed since they respond to and imitate both convergent and divergent call series. Imitation could thus serve both affiliative and agonistic functions depending on the context and timing of the interaction. Negotiations of dominance relationships in the ensuing interactions may well be agonistic, whereas addressing a specific individual prior to flock fusion would be affiliative.

Many of the experiments that suggest addressing of individuals through song type matching or call imitation have focused solely on dyadic interactions, only monitoring the response of the test subject [8], [14], [34], [35], [36], [37]. However, several experiments have now shown that non-intended receivers within a communication network may extract information from the interactions, i.e. eavesdrop [21], [22], [23], [24], [42], [43]. In this experiment, we created a simple three-member communication network (two live conures, plus playback stimuli) and monitored the response of both the addressed (imitated) and the non-addressed (non-imitated) member of the network. The responses of the addressed and the non-addressed test birds were positively related, indicating that the non-addressed bird attempted to associate either with the playback or the other flock member, although less actively than the addressed bird. This positive relationship is not due to the characteristics of the pair as the statistical model controls for differences between pairs. Our results suggest that future experiments on network communication should monitor the response of the network surrounding the focal individual, if possible.

Vocal pathway development in humans and ancestral birds is believed to have evolved independently [44]. Across taxa, however, several developmental and neural analogies exist such as an innate perceptual predisposition for vocal behavior, and a similar asymmetric brain structure with the left hemisphere specialised for language and song [44], [45]. In parrots, the analogies with humans seem even stronger as both have flexible vocal systems and can learn vocalisations throughout life. A common selection pressure that could give rise to such convergent evolution is the social system; both the hunter-gather life style of early humans [46] and the fission-fusion flock structure of orange-fronted conures [17] result in communication networks that change frequently. Such dynamics within the social networks necessitates flexible communication skills to enable addressing of specific individuals in the network to effectively mediate social interactions within and between groups or flocks. The current study thus demonstrates the use of such plastic communication system in a dynamic social environment, which may explain one reason for the evolution of vocal imitation in parrots.

Orange-fronted conures have the ability to immediately imitate the individually-distinctive contact calls of others. The response to being imitated by a new-comer in a flock context suggests that imitation serves as a way to address specific individuals. The ability to address using imitation probably evolved as a consequence of the complex fission-fusion structure of the orange-fronted conures. Addressing in an ever changing communication network that involve many different individuals necessitated a flexible vocal system to enable addressing of specific individuals in the network.

Acknowledgments

We would like to thank Roger Blanco and the staff of the Area Conservacion Guanacaste for all their help and support. Anne-Mette Wiberg, Judith Scarl, and Katrine Rolighed assisted with the field work. Danielle Adams and two anonymous reviewers gave valuable feedback on an earlier version of this manuscript. The trapping and playback experiment were done under permission from the ACG.