Introduction

In a rapdily changing world, it has become increasingly more important for us to understand why some organisms are successful adapters and invaders and the mechanisms behind these. We know that ecological cues can drive the evolution of phenotypic traits, but more recently we have shown that this, in turn, changes the environment. Life history traits have been a focus in eco-evolutionary dynamics, but behavior, and especially reproductive behaviors, have been understudied. Exploring sexually selected traits and courting behaviors will help us understand how organisms are adapting at a indiviual level, and how cascading effects emerge at the population level and beyond.

Trinidadian guppies are a model organism for studying rapid evolution. Guppies exist in two types of natural populations based on predator presence. High predation (HP) sites exist at the bottom of streams where large predators such as Crenicichla frenata and Hoplias malabaricus exist. Low predation (LP) sites, where only Anablepsoides hartii (a competitor) and guppies exist upstream after a series of barrier waterfalls. Guppies have diverged in a number of traits, including sexually selected traits. HP males are not as colorful and do more sneak mating towards females in order to avoid the risk of being predated upon. LP males are colorful and perform sigmoid displays, which take up more energy and time. LP females tend to be more choosy, whereas HP females are not. These traits are highly plastic and can be both innate and learned. It follows that sexual selection should play a role in eco-evolutionary feedbacks.

In this study, we take a subset of data from a 2021 mesocosm experiment (Yang et al. 2023) and investigate the effect of predation presence on male reproductive behaviors. We hypothesize that predation affects courtship effort and courtship type. Specifically, the presence of a predator decreases courtship effort (measured by sum of mating attempts) and increases sneak mating attempts.

Load the data.

mesodata <- read_csv("~/Desktop/Meso21_Behavior/data/Meso2021_Behavior.csv") %>%
  mutate(Predation_regime = case_when(
    `M_Pop` == 'GHP' ~ 'HP',
    `M_Pop` == 'AHP' ~ 'HP',
    `M_Pop` == 'ALP' ~ 'LP',
    `M_Pop` == 'LoL' ~ 'LP')) %>%
  mutate(Combined_population = paste(M_Pop, F_Pop, sep = "_")) %>%
  mutate(cichlid = ifelse(cichlid == "Y", "Present", "Absent")) %>%
  mutate(M_total = Sigmoid+Sneak) %>%
  mutate(Prop_Sneak = (Sneak/(Sneak+Sigmoid))) %>% 
  mutate(
    Round = factor(Round),
    Mesocosm = factor(Mesocosm),
    Block = factor(Block),
    cichlid = factor(cichlid),
    Date = factor(Date),
    Predation_regime = factor(Predation_regime),
    Combined_population = (factor(Combined_population)))
## Rows: 288 Columns: 20
## ── Column specification ────────────────────────────────────────────────────────
## Delimiter: ","
## chr   (7): Mesocosm, cichlid, M_Pop, F_Pop, Period, Date, notes
## dbl  (10): Round, Block, Time_from_sunrise, Sigmoid, Sneak, Peck, M_total, F...
## time  (3): Time, Sunrise, Solar_noon
## 
## ℹ Use `spec()` to retrieve the full column specification for this data.
## ℹ Specify the column types or set `show_col_types = FALSE` to quiet this message.
round1 <- subset(mesodata, Round=="1")
Methods and Materials
Experimental Design

In March 2020, we collected wild guppies from the Guanapo (HP) and Lower Lalaja (LP) streams in the Aripo drainage in Trinidad. We brought them to the laboratory at Washington University and reared them in Aquaneering systems. They were raised to F2 generation and separated before sexual maturity to ensure that we used adult virgin guppies in our experiment. Guppies were marked with a Visible Implant Elastomer (VIE) unique tag that allowed us to keep track of individuals.

In June and July 2021, we set up mesocosms at Tyson Research Center, Washington University in St. Louis. We used a 2 x 2 factorial design with male origin (HP/LP) crossed with female origin (HP/LP). We were originally interested in how male mating strategy correlates with female foraging strategy, and if the two have coevolved. The methods and results of that study are published (Yang et al. 2023). Here we are using a subset of that data in order to examine the effects of predator presence on male mating strategies across male and female origins.

We use behavioral data taken from the initial round of the experiment conducted on days 5-6. During observations, we turned off water flow and allowed the fish to acclimate for 5 minutes before starting. We randomly selected a female and recorded how many times a male did a sigmoid display or sneak mating attempt towards her. Observers were blind to the population and treatment of the mesocosms.

