DOI: 10.5061/dryad.1rn8pk0th
title: Data to: Carotenoids-based reddish pelvic spines in non-reproducing female and male sticklebacks (Gasterosteus aculeatus) – signalling social dominance?

description: Conspicuous ornaments are often considered a result of evolution by sexual
 selection. According to the social selection hypothesis, such conspicuous
 traits may also evolve as badges of status associated with increased
 boldness or aggression towards conspecifics in conflicts about ecological
 resources. This study tested predictions from the social selection
 hypothesis to explain evolution of conspicuous red colour of the pelvic
 spines of the three-spine stickleback (Gasterosteus aculeatus). Wild
 non-reproducing sticklebacks were presented to pairs of dummies which
 differed at their pelvic spines, having either (i) normal sized grey or
 red pelvic spines, or (ii) normal sized grey or large red pelvic spines.
 The experimental tank was illuminated by white or green light, since green
 light impedes the sticklebacks’ ability to detect red colour. The dummies
 moved slowly around in circles at each end of the experimental tank. We
 quantified the parameters (i) which of the two dummies was visited first,
 (ii) time taken before the first visit to a dummy, (iii) distribution of
 the focal sticklebacks in the two zones close to each of the two dummies
 and in the neutral zone of the tank, (iv) close to which of the two
 dummies did the focal fish eat its first food-piece, and (v) time spent
 until the first piece of food was eaten. This was carried out for 22
 females and 29 males sticklebacks. The results suggested no effect of the
 colour or size of the dummies’ pelvic spines, on none of the five
 behavioural parameters. Moreover, neither the colour of the pelvic spines
 of the focal sticklebacks themselves (as opposed to redness of the
 dummies’ spines) or their body length was associated with behaviour
 towards the dummies. Thus, this study did not support predictions from the
 social selection hypothesis to explain evolution of red pelvic spines in
 sticklebacks.
description: Wild sticklebacks were collected from the 140 m long and 50 m
 wide and landlocked freshwater Lake Pallvatnet, located at an altitude of
 140 m at 67°31’N, 14°40’E in Bodø, North Norway. Fish were caught by traps
 made of 1.5 L soda bottles deployed along the shore at 0.2 – 1.0 m depth.
 The traps fished for 24 hours 9 – 10 September 2019. Captured sticklebacks
 were transported to Mørkvedbukta Research Station in Bodø and kept in a 80
 l storage tank with continuously flowing water until further handling. The
 fish in the storage tank were daily fed frozen Chironomidae larvae
 (Akvarie Teknik, DeLang and Ekman AB, Filipstad, Sweden).
 The experimental trials were carried out 13 September to 4
 November 2019. Trials were run with a total of 37 males and 23 females.
 Twenty-four hours before each trial, one focal stickleback was isolated in
 a transparent Plexiglass tube (7.5 cm in diameter, 35 cm height),
 hereafter termed the “isolation tube”, and placed inside the storage tank
 with the other fish. The focal specimen could see its conspecifics in the
 storage tank and there was a continuous flow of water between the storage
 tank and the isolation tube. However, the isolated focal stickleback did
 not have access to food during these 24 hours in order to increase its
 motivation to forage during the upcoming trials. After 24 hours in the
 isolation tube the focal fish was transferred to an experimental tank
 which consisted of several devices as shown in Fig. 1. Two electric
 engines moved two dummies in circles in each end of the experimental glass
 tank. The circumference of the circles was 66 cm, the speed of the dummies
 was 2.7 cm s-1 which means that the dummies spent 24 s per lap. Two of the
 three different dummies (see below) were presented simultaneously in one
 trial. W.J. Rowland kindly molded the dummies in epoxy in 2003 as
 described in Rowland (1979). A 52.0 mm non-gravid female stickleback was
