Male and female guppies differ in speed but not in accuracy in visual. discrimination learning. Supplementary materials

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1 Male and female guppies differ in speed but not in accuracy in visual discrimination learning Tyrone Lucon-Xiccato and Angelo Bisazza Supplementary materials 1. Additional methods Ethical statement Experiments comply with the law of the country (Italy) in which they were performed (Decreto legislativo 4 marzo 2014, n. 26). The experimental procedures have been approved by the Università di Padova Ethical Committee (protocol n. 09/2012). The experimental tanks were provided with natural plants, bottom gravel and social companions to resemble maintenance tanks. Subjects spontaneously participated in the trials and they did not express distress during the experiments. Fish that refused to participate in the trials and fish that completed the experiment were released in maintenance tanks. Subjects maintenance Guppies used as subjects were bred in our laboratory at Dipartimento di Psicologia Generale (Università di Padova, Italy). Maintenance tanks were 20 aquaria (60 x 40 x 35 cm) with a gravel bottom, abundant natural and artificial plants, water filters and 15-w fluorescent 1

2 lamps (12:12 light/dark photoperiod). Subjects were randomly chosen from the different maintenance aquaria. Water temperature was kept constant at 26 ± 1 C. Three times per day guppies were fed commercial food flakes (Fioccomix, Super Hi Group, Ovada, Italy) and Artemia salina nauplii. Subjects measurement After completion of the experiment, we anaesthetised subjects in an MS-222 bath (Sigma Aldrich, St Louis, Missouri, USA), and then we photographed them to measure standard length. Experimental phases All the experiments were made up of five consecutive phases. Phase 1 Collective habituation to the apparatus: Two subjects, one male and one female, were moved from a maintenance tank to a tank identical to the experimental apparatus and left for three days to become accustomed to the new environment. Food was delivered as in the maintenance tanks. Phase 2 Collective habituation to the procedure: From here on, no food was delivered to the subject outside the experiment to ensure motivation to participate in the trials. This phase lasted three days, during which three times per day the subjects were led to the start box according to the procedure of the trial (described in the main manuscript) and their food ration (crumbled food flakes, Fioccomix, Super Hi Group, Ovada, Italy) was delivered in six, randomly chosen, holes of the plate. The subjects were then allowed to enter the main compartment and fed. On the third day of this phase, six yellow-coloured discs were placed so as to partially cover the holes with food, in a way that the subjects could reach the food by peeking and moving the discs. At the end of the third day, the two subjects were 2

3 moved individually to an experimental tank. The guppy is a social species and it is reported to habituate faster in the new tanks in presence of many social companions (Lucon-Xiccato et al. 2015; Miletto Petrazzini et al. 2015); thus, we provided five immature guppies (approximate standard length: 1 cm) in each experimental tank. To avoid any interference with the subject performance, social companions were moved in another tank during the occurrence of trials. Social companions were moved back to the experimental tank at the end of the block of trials. Phase 3 Training to dislodge the discs: After one day of habituation to the experimental tank, the subject was trained to dislodge the discs. Over two days we administered 16 trials in which one yellow disc was present on the plate. A small piece of food was placed in the hole under the disc. The disc covered 75% of the hole in the initial 12 trials. In the remaining four trials, the disc completely occluded the hole and the guppies had to dislodge the disc to reach the food. As observed in previous studies employing this setup (Bisazza et al. 2014; Lucon-Xiccato and Bisazza 2014; Miletto Petrazzini et al. 2015), most guppies rapidly familiarised with the new tank and the experimental procedure, and learned to dislodge the disc efficiently. A few guppies failed to learn the motor sequence necessary to dislodge the disk. This happened before the discrimination learning began and therefore should have no effect on the result of the experiment. We substituted a failing guppy with another guppy of the same sex to reduce the influence of this confounding variable on the experiments. The criterion for removing a subject was that it did not dislodge the disc in all the trials with holes completely occluded of phase 3. We discarded two males and three females in experiment 1; two males in experiment 2; and four males and one female in experiment 3. In experiment 1, one additional female stopped participating in the experiment after the third olfactory control cue trial; thus, its performance in this last phase was not 3

