NORTH-WESTERN JOURNAL OF ZOOLOGY 9 (1): 121-126 ©NwjZ, Oradea, Romania, 2013 Article No.: 131503 http://biozoojournals.3x.ro/nwjz/index.html

Lack of male mate choice in the Minshan’s ( gargarizans minshanicus)

Tong Lei YU1,* and Xin LU2

1. Department of Biology, College of Life Science, Xinyang Normal University, Xinyang Province, 464000, China 2. Department of Zoology, College of Life Sciences, Wuhan University, Wuhan, Province, 430072, China 3. Present Address: Department of Biology, College of Life Science, Xinyang Normal University, Xinyang Henan Province, 464000, China * Corresponding author, T.L. Yu, Phone: +8618790421879, E-mail: [email protected]

Received: 28. July 2012 / Accepted: 25. November 2012 / Available online: 13. January 2013 / Printed: June 2013

Abstract. We studied male mate choice and mating success in Minshan’s (Bufo gargarizans minshanicus) – a species in which female fecundity increases with body size, and the time invested in one mating is relatively long. Field data suggested that larger males had a mating advantage over smaller males, and that mating was size-assortative. We also tested sex recognition and mate choice in laboratory-based choice experiments. When a male B. g. minshanicus was allowed to choose between a male and a female of equal size, males did not discriminate between the sexes. When a male B. g. minshanicus was allowed to choose between a similar-sized gravid and non-gravid female, males preferably clasped the gravid female. When two different-sized gravid females were provided for males, the males did not discriminate females based on their body size. Although male B. g. minshanicus had an ability to distinguish between gravid and non-gravid females, under field conditions non-gravid female abundance is relatively low. Therefore, when the cost associated with making a wrong choice is low, male Minshan’s toads did not preferentially choose mates.

Keywords: Bufo gargarizans minshanicus, sex recognition, male mate choice.