 used as model. The dorsal part of the dummies was painted black (85 Coal
 Black Satin, AAA0655, Humbrol Enamel, Kent, UK) and the ventral part grey
 (64 Light Grey Matt, AA0713). We mounted artificial pelvic spines on three
 different dummies. Artificial pelvic spines of approximately normal length
 (10 mm) were mounted on two dummies, whereas larger (20 mm) artificial
 pelvic spines were mounted on the third dummy. On one of the dummies with
 small artificial pelvic spines and the dummy with large pelvic spines,
 part of the pelvic spines was painted red (60 Scarlet Matt, AAA0655). The
 red was painted along the entire spine from the base to the tip, covering
 about 180° of the pelvic spines with the red part directed towards the
 “body” of the dummies. The remaining part of the pelvic spine on these two
 dummies was painted with the same grey colour as the ventral part of the
 body of the dummies (see above). The pelvic spines of the remaining third
 dummy (with normal length spines) were painted grey all over. The
 artificial spines were installed spread out away from the body. This
 leaves us with three different dummies: one with grey pelvic spines of
 normal length (“Normal-grey” abbreviated “NG”), one with red pelvic spines
 and normal length (“Normal-red” abbreviated “NR”), and one with red and
 large spines (“Large-red” abbreviated “LR”). Two Chironomidae larvae were
 presented in each of two Petri dishes below each of the two dummies. The
 experimental tank was illuminated by either white or green light. The
 white light came from one light bulb (Anslut, E27 2.5 W 140 lm, rendering
 average (Ra) of 80, article number 421433 at www.jula.no), whereas green
 light came from three bulbs (Anslut, E27 0.7 W 30 lm, article number
 420698). Light intensity was 280 and 120 lux for white and green light,
 respectively, measured by a light meter (Amprobe LM-120 Light Meter,
 Glottental, Germany) inside the experimental tank where the dummies were
 located. Three sides of the experimental tank were covered by
 non-transparent green plastic foliage, whereas video-recordings were
 carried out through the fourth uncovered tank wall.  
 The behaviour of each fish was studied under four different
 combinations of pair of dummies and colour of light. The combinations were
 (i) “Normal-grey” and “Normal-Red” dummies illuminated by white light (ii)
 “Normal-grey” and “Normal-red” dummies illuminated by green light, (iii)
 “Normal-grey” and “Large-red” dummies illuminated by white light (iv)
 “Normal-grey” and “Large-red” dummies illuminated by green light. Green
 illumination prevents the sticklebacks from using their red cues which
 impedes their ability to see red colours (Milinski and Bakker, 1990). Each
 of the four combinations of dummies and light-colour is hereafter termed a
 “subtrial”, whereas the combination of the four subtrials with each
 individual focal fish is termed a “trial”. We randomized both the sequence
 of the four subtrials with each fish, and to which of the two zones (zone
 “A” or “B”, Fig. 1) each of the two dummies appeared in each
 subtrial. Five minutes before the actual start of a
 trial, the focal fish was transferred to the transparent Plexiglas tube
 (TPT) located inside the experimental tank (Fig. 1), in order to become
 familiar with the two dummies and the tank. A subtrial (the first subtrial
 of four in a trial) started by removing the Plexiglass tube by pulling a
 thread while hiding behind a tarpaulin to avoid disturbing the focal fish.
 The stickleback could then swim freely around in the “Neutral zone” or
 closer to the two dummies in “Zone A” and “Zone B”, and feed freely on
 Chironomidae larvae beneath any of the dummies (Fig. 1). Each subtrial was
 recorded by a GoPro (San Mateo, US) camera mounted on a tripod in front of
 the experimental tank. Between subtrials, the focal fish was kept in the
 isolation tube (in the storage tank) while preparing for the next subtrial
 with the same focal fish. The trials were carried out in a quiet room.
