DOI: 10.5061/dryad.jq2bvq871
title: Experimental evidence that acorn woodpeckers recognize relationships among third parties no longer living together

description: Triadic awareness, or knowledge of the relationships between others, is
 essential to navigating many complex social interactions. While some
 animals maintain relationships with former group members post-dispersal,
 recognizing cross-group relationships between others may be more
 cognitively challenging than simply recognizing relationships between
 members of a single group because there is typically much less opportunity
 to observe interactions between individuals that do not live together. We
 presented acorn woodpeckers (Melanerpes formicivorus), a highly social
 species, with playback stimuli consisting of a simulated chorus between
 two different individuals, a behavior that only occurs naturally between
 social affiliates. Subjects were expected to respond less rapidly if they
 perceived the callers as having an affiliative relationship. Females
 responded more rapidly to a pair of callers that never co-occurred in the
 same social group, and responded less rapidly to callers that were members
 of the same social group at the time of the experiment and to callers that
 last lived in the same group before the subject had hatched. This suggests
 that female acorn woodpeckers can infer the existence of relationships
 between conspecifics that live in separate groups by observing them
 interact after the conspecifics in question no longer live in the same
 group as each other. This study provides experimental evidence that
 nonhuman animals may recognize relationships between third parties that no
 longer live together and emphasizes the potential importance of social
 knowledge about distant social affiliates.
description: <b>Material and Methods</b> (a)
 <i>Study site and population monitoring</i> We collected all data at Hastings
 Natural History Reservation in central coastal California, where the acorn
 woodpecker population has been the subject of a long-term study since 1971
 and &gt;95% of the individuals are color-banded (MacRoberts and
 MacRoberts 1976; Koenig 1981a). Each year, approximately 50 social groups
 are monitored, and a census is conducted of each group approximately every
 8–10 weeks. Subjects for this experiment were 26 wild adult acorn
 woodpeckers, including 9 breeder females, 5 helper females, 7 breeder
 males, and 5 helper males from 18 social groups. Experimental trials were
 conducted from 19 Jul to 27 Nov 2017 and from 6 May to 8 Jul
 2018. (b) <i>Experimental
 design</i> We
 conducted a playback experiment with a violation-of-expectation paradigm
 following a previously published protocol (Pardo et al. 2018). Woodpeckers
 were presented with playback stimuli consisting of <i>waka</i>
 calls recorded from two different individuals, overlapped artificially to
 simulate two birds calling simultaneously. <i>Waka</i> calls
 are individually specific, affiliative vocalizations that are frequently
 given in an overlapping chorus between two or more members of the same
 group, but rarely given between individuals with no affiliative
 relationship (MacRoberts and MacRoberts 1976; Yao 2008). If the two
 overlapping callers in a playback stimulus had no affiliative relationship
 that the subjects recognized, the playback stimulus was expected to
 violate the expectations of the subjects and the subjects were expected to
 respond more strongly by reacting more rapidly and approaching the speaker
 more closely. Conversely, if the two overlapping callers in a playback
 stimulus had an affiliative relationship that the subjects recognized, the
 subjects were expected to respond less strongly (Pardo et al.
 2018). We presented
 subjects with playbacks from the following 5 treatment categories and
 attempted to present each subject with all 5 treatments, although this was
 not always possible (Supplementary Table S1). In all cases, the callers
 used in each playback stimulus were unrelated to and had never lived in
 the same group as the subject. The order of presentation was balanced
 using an incomplete Latin square design (Supplementary Table S1), and
 playbacks to the same group were spaced apart by 2–48 days (median = 4 d)
 to avoid habituation. Playbacks to groups within 250 m of one another were
 also spaced apart by at least 2 days. (T1) Related
 callers–currently live together: two related callers that lived
 in the same social group at the time of the experiment. (T2) Related callers–last
 together after subject fledged: two related callers that
 formerly lived in the same social group but ceased to do so because of
 dispersal or death of one individual 1.0­–5.8 years (mean 2.1 y) prior to
 the experiment, but <i>after</i> the subject had fledged.
