Acta Herpetologica 11(1): 69-73, 2016 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.13128/Acta_Herpetol-15529 No evidence for the ‘expensive-tissue hypothesis’ in the dark-spotted frog, Pelophylax nigromaculatus Li Zhao, Min Mao, Wen Bo Liao* Key Laboratory of Southwest China Wildlife Resources Conservation (Ministry of Education), China West Normal University, Nanchong 637009, P. R. China. *Corresponding author. E-mail: Liaobo_0_0@126.com Submitted on 2015, 10th February; revised on 2015, 9th November; accepted on 2016, 15th January Editor: Giovanni Scillitani Abstract. Increased brain size significantly contributes to the performance and fitness of organisms. The expensive-tis- sue hypothesis (ETH) based on studies of the correlation between brain size and size of the other energetically costly organs in mammals predicts that energy investment increased in one energetically costly tissue necessitates a decrease of investments in other costly tissues. Here, we test this hypothesis in an ectothermic species, the dark-spotted frog, Pelophylax nigromaculatus. We found that relative brain size was not correlated with relative sizes of testes, heart, liv- er, spleen, kidneys or limb muscles within each sex. Moreover, we also failed to find significantly negative correla- tion among the expensive organs (i.e., testes, heart, liver, spleen, kidneys or limb muscles) in this frog. However, we observed a significantly positive correlation between liver residuals and kidney residuals. Our finding suggests that energetic costs of one expensive tissue do not direct necessarily affect the investment in another expensive tissue, but rather may scatter its effect on all other expensive tissues. Keywords. Pelophylax nigromaculatus, brain size, expensive-tissue hypothesis. Brain size is an important characteristic that affects the performance and fitness of organisms (Allman, 2000; Liao et al., 2015a). Brain size is often used as an indicator of the brain’s evolutionary development state in response to cognitive benefits (see review, Striedter, 2005). Previ- ous studies have indicated that variations in brain size can be explained by the social brain hypothesis, the expensive-tissue hypothesis (ETH) and the sexual selec- tion hypothesis (Aiello and Wheeler, 1995; Pitnick et al., J 2006; Dunbar and Shultz, 2007; Barton and Capel- lini, 2011). However, the brain needs more energy per unit weight than the other somatic tissues, making it metabolically expensive (Mink et al., 1981). According to the ETH, investment in one metabolically costly tis- sue requires a decrease of investments in other tissues (Aiello and Wheeler, 1995). In the past two decades, the ETH has received mixed support in vertebrates (Aiello and Wheeler, 1995; Jones and Maclarnon, 2004; Isler and van Schaik, 2006; Pitnick et al., 2006; Barrickman and Lin, 2010; Navarrete et al., 2011). Some studies find that investment in a metabolically expensive tissue is negative significantly correlated with the other metaboli- cally expensive tissues, thus supporting the ETH (Aiello and Wheeler, 1995; Kaufman et al., 2003; Isler and van Schaik, 2006; Barrickman and Lin, 2010; Jin et al., 2015; Tsuboi et al., 2015). However, other studies found that there were either non-significant or even positive correla- tions between metabolically costly tissues (Isler and van Schaik, 2006; Lemaître et al., 2009; Barrickman and Lin, 2010; Navarrete et al., 2011; Liu et al., 2014). Most studies of the ETH have been performed among species, though some intraspecific studies have been con- ducted (fish, Warren and Iglesias, 2012; Liu et al., 2014; frogs, Jin et al., 2015). However, no consistent results have confirmed the patterns of intraspecific variation in brain size, and the ETH in anurans need further study. In this 70 Li Zhao et alii study, we tested the ETH in the dark-spotted frog Pelophy- lax nigromaculatus, a species widely distributed in plains, hilly paddy fields, ponds, lakes, rivers and mountains at an altitude below 2200m (Fei and Ye, 2001). Previous studies of the ETH have recognized brain, heart, kidneys, liver, testes and gut tissues as being metabolically costly (Aiel- lo and Wheeler, 1995; Pitnick et al., 2006; Isler and van Schaik, 2006; Barrickman and Lin, 2010; Navarrete et al., 2011; Warren and Iglesias, 2012). Our aim was to explore whether brain size was negatively correlated with any oth- er organs (i.e., heart, liver, spleen, kidneys, testes, and limb muscles mass) in P. nigromaculatus. We captured specimens by hand in ponds in Yingxi Town of Nanchong city (30°50’N, 106°07’E, 338 m a.s.l.) in Sichuan, China (Mao et al., 2014). All individuals were caught at night during the