Female polyandry and size-assortative mating in isolated local populations of the Japanese common toad Bufo japonicus

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1 bs_bs_banner Biological Journal of the Linnean Society, 2014, 113, With 2 figures Female polyandry and size-assortative mating in isolated local populations of the Japanese common toad Bufo japonicus KAZUKO HASE* and MASAKAZU SHIMADA Department of General Systems Studies, The University of Tokyo Graduate School of Arts and Sciences, Meguro, Tokyo , Japan Received 12 February 2014; revised 18 April 2014; accepted for publication 18 April 2014 In anurans, female polyandry under male harassment is distributed across taxa because of external aquatic fertilization. According to the sexual selection theory, male male competition for access to females is affected by the operational sex ratio (OSR) and population density. The Japanese common toad, Bufo japonicus, is widespread in mainland Japan, and like the European common toad, B. bufo, it engages in explosive breeding. In this study, we observed the breeding behaviour of B. japonicus in isolated local populations for over four years in two breeding ponds with different population sizes and densities: large-low (L-pond) and small-high (). We analysed the relative polyandry ratio in egg clutches laid by females and estimated the size-assortative mating pattern to be an indicator of male male competition in the two ponds. Both ponds tended to exhibit a sizeassortative mating pattern; however, the frequency of polyandry was different in the two ponds (L-pond = 20% and = 90%). Our results showed that polyandry could occur without multiple amplexus with a high population density, i.e. eggs were often fertilized by free-swimming sperm in the small shallow pond. We propose that high female polyandry ratios without continuous male harassment are generated because of a male-biased OSR and a high population density in the small pond The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113, ADDITIONAL KEYWORDS: Bufo japonicus free-swimming sperm male harassment male male competition operational sex ratio polyandry, population density size-assortative mating. INTRODUCTION The intensity of intra-sexual selection is mainly controlled by the operational sex ratio (OSR); consequently, male-biased OSR strengthens male male competition (Halliday, 1978; Arak, 1983; Jirotkul, 1999; Byrne & Roberts, 2004). In anurans, many toads in the family Bufonidae are explosive breeding species that exhibit strong male male competition (Wells, 1977, 2010; Arak, 1983). Male toads are persistent in their search for fertilization opportunities in breeding ponds. Therefore, females sometimes experience polyandry involuntarily via multiple amplexus (Sztatecsny et al., 2006). *Corresponding author. hasekazuko@gmail.com However, the mating pattern of Bufo may not be fixed. Some studies have indicated the occurrence of size-assortative mating (Höglund, 1989; Vogrin & Miklic, 2005), whereas others postulate random mating patterns (Olson, Blaustein & O Hara, 1986; Höglund & Robertson, 1987; Yu & Lu, 2012). In addition, some toads exhibit variation in mating patterns among populations. Bufo minishanicus and B. andrews showed size-assortative mating in two of three sites and one of five sites, respectively (Yu & Lu, 2010; Liao & Lu, 2012). These patterns may depend on the specific breeding situation, including OSR, male male competition and the local population density. Höglund (1989) suggested that continuous high male male competition increases the likelihood of size-assortative mating in B. bufo. There are two main reasons for this pattern. First, size-assortative 236

2 POLYANDRY AND MATING PATTERN IN BUFO JAPONICUS 237 mating is beneficial for both sexes. Although a female wants every egg she lays to be fertilized, it is costly to maintain amplexus between a large female or a small male (Lengagne et al., 2007). Thus, size matching during amplexus pairing is probably important for toads. Second, under strong male male competition, females paired with small males may be taken by large stronger males (Höglund, 1989; Liao & Lu, 2011). Thus, if a small male initially catches a large attractive female, a larger male may rob the female from the small male. Male male competition is positively enhanced by a male-biased OSR. Theoretical and empirical studies suggest that a high population density or a male-biased OSR increases the intensity of male male competition, which results in sperm competition and sperm allocation (Parker, 1990; Parker et al., 1996; Dziminski et al., 2010). The strength of male male competition is closely related to both the frequency of polyandry