 Thus, the subject would have had the opportunity to observe the callers
 living together in the past during extraterritorial forays to the callers’
 group (Barve et al. 2020). (T3) Related callers–last
 together before subject hatched: two related callers that
 formerly lived in the same group, dispersed into separate groups before
 the subject hatched in the nest and 2.2–6.4 years (mean 4.5 y) prior to
 the experiment, and were both confirmed to be alive after the subject had
 fledged. Thus, the subject would not have had the opportunity to observe
 the callers living together but could have observed them visiting one
 another post-dispersal.             (T4)
 Related callers–never lived together: two genetically
 related callers that never lived in the same group and were both confirmed
 to be alive after the subject fledged.             (T5)
 Unrelated callers–never lived together (control): two
 unrelated callers that never lived in the same group and were both
 confirmed to be alive after the subject fledged.
             Because of the difficulty of obtaining
 playback-quality recordings from a canopy-dwelling species with
 unpredictable calling patterns, for some playback stimuli we were forced
 to use the calls of individuals that had died or disappeared from the
 study area prior to the experiment. Preliminary analyses indicated that
 subjects responded more quickly to playback stimuli containing the call of
 a dead or missing individual (Supplementary Table S2; Figure S1).
 Therefore, we excluded all trials that used the call of a dead or missing
 individual from the analyses presented here (see Supplementary Materials
 for analyses including both live and dead callers). Most (19 of 24)
 playback stimuli for treatment T2 (related callers–last together after
 subject fledged) contained the call of a dead or missing individual, so
 this treatment was excluded from analysis. After excluding the
 aforementioned trials, there remained 24 playbacks of related
 callers–currently live together, 11 playbacks of related callers–last
 together before subject hatched, 13 playbacks of related callers–never
 lived together, and 13 playbacks of unrelated callers–never lived
 together, with a total sample size of 25 subjects (8 breeder females, 5
 helper females, 7 breeder males, and 5 helper males) from 17 different
 groups (Supplementary Table S1). Playback stimuli contained only
 recordings from callers of the same sex as the subject to increase the
 likelihood that the subjects would respond (Hannon et al. 1985). For the
 three treatment categories consisting of a pair of related callers,
 relatedness between the two callers in each playback stimulus was
 calculated using a pedigree, which was constructed from parentage
 assignments based on microsatellite markers (J. Haydock, unpublished
 data). Mean relatedness
 (<i>r</i><sub>(T1)</sub>=0.36,
 <i>r</i><sub>(T3)</sub>=0.38,
 <i>r</i><sub>(T4)</sub>=0.38) did not differ
 significantly across these three treatment categories (ANOVA,
 <i>F</i><sub>2,45</sub>=0.10,
 <i>P</i>=0.91). To increase the likelihood that
 subjects had the opportunity to become familiar with all the callers, we
 presented subjects with callers from territories as near as possible to
 the subject’s territory. The mean ± standard deviation (SD) distance
 between the territory centroids of the subjects and the callers was 435 ±
 233 m and did not differ significantly across treatment categories (ANOVA,
 <i>F</i><sub>3,57</sub>=0.43,
 <i>P</i>=0.73). As acorn woodpeckers make daily forays to the
 territories of other groups with a mean foray distance of 500–600 m (Barve
 et al. 2020), subjects were likely familiar with all or most of the
 callers with which they were presented. Whenever possible, we constructed
 each playback stimulus from a unique pair of call exemplars to minimize
 pseudoreplication (Supplementary Table S1). All of the playback stimuli
 for the treatments “related callers–last together before subject hatched”
 and “unrelated callers–never lived together” consisted of a unique pair of
 call exemplars that was only used once, although we sometimes used
 multiple different exemplars of the same callers. However, only 14 of 24
 stimuli for “related callers–currently live together” and 8 of 13 stimuli
 for “related callers–never lived together” consisted of a unique pair of
 call exemplars. The
