Acta Herpetologica 13(2): 141-146, 2018 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.13128/Acta_Herpetol-20920 The relationship between brain size and digestive tract length do not support expensive-tissue hypothesis in Hylarana guentheri Ya Ting Liu, Yi Luo, Jun Gu, Sha Jiang, Da Yong Li, Wen Bo Liao* Key Laboratory of Southwest China Wildlife Resources Conservation (Ministry of Education), China West Normal University, Nan- chong, 637009, China. *Corresponding authors. E-mail: Liaobo_0_0@126.com Submitted on: 2017, 11th September; revised on: 2018, 15th March; accepted on: 2018, 6th May Editor: Giovanni Scillitani Abstract. The brain is among the most energetically costly organs in the vertebrate body. The expensive-tissue hypothesis (ETH) predicts that increasing the size of another costly organ, such as the gut, should compensate for the cost of a small brain. To date, this hypothesis has mainly been tested in homoeothermic animals and in some ectothermic animals (e.g., fishes and anurans). Here, we undertake a test of the ETH by analyzing the relationship between brain size variation and length of the digestive tract in Hylarana guentheri. After controlling for geographical situation and body size, we did not find a correlation between brain mass and the length of the digestive tract in H. guentheri. Our findings suggest that the variation of brain size did not follow general patterns in this species and that the effect of diet quality cannot play a role in the variation of brain. Keywords. Brain size; energetic constraints; expensive-tissue hypothesis; Hylarana guentheri. INTRODUCTION As an important cognitive organ of animals, increased brains confirm to improve the cognitive abil- ity (Striedter, 2005; Jiang et al. 2015; Luo et al., 2017; Wu et al., 2016; Yu et al., 2018). However, brains are one of the most metabolically costly tissues in the vertebrate body (Mink et al., 1981). The high amount of energy related to maintaining brain tissues should constrain on evolution of brain size (Striedter, 2005; Isler and van Schaik, 2006), although the brains can confer cognitive ability (Kotrschal et al., 2013; Liao et al., 2015a; Zeng et al., 2016; Kotrschal et al., 2017; Samuk et al., 2018). The expensive-tissue hypothesis (ETH) predicts that the enlarged brains will inevitably decrease the sizes of oth- er metabolically costly tissues, such as gut (Aiello and Wheeler, 1995). There are evidences that the evolution of brain size follows the prediction of the ETH in ani- mal kingdom (Isler and van Schaik, 2006; Navarrete et al., 2011; Jin et al., 2015; Tsuboi et al., 2015; Liao et al., 2016a; Sukhum et al., 2016). Since the ETH was developed, primarily comparative studies have tested their predictions. While some stud- ies have shown that negative associations between brain mass and gut size support the ETH (Kaufman et al., 2003; Tsuboi et al., 2015; Liao et al., 2016a), other studies do not find such patterns (Lemaître et al., 2009; Barrickman and Lin 2010; Navarrete et al., 2011). Most studies on the cost of brain size evolution have mainly focused on birds and mammals (e.g., Aiello and Wheeler, 1995; Jones and MacLarnon, 2004; Isler and van Schaik, 2006; Pitnick et al., 2006; Navarrete et al., 2011). In recent years, the cost of brain size evolution in ectothermic vertebrates has been investigated at inter-specific species level (Tsuboi et al., 2015; Liao et al., 2016a; Sukhum et al., 2016) and intra-specific species level (Kotrschal et al., 2013; Liu 142 Ya Ting Liu et alii et al., 2014; Jin et al., 2015; Zhao et al., 2016; Gu et al., 2017; Yang et al., 2018). For instance, a negative correla- tion between brain mass and the length of the digestive tract have been found in Rana omeimontis (Jin et al., 2015) while Zhao et al. (2016) do not find any correla- tion between them in the dark-spotted frog (Pelophylax nigromaculata). Here we provide the test of the relationship between the brain mass and length of the digestive tract among Hylarana guentheri populations. Based on the ETH, we predict that a larger brain should accompany a smaller digestive tract in the frog. MATERIAL AND METHODS The Guenther’s frog, Hylarana guentheri is found in the subtropical forests in China at elevations ranging from 500 to 1100 m (Fei and Ye, 2001). The species exhibits a variable activ- ity period. Males actively look for females and used advertise- ment calls to attract them (Liu et al., 2011). In this species, mat- ing and egg-laying extends from March to May, as a prolonged breeder (Wells, 1977). We collected a total of 135 frogs from six populations along a 420-km latitudinal and 341-m altitudinal transect across the Hunan, Hubei and Sichuan provinces in China between June and August