4 analysed. In experiment 2, one female ceased to participate in the experiments on the ninth day of the training; thus, its performance was evaluated only up to that point. Phase 4 Experimental phase: In this phase, we evaluated discrimination learning performance of guppies. This phase varied according to the procedure of each experiment as described in the main manuscript. The experiments were conducted six day per week. Each day, the subject performed 10 trials subdivided in two blocks (five trials each) separated by almost five hours. Trials within a block were separated by five minutes. Phase 5 Olfactory cue control test: Previous experiments have shown that guppies are unable to use olfactory cues to find the food reward with this training procedure (Bisazza et al. 2014; Lucon-Xiccato and Bisazza 2014; Miletto Petrazzini et al. 2015). Yet, we repeated the control test for the use of olfactory cues in all the three experiments of this study. Other procedures performed tests for olfactory cues by interspersing control trials without food during the training (e.g., Miletto Petrazzini et al. 2015). However, the absence of reward in some trials might affect learning rate differently in the two sexes (e.g., Beatty et al. 1971); therefore in this work we performed our control test at the end of the experiment. Our control test consisted of 15 trials (corresponding to the 25% of the trials administered in the discriminations of experiments 1 and 3) in which identical yellow discs (two in experiments 1 and 2, and five in experiment 3) were placed on the plate. One of the discs, randomly chosen, hid the food. We performed only 15 control trials to avoid that, in absence of any visual cue, guppies could learn to exploit chemical cues during the control phase. The rationale of this test is that if during the main experiment guppies were using chemical cues to find food, they would continue to choose baited discs above chance during the control phase. If not, the performance would drop to chance level. To test these predictions, we compared the accuracy (proportion of correct choices) in the 15 trials of the olfactory cue control test with the accuracy in the last 15 trials of the discrimination learning phase with paired-sample t test 4

5 (see analysis below). Data were arcsine square root transformed before conducting parametric analysis (Sokal and Rohlf 1995) and reported in the text as mean ± standard deviation percentage. Details of statistical analysis The daily accuracy was calculated independently for each day of training according the formula: number of correct choices during the day / 10. Daily accuracy was therefore a repeated measures variable (12, 10, and six observations in experiments 1, 2, and 3, respectively). The overall accuracy was computed as: number of correct choices over all training / overall number of trials. Analysis was performed in R version (The R Foundation for Statistical Computing, Vienna, Austria, The linear mixed-effects models (LMMs) were fitted with the lme function, from the nlme R package. The generalised linear mixed-effects model (GLMM) were fitted with the glmer function, from the lme4 R package. Statistical significance of the effects in the models was assessed with F-tests and the Wald χ 2 test in LMMs and GLMMs, respectively. In our experiments, we aimed to compare the performance of male and female guppies. In case of nonsignificant differences between the two sexes in our analysis, the performance of males and females is likely to be equal. The null hypothesis significance testing allows to detect differences between groups, but it is mathematically incapable of supporting the hypothesis that two groups are equal (Barchard 2015; Dienes 2014). Therefore, we performed a Bayesian analysis that allowed us to estimate the strength of the evidence for the absence of difference. We calculated an approximated Bayes factor (BF) in favour of the absence of sex effect from the LMMs to analyse accuracy reported in the main manuscript (Schwarz 1978; Wagenmakers 2007). We computed the Bayesian information 5

6 criterion (BIC) of the LMMs with (BIC sex ) and without (BIC null ) the effect of sex. BF in favour of the absence of sex was calculated according to the formula: BF = exp((bic sex - BIC null ) / 2) (Schwarz 1978; Wagenmakers 2007). As an example, if BF = 5, our data are five times more likely to fit the model without the effect of sex than the model with the effect of sex. 2. Additional results Reliability test for decision time During the experiments, two different experimenters alternated in recording the decision speed of guppies (latency to dislodge the discs) with the chronometer. The reliability of this measurement was tested in a pilot experiment. We administered 24 discrimination trials to four guppies. One experimenter scored live the latency to dislodge the discs of the subjects and recorded the 24 trials with a digital camera. A different experimenter independently measured the latency to dislodge the discs from the recordings. The scores of the two experimenters were almost identical (2.03 ± 1.90% percentage difference, M ± SD). The two scores were also highly correlated (Spearman ρ= 0.999, p < ). Analysis of olfactory cues control test In the control test of experiment 1, subjects chose the baited disc in ± 10.71% of trials (chance level = 50%). This accuracy was significantly lower (paired-sample t test: t 18 = 4.129, p < 0.001) than the accuracy in the last 15 trials of the discrimination learning phase (65.67 ± 9.25 %), as expected if during the main experiment guppies did not use chemical cues to find food. 6