and the mating pattern. However, few studies have tested these relationships in anurans. The Japanese common toad, B. japonicus, is widespread in mainland Japan, and similar to the European common toad, B. bufo, it engages in explosive breeding, and it also exhibits a male-biased OSR (2:1 8:1; Davies & Halliday, 1979; Reading, 2001). In the present study, we investigated the frequency of polyandry using microsatellite DNA markers and analysed male male competition in two local populations of B. japonicus in Tokyo. To estimate the degree of male male competition, we compared the body sizes of paired and single males and assessed the mating patterns of amplectant pairs for three years. Our field sites (breeding ponds) were located in urban Tokyo, and the two local populations had a clear difference in their densities; one was a large deep breeding pond with a low density and the other was a small shallow breeding pond with a high density. The two local populations were virtually isolated from external areas, which reduced with toad migration and maintained the population densities. This unusual situation allowed us to detect the ongoing polyandry that existed with size-assortative mating in a small breeding pond with a high population density. Moreover, we suggest that polyandry without continuous male harassment can occur under the condition of the combination of male-biased OSR and high population density. MATERIAL AND METHODS STUDY SITES AND OBSERVATION OF INDIVIDUALS During , we monitored the breeding behaviour of B. japonicus at two different sized breeding ponds in Tokyo. One was a large pond in the campus of L-pond Figure 1. Photographs of the two breeding sites: and L-pond. the University of Tokyo (ID: L-pond, 455 m 2, mean depth: 44 cm, coordinates: N, E), and the other was a small artificial pond (ID:, 3 m 2, mean depth: 10 cm, coordinates: N, E) inside temple grounds (Fig. 1). The breeding cycle of B. japonicus, which is known as an explosive breeder, is usually completed within a few days or 1 week each year. Our field work was performed at the two ponds during the breeding period of B. japonicus, i.e. from late February to early March during In 2012, we conducted frequent observations throughout the breeding period to determine the correct OSR. All males and females in the two ponds were caught, and each individual was marked with a plastic knee tag (National Band & Tag Co.). COLLECTION OF EGG STRINGS AND ADULT TISSUES To assess the paternity of clutches, we collected 20 clutches (10 from ; 10 from L-pond) of the

3 238 K. HASE and M. SHIMADA longest female egg strings (30 50 cm) and 67 toes from breeding adults (27 from ; 40 from L-pond), which were used for genotyping during If we could follow the female spawning behaviour completely, we sampled the toe tips of a female and a male in amplexus before collecting the egg string. We avoided collecting egg strings laid by females who engaged in continual multiple amplexus throughout spawning to determine the potential for polyandry due to free-swimming sperm. Thus, all females we followed had engaged in single amplexus; hence, we could confirm the principal paired male. All egg strings were allowed sufficient time for fertilization overnight after spawning. Finally, offspring per clutch were randomly selected and genotyped from 20 clutches. All toads were treated carefully to minimize physical stress in compliance with ethical concerns. Body size was measured within 1 min; toe tip sampling for DNA extraction was limited to 1 2 mm and egg sampling was performed with care to minimize injury. ASSESSMENT OF THE INTENSITY OF MALE MALE COMPETITION To determine the sexual selection intensity in B. japonicus, we assessed the effect of male male competition in the two ponds during using the following two methods. First, to determine whether strong male male competition allowed larger males to pair with more females, we measured the body size [snout-to-vent length (SVL)] of males that participated in breeding aggregation and compared them between paired and unpaired (single) males using the Wilcoxon rank sum test. In this assessment, if a female was under amplexus by several males, we counted the individual located at the centre of her dorsum as the paired male. Second, to determine whether a pair in amplexus exhibited a sizeassortative relationship each year, we also measured SVL of breeding females and tested the correlation between SVLs of paired males and females using Spearman s rank correlation coefficient test. All statistical analyses were performed using R (R Development Core Team, 2009). GENOTYPING AND ESTIMATION OF THE FREQUENCY OF POLYANDRY All tissue samples were digested