DOI: 10.5061/dryad.5hqbzkhd2
title: Evidence for individual vocal recognition in a pair-bonding poison frog, Ranitomeya imitator

description: Individually distinctive vocalizations are widespread in nature, although
 the ability of receivers to discriminate these signals has only been
 explored through limited taxonomic and social lenses. Here, we asked
 whether anuran advertisement calls, typically studied for their role in
 territory defense and mate attraction, facilitate recognition and
 preferential association with partners in a pair-bonding poison frog
 (Ranitomeya imitator). Combining no- and two-stimulus choice playback
 experiments, we evaluated behavioral responses of females to male acoustic
 stimuli. Virgin females oriented to and approached speakers broadcasting
 male calls independent of caller identity, implying that females are
 generally attracted to male acoustic stimuli outside the context of a pair
 bond. When pair-bonded females were presented with calls of a mate and a
 stranger, they showed significant preference for calls of their mate.
 Moreover, behavioral responses varied with breeding status: females with
 eggs were faster to approach stimuli than females that were pair-bonded
 but did not currently have eggs. Our study suggests a potential role for
 individual vocal recognition in the formation and maintenance of pair
 bonds in a poison frog and raises new questions about how acoustic signals
 are perceived in the context of monogamy and biparental care.
description: This was a laboratory investigation of phonotaxis behavior and
 individual vocal recognition in a pair-bonding poison frog. Frogs were
 bred in a laboratory colony and virgin and pair-bonded (breeding) female
 subjects were assayed in an experimental arena within a soundproof chamber
 while male advertisement calls (also recorded in the laboratory) were
 broadcast from speakers. Video recordings of trials were scored blind in
 the behavioral analysis software, BORIS, and we quantified and compared
 the time spent in each arm of the arena to evaluate (1) how virgin females
 respond to a single acoustic stimulus (whether they preferentially spend
 time near the speaker), and (2) whether pair-bonded females show a
 preference for the speaker broadcasting calls of their mate as opposed to
 calls of a stranger. All behavioral data were analyzed in R. 
description: # Evidence for individual vocal recognition in a pair-bonding poison frog,
 Ranitomeya imitator \--- This dataset contains all raw video scoring
 tabulations and relevant trial information used in the quantification and
 comparison of phonotaxis behavior in virgin and pair-bonded Ranitomeya
 imitator, contextualized in experimental procedures and results. Five
 files (described in detail below) collectively serve as inputs for the
 script, Phonotaxis_Final.R, which performs the statistical analyses. ##
 Description of the data and file structure The file No Choice Trial
 Info.csv contains all information pertaining to the 40 no-choice trials
 conducted on virgin female R. imitator. Variables are as follows: * Trial
 Number: A numeric designation given to the trial at the time of
 completion, in the order of completion. * Female ID: ID of the female
 subject ('Ri.####') * Focal Female: Coded experimental ID given
 to each of 9 subjects ('j1-9') * Male ID: ID of male from which
 the stimulus call was recorded * Side: Side of arena from which stimulus
 was projected (L or R, randomized for each trial) * Date: Date on which
 trial was conducted * Time: Time at which trial was conducted *
 Acclimation Start: Time at which subject was placed under plastic cup to
 begin acclimation period * Acclimation End: Time at which the plastic cup
 was lifted, marking the end of the acclimation period (five minutes) and
 the start of the trial. * Trial Start: Time at which the trial began *
 Trial End: Time at which the trial ended (ten minutes later) The file Two
 Choice Trial Info.csv contains all information pertaining to the 28
 two-choice trials conducted on pair-bonded female R. imitator. Variables
 are as follows: * Focal Male: Coded experimental (letter) ID given to each
 of the pair-bonded male partners from which the stimulus calls were
 recorded. * Focal Female: Coded experimental (letter) ID given to each of
 9 female subjects. Each female letter designation mirrors the letter of
 her male partner, but in lower-case to distinguish. * Trial Number: A
 numeric designation given to the trial at the time of completion, in the
 order of completion. Note, the original study design included additional
 trial types which were excluded for the purpose of this analysis, leaving
 gaps in the numbering. * Block: Time block (1 or 2) during which the trial
 was conducted. Blocks are spaced nine weeks apart and facilitate repeated
 measures of some individuals. * Trial Type: 'Mate vs Stranger'
 or 'Stranger vs Mate' - two trial types signifying whether the
 speaker projecting the mate stimulus was on the left or right side,
 respectively. * Left Speaker: Letter ID of male stimulus presented from
 the left side speaker * Right Speaker: Letter ID of male stimulus
 presented from the right side speaker. * Date: Date on which trial was
 conducted * Time: Time at which trial was conducted * Acclimation Start:
 Time at which subject was placed under plastic cup to begin acclimation
 period. * Acclimation End: Time at which the plastic cup was lifted,
 marking the end of the acclimation period (five minutes) and the start of
 the trial. * Trial Start: Time at which the trial began * Trial End: Time
 at which the trial ended (ten minutes later) * Humidity (%): Level of
 humidity measured within the experimental arena, when recorded. * Temp
 (Farenheit): Temperature in degrees farenheit measured within the
 experimental arena, when recorded. * Breeding: Letter designation
 signifying whether the focal pair was breeding (i.e., had eggs and/or
 tadpoles; Y) or not (N) at the time when the trial was conducted. The file
 Two Choice IDs.csv deciphers the code used for individual females across
 time blocks in each time block for the two-choice experiments. Note that
 repeated females did not necessarily retain the same coded letter ID
 across time blocks. Variables are as follows: * Block: Time block (1 or 2)
 during which the trial was conducted. Blocks are spaced nine weeks apart
 and facilitate repeated measures of some individuals. * Male ID: ID of
 male from which the stimulus call was recorded. * Focal Male: Coded
 experimental (letter) ID given to each of the pair-bonded male partners
 from which the stimulus calls were recorded. * Female ID: ID of the female
 subject ('Ri.####') * Focal female: Coded experimental (letter)
 ID given to each of 9 female subjects. Each female letter designation
 mirrors the letter of her male partner, but in lower-case to distinguish.
