Test fish and holding conditions
Test fish were sourced from the Universität Hamburg and local suppliers. All individuals were held in family (sibling) groups (total = 10 families) under standardised holding conditions (100–200 l tanks, 25 ± 1 °C, aerated and filtered water, weekly water changes). Fish were fed with Artemia spp. once daily on 5 days per week in their holding tanks. During experimentation, individuals were kept individually in smaller tanks (25 × 50 × 25 cm) and fed daily to maintain constant conditions between trials. The latter tanks were equipped with an immersion heater, an internal filter and half a clay pot (4 × 8 × 8 cm) as shelter. Fish were uniquely marked on their dorsal side with Visible Implant Elastomers (VIEs, VIE-Northwest Marine Technology, Shaw Island, WA, USA) of different colours for individual identification. VIEs do not affect mate choice in our study species (Schuett et al. 2017). Before experiments, all individuals were measured for their standard length using ImageJ (Schneider et al. 2012) (mean ± SE standard lengths in experiment 1: males = 4.69 ± 0.02 cm, females = 3.98 ± 0.08 cm; experiment 2: males = 5.38 ± 0.04 cm, females = 4.30 ± 0.03 cm). For all experimental trials, the water in test tanks was changed after every trial; water level was 10 cm. Unless otherwise stated, the water temperature in experimental tanks was maintained at 25 ± 1 °C. During experimental trials, no humans were present in the experimental room to avoid disturbances and trials were video-recorded using an overhead video camera (Sony HDR-CX405). In both experiments, males were habituated to being in a clear Plexiglas cylinder (diameter = 8.0 cm) twice for 10 min, once on two consecutive days before the mate choice trials. Individuals became readily accustomed to these cylinders and did not show any behavioural signs of distress whilst in the cylinders. For all experiments, blinded methods were used. More specifically, all behavioural assessments were automated using a tracking software to minimise any observer bias. The experimenter was not aware of behavioural scores while conducting experimental trials.
Experiment 1: female choice for male apparent aggression
In experiment 1 (February–May 2017), we assessed female mating preference for the apparent level (N = 48 preference assessments) and apparent consistency (N = 48 preference assessments) of male aggression (see “Mate choice trials” section). Before the mate choice trials, all males (N = 96) and females (N = 48) were tested for their natural aggressive behaviour twice with 48 h (range of ± 15 min) between the two tests to determine the average level and inconsistency of behaviour (see “Aggression test”). We assessed personality differences using repeatabilities (for general principles, see e.g. Lessells and Boag 1987) and tested for sex differences in the level and inconsistency of behaviour (see Data analyses). Two days elapsed between the last aggression tests and the beginning of mate choice trials.
Male and female aggression was quantified separately and indirectly as the mean distance of approach (cm) towards a computer-animated, same-sex conspecific opponent, as outlined in Scherer et al. (2017a). To begin an aggression test, we introduced two focal individuals, matched for sex and standard length, each into one of two adjacent test tanks (visually isolated from each other) that were aligned to face a computer monitor on their shorter axis (Fig. 1a, left panel, set-up with a grey background). After a 10-min acclimation period, during which the computer monitor was visually separated from test tanks, focal individuals were exposed to a computer-animated and unfamiliar same-sex, same-size opponent (to prepare the animations we used: Nmales = 9, Nfemales = 7; size difference between the opponent and focal individuals < 3 mm) for a test period of 11 min. The simulated opponent was animated to swim back and forth horizontally along the width of a white computer screen (see Scherer et al. 2017a for details).
For all trials, the mean distance to the animated opponent was assessed for 10 min (we did not track the first minute of a video) using the tracking software Ethovision XT 11 (Noldus, Wageningen, The Netherlands). For each individual, we averaged the two mean distances to the opponent (obtained during the two aggression tests, see above) as a proxy measure of each fish’s individual level of aggressiveness. Further, we assessed behavioural consistency of each individual as inconsistency: the absolute value of the difference between its mean distance of approach towards the animated opponent in the repeated aggression tests (Scherer and Schuett 2018; Scherer et al. 2017b, 2018a). Please note that large values indicate low consistency.
