Larger is not better: No mate preference by European Common Frog (Rana temporaria) males

According to classical sexual selection theory, females are the choosy sex in most species. Choosiness is defined as the individual effort to invest energy and time to assess potential mates. In explosive breeding anurans, high intrasexual competition between males leads to a sexual coercion ruled mating system, where males could have evolved preferences for specific female traits. In the current study, we tested male mating preference in the explosive breeding European Common Frog without intrasexual competition. We hypothesized that males show preferences towards larger female body size in the absence of male competition. We conducted mate choice experiments, placing a male and two differently sized females in a box and recorded their mating behavior. Males did not show any preference considering female body size, neither in the attempt to grab a female nor during the formation of pairs. We witnessed a high failure rate of male mating attempts, which might make the evolution of mate choice too costly. However, small males are faster in attempting females, which could be an alternative strategy to get access to females, because their larger competitors have an advantage during scramble competition. Nonetheless, in successfully formed pairs, the females were on average larger than the males, an observation which deviated from our null-model where pairs should be of similar size if mating would be random. This indicates that selection takes place, independent from male mating preference or scramble competition.


Introduction 26
Research on sexual selection, exploring the mechanisms that lead to female/male mate choice and the evolution of 27 different mating systems that facilitate non-random mating, has increased considerably in recent years (Janetos 1980; 28 Ryan and Keddy-Hector 1992; Paul 2002; Edward and Chapman 2011). Studies addressing the theory of sexual 29 selection revealed that females are the choosy sex in most species. This is mainly based on one assumption, the 30 evolution of anisogamy, where males produce many small (cheap) gametes and females less but larger (expensive) 31 gametes. Thus, females invest more energy in the production of eggs than males invest in the production of sperm 32 (Trivers 1972). In consequence, reproduction is more costly to females and they should choose the 'fittest' male to 33 mate with. This includes those with the best possible genes to improve her offspring's fitness and/or those who can 34 provide vital resources (e.g. territory, nesting place, food, parental care) to increase offspring survivability and 35 attractiveness, thereby increasing the female´s personal fitness (Fisher 1958;Hedrick 1988; Møller and Alatalo 36 1999). Here, choosiness is defined as an individual's active effort to invest energy and time to assess potential mates, 37 whereas preference is defined as an intrinsic, passive attractiveness towards specific traits of the opposite sex 38 Preferences can enhance the evolution of different mating strategies and tactics to increase reproductive output with 41 behavioral plasticity; depending on sex, age, physiological state or operational sex ratio (Parker 1982;Gross 1996). 42 Nevertheless, newer studies suggest that males can be choosy too, if mate availability is high and simultaneous 43 sampling possible (Barry and Kokko 2010), if there is variation in female quality/fecundity (Krupa 1995;Johnstone 44 et al. 1996), and if the benefits of choosing between females is higher then the costs associated with assessing 45 females (Edward and Chapmann 2011, and references therein). Some prerequisites are the presence of males' ability 46 to detect differences and a preference for particular female traits. Body size can be such a trait, i.e. indicating 47 longevity based on good genes which could be heritable (Kokko and Lindström 1996;Møller and Alatalo 1999). 48 However, body size usually is based on a variety of genes and environmental processes, but might simply indicate 49 higher fecundity (Peters 1986;Shine 1988;Nali et al. 2014). Mating with a larger female thus may increase a male's 50 4 competition. On the other hand, costs associated with mate choice depend on male density and the frequency of 78 different mating tactics within a breeding aggregation (Arak 1983;Höglund and Robertson 1988), as well as for 79 instance male's individual predation risk (Magnhagen 1991;Bernal et al. 2007), all factors which may vary already 80 during a short breeding season (Olson et al. 1986;Vojar et al. 2015). 81 In this study, we investigate the mating preference of the European Common Frog (Rana temporaria) because it is an 82 excellent example of an explosive breeder with male-male competition. Although former studies suggest a lack of 83 male mate preferences in this species (Elmberg 1991), we observed non-random mating by body size and found 84 indications of male mate preference and different mating tactics in former experiments (Dittrich et al. 2018). Larger 85 females were paired more frequently than smaller ones and smaller sized males showed a different mating tactic to 86 get access to females (Dittrich et al. 2018). Here, we hypothesize that all males will prefer larger females 87 independent of their own body size, when intrasexual competition is absent and males are presented to differently 88 sized females. Additionally, we predict small males to be faster in attempting a female to increase their chances to 89 keep an exclusive access to the female during scramble competition. 90