overnight at 55 C in a solution (0.3% sodium dodecyl sulphate, 400 mm NaCl, 5 mm EDTA, 20 mm Tris-HCl, ph 8.0) that contained 200 μg ml 1 proteinase K. To estimate the frequency of polyandry, we genotyped all tissue samples on the basis of seven microsatellite loci: Bbufu11, Bbufu13, Bbufu23, Bbufu49, Bbufu39, Bbufu62 (Brede et al., 2001) and Bjap14 (Hase, Nikoh & Shimada, 2013) by PCR using ng DNA. Following the method described by Hase et al. (2013), the loci were amplified with two or three multiplex reactions using a Multiplex PCR Kit (Qiagen), according to the manufacturer s protocol. The PCR amplification cycles comprised an initial denaturation period at 95 C for 15 min, followed by cycles of 30 s at 94 C for denaturation, primer-specific touchdown annealing steps for 90 s (described by Hase et al., 2013) and an extension step at 72 C for 1 min. To determine the multilocus genotype of individuals, the fragment sizes of the PCR products obtained from each microsatellite locus were analysed using a CEQ 8000 Genetic Analysis System (Beckman Coulter) with a Genomelab Size Standard Kit 400 ( bp; Beckman Coulter). To estimate the frequency of polyandry, we analysed relative paternity using two programs: Cervus 3.0 (Kalinowski, Taper & Marshall, 2007) and Gerud 2.0 (Jones, 2005). When the parental (pairing female and male) genotypes were known, the relative paternity was analysed using Cervus 3.0 (Kalinowski et al., 2007). In Cervus 3.0, a male is assigned as a sire only if his likelihood score falls within an 80% confidence interval, which is derived from a simulation based on the observed population allele frequencies. When we lacked information about the parental genotypes, the relative paternity per clutch was estimated using Gerud 2.0 (Jones, 2005) to reconstruct the maternal genotype. In Gerud 2.0, which is based on the reconstructed maternal genotype, the minimum number of sires was determined for the offspring. If the offspring had more than one alternative paternal genotype, we considered that the most successful male sired the most offspring in the clutch. RESULTS ASSESSMENT OF MALE MALE COMPETITION The assessment of each pond in 2012 detected a male-biased OSR. The daily average male/female ratio in was 4.81 ± 3.86 (mean ± SD; approximately 4:1 in total) and that in L-pond was 2.74 ± 0.97 (approximately 3:1 in total). In total, 100 (17 pairs; 66 single males) individuals from over 6 days (7 12 March 2012) and 481 (120 pairs; 241 single males) individuals from L-pond over 5 days (22 26 February 2012) were caught. During breeding, we observed the displacement of two males in amplexus at and L-pond. In both cases, a smaller male (SVL: 8.55 cm, cm) was robbed of a female by a larger male (SVL: 9.85 cm, cm). In these cases, we counted the latter as a paired male.

4 POLYANDRY AND MATING PATTERN IN BUFO JAPONICUS 239 Female SVL (mm) No clear effect of body size on male male competition was observed between paired and single males in both ponds during ; the differences in the average SVL between paired and single males were not significant (: P = 0.345; L-pond: P = 0.145, Wilcoxon rank sum test). Annual comparisons of SVLs of paired and single males in both ponds are shown in supplementary Fig. S1. However, we detected significant size-assortative mating patterns in both ponds (Fig. 2). Although the values varied greatly among years, there was a tendency towards size-assortative mating, particularly in L-pond. The total values for three years indicated a significant correlation between male and female pairs in both ponds (: ρ = , P < ; L-pond: ρ = 0.400, P = 0.004, Spearman s rank correlation coefficient test). r = 0.54* y = 0.65x L-Pond r = 0.69 ** r = 0.30 *** r = 0.66 * y = 0.68x y = 0.41x y = 0.88x Male SVL (mm) 2013 Figure 2. Plot of the body size (snout-to-vent length, SVL) of each pair in amplexus in and L-pond during exhibited size-assortative mating (significant relationship between SVL in males and females) in 1 year (2011: N = 12, ρ = , P = 0.257; 2012: N = 17, ρ = 0.542, P = 0.024; 2013: N = 16, ρ = 0.429, P = 0.099, Spearman s rank correlation coefficient test) and the summed 3 years (N = 45, ρ = 0.497, P < ), whereas L-pond exhibited size-assortative mating every year (2011: N = 16, ρ = 0.694, P = 0.004; 2012: N = 120, ρ = 0.304, P = ; 2013: N = 12, ρ = 0.658, P = 0.020) and the summed 3 years (N = 148, ρ = 0.400, P = 0.004). The regression lines were calculated for each year with significant correlation coefficients. GENOTYPING AND ESTIMATION OF THE FREQUENCY OF POLYANDRY In total, we genotyped 67 adults and 424 offspring from the two ponds. None of the loci in either pond deviated significantly from Hardy Weinberg equilibrium after Bonferroni correction (detailed information for the microsatellite loci is provided in Table S1). The power of exclusion values for all seven loci combined in and L-pond was 0.89 and 0.88, respectively. The relative paternity results for each clutch are shown in two tables, i.e. Table 1 shows the results calculated using Cervus 3.0 (Kalinowski et al., 2007) based on paternal genotype information, and Table 2 shows the results calculated using Gerud 2.0 (Jones, 2005) without parental genotype information. According to our paternity analyses, 9/10 clutches (90%) in

