Variation and Sexual Dimorphism of Body Size in the Plateau Brown Frog along an Altitudinal Gradient

Xueyun FENG， Wei CHEN， Junhua HUand Jianping JIANG

1Chengdu Institute of Biology， Chinese Academy of Sciences， Chengdu 610041， China

2University of Chinese Academy of Sciences， 19A Yuquanlu， Beijing 100049， China

3Ecological Security and Protection Key Laboratory of Sichuan Province， Mianyang Normal University， Mianyang 621000， China

Variation and Sexual Dimorphism of Body Size in the Plateau Brown Frog along an Altitudinal Gradient

Xueyun FENG1，2， Wei CHEN3， Junhua HU1*and Jianping JIANG1*

1Chengdu Institute of Biology， Chinese Academy of Sciences， Chengdu 610041， China

2University of Chinese Academy of Sciences， 19A Yuquanlu， Beijing 100049， China

3Ecological Security and Protection Key Laboratory of Sichuan Province， Mianyang Normal University， Mianyang 621000， China

Variation in body size and sexual size dimorphism （SSD） can have important consequences for animal ecology， behavior， population dynamics and the evolution of life-history traits. Organisms are expected to be larger in colder climate （i.e.， Bergmann's rule） and SSD varies with body size （i.e.， Rensch's rule）. However， the underlying mechanisms are still elusive. The plateau brown frog （Rana kukunoris）， a medium-sized anuran species with femalebiased SSD， is endemic to the Qinghai-Tibetan Plateau （QTP）. From 1797 m （Maoxiang'ping） to 3453 m （Heihe'qiao）in the eastern margin of the QTP， we surveyed 10 populations of R. kukunoris and collected phalanges and snout vent length （SVL） data for 258 adult individuals （199 males versus 59 females）. Based on these data， we explored how body size and SSD varying along the altitudinal gradient and examined the corresponding effects of temperature. We found body size to be larger at higher altitude for males but not for females， with likely effects from the temperature on the variation in male body size. Sex differences in growth rates may be the main cause of the variation in SSD. Our results suggested that only males follow the Bergmann's rule and variation in SSD of R. kukunoris do not support the Rensch's rule and its inverse. Therefore， the variations of body size can be different between sexes and the applicability of both Bergmann's rule and Rensch's rule should depend on species and environment where they live.

Altitudinal gradient， Bergmann's rule， body size variation， Qinghai-Tibetan Plateau， Rana kukunoris，Rensch's rule， sexual size dimorphism

1. Introduction

Body size is important for understanding animal ecology，behavior， population dynamics and the evolution of life history of a species （Morrison and Hero， 2003； Roff，2002）. Spatial and temporal variation in intraspecifc body size is the basis of evolution and adaptation to different environments （Chown and Gaston， 2010； Yom Tov， 2001）. In addition， body size of species is considered to beinfuenced by life history traits such as growth rate （Cohen and Alford， 1993） and age at sexual maturity （Morrison and Hero， 2003）. Amphibians are sensitive to subtle changes in surrounding environments and temperature has been recognized as the major factor affecting their body size （Laugen et al.， 2005； Laugen et al.， 2002；Laurila et al.， 2002）. As predicted in Bergmann's rule（Bergmann， 1848）， organisms tend to be larger in colder climates than those in warmer climates. Bergmann's rule was originally detected in mammals （e.g.， Ashton et al.，2000； Blackburn et al.， 1999）. Subsequently， it has been found in ectotherms， including amphibians （e.g.， Ashton and Feldman， 2003； Olalla Tárraga and Rodríguez， 2007）. An generally adaptive explanation for Bergmann's rule is heat conservation in cold environment， but its underlying causes may be diverse and even elusive （Gardner et al.，2011； Stevenson， 1985）.