in 2016 (Figure 1). All individuals were captured by hand in paddy fields and pools at night and confirmed at the adult stage by direct observations of secondary sexual character- istics (see details in Liao and Lu, 2010; Jin et al., 2015; Liao et al., 2015b). We recorded the altitude, latitude and longitude of each population because environmental factors will affect morphology and physiology of organism (Chen et al., 2016; Yang et al. 2017; Liao et al., 2016b; Tang et al., 2018; Zhong et al., 2017; Wang et al., 2017; Liao et al., 2018; Qin et al., 2018; Wu et al., 2018). We took all individuals to the laboratory and kept indi- vidually in rectangular tanks (0.5 × 0.4 × 0.4 m) before being anesthetized with benzocaine. Each individual was killed by sin- gle-pithing and preserve in 4% buffered formalin in a phosphate buffer (Liao et al., 2016a; Lüpold et al., 2017; Mai et al. 2017a, b). We measured body size of all individuals (snout-vent length: SVL) to the nearest 0.01 mm with a caliper, and body mass to the nearest 0.1 mg with an electronic balance. All frogs were dissected, and their brains were removed (see detail in Liao et al., 2015a; Jin et al., 2016). We then weighed and measured all brains to the nearest 0.1 mg with an electronic balance. We used a Canon SX530 HS digital camera to take digi- tal images of digestive tract with a caliper. We then measured length of digestive tract using a Motic Images 3.1 digital camera mounted on a Moticam 2006 light microscope at a 400× mag- nification. All statistical analyses were performed using SPSS 22.0 (Statistical Product and Service Solutions Company, Chicago, USA). We ran a Linear Mixed Models (LMMs) with log10- Fig. 1. Topographic original maps showing the sampling locations of the six populations in Hylarana guentheri in China. 143No evidence for ETH in a frog transformed brain mass as a dependent variable, population as a random factor, and log10-transformed digestive tract length, latitude, longitude, sex and altitude as fixed effects, log10-trans- formed body size as covariate to test the original ETH. Within each sex, we also used LMMs to test the correlation between brain size and digestive tract length. RESULTS We at first analyzed the association between relative brain size and relative digestive tract length to evaluate the original ETH. We found that relative brain size was not correlated with relative size of digestive tract when controlling for the effect of the body size (Table 1). There was a non-significant variation in brain size among pop- ulations (Z = 0.656, P = 0.512). Moreover, sex, latitude, longitude and altitude did not affect the relative brain size (Table 1). The LMMs revealed that for males relative brain size was not correlated with relative length of digestive tract (Fig. 2; F1, 80.413 = 0.573, P = 0.451) and the environmental factors (e.g., latitude: F1, 1.97 = 1.069, P = 0.411; longitude: F1, 2.117 = 0.01, P =0.93; altitude: F1, 2.1 = 0.674, P = 0.494). However, there was a significant correlation between brain size and body size (F1, 79.354 = 96.174; P < 0.001). For females, the relative brain size was not correlat- ed with digestive tract length (Fig. 3; F1, 49 = 2.034, P = 0.16) and the environmental factors (e.g., latitude: F1, 49 = 0.002, P = 0.969; longitude: F1, 49 = 0.050; P = 0.943). However, we also found that brain size was positively correlated with the body size (F1, 49 = 57.732; P < 0.001). DISCUSSION Our results uncover a non-significant association between brain size and digestive tract size after control- ling for several environmental factors and body size in H. guentheri, which do not support the original “brain versus gut” prediction arising from the ETH. Thus, our study does not support the existence of energetic con- straints as important factors influencing patterns of brain size diversification among individuals in frogs. Below, we discuss our results in more detail considering the main hypothesis addressed. Although the data set on which the ETH is based refers to primates (Aiello and Wheeler, 1995), most con- Table 1. Regression models of brain size on digestive tract in rela- tion to various predictor variables in species when controlling for body size. Source d.f. Predictor F P Brain size 1, 133.961 Digestive tract 1.182 0.279 1, 134.993 Sex 1.104 0.295 1, 2.002 Altitude 3.517 0.201 1, 1.999 longitude 2.152 0.280 1, 1.981 Latitude 0.028 0.883 1, 110.113 Body size 150.942 <0.001 Fig. 2. Plot of relative brain size and relative digestive tract length of the sampled male individuals of Hylarana guentheri. Fig. 3. Plot of relative brain size and relative digestive tract length of the sampled female individuals of Hylarana guentheri. 