7 In experiment 2, subjects did not learn the discrimination during the training (accuracy during the 10 days of training: ± 4.88%). This provided evidence that guppies were not able to find food reward by using olfactory cues in this experiment. In the control test of this experiment, subjects chose the baited disc in ± 9.77% of trials (chance level = 50%). In the control test of experiment 3, subjects chose the baited disc in ± 9.04% of trials (chance level = 20%). This accuracy was significantly lower (t 19 = 4.026, p < 0.001) than the accuracy in the last 15 trials of the discrimination learning phase (42.67 ± %). Experiment 3: Accuracy of subjects showing recurrent and concept learning We compared the accuracy of subjects showing recurrent and concept learning. We fit the response of the subjects in each trial (correct or incorrect) in a generalised linear mixedeffects model (GLMM) with logit link function and binomial error distribution. We fitted day of training and trials of the day as fixed effects and subject ID as a random effect. We also fitted a two-level fixed effect indicating the strategy adopted by each subject (recurrent or concept learning; figure S2). Day of training had a significant effect in the GLMM (Wald χ 2 5 = , p < 0.001), but there was no significant effect of trial within day (Wald χ 2 9 = , p = 0.197). The strategy adopted by the subject had no significant effect (Wald χ 2 1 = 0.001, p = 0.973), suggesting the two groups achieved equal accuracy (recurrent learning: ± %; concept learning: ± %; figure S3). However, we found a significant strategy by day of training interaction (Wald χ 2 5 = , p = 0.001) and strategy by trial within day interaction (Wald χ 2 9 = , p = 0.049), providing further support that the two groups adopted different strategies to solve the task. Other interactions in the model were not significant. 7

8 3. Additional figures and table Day Pair of colours Figure S1 Pairs of colours used in oddity discrimination learning (experiment 3). Guppies had to choose the odd colour to find the reward. The rewarded colour was flipped for half of the subjects. 8

9 improvement rate across trials within days improvement rate across days Figure S2 Scatterplot of the improvement rate across trials within days versus the improvement rate across trials within days. Subjects that improve performance across trials within day (positive values on y axis) seem to be separated by the remaining group of subjects (dashed line). The two groups were composed from a roughly equal number of males (red dots) and females (black dots). 9

10 accuracy 50% 40% 30% 20% 10% 0% concept learning recurrent learning strategy Figure S3 Accuracy of subjects that apparently adopted concept learning and recurrent learning strategies in experiment 3. Data points represent mean ± SEM percentage of correct choices. Dashed line is chance performance. 10

11 Table S1 Analysis of individual accuracy. Data represents number of correct choices over all training / overall number of trials. Bold indicates performance significantly better than change according binomial test (50% of correct choices in experiments 1 and 2; 20% of correct choices in experiment 3). Data of the three experiments are independent; different subjects participated in each of the experiments. Subject Sex Experiment 1 Experiment 2 Experiment 3 1 female 67/120, p = /85, p = /60, p = female 81/120, p < /100, p = /60, p < female 67/120, p = /100, p = /60, p = female 58/120, p = /100, p = /60, p = 1 5 female 84/120, p < /100, p = /60, p = female 75/120, p = /100, p = /60, p < female 72/119, p = /100, p = /60, p < female 65/120, p = /100, p = /60, p < female 75/120, p = /100, p = /60, p = female 69/120, p = /100, p = /60, p < male 79/120, p < /100, p = /60, p < male 84/120, p < /100, p = /60, p < male 75/120, p = /100, p = /60, p = male 72/120, p = /100, p = /60, p < male 68/120, p = /100, p = /60, p = male 71/120, p = /100, p = /60, p < male 78/120, p = /100, p = /60, p = male 64/120, p = /100, p = /60, p < male 70/120, p = /100, p = 1 22/60, p = male 69/120, p = /100, p = 1 25/60, p <

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