Introduction selecting for a good quality female in several anu- ran species (Halliday 1983, Andersson 1994, Most work on sexual selection in ectotherms has Arntzen 1999, Liao & Lu 2009a, Chen & Lu 2011), focused on female mate choice and male-male absence of mate choice based on species, ovulation competition (Arak 1983, Ridley 1983, Andersson state or body size has been documented in anuran 1994, Martínez-Rivera & Gerhardt 2008, Brooks & species (Reading 1984, Arak 1983, Gergus et al. Gerhardt 2009, Ryan et al. 2010, Ursprung et al. 1999, Krupa 1995, Marco & Lizana 2002, Hettyey 2010, Gomez et al. 2011, Liao & Lu 2011). Based on et al. 2005, Yu & Lu 2010). Generally, males show a a qualitative model, Ridley (1983) predicted that preference for larger females in species where fe- male mate choice would occur in anuran species male fecundity increases with body size. This is where female fecundity is size-related, and the du- because greater male fitness can result from in- ration of amplexus is sufficiently long to impose a creasing the number of matings and thus offspring constraint on the number of times a male could sired (Halliday 1983, Andersson 1994). However, mate (Krupa 1995). However, even in species if the costs or probability of incorrect discrimina- without sex-role reversal, male mate choice can tion and the costs of mate searching are low, then also occur. The evolutionary implications of this discriminatory mechanisms may not be favored strategy were recently highlighted by Shine at al. (Marco & Lizana 2002, Yu & Lu 2010). (2003). Berven (1981) was the first to report female Minshan’s toad (Bufo gargarizans minshanicus) size-based male mate choice in Lithobates sylvati- is a medium-sized bufonid [adult snout–vent cus, and male mate choice subsequently has been length (SVL) = 55–98 mm] in forests, fields and shown to occur in several other anuran species as high pastures of southwestern China at elevations well (Bush et al. 1996, Marco et al. 1998, Arntzen between 1700 and 3700 m (Fei & Ye 2001). The 1999, Liao & Lu 2009a, Chen & Lu 2011). Sexual toad has an annual spawning period of approxi- selection has allowed species to evolve efficient mately 3 weeks, which makes it a typical explosive signals (visual, acoustic, tactile, olfactory and breeder (Wells 2007). Males arrive at the breeding chemical) and responses to allow individuals to ponds earlier than females and stay there locate potential mates (Halliday 1983, Paterson throughout the breeding season. During the 1985). breeding season, males do not produce advertise- Although there is substantial evidence for the ment calls to attract mates, but instead actively 122 T.L. Yu & X. Lu search for females (Fei & Ye 2001, Yu personal ob- proximately 12 cm of fresh water. Tank water was servation). Females are amplexed after they enter changed after each trial, and the tanks flushed twice with the water. After egg deposition females leave the fresh water to minimize olfactory contamination. In all trials, the two stimulus toads were place on each end of pond. Despite its wide distribution range, surpris- the tank with their legs tied to the tank ends with a 15 cm ingly little is known about the reproductive be- long string. This limits movement to a short distance, but havior of Minshan's toads. otherwise does not influence toad behavior. Each trial In the population, we found a long duration of was observed for 15 minutes. The trial began at the time amplexus (5–8 days), and a significant positive the test male was put into the tank, and ended when the correlation between fecundity and female body male toad clasped another individual. If there was no size (Fig.1), which met assumptions of Ridley’s amplexus after 15 min, the trial was terminated and we assumed that the male did not make a choice. These in- (1983) qualitative model. Therefore, we investi- stances were excluded from analysis. We recorded the gated whether male mate choice occurs in B. g. side of the tank towards which the male moved in his minshanicus. We used three choice experiments to first attempt. We also recorded the time that the test toad test whether male toads have the ability to dis- spent on each side of the tank. If the toad was in the mid- criminate between 1) females and males of similar dle of the enclosure, we used the position of the head to body size, 2) gravid and non-gravid females of assign its location. similar body size, and 3) gravid females of differ- For analyzing the relationship between female fe- cundity and body size, we allowed one of the males to ent body sizes. amplex with a given female after all mate choice experi- ments were completed. The amplexed pairs were put into 50 plastic containers (5 liter) until the eggs were depos- Materials and methods ited. After counting eggs for each clutch, the eggs and adults were returned to the pond from which they were Study site collected. The field study was carried out at Gahai Reserve in Gahai-Zecha National Nature Reserve of Luqu County, Data analysis , China (102°20′E, 34°12′N, total area 10,790 ha, ele- We used parametric tests when assumptions of these tests vation 3560 m). The mean temperature of the toads’ activ- were met. When assumptions were violated, non- ity season is 7.5°C with a mean annual precipitation of parametric tests were used. All values are presented as 637 mm. The reserve is characterized by wet meadows mean ± SE, and all statistical tests were two-tailed. and strong algal growth. Apart from some small and shal- low pools, a large plateau lake (Gahai Lake) provides adequate spawning sites for anurans. Results

Data collection Mating Pattern From 3–18 April 2009, we collected 548 individual toads On average, female SVL (mean = 7.30 ± 0.03 cm, N (349 males and 199 females), 396 of which were found in amplexus (i.e., 198 mating pairs). All specimens were = 200) was significantly larger than that of males caught by hand from two breeding ponds on days with (mean = 6.48 ± 0.02 cm, N = 349, t546 = 25.58, P < peak reproductive activity. We observed their mating be- 0.001). Paired males (mean = 6.54 ± 0.02 cm) were havior and recorded the snout–vent length (SVL) of each significantly larger than unpaired males (mean = individual to the nearest 0.5 mm using a ruler. In Experi- 6.44 ± 0.03) in the breeding ponds (t351= 3.10, P = ment 1, we attempted to determine whether males recog- 0.002). Hence, the males with larger body sizes in nize the sex of conspecifics (n = 38 trials). In Experiment the population have a mating advantage. The 2, we tested if males could discriminate between gravid versus non-gravid females (determined by lower abdo- population sex ratio of B. g. minshanicus was sig- men width or body mass after oviposition) of the same nificantly male-biased in the ponds (1.75:1 M: F, size, which would be expected if males show a preference Chi-square test: χ2 = 41.06, df = 1, P < 0.001). Av- for gravid females (n = 36 trials). In Experiment 3, we erage clutch size was 2788.11 ± 70.20 eggs (range: tested whether males could discriminate between large 1,972–3,908 eggs), and clutch size was positively and small gravid females, which would be expected if correlated with female SVL (Pearson correlation: r males show preference for larger females (n = 40 trials). = 0.82, n = 44, P < 0.001; Fig. 1). The correlation be- The experimental design was similar to that used by Marco et al. (1998). Test were isolated in separate tween the size of males and females in amplexus containers with fresh water for 20 min prior to the begin- provided weak evidence for size-assortative mat- ning of each trial. ing (r = 0.22, n = 198, P = 0.002; Fig. 2). All experiments were conducted in the laboratory during the mating period (12–15 April 2009) using a rec- Experimental Results tangular tank (90×50×30 cm; L×W×H) filled with ap- In Experiment 1, where males were offered a Lack of male mate choice in the Minshan’s toad 123

