NORTH-WESTERN JOURNAL OF ZOOLOGY 8 (2): 241-246 ©NwjZ, Oradea, Romania, 2012 Article No.: 121118 http://biozoojournals.3x.ro/nwjz/index.html

Mating pattern variability across three Asiatic ( gargarizans gargarizans) populations

Tong Lei YU1,* and Xin LU2

1. Department of Biology, College of Life Science, Xinyang Normal University, SD 464000, China. 2. Department of Zoology, College of Life Science, Wuhan University, SD 430072, China. * Corresponding author’s e-mail: [email protected]

Received: 18. November 2011 / Accepted: 11. April 2012 / Available online: 28. May 2012 / Printed: December 2012

Abstract. Mating patterns of the Asiatic toad, Bufo gargarizans gargarizans, were examined in three large breeding populations in China. Sexual dimorphism of body size was variable among the three populations, although males were always smaller than females. Amplectant males did not differ significantly from non- amplectant males with respect to body size and forelimb length in two of the three populations. However, in a southwestern population, amplectant males were larger than non-amplectant males, and there was evidence for positive size-assortative mating. Moreover, intraspecific displacement in which one male dislodges another already engaged in amplexus thereby usurping the mating, revealed that male-male competition resulted in a large-male mating advantage in the same population. Because mate choice appears to be lacking in Asiatic , we suggest that competition for females is a major behavioral mechanism shaping size-assortative mating.

Keyword: Bufo gargarizans gargarizans, body size, mating system, sexual selection, behavioral mechanism.

Introduction nisms to variation in mating patterns remains a major question in need of more detailed study Traditionally, behavioral ecologists have studied (Crespi 1989, Arnqvist et al. 1996). mating patterns from the individual’s point of Apart from a description of overwintering view and have asked how mate choice and compe- habitat and diet of Asiatic toad (Bufo gargarizans tition affect the fitness of females and males (re- gargarizans Cantor, 1842) (Yu et al. 2009, Yu & Guo viewed by Andersson 1994). Competition among 2010), there are few data concerning their repro- males for females and selection of a male by a fe- duction, beyond anecdotal reports that document male may lead to a size-dependent, or to a size- breeding behaviour and mating patterns (Zou, assortative mating pattern (Robertson 1986, Tsuji 1982). Therefore, we have studied the mating pat- & Matsui 2002, Hajer et al. 2011). terns in three distinct populations of B. gargarizans Although random mating patterns have also spanning a latitudinal gradient in China. The large been documented in some anuran species (Okuno sizes of our three study populations provide us 1986, Elmberg 1987, Höglund & Robertson 1987, with an excellent opportunity to examine mating Crump & Townsend 1990, Friedl & Klump 2005), pattern variation both among and within popula- there is substantial evidence for non-random mat- tions during the breeding season. We used field ing with respect to body size for several anuran observations and displacement experiments to of the genus Bufo (Gatz 1981, Wood- address the following questions: (1) Do amplec- ward 1982, Reading & Clarke 1983, Howard 1988, tant males constitute a random sampling from all Yu & Lu 2010, Liao & Lu 2011). In fact, mating pat- sexually mature males? (2) Does the size of a male terns differ widely both between and within spe- and female in amplexus correlate? (3) What behav- cies (e.g. Emlen & Oring 1977, Wittenberger 1979, ioural mechanisms are involved in shaping mating Clutton-Brock 1991, Olson & Blaustein 1986, Da- patterns? vies 1991). In addition to sexual selection, some of this variation in mating pattern has been ex- plained by the length of the breeding season Materials and Methods

(Wells 2007, reviewed by Sullivan et al. 1995), Study Site population density and operational sex ratio We observed B. gargarizans breeding activity at three (Lüddecke 2001) at different times within a popu- study sites in China from December 2007 to April 2008 lation, as well as in different populations (Elmberg (Fig. 1): (1) Site A, Fuxin (42°00′N, 121°45′E; elevation 203 1991, Lee & Park 2009). Nonetheless, the relative m based on GPS) from late-March to mid-April; (2) Site B, Xinyang (32°07′N, 114°07′E; elevation 119 m) from mid- importance of these various contributing mecha- 242 T.L. Yu & X. Lu

Figure 1. Maps for Bufo gargarizans gar- garizans, showing the positions of three sample sites (Site A, Fuxin; Site B, Xin- yang; Site C, Nanchong).

