Casanovas are liars: behavioral syndromes, sperm competition risk, and the evolution of deceptive male mating behavior in live-bearing fishes

Study organisms and their maintenance

The experiments reported here comply with the current laws of Germany (approved by Regierungspräsidium Darmstadt V-54-19c-20/15-F104/Anz.18) and the USA (approved by the Institutional Animal Care and Use Committee of the University of Oklahoma; AUS- IACUC approved protocols: R06-026 and R09-023).

Test subjects were lab-reared descendants of wild-caught fish. We included Atlantic mollies from the coastal lagoons around the Central Mexican city of Tampico (belonging to the subspecies P. mexicana limantouri); another population was collected in the Río Oxolotan in Tabasco, South México (P. mexicana mexicana). Recent phylogenetic analyses argue in favor of full species status of the two subspecies⁴⁵. We further included a locally adapted and genetically differentiated (i.e., independently evolving) ecotype from the P. mexicana mexicana clade: the hydrogen sulfide-adapted form inhabiting El Azufre, a tributary to the Río Oxolotan^46,47. As another representative of short-fin mollies^48,49 we included mangrove mollies (P. orri) from Roatán Island, Honduras. Two species of long-fin mollies were tested: sailfin mollies (P. latipinna) stemmed from the Comal River in Central Texas, USA, while Tamési mollies (P. latipunctata) were collected near Ciudad Mante in Tamaulipas, México. We further included guppies (P. reticulata) from Venezuela and a feral population from the San Antonio River, Texas, USA⁵⁰, as well as Venezuelan swamp guppies (P. picta). As representatives of the genus Limia, we included L. tridens and sulfur limia (L. sulphurophilia), both originating from the Dominican Republic. Gambusia sexradiatafrom the Río Teapa, and Grijalva mosquitofish (Heterophallus milleri) from the Río Oxolotán (both Tabasco, México) were included as representatives of mosquito fishes.

Test fish came from large, randomly outbred single-species stocks maintained at the Department of Ecology and Evolution of the University of Frankfurt (P. m. mexicana, P. m. limantouri, P. reticulata from Venezuela, P. picta, L. tridens), or at the Department of Biology at the University of Oklahoma in Norman (P. m. mexicana from El Azufre, P. latipinna, P. latipunctata, P. orri, feral P. reticulata, L. sulphurophila, G. sexradiata, H. milleri; Table 1). Fishes were reared as single-species, mixed-sex stocks in 200-l (Frankfurt) or 1,000-l (Norman) tanks at 25–27°C under an 12:12 hours light:dark cycle (Frankfurt) or under ambient light conditions in a greenhouse (Norman). At the University of Frankfurt, fishes were fed twice daily ad libitum with commercial flake food. Stock tanks in Norman contained naturally growing algae as well as a variety of naturally occurring invertebrates such as chironomid larvae, copepods and amphipods, on which the fishes could feed. In addition, fishes were supplied with flake food every two days. However, at least 1 week prior to the behavioral experiments, fishes were fed ad libitum at least once daily with flake food.

Aggressive behavior

We determined male aggressive behaviors during dyadic encounters by analyzing contests staged between pairs of males in a small test tank measuring 30 × 20 × 20 cm⁵¹. To avoid confounding effects of previously established dominance and/or familiarity^52,53, males were taken from different stock tanks. Males in a dyad differed by less than 15% in standard length (SL), which has previously been established as the threshold below which fights typically escalate⁵¹; nevertheless, size difference was included as a covariate in the statistical analyses (see below). We separated males by an opaque filter sponge while three sides of the test tank were taped with gray paper to minimize disturbances from the outside. The bottom of the tank was filled with black gravel, and water was aerated and maintained at 27–29°C. Males could habituate to the test tank overnight, and observations took place the next day between 09:00 and 13:00. To initiate a trial, the sponge divider was gently lifted, and we noted behavioral interactions for a maximum of 10 minutes, starting with the first interaction. We focused on three frequent aggressive behaviors⁵⁴: (1) S-position: this threat display usually initiates a fight. Males swim in a parallel or anti-parallel position and bend their bodies in an S-shaped manner with all unpaired fins erect; (2) tail-beats: S-positions are often followed or superimposed by tail-beats, which are fast movements of head and tail in opposing directions that either touch the opponent’s body or send shock waves to the opponent; and (3) bites: we defined all incidences of ramming and bite-like attacks⁵² as bites, because both these behaviors occur extremely quickly and thus are indistinguishable to the human eye. For some species examined in this study no formal description of aggressive behavior was available from the literature, and so we confirmed in pre-trials that the aforementioned behaviors are part of their behavioral repertoire.