5 240 K. HASE and M. SHIMADA Table 1. Relative paternity results for the offspring of seven Bufo japonicus females from and L-pond, which were estimated using Cervus 3.0 (Kalinowski et al., 2007); the females were engaged in amplexus with a single paired male (Male 1) during spawning Year N Male 1 Male 2 had more than two fathers, whereas only 2/10 clutches (20%) in L-pond were sired by at least two males. DISCUSSION Male 3 Others* Female S Female S Female S Female S L-pond Female L Female L Female L N, number of offspring analysed. *No candidate father (confidence < 80%). Table 2. Relative paternity results for 13 clutches collected from two breeding ponds ( and L-pond), which were calculated using Gerud 2.0 (Jones, 2005) Year N Father 1 Father 2 Clutch S Clutch S Clutch S Clutch S Clutch S Clutch S L-pond Clutch L Clutch L Clutch L Clutch L Clutch L Clutch L Clutch L N, number of offspring analysed. Our results indicate that had a high frequency of polyandry each year compared with L-pond. Thus, a high population density and a male-biased OSR led to female polyandry in B. japonicus. Our results agree with those of a previous report, which showed that polyandry in B. bufo is shaped by male density and OSR (Sztatecsny et al., 2006). In explosive breeding aggregation, many free-swimming sperm would be present at a high density in a small breeding pond, such as. Some males with high reproductive potential engage in repeated mating, which leads to sequential polygyny in Bufo (Davies & Halliday, 1979; Hettyey et al., 2009). Our paternity analyses failed to detect sperm of the same father derived from different clutches or the main male contributor to fertilization in. However, the clutches of at least four females were fertilized by more than two males without continuous male harassment (Table 1), which demonstrates the possibility of siring by freeswimming sperm. We also believe that floating males could participate in fertilization without paying the cost of entire amplexus. This suggests that all males operate under the control of a sperm economy rule and ejaculation strategies, which have been well studied in game theoretical approaches based on evolutionarily stable strategy models (Parker, 1990; Parker et al., 1996; Parker & Pizzari, 2010). Although females that engaged in multiple amplexus with over three males were sometimes observed in both ponds, this did not continue until spawning finished. Male toads possess innate variability in their mating behaviour, and they can switch between searching and interception (Arak, 1983). Males that disengage from participation in multiple amplexus may not want to become sexually exhausted before they secure mating, or they may give up mating because of their poor body condition. Some B. bufo males with a poor body condition maintain their reproductive potential because the sexual motivation of males varies and is determined by their sperm stocks (Hettyey et al., 2009). Males will avoid the risk of wasting sperm to maintain their reproductive value. Female polyandry in B. japonicus is controlled by the same mechanism as that found in B. bufo, i.e. it is related to the strength of male male competition based on OSR (Arak, 1983; Sztatecsny et al., 2006). However, similar to male, female fecundity varies in toads (Lengagne et al., 2007), e.g. a larger female that lays a longer egg string needs an adequate number of sperm to maintain a high fertilization rate. Thus, under a male-biased OSR, a female will accept multiple paternity. In addition, we detected clear size-assortative mating patterns in B. japonicus. However, the correlations varied between the two ponds and among years. In particular, the size-assortative mating pattern in was not robust but was only moderate; in short, a higher male-biased OSR did not strengthen size-assortative mating. This corresponds to previous studies of mating patterns in B. andrews (Liao & Lu, 2011, 2012). Size-assortative mating