Figure 1. Relationship between fe- male SVL and clutch size in a natu- ral population of Bufo gargarizans minshanicus.

Figure 2. Relationship between the body size (snout–vent length, SVL) of male and female Bufo gargarizans minshanicus found in amplexus.

choice between a male (mean SVL = 6.95 ± 0.05 on either side of the tank before amplexing (Mann- cm) and a similar female (6.94 ± 0.05 cm; paired t- Whitney, Z = -0.484, P = 0.628). test, t74= 1.71, P = 0.095), 16 of 38 males (6.55 ± 0.04 In Experiment 2, where males were given a choice cm, 42.1%) attempted to amplex with a male, 19 between a gravid (mean SVL =7.38 ± 0.04 cm) and (50.0%) amplexed with a female, and the remain- a non-gravid female (7.39 ± 0.04 cm; paired t-test, ing three males (7.9%) did not make any choice t70 = -0.79, P = 0.437), 21 of 36 males (56.50 ± 0.07 (Fig. 3). The percentage of males that attempted to cm, 8.3%) chose the gravid female. Ten males amplex with a male did not deviate significantly (27.8%) clasped a non-gravid female, and five from random (Chi-square test: χ2 = 0.26, df = 1, P males (13.9%) made no choice (Fig. 3). This per- = 0.612; Fig. 3). A post-hoc power analysis re- centage was significantly different from random vealed that the likelihood of detecting a difference (χ2 = 3.90, P = 0.048). In 27 of 31 trials, the first side between the groups given our sample size was of the chamber selected by the test male coincided 0.995, strongly suggesting that toads are not select- with the side in which the final choice was made. ing between males and females prior to initiating The time that test males spent on either side of the amplexus. The first side of the tank selected by test test arena was not significantly different (Z = - males apparently affected their final choice (χ2 = 0.862, P = 0.389). The time that it took test males to 15.11, P < 0.001). The time that it took males to at- attempt to amplex the gravid females was 37.04 ± tempt to amplex with a female was 29.46 ± 6.03 s 7.21 s, and that of the non-gravid females was and with males 39.89 ± 10.35 s. No significant dif- 55.21 ± 14.05 s. ference was found in the time that test males spent In Experiment 3, where males could choose 124 T.L. Yu & X. Lu

Figure 3. Male mate choice in Bufo gar- garizans minshanicus. Experiment 1: male toads selected between a size-matched female (open bar) or male toad (shaded bar; P = 0.10), or did not make a choice (diagonal bar). Experiment 2: male toads selected between a size-matched gravid (open bar) or non-gravid toad (shaded bar; P = 0.44), or did not make a choice (diagonal bar). Experiment 3: male toads selected between a large (open bar) or small gravid female (shaded bar; P < 0.001), or did not make a choice (diagonal bar).