 speaker was placed in a tree 40–50 m away from the focal bird, and
 playback volume was standardized at 100.1 ± 1.3 dB re 20 µPa at 1 m, which
 is near the upper end of the range of natural <i>waka
 </i>calls (Pardo et al. 2018). Playback stimuli consisted of 1 min
 of background noise with a fade-in, followed by the two overlapping calls,
 then 30 sec of background noise, then the same two overlapping calls
 again, and a final 10 sec of background noise with a fade-out. The trial
 was aborted if the focal bird flew away before the first set of
 overlapping calls began. As <i>waka</i> calls consist of a
 variable number of repeated notes, we could not standardize the duration
 of the playback stimuli without heavily modifying the calls. The duration
 of the overlapping call chorus within each playback stimulus ranged from
 3.7 to 7.0 sec and did not differ significantly among treatments (ANOVA,
 <i>F</i><sub>3,57</sub>=0.21,
 <i>P</i>=0.89). (c) <i>Measuring
 response to playback</i> We videotaped the subject during
 each trial and measured the following 6 response variables within a 3-min
 period beginning with the start of the playback: latency to the first
 “reaction” (vocalizing, flying to higher vantage point, or flying toward
 speaker), latency to the first “positive flight” (flying up to higher
 vantage point or toward speaker), latency to the first approach to the
 speaker, latency to the closest approach to the speaker, distance of the
 first approach to the speaker, and distance of the closest approach to the
 speaker. For latency variables, if the behavior of interest did not occur
 within the allotted 3 min, the latency was assigned the maximum possible
 value of 180 sec and marked as “censored”. We were blind to the
 experimental condition in each trial until all the videos had been scored.
 Before the trials, we measured distances between the speaker and various
 nearby landmarks using a transect tape and used these measurements to
 estimate approach distances to the nearest 5 m during the
 playbacks. We used
 only <i>latency to react</i>, <i>latency to positive
 flight</i>, and <i>distance of first approach</i> in the
 analysis, as the other response variables were highly correlated
 (Pearson’s <i>r</i> &gt; 0.80) with at least one of these
 variables. We used both <i>latency to react</i> and
 <i>latency to positive flight</i> because it was unclear
 whether reaction strength was best measured by flight behaviors and
 vocalizations or by flight behaviors alone. We could not measure
 <i>latency to vocalize</i> as a stand-alone variable because
 there were many trials in which the subject flew toward the speaker and
 out of sight before the first vocalization was heard, and thus it was
 impossible to know whether the vocalization was produced by the subject or
 another individual. (d) <i>Statistical
 analyses</i>             All analyses were
 conducted in R 3.6.3 (R Core Team 2020), and the significance level was
 set to 0.05 for all tests. We used mixed-effects Cox proportional hazards
 regression, which accounts for censored observations, in the package
 <i>coxme</i> (Therneau 2019) to analyze <i>latency to
 react </i>and <i>latency to positive flight </i>, and a
 linear mixed model in the packages <i>lme4</i> (Bates et al.
 2015) and <i>lmerTest</i> (Kuznetsova et al. 2017) to analyze
 <i>distance of first approach</i>. The residuals for <i>distance
 of first approach </i>were not normally distributed (Shapiro-Wilk
 test, W=0.95, <i>P</i>=0.02), so we rank transformed
 <i>distance of first approach</i> before running the linear
 model (Shapiro-Wilk test on rank-transformed model: W=0.97,
 <i>P</i>=0.11). Each model contained
 <i>treatment</i>, <i>sex</i>,
 <i>treatment*sex</i>,<i> order of
 presentation</i>, and <i>days since previous playback
 </i>as fixed effects, and <i>individual ID </i>as a
 random effect. <i>Days since previous playback </i>represented
 the number of days since the last playback to the same group and was coded
 as 0 for the first playback to a given group. We compared all treatments
 to the control (unrelated callers–never lived together) separately for
 females and males in the package “<i>emmeans</i>” (Lenth 2019)
 using Dunnett’s method to adjust for multiple comparisons within a given
 model.
description: The second spreadsheet explains each variable in the dataset.
 Some variables have some missing values.