See discussions, stats, and author profiles for this publication at: http://www.researchgate.net/publication/257160343

Effects of gestation temperature on offspring sex and maternal reproduction in a viviparous ( multiocellata) living at high altitude

ARTICLE in JOURNAL OF THERMAL BIOLOGY · OCTOBER 2012 Impact Factor: 1.51 · DOI: 10.1016/j.jtherbio.2012.03.002

CITATIONS READS 7 100

7 AUTHORS, INCLUDING:

Xiaolong Tang Feng Yue Lanzhou University Central China Normal University

17 PUBLICATIONS 60 CITATIONS 31 PUBLICATIONS 209 CITATIONS

SEE PROFILE SEE PROFILE

Cui Wang University of Helsinki

6 PUBLICATIONS 16 CITATIONS

SEE PROFILE

Available from: Xiaolong Tang Retrieved on: 21 October 2015 Journal of Thermal Biology 37 (2012) 438–444

Contents lists available at SciVerse ScienceDirect

Journal of Thermal Biology

journal homepage: www.elsevier.com/locate/jtherbio

Effects of gestation temperature on offspring sex and maternal reproduction in a viviparous lizard (Eremias multiocellata) living at high altitude

Xiao-Long Tang 1, Feng Yue 1, Xue-Feng Yan, De-Jiu Zhang, Ying Xin, Cui Wang, Qiang Chen n

Institute of Biochemistry and Molecular Biology, School of Life Science, Lanzhou University, Lanzhou 730000, China article info abstract

Article history: Temperature-dependent sex determination (TSD) is well studied in many species of , but little is Received 26 April 2011 known on how geographic distribution and altitude affect the sex ratio. In the present study, we Accepted 9 March 2012 focused on a population of a viviparous lizard with TSD (Eremias multiocellata) that lives at high Available online 17 March 2012 altitudes (E2900 m) in Tianzhu, Gansu province, China. Gestation temperature had a notable effect on Keywords: the offspring sex ratio, gestation period, and the mother’s body mass. The mothers produced female Viviparous lizard biased offspring at 25 1C but male biased offspring at 35 1C. All female lost weight during Eremias multiocellata pregnancy, and the least loss of the body mass was observed at 31 1C. The gestation period increased in Gestation temperature a non-linear fashion as ambient temperature was reduced. Average litter size was elevated with an TSD increase of gestation temperatures, reached a maximum at 31 1C, and then declined at 35 1C. Compared Maternal reproduction with a previous study on a Minqin population which lives at a lower altitude (E1400 m) and warmer climate, the present study obtained a less skewed sex ratio of offspring in the Tianzhu population. Geographic variations also affected offspring morphology between the two populations; females collected from Tianzhu produced larger litters but with a smaller body weight of offspring. These differences may be caused by the adaptive response to the cool climatic and high-altitude environ- mental conditions. & 2012 Elsevier Ltd. All rights reserved.

1. Introduction male offspring increasing in colder years (Wapstra et al., 2009). If cohort sex ratio bias at birth leads to adult sex ratio bias, long- Reptiles exhibit both genotypic (GSD) and environmental- term directional changes in thermal conditions may have impor- dependent sex determination (ESD) (Bull, 1983; Valenzuela tant effects on population dynamics. However, previous studies et al., 2003). The most common form of ESD is Temperature- also found viviparous lizards living at different extreme climatic dependent sex determination (TSD), a typical case of phenotypic conditions could regulate maternal behavior (Wapstra et al., plasticity, where sex is determined after fertilization is affected by 2004), or even alter their sex-determining mechanisms to avoid the environment temperature (Bull, 1983). The TSD phenomenon dramatic changes at population level (Pen et al., 2010). and its mechanisms have been studied in many reptiles. Since A great deal of work illustrated that geographic variations may Robert and Thompson (2001) first reported that TSD does indeed influence population dynamics through multiple pathways, includ- occur in a viviparous lizard, four live-bearing lizards were shown ing sexual selection (Blanckenhorn et al., 2006; Møller, 2004), to exhibit TSD (Ji et al., 2006; Wapstra et al., 2004; Zhang et al., reproduction (Angilletta et al., 2006; ChamaillE´ -Jammes et al., 2010). These studies were innovative and provide new arguments 2006; Dunn, 2004), and offspring phenotype (Du and Shine, for explaining the mechanism of TSD. It is also important to 2008; Du and Feng, 2008; Ji et al., 2006; Shine and Harlow, understand thermal adaptation of these TSD species under 1996). These variations may not only depend on the direct effects different environmental conditions. A recent study on a natural of environmental conditions (Angilletta et al., 2002), but also have population of viviparous lizard with TSD (Niveoscincus ocellatus) more complex causes such as adaptive phenotypic plasticity and found sex ratios varied significantly from year to year and closely the timing of life history events in response to local environmental tracked thermal conditions in the field, with the proportion of conditions (Pulido and Berthold, 2004; Wapstra et al., 2009). For instance, following Bergmann’s rule, a species of larger size were found in colder environments, and species of smaller size were n Corresponding author at: Institute of Biochemistry and Molecular Biology, found in warmer regions. But there exist many fields where inverse School of Life Science, Lanzhou University, 222 Tian Shui South Road, Lanzhou phenomenon appear, that the body size of ectotherm species may 730000, China. Tel./fax: þ86 931 8915316. E-mail addresses: [email protected], [email protected] (Q. Chen). change smaller as the ascending altitude and cooler temperatures 1 Both authors contributed equally to this work. (Ashton, 2001; Ashton and Feldman, 2003). In addition, under