between two gravid females differing in body size, son1989). Consequently, the cost of the absence of 17 of 40 males (mean SVL = 6.64 ± 0.05 cm, 42.5%) sex recognition in terms of lost time and energy is chose the larger female (7.81 ± 0.04 cm), whereas probably small, and results in a high chance of 16 chose the smaller female (6.85 ± 0.03 cm; paired mating success (Chen & Lu 2011). t-test, t78 = 21.80, P < 0.001). The remaining 7 indi- Studies on Anaxyrus boreas (Marco et al. 1998) viduals made no choice (Fig. 3). These percentages and Bufo andrewsi (Liao & Lu 2009a) have found were not significantly different from random (χ2 = that breeding males are unable to identify the re- 0.03, P = 0.862). A post-hoc power analysis re- productive state of females. Although B. g. Min- vealed that the likelihood of detecting a difference shanicus males were able to distinguish between between the groups given our sample size was gravid and non-gravid females, there is a lack of 0.987, strongly suggesting that toads are not select- adaptive significant to this finding for males in ing between larger and smaller females prior to natural population. One obvious reason is that the initiating amplexus. The first side of the tank se- probability of males’ amplexing non-gravid fe- lected by test males was the same side of the final males in nature is relatively low, because formerly choice in 84.8% of the trials. The time that test gravid females quickly leave the breeding site males spent on either side of the test arena did not immediately after laying eggs. For example, we differ (Z = -1.037, P = 0.300). only found one non-gravid female still at the breeding site. Additionally, we found that gravid females are more active than non-gravid females, Discussion so males prefer to mate with gravid females be- cause males are attracted to moving object. There- Previous studies have shown that some male an- fore, males probably do not adopt the strategy of urans do not discriminate between the sexes due discriminating between gravid and non-gravid to high population densities and strong scramble females when the density of non-gravid females is competition among males in populations (Marco very low in nature. et al. 1998, Marco & Lizana 2002, Mollov et al. In line with earlier reports from other species 2010, Kovács & Sas 2010, Liao & Lu 2009b, Yu & (Hettyey et al. 2005), our results indicate a positive Lu 2010). B. g. minshanicus males search actively correlation between female body size and fecun- for mates, and when females arrive at the breeding dity: larger females lay up to twice as many eggs sites they are quickly mounted by males. When a as smaller females. As males invest heavily into male attempts to amplex another male, the clasped mating in terms of time spent in amplexus, we ex- male gives release calls. As a consequence, male- pected males to show preference for larger fe- male amplexus only lasts a few seconds, leaving males. However, we found no evidence for male males free to continue their search for females. In mate choice based on female body size. Similar this context, males adopt the strategy of actively findings have also been reported in a few earlier searching for females and attempting to clasp any studies of other anuran species (Marco & Lizana nearby individual (Arak 1983, Brown 1977, Ol- 2002, Hettyey et al. 2005). We can think of at least Lack of male mate choice in the Minshan’s toad 125 two possible explanations for the lack of prefer- References ence for larger females in B. g. minshanicus. Firstly, Andersson, M. (1994): Sexual Selection. Princeton, New Jersey: as the sex ratio is male-biased (1.75:1 M: F), an in- Princeton University Press. tense scramble competition among males may Arak, A. (1983): Male-male competition and mate choice in anuran . In: Mate Choice. P. Bateson (ed.). Cambridge Univ. limit males’ mating opportunities and increase the Press, Cambridge. risk of ending up unpaired, thereby rendering the Arntzen, J.W. (1999): Sexual selection and male mate choice in the quality of potential mates of secondary impor- , Bufo bufo. Ethology Ecology and Evolution 11: 407-414. tance (Fawcett & Johnstone 2003). For instance, Berven, K.A. (1981): Mate choice in the wood , Rana sylvatica. Arntzen (1999) suggested that male B. bufo may be Evolution 35: 707-722. expected to be selective in their choice of females Bush, S.L., Dyson, M.L., Halliday, T.R. (1996): Selective phonotaxis by males in the Majorcan midwife toad. Proceedings of the only when the sex ratio is female biased and the Royal Society B 263: 913-917. probability of finding a more fecund female is Chen, W., Lu, X. (2011): Sex recognition and mate choice in male high. Similarly, Sullivan et al. (1995) suggested