We also recorded fight duration until dominance was established. Contest outcome could be inferred from behavioral differences between the contestants. Folded fins, head-down posture and a position at the periphery of the tank typically characterize contest losers, while winners constantly chase and further attack the loser with fins fully erect, occasionally performing S-positions or bites⁵¹. We met all requirements for animal well-being in behavioral experiments. Apart from occasional loss of single scales, no severe injuries were observed, as we separated males immediately once dominance was established. If no dominance was established within 10 minutes of the first interaction, we terminated the fight; those trials were discarded from the analysis of fighting durations, while fight durations were scored as “0” when no aggressive behavior occurred at all (those trials were terminated after a total of 15 minutes of observation). SL of both contestants was taken after a contest by laying the fish flat on plastic foil-covered millimeter paper (Table 1). Afterwards we transferred males back to their respective stock tanks. In total, we successfully completed N = 146 trials (Table 1).

Male mate choice

We reanalyzed previously published data on audience-induced changes in male mate choice (Table 1). Focal males were isolated in 25- to 38-l tanks for two to four days prior to the tests to ensure that they were motivated to mate¹². We tested each focal male only once; however, owing to the limited number of males available from our stocks, some males were also used as audience males after they had served as a focal male, but never on the same day and not in the same dyadic constellation. As familiarity among males affects the strength of audience effects in P. mexicana⁹, focal and audience males were taken from different stock tanks.

Each focal male was tested for its mating preference in a binary choice situation and was then retested with the same stimulus females either without audience (control treatment) or with an audience male present (50% of trials each). We were thus able to examine changes in focal males’ behavior from the first to the second part of the tests and could discern between effects induced by the audience and changes that would occur over the course of the experiment even without audience. In theory, we could have used an alternative design of presenting an audience in all trials while starting the tests with or without audience in alternating order; however, in such a design, prior exposure to the audience male (when presented during the first part) could still affect the focal males’ behavior during the second part of the tests⁵⁵.

The test tank (50 × 30 × 30 cm, length × width × height) was filled to 20 cm height with aged tap water. Water temperature was maintained at 27–28°C using an aquarium heater. In addition, the water was aerated between trials, but both the heater and the air-stone were removed for all trials. Black plastic covered all sides except the front. Prior to the tests, we choose two different-sized stimulus females (for SL see Table 1) from a stock tank and introduced them into the test tank. Poeciliid males prefer to mate with larger, more fecund females (e.g.^8,56–58, but see Ala-Honkola et al.⁵⁹). Afterwards, we introduced a focal male into a transparent Plexiglas cylinder (10 cm diameter) located in the center of the tank and left the fish undisturbed for 5 minutes. After the habituation period, we gently lifted the cylinder. During a 10-min observation period, we scored male sexual behaviors directed toward either of the two females and noted with which female the focal male interacted first. We decided a priori to terminate trials if the male did not show any sexual behavior during the first part of the test; N = 3 trials with P. orri, N = 5 (P. latipinna), N = 2 (P. latipunctata), N = 4 (P. reticulata, Venezuela), N = 1 (P. picta), N = 1 (P. reticulata, San Antonio), and N = 6 (H. milleri) were discarded from the statistical analyses based on this criterion.

Genital nipping is a typical pre-copulatory behavior in poeciliids, whereby the male approaches the female from behind and touches her genital region with his snout^54,60. During thrusting, males swing their gonopodium forward while attempting to introduce it into the female’s gonopore. Courtship behavior is absent in P. mexicana⁶⁰, P. orri, the examined Limia species (authors, personal observation) and Gambusia spp. (⁶¹ for G. holbrooki). Poecilia reticulata males court in front of females in an S-shaped body posture (sigmoid displays^62,63), while the primary courtship display of P. picta males consists of circling around the female (the so-called ‘orbit’^54,63), but males also court with their fins raised in front of the female (⁶³; D.B., personal observation). Heterophallus milleri males circle around the female and swing their gonopodium forward when in the female’s visual field⁵⁸. Large P. latipinna and P. latipunctata males occasionally court in front of females with raised dorsal fins^54,64. As not all species examined herein show courtship displays and courtship was by far the least frequent behavioral category, we excluded numbers of courtship displays from our main analyses.