Statistical Analysis

We first looked at if male mating effort (calculated as the sum of sigmoid displays and sneak mating attempts) differed due to male population origin. We use a negative binomial model with block and mesocosm as random effects and calculate the means.

model_total1a <- glmmTMB(M_total ~ M_Pop + (1|Block/Mesocosm), data = round1, family=nbinom2)

HP_total <- exp(1.9137)
LP_total <- HP_total * exp(0.7831)

HP_total
## [1] 6.778122
LP_total
## [1] 14.83219

We then looked at if this mating effort also differed due to differences from female origin.

model_total1b <- glmmTMB(M_total ~ Combined_population + (1|Block/Mesocosm), data = round1, family=nbinom2)
Table1<- kable(summary(model_total1b)$coefficients)
Table1
Estimate Std. Error z value Pr(>|z|)
(Intercept) 1.4768903 0.3006310 4.912635 0.0000009
Combined_populationGHP_LoL 1.1221514 0.4391567 2.555241 0.0106114
Combined_populationLoL_GHP 0.9432766 0.4418320 2.134922 0.0327674
Combined_populationLoL_LoL 1.3789868 0.4007409 3.441094 0.0005794

Finally, we tested if the presence of a cichlid changes mating effort between HP and LP males.

model_sneak <- glmmTMB(cbind(Sneak,Sigmoid) ~ Predation_regime*cichlid + (1|Block/Mesocosm), data = round1, family=binomial)

Table3<- kable(summary(model_sneak)$coefficients)
Table3
Estimate Std. Error z value Pr(>|z|)
(Intercept) 0.2171750 0.2819637 0.7702233 0.4411674
Predation_regimeLP 0.8739466 0.3569613 2.4482949 0.0143534
cichlidPresent 1.1320489 0.6605880 1.7136989 0.0865840
Predation_regimeLP:cichlidPresent -0.1011053 0.8183074 -0.1235541 0.9016683
Figures
Figure1a <- ggplot(round1, aes(x=M_Pop, y=(M_total), fill = M_Pop)) + geom_boxplot()
Figure1a +
  labs(subtitle="Figure 1a. Male origin on male mating effort",
       x="Male origin", y="Male mating effort") + 
  theme_classic()

Figure1b <- ggplot(round1, aes(x=F_Pop, y=(M_total), fill = M_Pop)) + geom_boxplot()
Figure1b +
  scale_fill_manual(values=c("#355834", "#E9B872")) +
  theme(axis.title.y = element_blank()) +
  labs(subtitle="Figure 1b. Female origin on male mating effort",
       caption="Both HP and LP males put more effort into mating to LP females.", 
       x="Female population", y="Male mating effort") + 
  theme_classic()

Figure2 <- ggplot(round1, aes(x=M_Pop, y=Prop_Sneak, fill=cichlid)) + geom_boxplot(width=0.4, position=position_dodge(0.5), alpha = 0.7)
Figure2 + 
  scale_fill_manual(values=c("#9DD1F1", "#133C55")) +
  scale_x_discrete(labels=c('HP','LP')) + 
  theme(plot.caption=element_text("plot", size=2)) +
  labs(subtitle="Figure 2. Predator presence on proportion of HP/LP sneak mating",
       x="Male population", 
       y="Proportion of sneak mating") + 
  theme_classic()
## Warning: Removed 17 rows containing non-finite outside the scale range
## (`stat_boxplot()`).

Results and Discussion

(1a and 1b) LP males do 2.1882453 times more mating attempts (sigmoid or sneak) than HP males in the absence of a predator (Figure 1). Mesocosms with males and females from HP populations exhibited the least male mating effort (p<0.01). This is contrasted by mesocosms with males and females from LP populations, who exhibited the most male mating effort (p<0.01). Mesocosms with HP males and LP females or LP males and HP females exhibited estimates in between (p<0.01).

This makes sense - LP guppies do not have to worry about predation and thus spend more time doing other behaviors such as foraging and reproducing. As for the mixed populations, it is interesting to note that HP males with LP females put more effort into mating than LP males and HP females. Overall, both HP and LP males put more effort into LP females (Figure 1b). One idea as to why these slight differences may matter is female receptivity. LP females tend to be more receptive (or choosy) towards male courtship. when females are receptive, males are invited to attempt more.

(2.) We looked at the proportion of sneak vs sigmoid displays on predation regime and predation and their interaction. LP males exhibited greater sneak mating attempts (Figure 2). Results due to HP males, the presence of the cichlid, and the interaction between LP males and cichlid presence was not significant (Table 3).

Contrary to our predictions, LP males did more sneak mating attempts than HP males in the absence of a predator. This could be a within-stream difference. Perhaps increased amounts of competition due to high densities in LP sites forces males to be more conservative with their energy expenditures. In other words, resource or energy constraints may also contribute to reproductive behaviors. However, what may be more surprising is that there was no difference in mating styles in both populations when a cichlid was present. This may have been expected for HP males, but not LP males. Additional experiments will be needed to test this.