Lixia ZHANG,Xiangyu YUAN,Yongsun SHENG,Fei YU,Xueting ZHONG,Xiaohong CHEN＊ and Dingqi RAO

1 Department of Ecology,College of Life Sciences,Henan Normal University,Xinxiang 453007,Henan,China

2 Key Laboratory of Vector Biology and Pathogen Control of Zhejiang Province,College of Life Sciences,Huzhou University,Huzhou 313000,Zhejiang,China

3 State Key Laboratory of Genetic Resources and Evolution,Kunming Institute of Zoology,Chinese Academy of Sciences,Kunming 650223,Yunnan,China

Abstract Theory predicts that well-developed limb musculature can result from sexual selection favoring strong males.We tested for this prediction in the Boulenger’s lazy toad (Scutiger boulengeri),a species that exhibits inguinal amplexus.As expected,we found that males had more massive forelimbs and hindlimbs than those of females.In addition,amplectant males had relatively more massive hindlimbs than non-amplectant males.This pattern can be explained by sexual selection,as the greater forelimb muscles allow males to hold females more tightly and massive hindlimbs likely confer a locomotor advantage in defending mates.This study contributes to an increasing body of literature clarifying the role of sexual selection in producing sexual dimorphism in anuran limbs.

Keywords inguinal amplexus,limb muscles,Scutiger boulengeri,sexual dimorphism,sexual selection

1.Introduction

In anurans,as in other vertebrates,the sexes often differ in a variety of traits (Shine,1979).The relative size of the limbs is one of which that exhibits sexual dimorphism consistently(Lee,2001; Mi,2012).Males tend to have more robust limbs,though females are substantially larger in body size (Peters and Aulner,2000; Lee and Corrales,2002).The cause of these sexual divergences is generally attributed to sexual selection,because males typically use their forelimbs to hold females and hindlimbs to kick attackers during amplexus (Duellman,1992; Duellman and Trueb,1994).Therefore,males with robust forelimbs are not only more able to resist take-over attempts by unpaired males,but are also able to resist females’ attempts to dislodge amplectant males by inflating their bodies (Bruninget al.,2010).In addition,the relatively massive hindlimbs of males are believed to enhance mating success in scramble competition(Vargas-Salinas,2005; Mi,2013).Many studies have been carried out to investigate this topic in anurans with axillary amplexus,however,none of the studies have been done in species that exhibit inguinal amplexus.

The Boulenger’s lazy toad (Scutiger boulengeri)is an anuran species endemic to the eastern Tibetan Plateau where it has an extensive altitudinal range (3300 to 5100 m) (Feiet al.,2009).During the mating period (June to August),males clasp females around their waist,exhibiting typical inguinal amplexus.However,little is known so far about the evolution of sexual dimorphism in limb muscles of this toad.The aims of this study were to assess the nature of sexually dimorphic muscles in limbs.Both forelimbs and hindlimbs were expected to be important in mating success and under sexual selection,thus,we predicted that the limb muscles mass of males significantly exceeded those of females and amplectant males significantly exceeded those of non-amplectant males.

2.Materials and Methods

2.1.Data collectionWe examined 37 preserved museum specimens (25 males and 12 females) ofS.boulengericollected during the breeding seasons of 2015 at Songduo (20.89°N,92.45°E,4351 m elevation),Medro Gongka County of Tibet.The specimens,12 of which were amplectant and 13 of which were non-amplectant males,had been stored in 10% neutral buffered formalin.The former was characterized as successful maters during breeding season,and the latter was defined as failed ones without regarding the fact that they may have been mated with a female prior to capture.The amplectant pairs were sampled first and then non-amplectant males,Therefore,differences between the two types of males are likely to reflect true variations in a given population.Their body size (snout to vent length,SVL) was measured to the nearest 0.1 mm with digital calipers.Then,five forelimb muscles (pectoralis,deltoideus,triceps branchii,flexor carpi radialis and extensor digitorum) and eight hindlimb muscles (triceps femoris,biceps femoris,semimembranosus,sartorius,adductor longus,gracilis major,gracilis minor and gastrocnemius) were separated from the right side of each specimen and dried for 48 hours to a constant mass using a thermostat drier of 60°C.The dry mass of these muscles was weighted using an electronic balance to the nearest 0.1 mg.We chose these muscles because they may act in clasping females and kicking rivals during scramble competition among anuran species engaging in amplexus.

