A Comparison of Somatic Condition and Testis Mass in Black-Spectacled Toad (Duttaphrynus melanostictus) between Two Populations at Different Altitudes

1Key Laboratory of Southwest China Wildlife Resources Conservation (Ministry of Education), China West Normal University, Nanchong, 637009, Sichuan, China 2Key Laboratory of Artificial Propagation and Utilization in Anurans of Nanchong City, China West Normal University, Nanchong, 637009, Sichuan, China 3Institute of Eco-adaptation in Amphibians and Reptiles, China West Normal University, Nanchong, 637009, Sichuan, China Article Information Received 18 March 2019 Revised 30 May 2019 Accepted 06 July 2019 Available online 01 June 2020


INTRODUCTION
T he same species in different geographical locations tend to have different morphology, physiology, and lifehistory traits (e.g., egg size, clutch size, sperm length and number) due to variations in climate, resource availability, competition or predation risk (Bergmann, 1847;Gould and Johnston, 1972;Atkinson and Sibly, 1997;Blanckenhorn and Demont, 2004;Liao et al., 2015a;Jin et al., 2016a;Lüpold et al., 2017;Kenthao and Jearranaiprepame, 2018;Samani, 2018;Yu et al., 2018;Liu et al., 2018;Yang et al., 2018;Zhong et al., 2018;Mai and Liao, 2019). Since poikilotherms are dependent on the relatively systematic climatic gradients along altitude and/or latitude to create chances for clinal variation in trait expression through local adaptation, they are often used as one of the major indicators of climatic variations (Morrison and Hero, 2003;Liao et al., 2014). For example, activity periods and breeding activity are often affected by seasonality and environmental temperature (Endler, 1977;Hjernquist et al., 2012;Alton et al., 2017).A relatively short activity period at high altitude and/or latitude is often associated with larger eggs but relatively smaller clutches (Dziminski and Alford, 2005;Lu, 2011, 2012). Differences in egg size and clutch size have been studied intensively across a wide range of taxa in females (Roff, 2002;Morrison and Hero, 2003;Liao et al., 2016a). However, the information on variation in testis mass remains poorly known in males.
Relative testis mass is widely used as a mea sure of male reproductive investment, and is considered under strong sexual selection (Gomendio and Roldan, 1991;Byrne et al., 2002;Zhou et al., 2011;Mi et al., 2012;Chen et al., 2016;Zeng et al., 2014;Tang et al., 2018;Cai et al., 2018). Therefore, when sperm competition is intense, males increase the investments in testes to enhance sperm production at inter-or intra-specific species level (Dziminski et al., 2010;. Recent works have focused on between-population variation in testis mass linked with environmental factors (Hettyey et al., 2005;Hettyey and Robert, 2006;Liao et al., 2013a;Jin et al., 2016a). Individuals inhabiting at high altitude/latitude have smaller relative testes mass because the total energy collection is limited due to the decreased environmental temperature and shortened breeding season, possibly O n l i n e

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allowing relatively less energy to be allocated to reproduction (Hettyey et al., 2005;Hettyey and Robert, 2006;Chen et al., 2014;Zhang et al., 2018).
Duttaphrynus melanostictus is a species widely distributed in South Asia, southeast Asia and southern China where they are found in ponds near grass clumps, kaleyard and stream near farmhouse with altitude ranging from 10 to 1700 m. The period of egg laying extends from March to July (Fei et al., 2010). So far, little information on difference in somatic condition and testis mass of this species across altitudinal populations is available. Here, we explored differences in somatic condition and testis mass in D. melanostictus between two populations at different altitudes. Previous studies have suggested that, at high altitude/latitude, lower environmental temperature and shorter breeding season decreased energy acquisition (Morrison and Hero, 2003), thereby limiting somatic condition and investments in sperm production (Chen et al., 2014;Jin et al., 2016b). Hence, we tested the hypotheses (1) that somatic condition and testis mass decrease with increasing altitude; (2) that testis mass increases with somatic condition.

Study sites
We visited two D. melanostictus populations located at different altitudes in Hunan and Yunnan Province in Southwest China, from July to August, in 2018. The low-altitude population was located at 120 m in Yuanling (110°24.41′E, 28°26.18′N) in which toads were collected in a stream at the distance of 1 km to farmhouse. The high-altitude population was located at 1673 m in Midu (100°28.51′E, 25°20.24′N), where toads are captured in a stream at the distance of 0.5 km to farmhouse.

