DOI: 10.5061/dryad.sv2k6s2
title: Reproductive control via the threat of eviction in the clown anemonefish

description: In social groups, high reproductive skew is predicted to arise when the
 reproductive output of a group is limited, and dominant individuals can
 suppress subordinate reproductive efforts. Reproductive suppression is
 often assumed to occur via overt aggression or the threat of eviction. It
 is unclear, however, whether the threat of eviction alone is sufficient to
 induce reproductive restraint by subordinates.Here,we test two assumptions
 of the restraint model of reproductive skew by investigating whether
 resource limitation generates reproductive competition and whether the
 threat of eviction leads to reproductive restraint in the clown
 anemonefish Amphiprion percula. First, we use a feeding experiment to test
 whether reproduction is resource limited, which would create an incentive
 for the dominant pair to suppress subordinate reproduction. We showthat
 the number of eggs laid increased in the population over the study period,
 but the per cent increase in fed groups was more than twice that in unfed
 groups (205% and 78%, respectively). Second, we use an eviction experiment
 to test whether the dominant pair evicts mature subordinates, which would
 create an incentive for the subordinates to forgo reproduction. We show
 that mature subordinates are seven times more likely to be evicted than
 immature subordinates of the same size. In summary, we provide
 experimental support for the assumptions of the restraint model by showing
 that resource limitation creates reproductive competition and a credible
 threat of eviction helps explain why subordinates forego reproduction.
 Transactional models of reproductive skew may apply well to this and other
 simple systems.
description: Rueger et
 al_Amhiprion_Egg Count
description: <b>Materials and Methods</b> <i>(a) Study population</i> This study was conducted in May-August 2017 on inshore reefs near Mahonia Na Dari Research and Conservation Centre, in Kimbe Bay, Papua New Guinea. All fieldwork was conducted using SCUBA. From May 26<sup>th</sup>-June 23<sup>rd</sup>, we identified 52 breeding groups occupying the anemone <i>Heteractis magnifica </i>on 10 reefs. Groups consisted of a breeding pair and zero to three non-breeders. Individuals were ranked (1-5) based on their size relative to other individuals within their anemone, with the largest being rank 1. Individuals were also photographed, and the photographs were used to identify individuals and confirm that they survived the entire study and did not move between anemones during the study (Nelson et al. 1994; Elliott & Mariscal 2001; Buston 2003b, 2003c).   We monitored the reproduction of all groups, every two days for three lunar months (May 26<sup>th</sup> – August 21st). Our monitoring spanned three lunar months rather than calendar months, because <i>A. percula</i> breed on a lunar cycle (Seymour et al. 2018). Breeding was readily detectable. In the days before spawning, the male selected a nest site next to the base of the anemone and cleaned it, and in the days after spawning the male spent much of his time tending the eggs (Barbasch & Buston 2018). The age of the eggs was determined using the colour of the yolk and the presence and developmental stage of the embryos’ eyes (Buston 2004b). The eggs hatched after 7 days. Each clutch was photographed on day 1 or 2 to provide an estimate of the number of eggs laid, and the number of eggs was determined using the Cell Counter plugin for ImageJ.  The study began shortly after a severe mass bleaching event. Many of the anemones studied were visibly affected by unusually high temperatures in Kimbe Bay from March to May 2017. We bore this in mind when it came to designing our two experiments: 1) a feeding experiment (see below) and 2) an eviction experiment (see below). For the feeding experiment, the same number of bleached and unbleached anemones were included in both treatment and control groups. For the eviction experiment, bleached anemones were not included in the experiment. <i>(b) Feeding experiment</i> To test the hypothesis that food resources for reproduction are limiting, we conducted a feeding experiment with all 52 breeding groups. We collected one lunar month of baseline data (May 26<sup>th</sup>-June 23<sup>rd</sup>), then two months of data in which we manipulated (fed) half of the groups (N=25) while keeping the other half (N=27) as controls (June 24<sup>th</sup>-August 21<sup>st</sup>). These groups were randomly stratified to ensure equal amounts of fed/control groups for the reefs closer to shore (n = 4 reefs, n= 32 anemones) vs the reefs further from shore (n = 6 reefs, n= 20 anemones), for bleached anemones (n = 10 anemones) vs unbleached anemones (n = 42 anemones). See Saenz-Agudelo et al. (2011), Beldade et al. (2017) and Chausson et al. (2018) for the rationale for stratifying by these two factors. Consequently, distance from shore and bleached status were not included as covariates in the analysis. The treatment groups received one vial (3ml) of food pellets (New Life Spectrum, Marine fish food 1mm pellets) and one vial (3ml) of dried brine shrimp (Omega One, Freeze dried brine shrimp). The food was kept dry in capped tubes until delivery. Brine shrimp (positively buoyant) were delivered first, by squirting the shrimp onto the anemone with a pipette. The pellets (negatively buoyant) were delivered second by opening the vial and tipping them onto the anemone. Through this method, most food was either immediately consumed by the fish, or it was stuck among anemone tentacles, where the fish could consume it. Some food was consumed by the anemone and other fish species present around the anemone. The control groups were treated in the same manner, by squirting water from an empty vial into the anemone and opening and tipping out another empty vial over the anemone, to control for the possible disturbance caused by feeding.  <i>(c) Eviction experiment</i> To test the hypothesis that the breeding pair will evict other mature individuals but not immature individuals, we conducted an eviction experiment between August 21<sup>st</sup> and 26<sup>th</sup>. Thirteen focal groups, which all consisted of at least three individuals and had bred in the preceding months, were chosen. Only groups consisting of at least three individuals were chosen, to ensure that the dominant breeders were predisposed to tolerating a non-breeding subordinate. Only groups that were observed breeding in the preceding months were chosen, to ensure the two dominant individuals were indeed breeding adults. All individuals in each group were caught and measured to the nearest 0.1mm.  At the beginning of the experiment, the rank 3 and other smaller individuals (if present) were removed from the focal group, leaving only the two dominant individuals, rank 1 and rank 2 (Figure 1). Then, a rank 2 (reproductively active male) or a rank 3 (non-breeding subordinate) from different groups were introduced, one at a time, on different days, in random order (Figure 1). While rank 3 are not reproductively active, they are capable of reproduction (Buston 2004a). The introduced rank 2 and rank 3 were smaller than the original rank 3 and they were size matched, within 1mm standard length of each other, so evictions would not be driven by size (Buston 2003b).  Introductions were left overnight and the following day we noted the presence/absence of the introduced individual and 5 min of observations were conducted. Introducees were considered evicted if they had either disappeared overnight or if they spent most of the observed time (≥3min) outside the anemone, i.e. with their full body length outside of the range of anemone tentacles (Figure 1). If present, the introduced individual was then removed from the focal anemone and returned to its host anemone.  <b>Figure 1</b>. Schematic diagram of the eviction experiment. <b>a) </b>Focal groups consisted of a breeding pair (R1 & R2) and at least one non-breeding subordinate (R3). The rank three individual was removed from the focal group and a size-matched rank three (R3’) and rank two (R2’) from other groups were introduced to the focal group one at a time, on separate days and in random order. <b>b)</b>After one day the introduced individual was scored as either evicted or not evicted. The individual was considered not evicted if it spent the majority of the 5 min observation period among the anemone’s tentacles. The individual was considered evicted if it was either not present, or spent the majority of the 5min observation outside of the anemone’s tentacles.