Study area and species 93
The European Common Frog, Rana temporaria Linnaeus, 1758, is an explosive breeder that forms dense breeding 94 aggregations in early spring (Gollmann et al. 2014). The males engage in scramble competition over temporally 95 available receptive females (Savage 1961). Here, larger males have an advantage in direct combat (Arak 1983), and 96 small males may apply a different mating tactic by being faster in amplexing females (Dittrich et al. 2018 for reproduction annually. In 2019 we fenced the four ponds with the largest known breeding aggregations for the 102 entire reproductive period (14 th to 28 th March). The fence consisted of plastic gauze (mesh size 2 mm, approx. height 103 60 cm) stretched between wooden poles and was monitored twice a day (morning and evening). 104 We collected individuals that sat at the fence or were on their way to the breeding pond, preferably collecting singles 105 to minimize differences in reproductive status. Amplected females could potentially be affected by the application of 106 amplexin, which was found in gland tissue under the nuptial pads of male R. temporaria. This is a protein similar to 107 the plethodontid modulating factor, a pheromone that influences courtship duration in salamanders (Willaert et al. 108 2013). So far, it is unknown if male R. temporaria are able to detect differences in the female's reproductive status 109 (Thomas 2011). We thus always took note if individuals were encountered as singles or in pairs and tested if the 110 former status influences preference behavior of males. 111 All individuals were sexed in-situ (males show characteristic dark nuptial pads during the reproductive period). We 112 measured snout-vent length (SVL in mm) using a caliper (to the closest 0.5 mm), and mass using a spring scale (1 -113 100 g, 1 g increments). For transport, we placed each individual singly in an opaque, 1 L volume plastic bucket with 114 lid, which contained leaf litter to hide and a thin layer of water to prevent desiccation. The animals were kept in these 115 buckets in the barn of the ecological field station in Fabrikschleichach (temperatures only marginally higher than at 116 the breeding sites) until the start of the behavioral experiments, which started after a maximum of 12 hours after 117 collection. Although this handling could cause stress, studies in Cane Toads, Rhinella marina, showed that stress 118 levels will decrease after 8 hours and with low temperatures (Narayan et al. 2012a(Narayan et al. , 2012b. All individuals were 119 released at their respective capture locations after completion of the experiments. 120

Behavioral experiments 121
We tested the hypothesis that males prefer the largest female in the absence of intrasexual competition with a mate 122 choice test, by placing two females of different body sizes in the same container with a single small/large male. The 123 size difference between females in each trial exceeded 10 mm, with small females SVL being below 70 mm (n = 48, 124 range: 48 -70 mm, mean ± SD: 63.0 ± 5.7 mm), and large females SVL over 71 mm (n = 48, range: 71 -89 mm, 125 mean ± SD: 77.3 ± 4.4 mm). In the containers, either a small male (n = 23, SVL range: 56 -70 mm, mean ± SD: 126 63.8 ± 4.5 mm) or a large male (n = 25, SVL range: 71 -89 mm, mean ± SD: 76.6 ± 5.5 mm) were introduced. The 127 allocation of individuals in the experiment was random, except the premisses of a minimum of 10 mm size difference 128 between the females. The experiments were conducted in plastic containers (40 x 60 x 40 cm), filled with 10 L of 129 rainwater (5 cm water depth). The species can form amplexus on the migration towards the pond in the terrestrial 130 habitat, as well as within the aquatic habitat in the pond. The 5 cm water depth are imitating the edges of the pond 131 were clutches are usually laid. Before starting the experiment, a non-transparent plastic sheet separated the male 132 from both females. We let the animals acclimatize in the container for 10 min, then removed the plastic sheet and 133 started the experiment. A web camera (Logitech C920) placed at 1.5 m height above the plastic containers recorded 134 each experiment for one hour, even if amplexus was formed earlier. 135 Before starting a new experimental run, we cleaned the respective container and changed the water completely to 136 minimize the risk of potential effects from residual chemical cues. Each animal was tested only once. If successful 137 amplexus did not occur within the one hour experimental time, the trial was terminated. In none of the experiments 138 spawning occurred. 139 We defined several variables that were recorded and analyzed: when and towards which female the male attempted 140 to clasp first, the number of successful and failed clasping attempts on each female, and with which female 141 successful amplexus occurred at the end of the experiment. The term attempt is defined by actively approaching a 142 female and trying to clasp her, it does not apply if animals are randomly bumping into each other. 