DOI: 10.5281/zenodo.16757294
title: Dataset and Code for - Punishment and appeasement in anemonefish societies: pay-to-stay promotes helping behaviour independent of kin selection

description: This repository contains the datasets and code associated with the manuscript:

Kirstin Gaffney, Megan E. Bartlett, Theresa Rueger (2025). Punishment and appeasement in anemonefish societies: pay-to-stay promotes helping behaviour independent of kin selection. [Manuscipt submitted for publication]

The dataset and code were archived on Zenodo prior to journal publication to ensure accessibility and reproducibility. Once published, the citation will be updated here.

Datasets included:

(1) PTS_baseline_helping.csv: baseline helping behaviour by rank 3 individuals, including anemone maintence, territory defence against predators and defence against food competitors.

(2) PTS_baseline_social.csv: baseline social behaviour in rank 3 individuals, including aggression recieved and submissive behaviour.

(3) PTS_experiment.csv: helping behaviour and social behaviour in rank 3 for each experimental condition

Variable definitions are provided in the attached README.txt file.

Code included:

(1) PTS_figures.R: R script to generate figures presented in the manuscipt

(2) PTS_Modelling.R: R script containing the statistical models used in the analyses
description: The evolutionary drivers of helping remain widely debated, particularly due to challenges disentangling the effects of different mechanisms. The pay-to-stay hypothesis proposes that helping aims to appease dominant breeders to avoid eviction. We tested pay-to-stay as an independent driver of helping by exploring its dynamics in anemonefishes (Amphiprion spp.) which lack prerequisites for other mechanisms, including kin selection. We experimentally prevented helping (anemone maintenance, territory defence) in A. percula, A. perideraion, and A. clarkii., and assessed two predictions: (1) helpers would be punished and/or (2) would compensate with increased cooperative effort (pre-emptive appeasement). We show that anemonefish non-breeders do pay-to-stay: punishment and/or appeasement occurred in all species, with differences reflecting variation in ecological and social constraints. We also highlight a novel trade-off between size and cooperative effort in pay-to-stay dynamics. Overall, this study enhances our understanding of helping evolution, demonstrating that pay-to-stay can drive helping independently of kin selection.
description: Study site and experimental groups:

This study was carried out on inshore reefs in Kimbe Bay, Papua New Guinea (5°30’ S, 150°05’ E) from February to April 2023. We used 12 groups of Amphiprion percula, 12 groups of Amphiprion perideraion and 6 groups of Amphiprion clarkii. Both A. percula and A. perideraion groups were comprised of either three (n = 10 and 9, respectively) or four (n = 2 and 3, respectively) individuals and A. clarkii groups were comprised of three (n = 3), four (n = 2) or five (n = 2) individuals. All A. percula and A. perideraion groups were associated with the anemone species Radianthus magnifica. For A. clarkii, most groups (n = 4) were associated with Stichodactyla mertensii and some (n = 2) were associated with Heteractis crispa. To exclude behavioural differences due to breeding, only non-breeding groups were selected.

Experimental protocol:

We experimentally prevented helping behaviours in the highest-ranked subordinate (rank 3) from each group. The experiment had a repeated-measures design with a treatment (i.e., helping prevention) and control condition [PTS_experimental_design.pdf]. All groups were tested once in each condition over consecutive days, with the order counterbalanced. In the treatment condition, we caught the rank 3 individual and placed them in a clear plastic box (15 x 15 x 15 cm) for approximately 25 minutes (20-33 minutes). After this period, the rank 3 was released back into their anemone host, the box was removed and behavioural recordings followed. The protocol for the control was identical, except the subordinates were immediately released back to their anemone host rather than being placed in the box. 

Behavioural recordings and analysis:

All groups were filmed on four occasions: two pre-treatment recordings (day 1 and 2) to measure baseline behaviour and two post-treatment recordings, one following each experimental condition (day 3 and 4, counterbalanced). Filming was carried out without divers present with a GoPro Hero 5 from a distance of 1.5 - 2.5 m for 17 minutes (excluding 1 minute from start and end to create a 15 minute observation period)

Behaviours were scored from videos using the BORIS programme (Friard & Gamba 2016), following an ethogram based on (Rueger et al. 2022 [Anemonefish_behaviour_ethogram.pdf]. We scored the following behaviours for rank 3 individuals: (i) aggression received from dominants, (ii) proactive submission (submission not directly in response to aggression) towards dominants, and (iii) ‘helping’ behaviours (anemone maintenance, defence against predators and food competitors). We calculated the duration of each behaviour and expressed it as a proportion of the total time the focal subordinate was visible during the observation period.

(See manuscript for full details)