 The files No Choice Scoring Data.csv and Two Choice Scoring Data.csv
 contain the tabulated 'time spent' data obtained from scoring
 trial videos with the behavioral analysis software, BORIS. Trials were
 scored without audio, and the observer was blind to side of the arena from
 which the stimulus/stimuli were presented. Variables are as follows: *
 Trial Number: A numeric designation given to the trial at the time of
 completion, in the order of completion. * Block: Time block (1 or 2)
 during which the trial was conducted. Blocks are spaced nine weeks apart
 and facilitate repeated measures of some individuals. * Observation Date:
 The date and time at which the analysis was performed. * Description: An
 automatically generated field in BORIS, which we left blank. * Media file:
 File path to the input video. * Total length: Total length of the video,
 in seconds. Videos longer than 600 seconds were analyzed only up to the
 600 s mark. * FPS: Frames per second * Focal Female: Coded experimental ID
 given to each of 9 subjects ('j1-9') * Behavior: The location of
 the frog within the arena - either 'center' (central starting
 square), 'R' (right arm), or 'L' (left arm). All frogs
 start the trial in the center and can move freely into the left or right
 arm. * Behavior Type: A categorical variable within BORIS to signify
 whether behaviors were instantaneous or continuous. All of our behaviors
 were recorded as 'State' behaviors, meaning once started they
 continued until the user specified 'stop'. The frog begins the
 trial in center by default, and with the first click the user records the
 time of first movement, which is always within center because the central
 starting square spans ~10 frog body lengths. Thus, the first two behaviors
 are always 'center,' whereas the third denotes the first time
 the frog crosses into the left or right arm. Each trial may have as few as
 1-2 rows (meaning the frog never left center) or &gt;2 rows (meaning the
 frogs explored the arena). * Start (s): Time at which the focal behavior
 commenced, expressed in seconds into the video. * Stop (s): Time at which
 the focal behavior ceased, expressed in seconds into the video. * Duration
 (s): Total duration of the behavior, calculated as Stop (s) - Start (s).
 ## Code/Software All statistical analyses were performed in R Studio v.
 2023.03.0+386 (R Core Development Team, 2020). Analyses are divided into
 two sections for the two experimental types: No Choice and Two Choice
 experiments. We first read in and merge associated trial info and blind
 scoring data and expand data frames to include rows for behaviors not
 observed. We then summarize time spent data for each trial by summing the
 total time spent in each behavior, or section of the arena, and recoding
 BORISÕs coded behaviors (center, L, R) in the context of the experiment:
 For No-Choice experiments, L and R are recoded as 'Toward
 Stimulus' and 'Away from Stimulus', contingent on the side
 of the arena from which the stimulus was presented. For Two-Choice
 experiments, L and R are recoded as 'Toward Mate' and
 'Toward Stranger' contingent on the Trial Type, which designates
 whether the Mate stimulus was presented on the left or right side.
 Following data wrangling steps, we compare total time spent on each side
 of the arena using Type III ANOVAs. Model testing was carried out using
 the packages 'lmerTest', with breeding status specified as
 covariates (for two-choice trials) and female ID, speaker position, and
 block (for two-choice trials) specified as random effects in all models.
 For no-choice experiments, we also ran a model including Male ID as a
 fixed effect. For each trial type, we next evaluated the latency of
 subjects to exit center (if they exited center) and the overall temporal
 pattern of space use within the arena. These resulted in additional models
 including latency to approach as an independent and dependent variable
 (see script and methods for details of model structure). This script
 requires the following packages: lme4, lmerTest, plyr, dplyr, stats,
 ggplot2, and ggpubr.