 Care was taken not to disturb the fish during the trials and the focal
 fish saw no humans from being placed in the TPT-tank until the end of the
 recordings in each subtrial. After the termination of
 all four subtrials of a trial, the specimen used in this experiment was
 killed by an overdose of MS-222 and frozen in –20°C in darkness awaiting
 further analysis. Each individual was measured for total length to the
 nearest mm and weight to the nearest 0.001 g, and the sex was determined
 by inspection of the gonads. The ventral part of each fish was
 photographed using an Olympus E-M10 with a M.Zuiko ED 60 mm 1:2.8 macro
 lens and a Nissin i40 flash, with its pelvic spines erected and together
 with a standardized colour palette, for later quantification of the
 redness of the pelvic spines (see below). The water in the experimental
 tank was removed and replaced with fresh water before a new trial started
 with another focal stickleback. Intensity of the red colour (IR) of the
 pelvic spines of each stickleback and the reddish part of the colour
 palette were quantified separately from the digital photos in RGB mode by
 Adobe photoshop version 13.1. This method has previously been applied to
 quantify colour by several authors (Skarstein and Folstad, 1996, Skarstein
 et al., 2005, Villafuerte and Negro, 1998, Nordeide, 2002, Nordeide et
 al., 2006, Nordeide et al., 2008, Amundsen et al., 2015). In the
 quantifications, we encircled the pelvic spines and the reddish part of
 the colour palette and estimated the average density values for all three
 primary colours R, G, B (red, green, and blue) from the pixels enclosed in
 each of the areas (Villafuerte & Negro 1998). Intensity of red
 (IR) of the two pelvic spines and of the reddish cardboard was calculated
 as:  (IR) = (R/(R+G+B). The average IR-value for both the pelvic spines
 was used in the analyses, after correcting for differences between photos
 using the IR-value from the reddish cardboard (see Nordeide et al., 2006
 for details). Repeatability of IR in previous studies has been high (0.99
 in Nordeide et al., 2008, see also Nordeide, 2002, and Nordeide et al.,
 2006). The first five minutes of each subtrial,
 starting when the focal fish was released from the transparent Plexiglas
 tube (see Fig. 1), were analysed using the VLC Media Player. The monitor
 was set in black and white mode during this analysis to reduce potential
 subjectivity from the analyser, since this step hindered distinguishing
 between both white and green light and between the Normal-grey and the
 Normal-red dummies. A total of 60 trials were carried out of which 9 were
 removed from the final data-set due to the sticklebacks being either
 infected by the endo-parasite Schistocephalus solidus (Eucestoda) (3
 individuals), or due to technical problems during recording (2 specimens)
 or opening of the video-files of sub-trials (4 specimens). This left us
 with data from all four sub-trials from 51 trials, 29 males with total
 length 55.2 (S.D. ± 4.84) mm and 22 females with total length 55.6 (± 7.69
 mm), respectively. During the video-analyses we quantified seven different
 parameters in each subtrial. First, we quantified to which of the two
 dummies in a pair of dummies (Normal-grey (NG) vs. Normal-red (NR) or
 Normal-grey (NG) vs. Large-red (LR)), each stickleback approached first
 after being released from the transparent Plexiglas tube (TPT). This
 preference was defined by which of the zones “A” or “B” containing one of
 the dummies - the focal fish entered first (see Fig. 1). Second, we
 quantified the time spent in the neutral zone before the focal fish for
 the first time swam into one of the two zones (Zone A or B in Fig. 1)
 containing a dummy. Third, we quantified near which of the two dummies the
 focal stickleback spent the most time. This was done by noting in which of
 the zones “A” and “B” the focal fish was located every 15 s during the
 first five min (a total of 20 observations) after being released to swim
 freely in the experimental tank. Fourth, we quantified close to which of
 the dummies the focal specimen preferred to pick its first
 Chironomidae-larvae. Fifth, we quantified the time spent before this first
 feeding (in the previous point). Finally, we quantified the Intensity of
 red (IR) of the sticklebacks’ pelvic spines and the body length of each
 stickleback. Most statistics were carried out with IBM
 SPSS Statistics version 27, whereas Cohen’s d and Cohen’s h and
 statistical power were estimated using the pwr package in R version 4.0.2
 (R Core Team, 2020). Significance level was set to 0.05, and all p-values