breeding season from April 11 to 19 in 2010. We collected a total of 84 individuals (45 males and 39 females), and then brought them to the laboratory. The sex was determined by observing the dif- ferences in secondary sex characteristics. Before process- ing, the frogs were kept in rectangular tanks (1.0*0.5*0.4 m; L*W*H) with a water depth of 5 cm at room tempera- ture. Animals were sacrificed by using double-pithing. We measured body size (snout to vent length, SVL) to the nearest 0.01 mm by a caliper, and body mass to the nearest 0.1 mg by an electronic balance. Frogs were dis- sected and all tissues (i.e., brains, livers, hearts, spleen, kidneys, testes, and limb muscles) were removed and weighed. We analyzed the male and female data sepa- rately. All data were log-transformed prior to analysis. In order to control the effect of body mass on energeti- cally expensive tissues, we used relative size of expensive tissues to analyze correlations between brain size and the other organs. The relative size of the expensive tissue was the residual of observed expensive tissues size to that predicted on the basis of the regressions of brain, heart, spleen, kidneys, liver, testes mass and limb muscles on body mass. We estimated body condition using the resid- uals from a regression of body mass on SVL. Some tis- sues were affected by body condition. If the effect of body condition on each of the expensive tissues was significant, we then used a partial analysis and body condition as a covariate to test relationships between brain mass and each of the expensive tissues. Statistical tests were para- metric, using Type III sums of squares tests with the SPSS (22.0) statistical package. In the male dark-spotted frog, P. nigromaculatus, the results showed that brain residuals did not negatively correlate with residuals of testes, heart, liver, spleen, kid- neys or limb muscles (Table 1). For other tissues (testes, Table 1. Regressions of brain mass residuals and body condition on other organ size residuals in male Pelophylax nigromaculatus. Coef- ficient estimates from regressions are given with 95% CI in brackets, and β and P-values associated with each regression are also provided. Organ size Brain mass Body condition Estimates [±95% CI] β P-value Estimates [±95% CI] β P-value Testes 0.010[-0.205,0.224] 0.015 0.927 0.017[-0.082,0.115] 0.056 0.731 Heart -0.051[-0.388,0.286] -0.047 0.761 0.111[-0.265, 0.043] -0.217 0.152 Liver 0.086[-0.223,0.394] 0.085 0.578 0.104[-0.038,0.055] 0.220 0.147 Spleen -0.027[-0.144,0.090] -0.071 0.644 -0.009[-0.064,0.046] -0.052 0.733 Kidneys 0.158[-0.158,0.473] 0.152 0.320 -0.003[-0.153,0.146] -0.007 0.965 Limb muscles 0.428[-0.324,1.179] 0.172 0.257 0.048[-0.309,0.405] 0.041 0.788 Table 2. Regressions of brain mass residuals and body condition on other organ size residuals in female Pelophylax nigromaculatus. Coef- ficient estimates from regressions are given with 95% CI in brackets, and β, P-values associated with each regression are also provided. Organ size Brain mass Body condition Estimates [±95% CI] β P-value Estimates [±95% CI] β P-value Heart -0.080[-0.393,0.239] -0.085 0.605 -0.292[-0.308,-0.277] -0.252 0.121 Liver 0.050[-0.193,0.293] 0.069 0.678 -0.083[-0.238,0.073] -0.175 0.287 Spleen 0.012[-0.111,0.135] 0.032 0.847 -0.063[-0.140,0.014] -0.264 0.104 Kidneys 0.215[-0.058,0.489] 0.254 0.119 -0.023[-0.206,0.161] -0.041 0.804 Limb muscles 0.063 [-0.591,0.716] 0.032 0.847 -0.370[-0.775,0.036] -0.290 0.073 71The expensive-tissue hypothesis lacking in a frog liver, spleen, kidneys, limb muscles), we found only one significant positive correlation between the liver residu- als and the kidney residuals (Fig. 1a). We found the same relationships between metabolically expensive tissues in females (Table 2, Fig. 1b). The results of our study did not find a significant correlation between body condition and other organs either in males or females (Table 1, Table 2). However, a significantly positive correlation between liver residu- als and kidney residuals still existed when controlling for the effect of body condition (male: r = 0.442, P = 0.005; female: r = 0.527, P < 0.001). The expensive tissue hypothesis states that  organisms can reduce the size of other expensive tissues in their body to maintain a relatively larger brain size (Aiello and Wheeler, 1995). Previous most convincing studies in favor of the expensive tissue hypothesis result from ecto- thermic animals such as the elephant nose fish Gnatho- nemus petersii (Kaufman et al., 2003), or the guppy Poe- cilia reticulata (Kotrschal et al., 2013), 73 species of Lake Tanganyika cichlids (Tsuboi et al., 2015), the Omei wood frog Rana omeimontis (Jin et al., 