January to late-January; (3) Site C, Nanchong (30°48′N, trials). We observed each pair at least once an hour. All 106°06′E; elevation 268 m) from late-December to mid- observations ceased after 24 hours because single males January. rarely displaced amplectant males after that length of time. Field Procedures Breeding ponds at the study sites were searched exhaus- Statistical Analysis tively approximately once a week during daylight hours. To test whether the magnitude of sexual size dimorphism Toads were captured by hand and were individually and mating patterns differed across the three populations, marked by toe-clipping (Lüddecke & Amézquita 1999). we first compared SVL with two-way ANOVA, compar- The development of secondary sexual characters of male ing sexes, and populations as factors. Subsequently, for and eggs of females begins before the breeding season. males, we used a similar two-way ANOVA to examine This allowed us to identify the toads as sexually-mature the effect of mating status (amplectant or non-amplectant) males if they displayed nuptial pads on the fore digits, or and population (A, B, or C). We conducted ANCOVAs for sexually-mature females if they had well-developed oo- forelimb length, but also included SVL as a covariate. To cytes (which were readily visible through the skin of the test for size-assortative mating, we added population as a abdomen). All individuals were released at the site of factor, female size as a covariate and male size as a de- capture, with the exception of 145 individuals from site C pendent variable based on a general linear model and that were transported to the laboratory to test for dis- analyzed the interaction between population and female placement during amplexus. The snout-vent length (SVL) size. Statistical analyses were performed with SPSS ver. of each toad was measured to the nearest millimeter with 13 (SPSS institute, Inc. 2002-2003), and all P-values given a ruler while holding the against a flat surface. are two-tailed, with values presented as means ± stan- The length of each breeding season was determined ac- dard error. cording to the occurrence of amplectant pairs and eggs in the breeding ponds.

Laboratory Procedures Results From 9–15 February 2008 we ran male displacement trials in 45 plastic wading pools (50 cm in diameter, 40 cm Field Comparisons deep) filled to a depth of 15 cm with pond water. To pro- B. gargarizans is a typical "explosive" breeder, hav- vide cover for the and substrate for egg- ing a relatively short breeding season (6–24 days). deposition, a handful of sedge-leaves collected from the More than 190 breeding adults were captured at pond were placed into each pool. We divided our ex- each study site (Fig. 1). The sex ratios of breeding perimental animals into 3 groups consisting of: (1) a lar- adults were male-biased at all three sites ger single male and smaller amplectant male (experiment (male/female: 1.35 at site A, 3.17 at site B, 1.43 at 1; 15 trials), (2) a single male and an amplectant male of the same size (experiment 2; 15 trials) and, (3) a smaller site C). Males actively searched for females on the single male and larger amplectant male (experiment 3; 15 water surface, clasped other toads regardless of Mating pattern variability 243 sex, and neither defended territories nor gave mat- population interaction (F2, 602 = 1.00, P = 0.369). ing calls. Males emitted release vocalizations when Thus, we found that while paired males were sig- clasped by males. When examining the body size nificantly larger than unpaired males during of all samples, we found that sex (F1, 979 = 266.00, breeding season at site C (t = 5.87, df = 172, P = P < 0.001) and population (F2, 979 = 214.70, P < 0.007, Fig 2b), white at sites A and B, there was no 0.001) influenced SVL, but there was no significant significant difference between the size of paired sex × population interaction (F2, 979 = 2.70, P = and unpaired males (both P > 0.05). Mating status 0.067). These results show that males were smaller did not influence forelimb length, using SVL as a than females between populations, and SVL dif- corvariate (F1, 545 = 3.03, P = 0.143), though it did fered among populations (Fig. 2a). vary significantly between populations (F2, 545 = 108.85, P < 0.001, Fig 2c). Mating Patterns When examining size-assortative mating, we When examining male body size, we found that found a significant interaction between female mating status (F1, 602 = 5.95, P = 0.015) and popu- SVL and population (F2, 325 = 6.70, P < 0.001). lation both influenced SVL (F2, 602 = 139.15, P < These results show that the relationship between 0.001), but there was no significant mating status ×

Figure 2. Comparison of mean morpho- logical traits of Bufo gargarizans gargari- zans from three latitudinal populations. (a), all samples (filled circles represent males, open circles females); (b), paired males (open circles) and unpaired males (filled circles); (c) forelimb length with paired males (open circles) and forelimb length with unpaired males (filled cir- cles). Sample size is given above or be- low the 2 standard error bars. 244 T.L. Yu & X. Lu

Figure 3. Plots of snout– vent length (SVL) of am- plectant male and female Bufo gargarizans gargari- zans from three latitudinal populations.

amplectant female and male body size varied sig- (80%) succeeding. In the second experiment, when nificantly between populations. Hence, we found the test male (9.29 ± 0.05cm) and the amplexing a significant correlation between male and female male were of similar size (9.30 ±0.14cm; P = 0.89), body size at site C (Pearson's correlation: r = 0.589, ten of the 15 males made a displacement attempt. n = 132, P < 0.001, Fig 3c), but this pattern was not Only three test males (20%) were observed to dis- evident in the remaining populations (both P > place the amplectant males. In group three, when 0.05, Fig 3a, b). a smaller male (8.71 ± 0.17cm) was placed with a larger male already in amplexus (10.13 ± 0.13cm; P Amplexus Displacements = 0.001), only two displacements (13.33%) took In the first experiment, all large males (mean SVL: place in 15 trials. Male takeovers were differen- 10.07 ± 0.15cm) attempted to displace smaller am- tially distributed across the three experiments plexed males (8.75 ± 0.18 cm; Wilcoxon signed- (Chi-square test: χ2 = 10.71, df = 2, P = 0.005). ranks test: P = 0.03), with 12 of 15 larger males Mating pattern variability 245