2.2.Statistical analysesThe body size between the sexes and between amplectant and non-amplectant males were examined using independent-samplesttest.The differences of muscle mass between the sexes and between the two types of males were assessed by general linear models with muscles mass as dependent variables,sexes/male mating category as a fixed factor,and SVL as a covariate.The total muscle mass of both sexes and for the two type males were regressed on SVL.In addition,we tested the significance of differences in adjusted means by analysis of covariance (ANCOVA).Before analysis,all morphometric variables were transformed to their natural logarithm to correct for normality and improve homogeneity of variances.All probabilities were two-tailed,and the significance level was set atP=0.05.Data are presented as mean ± SD.

3.Results

3.1.Comparison between males and femalesOn average,females significantly exceeded males in SVL (females,(62.50± 6.12) mm,n=12; males,(52.61 ± 3.70) mm,n=25;t=6.12,df=35,P＜ 0.001).Four forelimb muscles (pectoralis,deltoideus,triceps branchii and flexor carpi radialis) and the total forelimb muscle mass differed significantly between the sexes when the influence of SVL was controlled,but the mass of the extensor digitorum muscle did not differ between the sexes (Table 1).Linear regression of total forelimb muscle mass on SVL was highly significant for both sexes (Figure 1A,males:r2=0.157,F=4.28,P=0.050; females:r2=0.671,F=20.39,P=0.001).The differences between the adjusted means of males and females are highly significant by ANCOVA for the size of four muscles and for the total forelimb mass (muscle pectoralis:F1,34=5.27,P=0.028; muscle deltoideus:F1,34=10.24,P=0.003; muscle triceps branchii:F1,34=5.54,P=0.024; muscle flexor carpi radialis:F1,34=44.38,P＜ 0.001; total forelimb muscle mass,F1,34=22.13,P＜ 0.001).

Regarding hindlimbs,the gender differences presented in seven muscles (triceps femoris,biceps femoris,semimembranosus,adductor longus,gracilis major,gracilis minor,and gastrocnemius) and the total hindlimb muscle mass when body size was controlled,and only one muscle (sartorius)mass did not differ between the sexes (Table 1).The total hindlimb muscle mass regressed significantly on SVL within each sex (Figure 1B,males:r2=0.261,F=8.12,P=0.009; females:r2=0.655,F=19.02,P=0.001).By ANCOVA analysis,the adjusted means of males significantly exceeded those of females for the size of seven muscles and for total hindlimb mass when the influence of SVL was controlled (muscle triceps femoris:F1,34=8.34,P=0.007; muscle biceps femoris:F1,34=8.68,P=0.006; muscle semimembranosus:F1,34=25.41,P＜ 0.001; muscle adductor longus:F1,34=10.81,P=0.002; muscle gracilis major:F1,34=30.37,P＜ 0.001; muscle gracilis minor:F1,34=17.34,P＜0.001; muscle gastrocnemiusF1,34=28.06,P＜ 0.001; total hindlimb muscle mass,F1,34=31.72,P＜ 0.001).

For 11 of the 13 muscles (both forelimbs and hindlimbs)examined,the adjusted means of males significantly exceeded those of females.A broad range of dimorphism existed in both the forelimb and hindlimb muscles (35%-108%),and the flexor carpi radialis and the gastrocnemius were the most dimorphic muscles in the forelimb and hindlimb,respectively (Table 1).

3.2.Comparison between amplectant and non-amplectant malesThe mean body size did not differ significantly between amplectant and non-amplectant males (amplectant males,(53.05 ± 3.41) mm,n=12; non-amplectant males,(52.20 ± 4.04)mm,n=13;t=0.57,df=23,P=0.576).When controlling for the influence of body size,only muscle deltoideus differed significantly between the amplectant and non-amplectant males (Table 2).Linear regression of the total forelimb muscle mass on SVL was highly significant for non-amplectant males,but not for amplectant males (r2=0.485,F=10.37,P=0.008;r2=0.012,F=0.12,P=0.735; respectively).The ANCOVA analysis revealed that the differences between the adjusted means of amplectant and non-amplectant males are highly significant for muscle deltoideus (F1,22=7.91,P=0.010).

