Male secondary sexual traits do not predict female preference in Caribbean livebearing fishes (Limia)

1 INTRODUCTION

Sexual selection is a mechanism of evolution in which fitness is determined by an individual's ability to secure matings. Mate choice is one of the primary drivers of sexual selection (Andersson, 1994; Rosenthal, 2017) and is typically defined as any behaviour or trait that leads to non-random mating (Edward, 2015). Pre-copulatory female preference is amongst one of the most well-studied and prevalent aspects of mate choice (Ryan & Keddy-Hector, 1992; Varela et al., 2018; Wilson et al., 2017). Although female preference is widely described, the underlying mechanisms that shape preferences are not fully understood, hence these are still a major focus of sexual selection studies (Candolin et al., 2007; Kirkpatrick & Ryan, 1991; Mitoyen et al., 2019). Our lack of knowledge is most apparent when examining species where males contribute little or nothing to the offspring beyond sperm to fertilize eggs (Achorn & Rosenthal, 2020).

In species where males contribute minimally to offspring, males more readily re-enter the breeding population after mating than females causing a male-biased operational sex ratio (OSR) (Ahnesjö, 1996). The abundance of males allows females to mate selectively, with female preferences selecting for elaborate male secondary sexual traits (Clutton-Brock, 2007). Often secondary sexual traits take the form of ornaments such as fluorescent colours, movement patterns and body size (Maynard-Smith and Harper, 2003). Although it is well-established male secondary sexual traits are commonly a product of female preference (Fisher et al., 2009; Mitoyen et al., 2019), the impact of male secondary sexual traits on female preference is less well-understood (Ptacek & Travis, 1997). Given that the intensity of male secondary sexual traits is often attributed to Fisherian runaway selection, whereby male SST become more exaggerated due to directional female choice, we hypothesized that female preference should scale with the intensity of male secondary sexual traits as well (Fisher, 1915).

We determined if female preferences scaled with male secondary sexual traits by comparing female preferences between three species of Limia. Limia is a genus of livebearing fishes in the family Poeciliidae, endemic to the Greater Antilles (Weaver et al., 2016). The species of Limia are closely related and share similar life histories (Cohen et al., 2015); however, they vary considerably in male sexual strategies (Farr, 1984). Specifically, species differ in their degree of ornamentation and the use of courtship displays. Here, we compared female preference between L. perugiae, L. dominicensis and L. zonata. The species serve as treatment groups: high, intermediate and low degrees of secondary sexual traits, respectively. Species degree of secondary sexual traits was based on ornamentation indices using dorsal fin size as described by Goldberg et al. (2019) (L. perugiae 0.36, L. dominicensis 0.09 and L. zonata −0.11) as well as the presence of courtship, which is only present in L. perugiae (Erbelding-Denk et al., 1994). These differences in mating strategies make them ideal for our comparative study.

However, comparing mating preferences between species presents challenges. The primary challenge being mate preference data gathered via mate choice designs (i.e. binary choice tests) cannot be directly compared between species (Wagner, 1998). We circumvented this problem by using absolute preference functions to measure preferences in lieu of binary choice tests. Preference functions describe the correlation between a phenotypic character expressed by males and the resource(s) expended by females (Lande, 1981). Here we elected to measure female preference for male body size in Limia because preference for large males is widely described in poeciliids and correlated with other male secondary sexual traits. For example, female guppies (Poecilia reticulata) prefer highly ornamented males (Houde & Endler, 1990), and swordtail (Xiphophorus pygmaeus) females show strong preferences for large male body size (Hankison & Morris, 2002; Morris, 1998). Moreover, other male secondary sexual traits, such as ornamentation and courtship, are known to scale with male size in poeciliids, including Limia (Farr, 1984). Therefore, by using male body size as an SST as well as a proxy for ornamentation, we were able to easily ascertain female preference in Limia.