Discussion ing amplexus demonstrated a large- male mating advantage, suggesting that this behaviour may be Anuran mating patterns are often non-random responsible for the observed non-random mating with respect to body size (reviewed by Halliday & patterns in a natural population. Additionally, fre- Tejedo 1995). By investigating natural pairing pat- quent male combat was observed with some in- terns in B. gargarizans a over three sites, we dem- stances of takeovers in the field being recorded, onstrate that species subscribe to a size-related even found abnormal amplexus (e.g. multiple am- non-random mating pattern and show consider- plexus; amplexus between alive male and dead able among-population variation in pairing pat- female). Similarly, Mollov et al. (2010) reported terns. Of the 3 sites studied, sexual dimorphism cases of abnormal amplexus in anurans. Amplexus was observed between sexes, and females were displacement is a common method employed by larger than males (Çiçek et al. 2011). Moreover, many anuran males to obtain mating success (Ber- amplectant males of B. gargarizans were signifi- ven 1981, Reading & Clarke 1983, Höglund 1989, cantly larger than non-amplectant males at site C, Tsuji & Matsui 2002). Therefore, we speculate a consistent with a large-male mating advantage. size-assortative mating process resulting from fre- We also found size-assortative mating for B. gar- quent takeovers of the original smaller males dur- garizans at the same site as SVL within am- ing a pairing period. plec¬tant pairs were positively correlated (Fig 3c). Mating patterns may be influenced by opera- Our results give some evidence for mating tional sex ratio (OSR) and the length of the breed- success biased toward larger males for B. gargari- ing period. The most anuran amphibians showed zans, and parallels findings on other Bufo species highly male-biased OSR and short breeding peri- (Howard 1988, Howard & Young 1998, Lee & Park ods in a natural population (Kovács & Sas 2010). 2009). Similarly, a study on a different subspecies In our study, breeding periods is 13 days at site B. of the same species, Bufo andrewsi, (Liao & Lu The time window available for large males to en- 2011) suggested that the large male mating advan- counter all possible female mates is relatively lim- tage observed in the field and laboratory is caused ited. Moreover, strong male competition may limit by competition among males rather than by fe- the opportunities for large males to exhibit contest male choice. Our studies also provide evidence for advantages in mating and for both sexes to choose size-assortative mating in B. gargarizans. Females a large mate (Lu et al. 2009). However, at site C, of other species have also been shown that the size the breeding period was slightly longer (19 days) of amplectant males varied relative to their own and the operational sex ratio was intermediate be- body size, e.g. Bufo americanus (Licht 1976), Bufo tween site A and B. Here, sufficient time is avail- bufo (Davies & Halliday 1977), and Bufo stejnegeri able for competition to occur, with large males (Lee & Park 2009). consistently out-competing small males for access Observed mating patterns in B. gargarizans to females (Wilbur & Rubenstein 1978, Hillis & may result from female choice, male–male compe- Hillis 1984, Olson & Blaustein 1986). tition, male choice, or a combination of these Among anurans, considerable variation exists (Wilbur & Rubenstein 1978, Howard & Kluge in mating patterns, and different populations of 1985). In explosively breeding species that congre- the same species may exhibit different mating tac- gate in masses for short periods, the opportunity tics (Gittins et al. 1980, Höglund & Robertson for intense male–male competition is significant 1987). Tactics can also vary among different breed- and, as a result, there is typically little occasion for ing seasons in the same population (Olson et al. females to exercise choice. Mating, instead, may 1986, Morris, 1989, Wagner & Sullivan 1995). involve a scramble competition among males for Therefore, the relative importance of various con- females (Emlen & Oring 1977). In this case, devia- tributing mechanisms to the variation in mating tions from random mating may be expressed as a tactics remains a major question in need of study. large-male mating advantage. At our three study sites, female choice based on male vocalizations is unlikely because males do not produce mating Acknowledgments. We would like to thank the teachers calls. Moreover, we also found male mate choice of Normal University for providing the laboratory and lacking in B. gargarizans by means of laboratory equipment for this research. I would also like to thank experiments at site C (Yu & Lu 2010). However, W.H. Qi, Y.L. Han, and J.Z. Ning for their assistance in the field, especially to R. Tingley, J. Malone and D. Pike’s our laboratory experiments of displacement dur- comments and revision on the manuscript. 246 T.L. Yu & X. Lu

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