Mate choice trials
A mate choice trial consisted of an initial observation phase, followed immediately by a choice phase (Fig. 1). During the observation phase, a female was allowed to observe two stimulus males concurrently, with the males either differing in their apparent level of aggression (high vs. low) with consistency held constant (both fish consistent; Fig. 1a) or differing in their apparent consistency of aggression (consistent vs. inconsistent; Fig. 1b) with the average level of aggression held constant (intermediate aggression level for both males). Males were made to appear highly or less aggressive (or alternatively consistent or inconsistent) by manipulating their distance to an animated opponent moving on a computer monitor screen (Scherer et al. 2017a). The spatial position of each stimulus male was standardised by introducing them into separate clear Plexiglas cylinders (diameter = 8.0 cm) that were placed on the bottom of their test tanks either close to (4 cm), intermediate to (24 cm) or far from (44 cm) the animated opponent so as to simulate high, intermediate or low aggression level in the stimulus male, respectively. Consistency was manipulated by changing (inconsistent aggression) or maintaining (consistent aggression) the distance to the animated opponent between two periods of the observation phase as follows.
Following10 min of acclimatisation to the experimental tanks (Fig. 1a, b), we started the observation phase (22 min), which consisted of two consecutive 11-min periods. After the first observation period, we either changed or maintained the positions of the paired stimulus males according to their respective manipulation and allowed the fish to acclimatise for another 5 min. When testing female preference for the apparent level of male aggression, both males differed in their apparent level of aggression but showed the same apparent behavioural consistency. During both observation periods, one of the paired stimulus males was placed in close proximity to the virtual opponent (apparent high-aggression male) and the other one further away from the opponent (the apparent low-aggression male) (Fig. 1a). Conversely, when testing female preference for consistency, we altered the position of one of the two stimulus males relative to the animated opponent between the two observation periods (thus simulating inconsistency in his aggression level), while keeping the position of the other male (thus simulating consistency in his aggression level) (Fig. 1b). We sham-changed the position of the consistent male, i.e. we moved the male but placed him back into his original position to control for potential effects of handling. We placed the apparently inconsistent male close to the opponent during one observation period and far from the opponent during the other observation period (in randomised order). The apparently consistent male was placed at an intermediate distance from the opponent during both observation periods (Fig. 1b). Thus, both stimulus males showed on average the same apparent level of aggression, but differed in their apparent behavioural consistency. Throughout the observation phase, the female was placed in a clear Plexiglas cylinder (diameter 20 cm, placed in the centre of her tank) so that both males remained visible to her. During acclimatisation periods, removable opaque screens visually blocked the female observer tank, the stimulus males’ tanks, and the computer monitor from each other.
We also carried out control mate choice trials (for both level and consistency of male aggression, respectively) in a similar manner to that described above, except that the computer screen monitor did not display a virtual conspecific opponent but only a static white background during the observation phase (Fig. 1a, b). Control trials were used to account for the possibility that differences in the manipulated distances between the observer female and the stimulus males per se could account for any subsequent female preference for either stimulus male. Hence, there were four different treatments for the observation phase: level, level control, consistency, and consistency control. Each female was tested for her mating preference four times, once in each treatment (resulting in N = 192 mate choice trials, randomised testing order, 48 h between consecutive trials). A difference in female preference between trials with the presence of a virtual opponent (level and consistency treatment) vs. absence of such opponent (level control and consistency control treatment) would validate that female preference is related to a male’s distance to an opponent (i.e. apparent aggression) and not simply to male spatial position per se. However, such an effect was expected only if there was an effect of male apparent aggression on female mating preference in trials with a virtual opponent present during the observation phase. As this was not the case (see “Results”), an analysis of the control trials would be redundant and not informative. For completeness, we nonetheless present an analysis of the results for control trials in Online Resource 1.
Immediately following the observation phase, the paired stimulus males and the observer female were transferred to a dichotomous mate-choice arena with the female in a central compartment (Fig. 1c) to test for the female’s mating preference (e.g. Thünken et al. 2007; Dechaume-Moncharmont et al. 2011). After a 10-min acclimatisation period, the female was allowed to choose between the two stimulus males during a 22-min mate choice phase that was divided into two recording periods of 11 min each. In between these two recording periods, we switched the two stimulus males in their position (followed by another 5 min of acclimatisation) to control for any potential female side bias (e.g. Poschadel et al. 2009; Scherer et al. 2017b). To control for male activity, males were kept in clear Plexiglas cylinders (diameter = 8.0 cm), located in the middle of their compartments throughout mate choice trials. During recordings, the compartments of the mate choice arena were physically separated (clear Plexiglas), and they were additionally visually separated from each other during the acclimatisation period.