When controlling for the influence of body size,three hindlimb muscles (triceps femoris,semimembranosus,and gracilis major) and the total hindlimb muscle mass differed significantly between the amplectant and non-amplectant males (Table 2).Linear regression of the total hindlimb muscle mass on SVL was significant for non-amplectant males,but not for amplectant males (r2=0.401,F=7.36,P=0.020;r2=0.110,F=1.24,P=0.292; respectively).The ANCOVA analysis revealed that the adjusted means of amplectant males significantly exceeded those of non-amplectant males for the mass of three muscles and for the total hindlimb mass when the influence of SVL was controlled (ANOVA:muscle triceps femoris:F1,22=10.09,P=0.004; muscle semimembranosus:F1,22=5.64,P=0.027; muscle gracilis major:F1,22=4.97,P=0.036; total hindlimb muscle mass,F1,22=5.23,P=0.028).

4.Discussion

The results of this study demonstrate that adult males ofS.boulengerisignificantly exceed adult females in the mass of both forelimb and hindlimb muscles,though females are larger than males.This is consistent with previous works that have shown that male anurans have relatively more robust limbs than female anurans under sexual selection.In addition,the total mass of hindlimb muscles between amplectant and nonamplectant males differed significantly inS.boulengeri.

Life-history models suggest that many factors can influence the evolution of sexual dimorphism,such as mating behavior(Roff,2002).Amplexus is a common reproductive behavior in anurans and the male-biased sex ratio is normal,so males often engage in scramble competition for mates (Wells,2010; Loman and Madsen,2010; Liao and Lu,2012).Under such context,the robust forelimbs can allow males to retain a firm grip on the female in amplexus and the relatively larger hindlimbs likely confer a locomotor advantage in defending mates (Lee,2001; Clark and Peters,2006).So,males with massive limbs can function efficiently,and thus the dimorphism emerges(Lee and Corrales,2002; Liaoet al.,2012; Mi,2013).These predictions were confirmed by our study.In addition,the extent of dimorphism is an important employee in functional roles(Andersson,1994).InS.boulengeri,a broad range of dimorphism existed in both forelimb and hindlimb muscles.Muscles involved in the clasping action and fighting among males were predicted to be the most dimorphic of the muscles (Warburtonet al.,2013; Richardset al.,2015).Indeed,the flexor carpi radialis and the gastrocnemius are the most dimorphic muscles in the forelimb and hindlimb,respectively.The former is a primary forelimb flexor and its essential function is to flex the wrist.The latter is a premier muscle crossing the knee and it plays an important role in biomechanics.These results suggest that these muscles are likely important in inguinal amplexus.

Figure 1 Linear regression of the total mass of forelimb (A) and hindlimb muscles (B) on SVL for males (close circles,solid line) and females (open circles,broken line) in the Boulenger’s lazy toad S.boulengeri in eastern Tibetan Plateau.

Table 1 Comparisons of mass of male and female limb muscles in S.boulengeri using general linear models (GLMs) analysis.

Table 2 Comparisons of mass of amplectant and non-amplectant male limb muscles in S.boulengeri using general linear models (GLMs)analysis.

In vertebrate animals,muscle mass has significant positive associations with physical activity (Baumgartneret al.,1999;Nakaoet al.,2006).For anurans,the positive relationship between the tetanic force and muscle mass has been confirmed,and certain limb muscles of males were less fatigable than those of females (Peters and Aulner,2000; Navas and James,2007).So,the greater limb muscle masses attained by males can allow them to hold and defend mates more firmly and extendedly.ForS.boulengeri,the pattern of sexual dimorphism in limb musculature was recapitulated by comparing the limb muscles of amplectant and non-amplectant males,and amplectant males have relatively more robust hindlimbs than nonamplectant males.Proximate reasons for this were mainly due to the relatively more robust thigh muscles in amplectant males.This finding is generally true in anuran species,indicating that sexual selection is operating on males.

In conclusion,the results of our study revealed that the sexual dimorphism in limbs musculature was evident inS.boulengeri,with males having more massive limbs than females.To verify the conclusion,more studies are required to explore the inter-sexual differences in limb musculature of other species with inguinal amplexus.In addition,studies that compare sexual dimorphism in musculature for species that exhibit different types of amplexus and that reveal functional attributes of limb muscles in living anurans can shed lights on this issue.The prediction that the intensity of sexual selection positively covaries with the degree of sexual dimorphism in anuran limbs remains to be tested by future research efforts.

AcknowledgmentsWe thank Xin LU,Bohao FANG,Zhibing LI and Yuxiao HE for field and laboratory assistance and thank Jianping JIANG for improving the language and giving some valuable suggestion to the early draft.This study was supported by the Joint Funds for Fostering Talents of NSFC and the People’s Government of Henan Province (No.U1304309) and National Sciences Foundation of China (No.31501870 and No.31872216).