Because we hypothesize female preference should scale with male secondary sexual characters, we predicted 1) L. perugiae females possessed the most pronounced preferences for male body size of the three species. 2) Limia zonata to display the least exaggerated preference, 3) whilst L. dominicensis showed an intermediate preference. However, we expected females of all species to show a preference for large males because of the purported indirect genetic benefits associated with mating with large males (Hankison & Morris, 2002; Morris, 1998). Support for the null hypotheses was determined as female preference being indistinguishable between species and a lack of preference for larger males. In addition to testing our hypothesis, our results provide some of the first descriptions of female preference in the Limia.

2 MATERIALS AND METHODS

2.2 Experimental setup

We used an absolute preference function assay to measure female preference in all three species. Assays were conducted in a 76-l tank that was divided lengthwise into three equal zones (Figure 1). Two vertical lines were drawn on the front pane of the tank to designate zones. The centre zone was deemed the neutral zone, with the two outer zones designated preference and non-preference zones. In each zone, we placed a clear Plexiglas tube (8.5 cm × 8.5 cm rectangular prism), which was used to restrict a male within its assigned zone and to reduce the transmission of mechanosensory and chemical signals (Figure 1). In the preference zone, one male fish was placed in a Plexiglas tube, and in the non-preference zone, there was no male present (i.e. an empty clear Plexiglas tube to control for any bias the female might show toward the tube). The preference and non-preference zones were randomized to control for side bias.

During absolute preference function assays, females of each species (N = 20 per species) were sequentially presented with three conspecific males of varying size (small, medium and large) in a randomized order. Females were randomly selected by ID number and placed in a clear Plexiglas tube in the centre of the neutral zone after her assigned male was first placed into the preference zone. Both individuals were then given 300 s to acclimate to the environment. To begin the trial, we released the female from the tube, and after she was swimming freely around the tank (i.e. showing no signs of stress-like behaviours), we recorded for 300 s the amount of time (s) the female spent in the preference or non-preference zone. Females were defined to be in the preference or non-preference zone if their entire head passed the vertical line on the front pane of the tank. After the 300 s passed, we gently placed the female back in the tube in the centre of the neutral zone, and the male was removed. Then a male of another size class was presented to the focal female. The process was repeated until the female was exposed to one male from each of the three size classes. Once a female was exposed to three males, she was returned to her home tank, and males were placed in size-specific recovery tanks labelled “used.” Males remained in recovery tanks for 3 days before haphazardly being used in another trial with a different conspecific female. After a trial, the testing tank received a 50% partial water change to reduce any lingering chemical signals from affecting future trials. All trials for any given species were conducted over the course of 30 days. Trials began in November 2017 and were concluded in January 2018.

3 RESULTS

The variance between species had the largest impact on the amount of time females associated with males (Table 1). Limia zonata, the low male SST species, associated with males more than both L. perugiae, the high male SST species (p = .03), and L. dominicensis, the intermediate male SST species (p = .0002; Figure 2). Species-specific linear mixed-effects models revealed L. perugiae females possess preference. Using a post hoc Tukey's test, we observed L. perugiae females prefer small males over both intermediates and large males (p = .02; p = .04; Figure 3a). Neither L. zonata nor L. dominicensis displayed a preference for male body size (p = .94; Figure 3b; p = .90; Figure 3c). In all three species, neither presentation order, female identity, nor female size significantly influenced female preference. The Bayes factor analysis revealed substantial evidence for L. perugiae's preference for small males (BF = 4.81), as well as anecdotal evidence for no relationship between female preference and male size in both L. dominicensis (BF = 0.51) and L. zonata (BF = 0.35).

4 DISCUSSION

When comparing female preference for male size, we expected female preference to scale with male secondary sexual traits. Female L. zonata associated with males more than the other two species; however, they did not demonstrate a preference for male size. Limia perugiae, the species with the most secondary sexual traits, was the only species to display female preference, providing support to our hypothesis that female preference should scale with male SST. However, L. perugiae females preferred small males, which tend to be minimally ornamented and rely on coercion to secure matings (Farr, 1984). Together with our results, this suggests that pre-copulatory female preferences are not maintaining male ornaments in these species (Figure 3).