Moreover， intraspecific difference in body size between males and females has been defined as sexual size dimorphism （SSD） that is likely a result of sexual and/or natural selection （Fairbairn， 1997）. Sexual selection can affect SSD through intra-sexual competition or inter-sexual mate choice favouring larger or smaller size in one sex （Andersson， 1994） and SSD may arise through natural selection when the sexes evolve to utilize resources in different way （Székely et al.， 2000）. Additionally， SSD also acts as a consequence of differences in age， growth rate and age at maturity between sexes （Fairbairn et al.， 2007； Monnet and Cherry，2002）. Rensch's rule predicts that SSD increases with increasing body size when males are larger than females（male-biased SSD）， but SSD decreases when females are larger than males （female-biased SSD； Fairbairn， 1997；Rensch， 1950）. In regards to Rensch's rule， interspecifc patterns with male-biased SSD have frequently been confirmed across different taxa， while female-biased SSD attracts relatively fewer attention （Fairbairn， 1997；Liao et al.， 2014； Stuart Fox， 2009）. In recent years，the intraspecific pattern of Rensch's rule have been examined （e.g.， Pungitius pungitius） （Herczeg et al.，2010）， and focused on male-biased SSD. This may result from the fact that the positive selection on body size for females was stronger than that for males （Herczeg et al.，2010； Teder and Tammaru， 2005）. Although females were suggested to be larger than males in 90 % anuran species（Shine， 1979）， SSD in amphibians always do not follow Rensch's rule （De Lisle and Rowe， 2013； Liao et al.，2013， 2014）.

The Plateau brown frog Rana kukunoris， a mediumsized anuran species with female-biased SSD， is endemic to the Qinghai-Tibet Plateau （QTP）， which distributed from 700 to 4400 m a.s.l. （Chen et al.， 2011； Fei et al.，2009）. Male frogs prefer large females as mates and males discriminate between the sexes based on their release calls（Fei et al.， 2009）. Although female fecundity is positively related to body size， females at higher altitudes do not produce larger clutch size with the reproductive strategy for producing high offspring viability rather than having massive clutches （Chen et al.， 2013a）. The eastern margin of the QTP is unique for the steep variation of altitude from 1200 to 3800 m a.s.l. within 50 kilometers and harsh environmental conditions （annual mean temperature：14.1-1.1°C； annual total precipitation： 1332-649 mm；frost-free period： 269-120 days； http：//www.maoxian. gov.cn/； http：//www.ruoergai.gov.cn/）. Therefore， it is important for us to understand how a frog species may adapt to a specifc environmental conditions on the QTP.

In this study， we aim to： 1） explore how body size of R. kukunoris varies along the altitudinal gradient and the effect of temperature on body size； 2） examine whether age， growth rate and temperature affecting the variation in SSD， and 3） test both the Bergmann's rule and the Rensch's rule for R. kukunoris.

2. Materials and Methods

We surveyed 10 populations of R. kukunoris from 1797 m a.s.l. （Maoxiang'ping， Maoxian County） to 3453 m a.s.l.（Heihe'qiao， Zoige County） in the eastern margin of QTP（Figure 1）. A total of 258 adult individuals （199 males versus 59 females） were collected from late March to early May 2014 （Table 1）. Frogs were randomly caught by hand in their spawning habitats. We identifed individuals as sexually-mature males and females if they displayed nuptial pads on the fore digits and had well-developed oocytes （through visible externally）， respectively （Fei et al.， 2009）. We measured body size （snout vent length；SVL） of each adult to the nearest 0.02 mm with digital calipers. The longest phalange of the hindlimbs was removed and stored in 10% neutrally buffered formalin for subsequent age determination. Altitude of each population was recorded in the field by the Magellan eXplorist 310 GPS and average annual temperature was extracted from the data of Hijmans et al. （2005）.

Age was estimated following the method adopted by Liao and Lu （2010）. Cross-section of 12 μm of the mid-shaft diaphysis of the second phalanx with the smallest medullar cavity was selected and mounted on glass slides. The lines of arrested growth （LAGs） were indentifed under a microscope by two persons with equal experiences with agreed criteria for characterization. Due to the distinct temperature cycles through the year within the study area， the number of LAGs corresponds to the age of frogs （Chen et al.， 2011）. Since the phalanges were collected during the breeding season， we regarded the surface of the bone as an additional LAG （Lai et al.，2005； Morrison et al.， 2004）. According to the protocol of Wake and Castanet （1995）， we identifed the endosteal resorption of LAGs based on the presence of the Kastschenko line （KL； the interface between the endosteal and persiosteal zones； Rozenblut and Ogielska， 2005）.

The index of SSD was calculated as log10（mean female size） - log10（mean male size）. The index of sexual age dimorphism （SAD； the difference between mean male age and mean female age） of each population was calculated as log10（mean female age） - log10（mean male age） （Liaoet al.， 2014； Smith， 1999）.