Using Ethovision XT 11, we quantified female association time (time spent within 10 cm of either male compartment, hereafter preference zone; Fig. 1c) for the two males over both recording periods (videos were analysed for 10 min, no tracking of the first minute) as a proxy for her mating preference (Thünken et al. 2007; Jeswiet and Godin 2011). Female preference for a particular male was calculated as her total association time with that male divided by her total association time spent with both males (e.g. Schlüter et al. 1998; Schlupp et al. 1999; Poschadel et al. 2009). Females that showed an obvious side bias (i.e. spent >80% of total association time in a particular preference zone over both recording periods) were excluded from statistical analyses (excluded trials: Nlevel = 15, Nconsistency = 5, Nlevel control = 8, and Nconsistency control = 8; resulting in Nlevel = 33, Nconsistency = 43, Nlevel control = 40, and Nconsistency control = 40 remaining trials for analyses). The removal of side-biased females is common practise in mate choice studies (e.g. Schlüter et al. 1998; Schlupp et al. 1999; Dosen and Montgomerie 2004; Hoysak and Godin 2007; Poschadel et al. 2009; Williams and Mendelson 2010; Kniel et al. 2015; Scherer et al. 2018a, b) and is important to control for females that either did not show interest in stimulus males, were frightened, and/or remained motionless in one corner of the experimental tank (Scherer et al. 2016, 2017b). The relatively high threshold of 80% was chosen to ensure that only females showing a strong side preference were excluded and to be in line with the existing literature (e.g. Hoysak and Godin 2007; Scherer et al. 2018a, b). To validate that the 80% threshold is biological meaningful, we tested whether side-biased females were less active, and thus more anxious, during mate choice trials compared to females not showing a side bias. We fitted a linear mixed-effects model (LMM) with female activity (total distance moved in cm, sum of both test periods) as the dependent variable and female side bias (yes or no) as predictor variable. We included female ID, treatment (level, level control, consistency, and consistency control), and female mate choice trial number as random terms. Side-biased females were significantly less active than females not exhibiting a strong side preference (χ21 = 19.887, P < 0.0001, estimate side-biased females ± SE = 2176.4 ± 124.9 cm, estimate not side-biased females ± SE = 2750.2 ± 159.1 cm; Ntrials = 192; N = 48 trials per treatment).
For each mate choice trial, the focal female was unfamiliar with the stimulus males (i.e. she had not seen them before). Stimulus males were used once in each treatment and not used more than once per day. We matched paired stimulus males for family (i.e. male pairs consisted of brothers), body size (standard length difference < 5%; mean ± SE = 0.216 ± 0.011 cm), natural aggression level (male difference in their distance to virtual opponent; mean ± SE = 1.42 ± 0.12 cm) and natural consistency of aggression (male difference in their consistency in distance to virtual opponent; mean ± SE = 1.559 ± 0.138 cm).
Experiment 2: female choice for male apparent boldness
In experiment 2 (February–April 2018), we tested for an effect of the apparent level (N = 60 preference assessments) and apparent consistency (N = 60 preference assessments) of male boldness on female preference. Before mate choice trials, we tested all males (N = 71) and females (N = 60) for their boldness level twice, with 48 h (± 15 min) elapsed between tests (see “Boldness test”). We tested for repeatability of boldness and for a sex difference in the level and consistency of behaviour (see “Data analyses”). We started mate choice trials three days after the boldness typing was completed.