The results of the present study may have been influenced by the design of our experiment. Absolute preference function assays introduce males sequentially to females and remove social information females may use to evaluate mates. However, we aimed to understand the preferences underlying female mating decisions, and by controlling for social information via sequential exposure of males, we removed the element of choice present in other methodologies. Our experiment would have benefited from allowing sensory cues other than only visual. Poeciliiids such as Poecilia chica and P. sphenops use chemical and tactile feedback, respectively, when assessing mates (Brett & Grosse, 1982; Schlupp et al., 2010). Although, the bulk of poeciliid studies have found very strong responses using only visual cues. Indeed, one study found that visual information was the strongest when comparing multiple sensory channels (Makowicz et al., 2016). Finally, live males introduce variation in individual stimuli which is difficult to control. For example, L. perugiae females' preference for small males may be indicative of an increase of reproductive effort on behalf of males via courtship in an environment where predation risk is minimal. Despite this possibility, studies using live individuals in preference assays have demonstrated live individuals do not compromise the integrity of preference studies (Fisher et al., 2009).

In the bigger picture, it is well-documented in livebearing fishes and beyond that females tend to prefer large male body size and heavily ornamented males (Bisazza & Marin, 1991; Fernandez & Bowser, 2010; Hughes, 1985; Plath et al., 2004). Moreover, females across many taxa show a consistent pattern of preference for larger mates (Ryan & Keddy-Hector, 1992). Females of a closely related species, also from Hispaniola, Limia nigrofasciata, show a clear preference for large males (Holz, 2015), which makes our result stand out as very unusual. The lack of pre-copulatory female preference for larger males within the Limia species studied here raises the important question: How are male ornaments seemingly uninvolved in female preference evolved and maintained? We offer two hypotheses for the maintenance of ornamentation in the absence of female pre-copulatory preference. First, females may exhibit cryptic preferences, as opposed to pre-copulatory choice, wherein females seemingly mate indiscriminately and bias male paternity towards preferred males via selecting sperm from preferred males. Secondly, male secondary sexual characters, like body size, could be the product of alternative selective forces or may not even be adaptive.

Cryptic female choice in livebearing fishes tends to be expressed as a bias towards the sperm of the preferred male being retained in the female reproductive tract (Firman et al., 2017). The higher retention of preferred male sperm leads to biases in offspring paternity toward preferred mates. This mating strategy is particularly effective in species where female pre-copulatory preferences are suppressed due to male harassment and forced copulations, like in many livebearing fishes (Plath et al., 2007). We hypothesize that cryptic female choice is a mechanism maintaining male secondary sexual traits in Limia. Initial work by Schartl et al. (1993) found that L. perugiae non-dominant males are more reproductively successful than dominants. However, it is inconclusive whether this is a product of cryptic female preference or male behaviour. Therefore, we suggest an experimental design that tests if male secondary sexual traits are positively correlated with a bias in sperm retention in the female reproductive tract in Limia.

Given the relatively recent diversification of Limia found on Hispaniola (Weaver et al., 2016), we would be remiss in not acknowledging that there are a variety of other evolutionary alternatives we are unable to mention here, which could result in sexual dimorphism in Limia (Bisazza, 1993). Our study assumed, as with many other taxa, that females were evaluating male secondary sexual characters. Therefore, male secondary sexual characters are maintained through mate choice. Our results suggest this may not be the case. Instead, the sexual dimorphism found in Limia could be a means of hybrid avoidance, male–male competition, or even a vestigial trait (Berglund et al., 1996; Liou & Price, 1994). Further study of female L. perugiae’s preference for small males, in particular, may shed light on the unusual preferences we find. It is worth-noting that the consideration of these alternative research opportunities would not be possible without the use of an animal system where there is limited knowledge. Future work in Limia provides an excellent opportunity to assess the ubiquity of the phenomena described in well-researched taxa.