Upon completion of the first preference test, we immediately repeated measurement of male mating preferences, but in one half of the trials, an audience male was presented, while the other half of the trials was repeated without audience (control). To initiate this second part of a trial, we reintroduced the focal male into the acclimatization cylinder. An audience male was placed in another transparent cylinder in the central back of the tank, while for the control only an empty cylinder was presented. The audience male was confined in his cylinder throughout the test. After another 5 minutes of habituation (during which all four fish could interact visually), measurement of male preferences was repeated, as described above. Interactions between males were not quantified, but aggressive displays were not observed. In total, we successfully completed N = 408 trials (Table 1). Once a trial was completed, all fish were measured for SL to the closest millimeter (Table 1).

Aggressive behavior

We tested for variation and compared the magnitude of differences in male aggressiveness across species. First, we employed Principal Component Analysis (PCA) to reduce the number of dependent variables (numbers of S-positions, tail-beats and bites per male dyad) and extracted one independent component (PC1; eigenvalue = 2.47) that explained 82.3% of the variance. The three aggressive behaviors had axis loadings of 0.85 (S-positions), 0.93 (tail-beats) and 0.94 (bites). PC1 was checked for normal distribution using a Kolmogorov-Smirnov test and used as a dependent variable in a univariate General Linear Model (GLM) with ‘species’ as a fixed factor. We included ‘mean male SL of a dyad’ (nested within species) as a covariate because larger males tend to be more aggressive⁵¹. Moreover, the opponents’ body size difference influences fight intensity⁵¹, and so we included arcsine (square root)-transformed body size difference (SL_small/SL_large) as another covariate as well as its interaction with the fixed factor in our final model. If a covariate had a significant effect, we employed Pearson’s correlation on unstandardized residuals to explore the direction of the effect.

Fight durations were analyzed in a separate univariate GLM with the same factor and covariate structure. Both covariates and the interaction term, however, were removed from the final model since none was significant (‘mean male SL of a dyad’: F_13,67 = 1.62, P = 0.24; ‘opponent body size difference’: F_1,80 = 2.84, P = 0.09; ‘opponent body size difference × species’: F_12,55 = 1.04, P = 0.37).

Male sexual behavior

As described for the analysis of aggressive behavior, we first used PCA to condense sexual behavior (genital nipping and thrusting) to one principle component (PC1, eigenvalue = 1.79) that explained 89.7% of the variance. Both variables had equal axis loadings of 0.95. We used PC1 as dependent variable in a univariate GLM and included ‘species’ as a fixed factor. Small males show more sexual behaviors than larger ones in at least some of the species examined here as part of a ‘sneak-like’ alternative mating strategy⁶⁵, and so we included focal males’ SL as a covariate (nested within species). Also, poeciliid males typically prefer to mate with large females (see above), and we thus included the mean SL of each stimulus female dyad (nested within species) as another covariate. However, both covariates had no significant effect (‘focal male SL’: F_13,382 = 1.22, P = 0.26; ‘mean stimulus SL’: F_13,369= 0.97, P = 0.48) and were removed from the final model.

Audience-induced changes in preference expression

To compare the magnitude of audience-induced changes in individual male mate choice behavior across species, we calculated a preference score⁴¹ as:

(fraction of sexual behaviors with the initially preferred female during the second part of a trial) – (fraction of sexual behaviors with the same female during the first part),

such that negative values would indicate that individual preferences decreased. Scores were included as the dependent variable in a univariate GLM with ‘species’ and ‘treatment’ (with or without audience) as fixed factors. Beside ‘focal male SL’ (nested within species), we also included ‘stimulus female SL difference’ [arcsine (square root)-transformed SL_small/SL_large] since one could predict that males would show stronger audience effects the larger the size difference, as large females represent high-quality mates. All possible interactions were included in the initial model. However, neither the covariates themselves (‘focal male SL’: F_13,369 = 0.48, P = 0.93; ‘stimulus female size difference’: F_1,380 = 0.93, P = 0.34) nor their interaction terms were significant (P > 0.22 in all cases) and were removed from the final model.

Qualitatively, H. milleri did not follow the pattern of reduced preference expression shown by other poeciliids (see also Bierbach et al.⁵⁸), so we re-ran all analyses while excluding H. milleri, but the direction of the results was not affected (results not shown).

Deceptive mating behavior

In the context of deceptive mating behavior, the first sexual approach of focal males is of interest, as interacting first with the previously non-preferred female has been interpreted as an attempt to mislead the rival⁴¹. Thus, we analyzed the fraction of males that first interacted with the opposite (“1”) or same female during the second part (“0”) using a binary logistic regression, with ‘species’, ‘treatment’ and their interaction term as categorical independent variables. We also included ‘focal male SL’ and ‘female SL difference’ as covariates and used a step-wise backwards elimination approach, based on likelihood ratios, to remove effects if P > 0.1.