144 Ya Ting Liu et alii vincing evidences favoring the ETH come from ectother- mic animals (e.g., Gnathonemus petersii: Kaufman et al., 2003; Rana omeimontis: Jin et al., 2015; Poecilia reticulata: Kotrschal et al., 2013; Tanganyika cichlids: Tsuboi et al., 2015; anurans: Liao et al., 2016a). The negative relation- ship between brains and guts is not observed in homo- thermic animals (Isler and van Schaik, 2006, Jones and MacLarnon, 2004; Barrickman and Lin, 2010; Navar- rete et al., 2011). Hence, the ETH might explain cases of encephalization in specific lineages of homoeotherms but it is not valid in overall these groups (Aiello et al., 2001). In contrast to the homoeotherms where brain mass corresponds to 1-2% of total body mass (Striedter, 2005), frogs and toads have smaller brain mass that, on average, corresponds to 0.3% of body mass (Liao et al., 2016a). The robust demonstrations of negative associa- tions between brain and gut across species suggest that energetic constraints play an important role in vertebrate, especially ectotherm brain evolution. For H. guentheri, the energetic constraints cannot explain brain size varia- tion because of the lack of correlation between brain size and digestive tract length. There is at first an inverse correlation between one very costly organ, the brain, and the very organ that pro- cures energy, the gut. Potentially this correlation may materialize if an increased brain enables a potentially cognitively driven shift towards a more nutritious diet (Liao et al., 2016a). There is evidence that the smallest- brained species are primates, with the largest guts feed on plant matter, while the larger-brained species are more omnivorous and humans, with the largest brain and smallest gut, even outsourced part of its gut function to cooking pots (Aiello and Wheeler, 1995). Previous studies have argued to find support for the ETH in primates by using diet quality as a proxy of gut morphology (Fish and Lockwood, 2003). However, diet quality does not corre- late with brain size in lemurs and lorises (Gonzalez-Voyer et al., 2009; Allen and Kay, 2011). Within the anurans, because most are insectivorous as adults, the potential for dietary divergence may be limited. However, anurans may feed on insects of different nutritive value, which may demand varying levels of cognitive ability to catch (Liao et al., 2016a). Hence, diet quality might affect the brain size variation in anuran species. The energetic constraints play an important role in promoting brain size variation within single species because there are negative associations between brains and guts (Kotrschal et al., 2013; Jin et al., 2015). However, inconsistent with the ETH, the relative brain size did not exhibit a negative correlation with digestive tract length in H. guentheri. Similar pattern has been supported in the other two frogs (e.g., P. nigromaculata: Zhao et al., 2016; Fejervarya limnocharis: Yang et al., 2017). The discrep- ancy among above studies also may result from the diet quality (Isler and van Schaik, 2006). There is evidence that a high diet quality combined with a large brain and a low diet quality combined with small brain has been reported in a frog R. omeimontis (Jin et al., 2015), which suggests that more clever individuals (larger brains) make use of better food and therefore develop smaller guts. However, we did not find a correlation between brain and gut in H. guentheri, suggesting that diet quality is not combined with brain size variation. In conclusion, our findings suggest that the expen- sive-tissue hypothesis is not supported in H. guentheri because brain size and gut length are not inversely related. Thus, the energetic constraints do not play an important role in promoting brain size variation for this species. ACKNOWLEDGEMENTS Financial support was provided by the National Nat- ural Sciences Foundation of China (31471996; 31772451) and the Key Cultivation Foundation of China West Nor- mal University (17A006) and Talent Project of China West Normal University (17YC335), and Sichuan Prov- ince Department of Education Innovation Team Project (15TD0019) and the Students Science and Technology Innovation Foundation of China (201610638049). The sacrifice of animals was approved by the Animal Ethics Committee at China West Normal University. The report- ed experiments comply with the current laws of China concerning animal experimentation, and permission to collect frogs was received from the Ethical Committee for Animal Experiments in China Council on Animal Care (CCAC) guidelines and Wildlife Protection Section of Sichuan Forestry Department. REFERENCES Aiello, L.C., Bates, N., Joffe, T. (2001): In defense of the expensive tissue hypothesis. 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