Sex differences in mean body size and age among populations were examined using the generalized linear mixed models （GLMMs） with SVL and age as dependent variables， sex as a fixed factor， and population as a random effect. To control the effect of age on body size，age was added as covariate together with the interactions of sex × age and sex × population. The signifcant sex × age interaction would be suggestive of sex differences in growth rates and the significant sex × population interaction would provide evidence for variation in SSD among populations. Pearson's correlation analysis was used to evaluate the relationship between body size and altitude as well as the relationships between SSD and age-at-maturity， sex ratio， altitude， SAD and temperature. Partial correlation analysis was also adopted to decrease the effect of age on body size. General linear model（GLM） was conducted for testing the relationships between body size and both temperature and altitude，with SVL as a dependent variable， sex as a fxed factor，temperature， altitude and age as covariates.

To test the Rensch's rule， we conducted a regression for body size of females versus males. Least squares regression is inappropriate for testing the allometry because it assumes that the independent variable （here female size） is measured without error （Fairbairn， 1997）. The reduced major axis （RMA） slope can be estimated as the ratio of male to female standard deviations（Sokal and Rohlf， 1995）. Therefore， we used RMA regression of log10（male size） against log10（female size）using the population means to test the Rensch's rule. When log10（female size） was plotted on the x-axis and log10（male size） plotted on the y-axis， a slope larger than 1.0 provided evidence for Rensch's rule （Fairbairn， 1997）. The RMA regressions were performed in RMA software（http：// www.bio.sdsu.edu/ pub/ andy/ rma.html； Bohonak and van der Linde 2004）.

Our statistical analyses were performed in SPSS 19.0 software （IBM Corporation， Chicago）. All variables were log-transformed to meet the assumption of normality of variances. All probabilities were two tailed， and the signifcant level was at P ＜ 0.05.

This study did not involve endangered or protected species. All the animal operations were approved by the Institutional Ethical Committee of Animal Experimentation of Chengdu Institute of Biology，Chinese Academy of Sciences， and strictly complied with the ethical conditions by the Chinese Animal Welfare Act（20090606）.

3. Results

Age at sexual maturity was 2-3 years across populations without difference between males and females （Table 1）.Mean age of individuals differed among populations （Z = 10.71， P ＜ 0.01） but not between sexes （F1，1，197.57= 3.28， P = 0.07）.

Table 1 Studied populations of Rana kukunoris along an altitudinal gradient with body size （snout vent length； mean ±SD） and age of males and females for each population respectively.

Body size signifcantly differed among populations （Z = 10.70， P ＜ 0.01） and females were larger than males（F1，1，197.57= 46.88， P ＜ 0.01）. When the effect of age（F1，197.57=11.11， P = 0.01） was controlled， body size still significantly differed among populations （Z = 10.68， P＜ 0.01） and between sexes （F1，1，197.57= 43.26， P ＜ 0.01）. The signifcant sex × age interaction （F2，1，197.57= 13.22， P＜ 0.01） showed that the relationship between body size and age （≈ growth rate） differed between sexes. The significant interaction between population and sex （Z = 10.4， P ＜ 0.01） indicated that the degree of SSD differed among populations. Moreover， both temperature and altitude had signifcant effects on body size （temperature：F3，254= 41.54， P ＜ 0.01； altitude： F3，254= 52.72， P ＜ 0.01）. Males tended to be larger in higher altitude but not for females （males： r = 0.28， P ＜ 0.01； females： r = 0.16， P = 0.22）. These trends of body size along the altitudinal gradient were similar when controlling the effect of age（males： r = 0.23， P = 0.01； females： r = -0.18， P = 0.17）.

The degree of SSD did not correlate with age at sexual maturity （r = -0.15， P = 0.69）， operational sex ratio （r = -0.01， P = 0.99）， altitude （r = -0.30， P = 0.43）， SAD （r = 0.41， P = 0.27） and temperature （r = 0.17， P = 0.66）. The slope of the RMA regression of log10（male size） on log10（female size） across populations was not signifcantly different from 1 （slope = 0.701， 95% CI = 0.21 - 1.20， R2= 0.379， P = 0.071）.