 were two-tailed except the χ2 = tests which are one-tailed (Sokal and
 Rohlf, 1981). Statistical tests were carried out on females and males
 separately, and additionally on pooled data from both sexes. Exceptions
 were tests of “First feeding close to a dummy” (see Results, and Table 1
 and Fig. 4) where we only tested after pooling data from both sexes due to
 the relatively low sample size (n), and test of “Effect of the intensity
 of red pelvic spines of the focal sticklebacks” (Fig. 5) where the
 response variable (IR) differed significantly between the sexes and thus
 data were not pooled. Binomial tests were used for frequency data. T-tests
 or Mann-Whitney U-tests were used to compare measurement variables (time
 spent and the intensity of red pelvic spines) relative to the choice
 between different dummies, after testing data for deviation from normal
 distribution using the Kolmogorov-Smirnov-test. Box-Whiskers plots show
 10th and 25th, median value, and 75th, 90th percentiles. A reviewer
 suggested that we carry out a linear model for each response variable like
 : “Behavior ~ sex + dummy treatment + light treatment + spine intensity +
 body size”. Our main argument why this is probably not a good idea are:
 (i) There are two pairs of dummies involved in this experiment: NG – NR,
 and NG – LR. A single multivariate linear model for each response variable
 would also compare the behaviour of the focal fish towards one dummy in
 one pair of dummies with the behaviour towards another dummy in the other
 pair of dummies. This does not make sense, and it would flaw our results.
 Additional challenges are: (ii) Which of several models to pick for
 presentation when five predictors and their interaction terms are involved
 and a 0-model being the best model when using an AIC-approach. (iii)
 Collinearity is involved, and (iv) we would have to remove the
 power-analyses. The null hypotheses were not rejected
 in most of the statistical tests: t-tests, Mann-Whitney U-tests and
 binomial tests, in this study (see Results). Thus, we estimated
 statistical power (the probability 1 – β of correctly rejecting a false
 null hypothesis) for some of the tests (see “Results” and “Power analysis”
 below). Power is a function of effect size, and a larger difference
 between the two means to be compared in t-tests, or more specimens
 preferring one particular of the two zones with the dummies at the expense
 of the other zone in the binomial tests, would increase the power of our
 tests. Thus, we iteratively increased the effect size until the
 statistical tests turned out as marginally significant (p &lt; 0.05)
 and then we re-estimated statistical power using this new effect size. (i)
 When increasing the effect size in the t-tests we kept the sample sizes
 and standard deviations from actual data sets unchanged. We also kept one
 of the estimated sample means constant, whereas the other mean was
 gradually changed until the difference between means became marginally
 significant (p &lt; 0.05), when tested by t-test. We then estimated
 Cohen’s d based on this new simulated mean difference and pooled sample
 standard deviations, in accordance with Cohen (1988). Finally, 
 statistical power was calculated for two-sided t-tests, using the
 pwr.t2n.test of the pwr package, by inserting actual sample sizes, the
 newly calculated Cohens d and a significance level of 0.05. (ii)
 Similarly, to estimate statistical power of the binomial tests we
 gradually increased the difference in number of observations between the
 two groups while keeping the total sample size (n) constant, until the
 p-value from the binomial test turned out to be slightly significant. Then
 we used these new counts (which were now significantly different) to
 estimate Cohen’s h and statistical power.  By these
 simulations we were able to estimate the magnitude of effect sizes
 (difference between two means in t-tests, or counts in binomial tests) to
 detect significant differences between the groups. Effect sizes of Cohen’s
 d and Cohen’s h less than 0.5, between 0.5 and 0.8, and above 0.8, are
 considered as “small, “medium”, and “large”, respectively (Cohen, 1988).
 Finally, statistical power was estimated using the new simulated
 parameters. The
 study was carried out in accordance with ethical guidelines stated by the
 Norwegian Ministry of Agriculture and Food through the Animal Welfare Act.
 According to these guidelines, we were not supposed to - and therefore do
 not - have a specific approval or approval number.
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