if Rana kukunoris. Herpetological Journal 21: 141–144. Davies, N.B., Halliday, T.R. (1977): Optimal mate selection in the females were a limited resource for male repro- toad Bufo bufo. Nature 269: 56-58. ductive success, temporal constraints may increase Davies, N. B., Halliday, T.R. (1979): Competitive mate searching in the costs of highly discriminating behavior by male common toads, Bufo bufo. Behaviour 27: 1253-1267. Fawcett, T.W., Johnstone, R.A. (2003): Mate choice in the face of males. Secondly, the matching by size may im- costly competition. Behavioral Ecology 14: 771-719. prove fertilization success (i.e. Davies & Halliday Fei, L., Ye, C.Y. (2001): The colour handbook of amphibians of 1977). Thus, some males may make a choice ac- . China Forestry Publishing House, Beijing. Gergus, E.W.A., Malmos, K.B., Sullivan, B.K. (1999): Natural cording to their own body size and fertilizing abil- hybridization amongdistantly related toads (Bufo alvarius, Bufo ity, rather than blindly pursuing females of greater cognatus, Bufo woodhousii) in central Arizona. Copeia 1999: 281- body size, which would lead to a size-assortative 286. Gerhardt, H.C., Brooks, R. (2009): Experimental analysis of mating pattern. multivariate female choice in gray treefrogs (Hyla versicolor): In some explosive breeders, larger males take evidence for directional and stabilizing selection. Evolution 63: females from smaller males by aggressively dis- 2504–2512, Gomez, D., Théry, M., Gauthier, A.L., Lengagne, T. (2011): Costly placing them from amplexus (Davies & Halliday help of audiovisual bimodality for female mate choice in a 1979, Lamb 1984, Howard & Kluge 1985, Loman & nocturnal anuran (Hyla arborea). Behavioral Ecology 22: 889-898. Madsen 1986). We observed frequent male combat Halliday, T. (1983): The study of mate choice. pp.3–32. In: Bateson, P. (ed.), Mate Choice. Cambridge: Cambridge University Press. and recorded a few instances of takeovers in the Hettyey, A., Török, J., Hėvizi, G. (2005): Male Mate Choice Lacking field. Therefore, we speculated that a size- in the Agile Frog, Rana dalmatina. Copeia 2: 403-408. assortative mating process resulted from frequent Howard, R.D., Kluge, A.G. (1985): Proximate mechanisms of sexual selection in wood . Evolution 39: 260-277. takeovers of the original smaller males during the Kovács, É-H., Sas, I. (2010): Aspects of breeding activity of Bufo pairing period. In fact, among anurans, consider- viridis in an urban habitat: a case study in Oradea, Romania. able variation exists in mating patterns, and dif- Biharean Biologist 4: 73-77. Krupa, J.J. (1995): How likely is male mate choice among anurans? ferent populations of the same species may exhibit Behaviour 132: 643-664. different mating tactics (Wells 2007, Liao & Lu Lamb, T. (1984): Amplexus displacement in the southern toad, Bufo 2012, Yu & Lu 2012). Therefore, male B. g. min- terrestris. Copeia 1984: 1023-1025. Liao, W. B., Lu, X. (2009a): Male mate choice in the Andrew's toad shanicus exhibited the preferences which could be Bufo andrewsi: a preference for larger females. Journal of influenced by particular conditions of the popula- Ethology 27: 413-417. tion tested (e.g., density, time course of the breed- Liao, W.B., Lu, X. (2009b): Sex recognition by male Andrew’s toad Bufo andrewsi in a subtropical montane region. Behavioural ing activity, rainfall). Processes 82: 100-103. Liao, W.B., Lu, X. (2011): Proximate mechanisms leading to large male-mating advantage in the Andrew's toad Bufo andrewsi. Behaviour 148: 1087-1102. Liao, W.B., Lu, X. (2012): Variation in mating patterns in the Acknowledgments. We are very grateful to the staff of Andrew’s toad Bufo andrewsi along an elevational gradient in the Gahia-Zecha National Nature Reserve for providing southwestern China. Ethology Ecology and Evolution 24: 174- the laboratory and equipments for this research; to W. 186. Chen and C. Wang for their assistance in the field; to J. Lu, X., Ma, X.Y., Li, Y., Fan, L.Q. (2009): Breeding behavior and mating system in relation to body size in Rana chensinensis, a Merilä, R.E. Diaz, R. Taylor and D. Pike for comments on temperate frog endemic to northern China. Journal of Ethology the earlier drafts; and to anonymous referee for 27: 391-400. suggestions and comments on our manuscript. The study Loman, J., Madsen, T. (1986): Reproductive tactics of large and was funded by Joint Funds for Fostering Talents of NSFC small male toads Bufo bufo. Oikos 46: 57-61. and the People's Government of Henan Province (Grant Marco, A., Kiesecker, J.M., Chivers, D.P., Blaustein, A.R. (1998): Sex no. U1204306). recognition and mate choice by male western toads, Bufo boreas. Animal Behaviour 55: 1631-1635. 126 T.L. Yu & X. Lu