4. Discussion

For amphibians， variation in body size is frequently shown to follow the Bergmann's rule and is sometimes suggested to follow its inverse （e.g.， Ashton， 2002； Iturra Cid et al.， 2010； Liao and Lu， 2010）. For R. kukunoris，our results showed that the body size variation of males supported the Bergmann's rule， but not females （Figure 2）. This is consistent with the different patterns of altitudinal variation in body size between sexes （Hsu et al.， 2014）. Comparing with Chen et al. （2013a），we further found that the female body size tend to be constant along the altitudinal gradient. However， Chen et al. （2011） indicated that both males and females tend to be smaller at higher altitudes. This divergence may be induced by the different populations examined between this study and Chen et al. （2011）.

Life history theory predicted that female body size is often related to fecundity （Monnet and Cherry， 2002）. Fecundity hypothesis predict that female should invest more energy into somatic growth and delay sexualmaturity to reach a larger body size and to attain higher fecundity （Iturra Cid et al.， 2010； Sinsch et al.， 2010）. However， females' body size in this study did not vary among populations， and clutch size of them also did not increase with increasing altitude （Chen et al.，2013a）. Shorter activity time periods， combined with physiological limits on prey digestion may constrain female body size （Morrison and Hero， 2003； Roff， 2002）. The fact that larger individuals may be unable to both recover energy spent on reproduction and store necessary energy to survive the coming winter （Chen et al.， 2013a）may explain why there was no altitudinal trend of female body size.

Body size can be strongly affected by environment conditions （Teder and Tammaru， 2005）. For example，anurans at low temperatures grow slower towards the asymptotic body size and reach maturity later than those exposed to warmer temperatures （Morrison and Hero，2003）. In this study， the temperature gradient was not only the factor affecting the body size variation in R. kukunoris. At higher altitudes， the shorter activity time restricts the opportunity for energy accumulation. Hence，the frogs， especially females with a larger body size could be costly at higher altitudes， where shorter activity seasons restrict the amount of energy that an individual can accumulate and store prior to overwintering （Chen et al.，2013b）. Due to lower temperatures， individuals from higher altitudes reach sexual maturity later than those in lower altitudes. Moreover， higher ones could allocate more energy to reach larger body size （Morrison and Hero， 2003）.

Sex differences in growth rate have potential effects on the variation in SSD （Fairbairn et al.， 2007； Monnet and Cherry， 2002）. However， we find that variation in SSD among populations is signifcant when the effect of age is controlled and insignifcant correlation relationship between SSD and SAD. These suggest that age or age structure is unlikely to explain variation in SSD for R. kukunoris. Moreover， differences in age at maturity， sex ratio， altitudes and temperature among populations could not explain variation of SSD as they were not correlated with SSD. In contrast， a significant difference in the relationship between body size and age of males and females across populations is found. This suggests that the differences in growth rates between sexes may explain the variation in SSD.

In R. kukunoris， variation in SSD do not support the Rensch's rule （Figure 3） as the allometric relationship between male size and female size is not significantly differed from the isometric relationship. This pattern has also been found in other taxa with female-biased SSD（e.g.， Liao et al.， 2013）. A potential explanation for this is that fecundity selection on females favoring size balancesrather than sexual selection in favor of larger males（Liao et al.， 2013）. However， the limited sample size for females in this study may have effects on the insignifcant results.

Acknowledgements We thank Song HE and Donghan HOU for their assistance in the field survey. We also thank Zhiping MI， Shangling LOU， Janak Raj KHATIWADA， Yan HUANG and Chen YANG for their help in skeletochronology method and/or the data analysis. We are grateful to the two reviewers for providing constructive comments on an early version of this paper. This study was supported by the National Natural Science Foundation of China （Nos. 31270568；31572290）， the Youth Innovation Promotion Association CAS （2015304）， and the Sichuan Province Distinguished Youth Fund （2014JQ0056）.

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Prof. Junhua HU， from Chengdu Institute of Biology， Chinese Academy of Sciences， Chengdu， China， with his research interest in understanding the mechanisms of patterns for species richness， functional traits and phylogeny across scales， and the impacts of environmental change on wildlife； Prof. Jianping JIANG，from Chengdu Institute of Biology， Chinese Academy of Sciences，Chengdu， China， with his research focusing on taxonomy， systematics and evolution of amphibians and reptiles.

E-mail： hujh@cib.ac.cn （J. H. HU）； jiangjp@cib.ac.cn （J. P. JIANG）

13 April 2015 Accepted： 29 June 2015