Samplings collection
A total of 45 adult males were collected. We captured all individuals by hand at night through using a 12-V flashlight in streams. Before processing, the toads were kept in individual rectangular tanks (1.0×0.5×0.5 m; L×W×H) with a water depth of 2 cm (Jin et al., 2015). All the individuals were killed by using singlepithing (Mai et al., 2017a;Liao et al., 2015b). Body size (snout-vent length: SVL) was measured to the nearest 0.1 mm with a vernier caliper and body mass to the nearest 0.1 mg using an electronic balance (Zhong et al., 2016). Toads were dissected, and testes were removed and weighed to the nearest 0.1 mg using an electronic balance Wu and Liao, 2017).

Statistical analyses
Body mass, soma mass (i.e., body mass-testis mass), testis mass, and body size were log-transformed to achieve normality and improve homogeneity of the variances. We ran a linear regression treating log10 (body mass) as a dependent variable and log10 (body size) as an independent variable, and residual mass was used as an index to measure body condition (Schulte-Hostedde et al., 2001Jin et al., 2016a). Differences in body mass, testes mass and somatic condition (i.e., log 10 (soma mass) / log 10 (SVL)) between populations were tested using One-way ANOVA. To test statistical differences in relative testis mass (i.e., log 10 (testis mass) / log 10 (soma mass)) between the low-and high-altitude populations, we ran a generalized linear models (GLMs) treating testis mass as a dependent variable, population as fixed factors, and soma mass or somatic condition as covariate. All statistical tests were two-tailed, and the nominal significance level was set at P = 0.05. All analyses were performed by SPSS 22.0.

DISCUSSION
The results demonstrate that the black-spectacled toad exhibits striking differences in somatic condition between different populations, and this somatic condition displays an increase with altitude. In addition, testis mass significantly differed between the populations, and this testis mass displayed an increase with increased altitude. Moreover, testis mass was positively correlated with soma mass and somatic condition, suggesting that males invest more in their gonads in good somatic condition.
Testis mass relative to soma mass is widely used as a measure of reproductive investment in males O n l i n e  (Jin et al., 2016b;Tang et al., 2018). It has been suggested by previous studies that there is a condition-dependent testis mass across a wide range of taxa (Byrne et al., 2002;Schulte-Hostedde and Millar, 2004;Burness et al., 2008;Jin et al., 2016b;Tang et al., 2018). Males with good somatic condition have relatively large relative testis mass than males in poor somatic condition (Simmons and Kotiaho, 2002;Jin et al., 2016b). In the study, testis mass was significantly positive associated with soma mass and somatic condition, suggesting that males in good body condition may have larger reproductive investment than males with poor body condition. Similar results have been reported previously in other studies (Simmons and Kotiaho, 2002;Mai et al., 2017b;Zhou et al., 2011;Chen et al., 2014;Lüpold et al., 2017;Tang et al., 2018). Life-history strategy with respect to resource allocation for reproduction reflects a trade-off between current reproductive effort and future reproductive success and/or survival (Lack, 1966;Roff, 2002). In highaltitude or latitude, individuals experience less time for reproduction and the extended re-acquisition of resources necessary for survival, as a result, there was a negative relationship between testis mass and altitude or latitude in male frogs (Hettyey et al., 2005;Chen et al., 2014). However, we found a positive correlation between relative testis mass and altitude in this species, although there was a lower environmental temperature and shorter time available for reproduction in high-altitude population. Our evidence suggests that the increased resources required for survival in high-altitude cannot decline the energetic investment allocated to testis mass in D. melanostictus. Similar result has been reported in Dianrana pleuraden (Mai et al., 2017b) and Feirana quadranus (Tang et al., 2018). In addition, somatic condition displays an increase with altitude in this study suggesting that males with good somatic condition in high-altitude population have more resources available to invest in testes.

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In general, we observed significant variation in O n l i n e

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somatic condition and testis mass in D. melanostictus between different populations. In addition, we found that somatic condition and testis mass displayed an increase with altitude and testis mass was positively correlated with somatic condition, suggesting that males with good somatic condition have more energy available to invest in testes.

ACKNOWLEDGEMENT
The study was supported by the National Natural Sciences Foundation of China (31772451), the Science and Technology Youth Innovation Team of Sichuan Province (19CXTD0022), the Key Cultivation Foundation of China West Normal University (17A006) and the Talent Project of China West Normal University (17YC335).

Ethical approval
All experiments involving the sacrifice of live animals were approved by the Animal Ethics Committee at China West Normal University.

Statement of conflict of interest
The authors declare that there is no conflict of interests.