Correlations of behavioral types at the species level

The central question of our present paper was whether there are correlations between the four aforementioned behaviors at the species level. Owing to the limited sample size (N = 13 groups), we used non-parametric, pair-wise Spearman rank order tests to correlate species means for (1) aggressiveness (sum of all aggressive interactions per fight), (2) sexual activity (sum of nipping and thrusting behavior during the first part of the tests), (3) the strength of changes in preference expression in male mate choice (score from audience treatment minus mean score from control treatment) and (4) the occurrence of deceptive behavior (fraction of males that changed their first interaction in the audience treatment – fraction in the control treatment). To avoid error inflation due to multiple comparisons, we used Bonferroni corrections and inferred statistical significance only if P < α = 0.017.

Male aggressive behavior

There was pronounced variation in aggressive behavior among species (for univariate GLM, see Table 2a; Figure 1a). Both covariates (‘mean male dyad SL’ and ‘opponent body size difference’) had significant effects (Table 2a), but species-wise post hoc Pearson correlations confirmed the predicted pattern of larger males fighting more intensely only in P. m. mexicana (sulfide ecotype, see Table 3). Only in the highly aggressive Atlantic mollies (P. m. mexicana, both ecotypes, and P. m. limantouri; Figure 1) was the predicted negative relationship between opponent size difference and mean aggressiveness uncovered (Table 3), i.e., the larger the size difference the less intense fights became.

Figure 1.

Means (+ SE) of (a) numbers of aggressive interactions per male fight and (b) fight duration in the different poeciliid species examined.

The GLM on mean fight durations also detected significant species differences (Table 2b; Figure 1b).

Male sexual behavior

GLM detected a significant effect of the factor ‘species’ (Table 2c), indicating that male sexual activity also showed pronounced variation among species (Figure 2a).

Figure 2.

(a) Mean (+ SE) numbers of male sexual behaviors during a 10 min observation period. (b) Changes in individual focal males’ mate choice behavior in the presence of an audience male. Depicted are mean (+ SE) preference scores (see main text), whereby negative values indicate that male preferences decreased in strength. (c) Proportion of males that first interacted with the opposite female when released from the acclimation cylinder in the second part of the tests. Open bars in (b) and (c) represent the control treatment (no audience) while gray bars represent the audience treatment.

Audience-induced changes in preference expression

When comparing the change in individual males’ mating preferences from the first to second part of the tests (preference score), we detected a significant main effect of the factor ‘treatment’, suggesting that focal males responded to an audience male with altered mate choice behavior (Table 2d). No significant effect of the interaction term ‘species by treatment’ was uncovered (Table 2c), suggesting that species did not overall differ in their response to the audience treatment (Figure 2b). A significant effect of the main factor ‘species’ (Table 2c), by contrast, can be interpreted as species differing in the consistency of their mate choice over the course of the experiment (note that this main effect considers changes in both the audience and control treatments).

Deceptive mating behavior

When comparing the number of trials in which the focal males first approached the same (“0”) or different (“1”) female during the second part using logistic regression, the interaction term of ‘species by treatment’ was excluded already during the first step of the step-wise elimination process (B = -0.031, SE = 0.06, Wald = 0.233, df = 1, P = 0.629), indicating that male responses to the audience treatment did not differ among species (Figure 2c). In the final model, only the factors ‘treatment’ (B = 1.39, SE = 0.27, Wald = 34.76, df = 1, P < 0.001) and ‘female body size difference’ (not significant: B = 1.49, SE = 0.83, Wald = 3.27, df = 1, P = 0.071) were retained. Thus, focal males were more likely to change their initial interaction when an audience male was presented (Figure 2c).

Correlations of behavioral types at the species level

In line with our prediction derived from the interpretation that SCR explains the occurrence of audience-induced behavioral changes, we found a strong, positive correlation between sexual activity and the amount of deceptive behavior at the species level (Figure 3e). The alternative prediction, that avoidance of aggressive behavior drives audience effects (leading to a positive correlation between both variables), received no support, as the correlation between mean aggression and strength of audience-induced changes in preference expression even yielded a negative correlation coefficient (not statistically significant; Figure 3b). Finally, there was a significant positive correlation between aggression and sexual activity (Figure 3a).

Figure 3. Correlations between species-level means (± SE) of male aggressiveness (log scale), sexual activity (log scale), the strength of audience-induced changes in preference expression, and the level of deceptive mating behavior.

Shown are results from Spearman’s rank order tests; α’ indicates the alpha error level after Bonferroni correction.