2015). In contrast to the ETH, we did not find clear evidence to support this hypothesis in P. nigromaculatus. There is a similar study investigated ETH in a fish which did not find support for ETH (Liu et al., 2014). The energy trade-off hypothesis predicts that organ- isms, in order to maintain relatively larger brain, will reduce investment in reproduction (Isler and van Schaik, 2006). Pitnick et al. (2006) found significantly negative correlations between investment in testes and brains in bats, supporting this theory. However, we did not find a negative correlation between the size of the brain and tes- tes in the dark-spotted frog. There are two possible evo- lutionary paths to fuel energetic requirements for brain enlargement in animals: (i) increase overall energy budg- et and (ii) re-allocate energy budget. The metabolic con- straints hypothesis concerns the first possibility, and ETH and the energy trade-off hypothesis concern the second possibility. The lack of support for the ETH found in this study suggest that energetic constraints operates in frog brain size evolution, despite copious evidence that brain tissue is metabolically costly to develop and maintain in another frog (Jin et al., 2015). This difference in the ETH between the two frogs’ species might relate to difference in their habitat use (Fei and Ye, 2001). Muscle tissue can consume a considerable pro- portion of the organism’s energy at rest and it must therefore be included in the ETH (Aiello and Wheeler, 1995). For amphibians, individuals with high energy costs of locomotive capability have strong ability to search for mates and to avoid predation (Duellman and Trueb, 1986; Liao et al., 2012; Jin et al., 2015; Liao et al., 2015b). As a result, individuals with greater reproductive fitness selected larger brains are associ- ated with an elevated cognitive ability (i.e., the ability to process information; Striedter, 2005). However, we did not find an increase brain mass increasing with the mass of limb muscles, as proxy for the costs of locomo- tion. The kidneys are the primary organs for excreting metabolic waste and maintaining pH balance in organ- isms (Moore, 1995; Ganong, 2005). The liver is one of the most important energy storage organs in animals (Ji et al., 2002; Yang and Wu, 2006). The ETH predicts a significant negative correlation between the size of the brain and both the liver and the kidneys. Nonetheless, our findings demonstrated that there were no significant relationships among them in P. nigromaculatus. The critically important organs (testes, liver, spleen, kidneys, and limb muscles) can change in size and met- abolic activity in different life-cycle periods (Piersma, Fig. 1. Correlations between residuals liver mass and residuals of kidneys mass in Pelophylax nigromaculatus, controlling for body condition (a: male; b: female). 72 Li Zhao et alii 2002). In this study, we did not find significant relation- ships between brain mass and organ mass of any of the major metabolically expensive organs. This lack of cor- relation supports the hypothesis that energetic costs of one expensive tissue do not necessarily directly affect the investment in another expensive tissue, but rather may scatter its effect on all other expensive tissues (Lemaî- tre et al., 2009). Moreover, we did not find evidence that there were negative correlations among testes, spleen, limb muscles, liver or kidneys, suggesting that there was no trade-offs between metabolically expensive tissues due to differences in activity level or growth among individu- als. However, we found a positive correlation between liver and kidneys in both sexes. ACKNOWLEDGEMENTS We thank the National Sciences Foundation of China (31471996), Sichuan Province Outstanding Youth Aca- demic Technology Leaders Program (2013JQ0016) and Sichuan Province Department of Education Innovation Team Project (14TD0015; 15TD0019) for providing the financial support. The reported experiments comply with the current laws of China concerning animal experimen- tation, and permission to collect frogs was received from the Ethical Committee for Animal Experiments in China West Normal University. REFERENCES Aiello, L.C., Wheeler, P. (1995): The expensive-tissue hypothesis – the brain and the digestive system in human and primate evolution. Curr. Anthropol. 36: 199-221. Allman, J. (2000): Evolving Brains. Scientific American Library, New York. Barrickman, N.L., Lin, M.J. (2010): Encephalization, expensive tissues, and energetics: An examination of the relative costs of brain size in Strepsirrhines. Am. J. Phys. Anthropol. 143: 579-590. Barton, R.A., Capellini, I. 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