6 POLYANDRY AND MATING PATTERN IN BUFO JAPONICUS 241 patterns in toads are generated not only by continuous male male competition but also by cryptic female choice (Höglund, 1989; Lengagne et al., 2007). Aggregations of males generally tend to reduce their male male competition when there is a high population density, and the males shift to alternative tactics (Jirotkul, 1999; Dziminski et al., 2010). If the population density increases, male toads will avoid exhausting battles to take females that have already mated with another male. Moreover, if the male toad can ejaculate for any length of time, his freeswimming sperm may fertilize eggs in a small water body such as. Although long-term amplexus while avoiding rivals is a hard task, amplexus is supposedly required for male ejaculation in B. japonicus [we never observed any of the piracy acts reported in Rana temporaria by Vieites et al. (2004), where a male ejaculates onto the egg strings after the female leaves]. However, a floating male could grasp a post-spawning female; nevertheless, amplexus between a male and a thin female, that had completed spawning, was infrequently observed during the late breeding period in both ponds (K. Hase, unpubl. data). It will be one of the male s alternative tactics. may have given single males the opportunity of paternity via short-term ejaculation. Hence, engaging in amplexus from the beginning to the end of female spawning would not be as important in a small shallow pond. Usually, polyandry is costly to a female toad because she is in danger of being drowned through multiple amplexus (Davies & Halliday, 1979). Overly aggressive male male competition negatively affects females. Conversely, if polyandry works positively towards increasing reproductive success for both sexes, there is no reason to stop it. High-frequency polyandry without male harassment may persist in a small pond with a high population density. ACKNOWLEDGEMENTS This study was supported by The University of Tokyo. We thank R. Yamada and H. Hase for helping with the field work and Dr T. Iwasaki for advising on the paternity analyses. REFERENCES Arak A Male male competition and mate choice in anuran amphibians. In: Bateson P, ed. Mate choice. Cambridge: Cambridge University Press, Brede EG, Rowe G, Trojanowski J, Beebee TJ Polymerase chain reaction primers for microsatellite loci in the common toad Bufo bufo. Molecular Ecology Notes 1: Byrne PG, Roberts JD Intrasexual selection and group spawning in quacking frogs (Crinia georgiana). Behavioral Ecology 15: Davies NB, Halliday TR Competitive mate searching in male common toads, Bufo bufo. Animal Behaviour 27: Dziminski MA, Roberts JD, Beveridge M, Simmons LW Among-population covariation between sperm competition and ejaculate expenditure in frogs. Behavioral Ecology 21: Halliday TR Sexual selection and mate choice. In: Krebs JR, Davies NB, eds. Behavioural ecology: an evolutionary approach. Oxford: Blackwell, Hase K, Nikoh N, Shimada M Population admixture and high larval viability among urban toads. Ecology and Evolution 3: Hase K, Shimada M Data from: Female polyandry and size-assortative mating in isolated local populations of the Japanese common toad Bufo japonicus. Dryad Digital Repository. doi: /dryad.255qd. Hettyey A, Vagi B, Hevizi G, Toeroek J Changes in sperm stores, ejaculate size, fertilization success, and sexual motivation over repeated matings in the common toad, Bufo bufo (Anura: Bufonidae). Biological Journal of the Linnean Society 96: Höglund J Pairing and spawning patterns in the common toad, Bufo bufo: the effects of sex ratios and the time available for male male competition. Animal Behaviour 38: Höglund J, Robertson JG Random mating by size in a population of common toads (Bufo bufo). Amphibia- Reptilia 8: Jirotkul M Population density influences male male competition in guppies. Animal Behaviour 58: Jones AG Gerud 2.0: a computer program for the reconstruction of parental genotypes from half sib progeny arrays with known or unknown parents. Molecular Ecology Notes 5: Kalinowski ST, Taper ML, Marshall TC Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment. Molecular Ecology 16: Lengagne T, Arthaud F, Cormier M, Joly P Cost of sexually embracing a large female offset by the number of eggs fertilized for small male Bufo bufo L. Biological Journal of the Linnean Society 92: Liao WB, Lu X Proximate mechanisms leading to large male-mating advantage in the Andrew s toad, Bufo andrewsi. Behaviour 148: Liao WB, Lu X Variation in mating patterns in the Andrew s toad Bufo andrewsi along an elevational gradient in southwestern China. Ethology Ecology & Evolution 24: Olson DH, Blaustein AR, O Hara RK Mating pattern variability among western toad (Bufo boreas) populations. Oecologia 70: Parker GA Sperm competition games: sneaks and

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