143 We used χ 2 tests to analyze male mate preferences. First, we tested if males approaches towards the small/large 144 female were random and second, if a successful amplexus with a small/large female was random. Additionally, we 145 tested size-assortment in the pairs with a Pearson correlation. To account for the experimental design -males can 146 only choose from two females, thereby limiting choice -we run a bootstrapped null-model with 1000 iterations of 147 random choice for one of the females in the experimental pairs. For those random pairs we calculated the Pearson 148 correlation coefficients and plotted them in a histogram. We than calculated the z-score for the Pearson correlation of 149 the observed pairs with the mean and standard deviation of the bootstrapped null-model. The same approach was 150 used to investigate the body size difference between the approached females and successfully formed pairs. The size 151 difference between the male and a randomly chosen females in the experimental run was calculated with 1000 152 iterations and plotted as the distribution of the null-model. We calculated the mean size difference for attempted 153 pairs and for pairs in amplexus. The observed data were compared to the mean and standard deviation of the null-7 model. The deviation from the null-model was significant when the z-score of observed values was above 1.96. For 155 all analysis and graphs we used the R statistical environment (R Core Team 2020, version 3.6.3). We used the 156 packages effsize (Torchiano 2019) to calculate Cohen's D, ggplot2 (Wickham 2009) for drawing graphs and plyr to 157 count number of occurrences (Wickham 2011). 158

Results 159
First, we tested if female and male status (caught as a single or in amplexus) had an influence on amplexus during 160 the experimental runs. The proportion of females being in amplexus was not significantly different between females 161 caught as single or paired (χ 2 = 0.05, df = 1, p = 0.83). The same was observed for the males (χ 2 = 0.17, df = 1, p = 162 0.68). Therefore, we pooled the data. 163 In five experiments, males failed to grasp a female because she swam or jumped away. Second, in nine cases, males 167 were successful in amplexing a female, but females showed avoidance behaviors and escaped the male grip (unp. 168 data). Third, in two cases the males did not even tried to grab a female and showed no interest at all. 169 The proportion of females in amplexus was not significantly different between small (n = 13) and large females (n = 170 19, binomial test, p = 0.38). The proportion of males in amplexus was not significantly different between small (n = 171 15) and large males (n = 17, binomial test, p = 0.49). There was no indication for a preference of any female body 172 size category by either small or large males (χ 2 = 0.18, df = 1, p = 0.67; Table 1). Additionally, during their first 173 approach large and small males did not show any preference for a specific female body size category and tried to 174 clasp large and small females almost equally (χ 2 = 0.01, df = 1, p = 0.96; Table 1). A total of 31 males approached 175 both females during the experiment (65%), 15 approached only one of the females (31%), and two males did not 176 show any interest in any female (4%). The number of total attempts per male, which could be a proxy for the effort 177 invested, were not influenced by being paired before the experiment (Welsh two-sample t-test, t = -0.41, p = 0.69), 178 nor did it correlate with male body size (Pearson correlation, r = 0.13, df = 46, p = 0.37). 179 Table 1 Counts of small and large males first approach towards small and large females and counts of males in The only difference between small and large males, was the time till they attempted a female. Small males were 183 faster in approaching a female in the first attempt (n = 21, mean± SD = 6.83 ± 6.73 min), compared to large males (n 184 = 25, mean ± SD = 13.62 ± 13.3 min, Welsh two-sample t-test, t = 2.24, p = 0.032, Fig. 1)

line) and outliers (black dots). 191
For the following analyses we did not use the size categories but took the original SVL values in mm. We observed a 192 correlation of body sizes for final pairs in amplexus (Pearson correlation, r = 0.41, df = 30, p = 0.02), and also 193 towards the first females that were attempted (Pearson correlation, r = 0.34, df = 44, p = 0.02). We compared these 194 findings with a simulation of random pairings with a bootstrapped null-model. Although, allocation to the 195 experimental pairs was random, the null-model showed a mean correlation coefficient of 0.3 and a standard deviation 196 of 0.1. Therefore, our observed correlations were not statistically different from the random null-model (Fig. 2). Nevertheless, it seemed that body size difference between males and females played a pivotal role in pair formation 205 and staying amplexed. Pairs in amplexus had a negative size difference to each other which means that the female 206 was on average larger than the male (mean ± SD = -2.22 ± 8.42 