Marco, A., Lizana, M. (2002): The absence of species and sex Ryan, M. J., Bernal, X.E., Rand, A.S. (2010): Female mate choice and recognition during mate search by male common toads, Bufo the potential for ornament evolution in túngara frogs bufo. Ethology Ecology and Evolution 14: 1-8. Physalaemus pustulosus. Current Zoology 56: 343-357. Marquez, R., Bosch, J. (1997): Male advertisement call and female Shine, R., Phillips, B., Waye, H., Lemaster, M., Mason, R.T. (2003): preference in sympatric and allopatric midwife toads. Animal The lexicon of love: What cues cause size-assortative courtship Behaviour 54: 1333-1345. by male garter snakes? Behavioral Ecology and Sociobiology 53: Martínez-Rivera, C.C., Gerhardt, H. (2008): Advertisement-call 234-237. modification, male competition, and female preference in the Sullivan, B.K., Ryan, M.J., Verrell, P.A. (1995): Female choice and bird-voiced treefrog Hyla avivoca. Behavioral Ecology and mating system structure, pp. 469-517. In: Heatwole, H., Sullivan Sociobiology 63: 195-208. B.K., (eds), Biology, Vol. 2: Social behaviour. Mollov, I.A., Popgeorgiev, G.S., Naumov, B.Y., Tzankov, N.D. Chipping Norton, Surrey Beatty & Sons. Stoyanov, A.Y. (2010): Cases of abnormal amplexus in anurans Ursprung, E., Ringler, M., Jehle, R., Hödl, W. (2011): Strong (Amphibia: Anura) from Bulgaria and Greece. Biharean male/male competition allows for nonchoosy females: high Biologist 4: 121-125. levels of polygynandry in a territorial frog with paternal care. Olson, D. H., Blaustein, A.R.,Óhara, R.K. (1986): Mating pattern Molecular Ecology 20: 1759-1771. variability among western toad (Bufo boreas) populations. Wells, K.D. (2007): The Ecology and Behavior of Amphibians. Oecologia 70: 351-356. University of Chicago Press. Paterson, E.H. (1985): The recognition concept of species. pp. 21–29. Yu, T. L., Lu, X. (2010): Sex recognition and mate choice lacking in In: Vrba, E.S. (ed.), Species and Speciation. Vol. 4. Pretoria: the male Asiatic toads (Bufo gargarizans). Italian Journal of Transvaal Museum Monograph. Zoology 77: 476-480. Pfennig, K.S. (2000): Female spadefoot toads compromise on mate Yu, T. L., Lu, X. (2012): Mating pattern variability across three quality to ensure conspecific matings. Behavioral Ecology 11: Asiatic toad (Bufo gargarizans gargarizans) populations. North- 220-227. Western Journal of Zoology 8: 241-246. Reading, C.J. (1984): Interspecific spawning between common frogs (Rana temporaria) and common toads (Bufo bufo). Journal of Zoology 203: 95-101. Ridley, M. (1983): The Explanation of Organic Diversity: the Comparative Method and Adaptations for Mating. Clarendon Press, Oxford, UK.