Male and female boldness was assessed as activity under simulated predation risk (total distance moved in cm, hereafter: APR) using animated individuals of Parachanna obscura (N = 4, mean ± SE standard length = 19.3 ± 0.3 cm), a naturally occurring sympatric fish predator of P. pulcher (Scherer et al. 2017a, 2017b). Boldness tests and the subsequent calculation of the average level and inconsistency of behaviour were performed as described in the “Aggression test” in “Experiment 1: female choice for male apparent aggression”. Here, we used a 6-min test period and tracked individuals for 5 min (no tracking of the first minute). For all individuals, the boldness tests were carried out using a virtual predator specimen that focal fish had not seen before. Different to the above protocol, individuals were transferred to the test tanks without their housing pot. Further, we here aligned two observer tanks behind the test tanks (Fig. 2a, b). We included observer tanks in order to perform the boldness tests and the observation phase of mate choice trials under the exact same conditions, minimising effects that may interfere with our prediction of male behaviour exhibited during the observation phase of mate choice trials. During mate choice trials, observer tanks allowed test females to view the apparent boldness of stimulus males (see “Mate choice trials”). During the boldness test, we introduced opposite-sex observers, which were not further used in this experiment into the observer tanks. Observer conspicuousness was reduced using reflecting lighting (LED lights; I-SY-TL5P01; Soaiy Stick & Push Lamp; Shenzhen, China) and black plastic surrounding of the observer tanks (see Fig. 2a, b).
Mate choice trials
Similar to the above experiment 1, females could choose between two paired stimulus males after prior observation of apparent male behaviour (Fig. 2). That is, mate choice trials consisted of an observation phase and a subsequent choice phase (both observation and choice were divided into two test periods, see below). During the observation, paired stimulus males were manipulated to appear either shy or bold to an observer female by placing them in tanks of different ambient water temperatures, whilst viewing a virtual fish predator moving on a nearby computer screen (Fig. 2a, b). We used three different temperature treatments: low (21 °C), medium (25 °C) and high (29 °C) (for all treatments: range of ± 1 °C). We created an apparent difference in male level of APR by keeping one of the paired stimulus males in medium water temperature (apparent moderate APR) and the other one in either low water temperature (apparent low APR) (Fig. 2a) or high water temperature (apparent high APR) (Fig. 2b). To test female preference for apparent consistency of male boldness, we performed a second mate choice trial using the same pair of males (48 h between repeated tests, range of ± 15 min). During second mate choice trials, the apparent low-level (or high-level) male was now kept in high (or low) water temperature (apparent high APR) making it appear inconsistent, while the male being previously kept in medium water temperature was again concurrently presented in the same (medium) temperature treatment making it appear consistent.
For efficiency of time, we tested two females simultaneously for their mating preferences (Fig. 2). During the observation phase, each of the two females could only view one male at a time, we therefore divided the observation phase into two test periods (6 min each) with the female observer tanks being switched in their position in between the two test periods of an observation phase (Fig. 2a, b). This way, the two females could observe both males (in succession not simultaneously). All fish were allowed to acclimatise for 10 min before the first observation period and for another 2 min after female tanks were switched. During acclimatisation periods, the female observer tanks, the stimulus male tanks, and the computer monitor were visually separated from each other using removable opaque screens. Different to the above experiment 1, we did not change male treatments in between the two test periods of an observation phase of a single mate choice trial (Fig. 2a, b) (behavioural consistency was manipulated by performing a second mate choice trial, see above). The order in which males were presented during the observation phase did not affect female preference. To test this, we fitted a LMM to the data with female preference (see below) for the first male as dependent variable and included male ID, female ID, mate choice trial number (first or second), and male treatment temperature as random effects (no fixed effects included, aka null model, see below). There was no bias in female preference towards (or against) the male they saw first (intercept ± SE = 0.497 ± 0.000; 95% CI = [0.470, 0.526]; Ntrials = 120).
Similar to the mating preference test in experiment 1, the dichotomous choice test (where both stimulus males were presented simultaneously; Fig. 2c) was performed with two test periods of 11 min, with the males being switched in their position between test periods; initial acclimatisation was 10 min, and acclimatisation before the second test period was 5 min. During test periods, the tanks of the mate choice arena were physically separated (clear Plexiglas), whereas they were additionally visually separated using opaque screens during the acclimatisation periods. During the two test periods, males were kept in clear Plexiglas cylinders (diameter = 8 cm), positioned in the centre of their respective tanks, ensuring they remained visible to both females throughout the test phase and controlling for male activity. We assessed female preference and female side bias from the association time spent with the two males, as described for experiment 1. Side-biased females were excluded from preference analyses (excluded trials: Nlevel = 3, Nconsistency = 4).