mm). Contrary, the size difference between the male 207 and the female that was approached first was positive, which means that the female was on average smaller than the 208 male (mean ± SD = 1.72 ± 9.99 mm). The distribution of a bootstrapped null-model had a mean of 0.26 mm size 209 difference between pairs and a standard deviation of 1.03 mm, if pairs were formed randomly. Therefore, the size 210 difference of pairs in amplexus was non-random (z-score = -2.41, Fig. 3). temporaria) to prefer larger, more fecund females for mating. Contrary to our expectations R. temporaria males did 221 not prefer any female based on body size and seem to randomly attempt females. However, as expected, small males 222 tried to clasp females faster, which indicates a different mating tactic to increase mating chances during scramble 223 competition. In general, the size difference between formed pairs was on average negative and significantly larger 224 than the expected values from a bootstrapped null-model. Hence, females in successfully formed pairs were larger 225 than the males. Because size difference between first attempted females and males was positive in our experiments, 226 i.e. males were larger than the attempted female, a selective mechanisms other than male competition and male mate 227 preferences seems to be responsible for this pattern. 228 One prerequisite for mate choice to evolve would be existing preference for specific traits, which we could not detect 229 for female body size. This confirms Elmberg (1991), who shows that there is no male mate preference for larger 230 body size in R. temporaria. However, body size alone should not be a trait under sexual selection in anurans anyhow, 231 because it is age and resource dependent (Halliday and Verrell 1988; Lodé et al. 2004) and cannot be considered a 232 true sexual secondary trait that provides an honest signal concerning mate quality, because heritability should be low. 233 We did not find direct tests for heritability of body size in anurans. Although, larger body size could be an indirect 234  In most species females grow larger than males, but males reach maturity earlier (Monnet and Cherry 2002). Indeed, 241 within amplected pairs the females had been on average larger than the males. Although, one could argue that 242 females are in general larger than males, in our experiments larger males approached smaller females first and the 243 null-model indicated that pairs should be of similar size, if pair formation would be random. The explanation why 244 this pattern is seen in the pairs which finally formed successfully, could be mechanical and independent from male 245 preferences. If females are smaller than the amplexing male, males' may not be able to hold them tight enough to 246 maintain amplexus. In Cane Toads (Rhinella marina) it was shown that males with shorter arms could cling better to 247 females compared to males with longer arms, the latter being replaced more often as they could not hold the females breeding sites (Woodward 1982). However, if males spend too much time with selecting particular females or 275 amplexing/defending a non-receptive female, males' chances to reproduce in a given year decrease over time. Thus, 276 males should minimize female selection time in order to increase the probability to reproduce in a given year and in 277 the best case, reproduce more than once. That small males are faster in amplexing a female was shown before 278 (Dittrich et al. 2018), and here we show that they are also faster in attempting to clasp a female. Small males are less 279 competitive during scramble, but in our experiment, we excluded intraspecific competition, and thus this behavior 280 appears to be intrinsic to smaller males and independent from other males presences. Therefore, this tactic seems to 281 follow an individual, condition dependent strategy based on male body size (Gross 1996;Brockmann 2001). This 282 mating system implies that until a specific body size is reached, the tactic of fast clasping is probably more 283 successful than participating in scrambling and that a switch point exists were the reverse is true for larger body size. 284 It would be interesting to test this assumption with the same males at different size and age. However, it should be 285 always beneficial to get access to a female as soon as possible and to stay with the female until spawning occurs, 286 independent of the males' body size. Hence, the size dependent 'speed'-hypothesis needs to be tested, including 287 frequency and density dependent approaches, to examine reproductive success of alternative tactics (Brockmann 288 2001). 289 In conclusion, we could show that male European Common Frogs do not prefer larger females and seem to mate 290 randomly. Smaller sized males seem to follow an individual, condition dependent strategy to get access to females 291 compared to their larger competitors that benefit from scramble competition. However, there is a non-random mating 292 pattern of males being in amplexus with larger females, which indicates that there are selective mechanisms that do 293 not depend on male mate prefernces or male-male competition. Probably there are other selective forces which shape 294