Male treatment temperatures were induced in their individual housing tank 2 days prior to a mate choice trial (using submerged heaters), ensuring sufficient acclimation time (0.17 °C change/h) to the new temperature regime. Males did not show any signs of distress in response to temperature changes induced. To ensure that temperatures remained constant throughout experimental trials, all experimental tanks were covered externally with polystyrene (apart from tank sides needed to see through; see Fig. 1). The room temperature was set to 20.0 °C using air conditioning. The water temperature in the female tanks (housing and experimental tanks) was maintained at 25 ± 1 °C (equivalent to male medium temperature treatment). In order to avoid an effect of natural male behaviour on mate choice, male pairs were matched as closely as possible for natural inconsistency (mean ± SE; inconsistency = 193.28 ± 18.25 cm, within-pair difference in inconsistency = 107.44 ± 15.99 cm) and natural level (mean ± SE; average APR for all males = 684.21 ± 41.67 cm, within-pair difference of APR = 112.27 ± 18.93 cm) in APR. For male pair formation, we did not use the males showing the highest inconsistency values during boldness tests (N = 11) in order to efficiently manipulate male behaviour. Therefore, the number of males tested for boldness (Nmales = 71) was higher than the number of males used to form male pairs (Npairs = 30, Nmales = 60). Males were further matched for standard length as closely as possible (mean ± SE difference in standard length = 0.11 ± 0.02 cm) and for family.
Our manipulations during the observation phase were effective in manipulating behavioural inconsistency: the inconsistent male showed significantly higher inconsistency than the consistent male in a pair (see Online Resource 2 for statistical analysis and Online Resource 3 for graphical illustration). Further, apparently consistent and inconsistent males did not differ in their apparent level of APR (see Online Resource 2 for statistical analysis and Online Resource 3 for graphical illustration). For our manipulation of the behavioural level, we could confirm that males in the high temperature treatment showed higher APR compared to males in the medium temperature treatment. However, the low and medium temperature treatment males did not differ in their apparent level of APR. Therefore, we restricted the analysis of female preference for the apparent level to mate choice trials where males in the high vs. medium temperature treatment were presented (during the first mate choice trial); that is, all first trials containing low vs. medium temperature treatments were excluded (N = 30 preference assessments were excluded; resulting in N = 30 remaining preference assessments). Further, we removed all mate choice trials from the data set where the behavioural manipulation via ambient water temperature was not successful, i.e. in some mate choice trials, the apparently bold male showed a higher level of APR than the apparently shy male (excluded trials: N = 10 out of 30 trials; N = 20 remaining trials; total N after removing side biases is Nlevel = 17) or the apparently consistent male showed higher inconsistency than the apparently inconsistent male (excluded trials: N = 22 out of 60 trials; N = 38 remaining trials; total N after removing side biases is Nconsistency = 34).
The apparent level of male APR (assessed as outlined in “Boldness test”) exhibited during the first test period of the observation phase highly correlated with their apparent level during the second test period of the observation phase (mean ± SE APR; first test period: 963.346 ± 30.176 cm; second test period: 875.683 ± 26.046 cm). To test this, we performed an LMM with male APR of the first test period as dependent variable, male APR of the second test period as fixed effect, and male ID as well as mate choice trial number (first or second) as random terms: χ21 = 56.918, P < 0.0001, intercept ± SE = 333.278 ± 76.314 cm, coefficient ± SE = 0.720 ± 0.083 cm; N = 120 test periods of 60 mate choice trials. However, males were significantly more active (exhibited higher APR values) during the first test period of the observation phase compared to the second test period (possibly due to curiosity or excitement). To test this, we performed an LMM with male APR as dependent variable, test period (first or second) as fixed effect, and male ID and mate choice trial number (first or second) as random terms: χ21 = 7.086, P = 0.0008, intercept ± SE = 1051.01 cm, coefficient ± SE = −87.66 ± 32.61 cm; N = 240 test periods of 60 mate choice trials. Due to this behavioural difference, we did not use the average male APR over both test periods but kept these two scores of an observation phase separately for analyses.
We calculated male apparent inconsistency (absolute difference in apparent APR between first and second mate choice trial) from the female’s perspective. That is, we calculated two different scores of male apparent inconsistency, one score for each of the two females that saw the male. Each score was based on the very behaviour the female could observe (a female could only observe one male during the first test period and the other one during the second test period of an observation phase). For each male, we thus calculated one score of apparent inconsistency based on the two first observation phase test periods of each mate choice trial and the other score based on the second observation phase test periods (the order in which a female could observe a male was consistent between the two mate choice trials).