0306-4565/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jtherbio.2012.03.002 X.-L. Tang et al. / Journal of Thermal Biology 37 (2012) 438–444 439 different environmental conditions, the pregnant females may 2.2. Constant gestation temperature experiment change output strategies. For example, some pregnant females produce ‘‘small clutches with big eggs’’ but the reverse situations The lizards were captured in late May and brought to our are found in some other species (Sinervo, 1990). However, most of laboratory in Lanzhou University. For each lizard, sex was identi- these studies were focused at the species level and investigated the fied by hemipene eversion and the morphological traits (body relationship between offspring sex ratio and varied incubation mass and SVL) were measured. In some species, females temperature. How thermal environments affect female reproduc- could adjust their offspring sex ratios in response to imbalances in tive characters and offspring sex ratio in different populations the population adult sex ratio, which is known as operational sex remain an eristic question (Escobedo-Galva´netal.,2011; ratio (OSR) (Thompson et al., 2003; Warner and Shine, 2007). In Kallimanis, 2010; Witt et al., 2010). order to exclude the influence of the OSR, we collected the same Global warming is being shown to significantly affects the numbers of adult male and female lizards in the present study. 48 ecosystem (Arago´ n et al., 2010; Parmesan and Yohe, 2003) and female and 48 male lizards were divided equally into four groups climatic conditions and this could drive offspring sex ratios in using random sampling methods. Each group was comprised of species with TSD, which could potentially lead to local extinction six cages, two females and two males were maintained in one (Janzen, 1994). However, if ectotherms live in considerably cooler cage (60 30 40 cm3). A cut a toe method was used environments than their optimum, they may take advantage of for individual identification. Four constant temperatures were warming temperature by basking and would benefit more from a provided (25, 29, 31 and 35 1C) from 0900 to 1700, and during the warmer climate (Huey and Tewksbury, 2009). In order to learn rest of the time, the temperature inside the cage was consistent more about the possible variations on offspring sex ratio and with the room temperature. The room lights were programmed to female reproductive characters in cool environmental conditions, a 12 h lighting: 12 h dark cycle (0700–1900 h) in order to we chose a high altitude-inhabiting viviparous lizard Eremias simulate natural light. The bottom of cage was covered with multiocellata in Tianzhu, Gansu province, China. This population 5 mm silver sand and cage temperature (set temperature70.3 1C) lives vibrantly in mountain area, although the mean temperatures was kept constantly by electronic temperature control device at reproductive season were lower than its optimal temperature (WMZK-10, Shanghai Medical Instrument). The room tempera- (Yan et al., 2011). Our previous study on Minqin population ture was controlled at 1870.5 1C, and the temperature inside the showed that E. multiocellata is a TSD species (Zhang et al., cage fell to room temperature at night. Sufficient mealworms and 2010). The present study aims to investigate (1) whether effects water were provided every two days during entire experiment. of gestation temperature on offspring sex ratios are different in high- and low-altitude populations and (2) possible adaptive 2.3. Field experiment as control group characteristics of Tianzhu population in offspring phenotypic plasticity and female fecundity. The control groups consisting of 12 female and 12 male lizards raised in semi-natural enclosures. The field enclosure (4 3.5 m2) was constructed of 100 cm high zinc-gilt iron sheet with 50 cm buried underground to prevent lizards escaping. A fine wire net 2. Materials and methods was placed over the enclosure to keep lizards from being eaten by predators. Bumpy ground with sparse grass was artificially built 2.1. Study species and several bricks were placed in each enclosure to give lizards opportunity to choose their optimal temperature or basking E. multiocellata have been studied extensively over the past places. In addition, sufficient water and mealworms were pro- decades, with particular attention on differences in some physio- vided. Before the birth of offspring, female lizards in the field logical and ecological characters among populations (Li et al., were return to the laboratory at the end of June, and maintained 2011; Yan et al., 2011; Zhang et al., 2010). It is a small terrestrial in cages (200 60 50 cm3) with a 10 cm depth of silver sand. A viviparous lizard (mean adult snout-vent length E65 mm) and 225 W infrared light bulb was suspended over the top of the cage the mating behavior occurs in May. The female lizards produce a to provide a thermal gradient ranged 18–38 1C in a daily 8 h single litter between late July and August with a litter size of 2–5 on:16 h off cycle (0900–1700 h). Thus, lizards in these cages could (Zhao, 1999). It has a wide distribution (Mongolia, Kazakhstan, choose the gestation temperatures whenever the heating bulbs Kyrgyzstan and northern China) and is considered as an appro- were switched on. Fluorescent lamps were used to simulate the priate model for the study of geographic variation in female natural light on 12 h lighting: 12 h dark cycle (0700–1900 h). reproduction and offspring phonotype (Zhang et al., 2010). The The room temperature was controlled at 1870.5 1C, and the E. multioceallata used in this study lives in Tianzhu (3716’N, temperature inside the cage fell to room temperature at night- 10319’E, altitude E2900 m), Gansu province, China, which is time. All of these conditions were designed to simulate a wild the highest known distributed E. multioceallata population. The environment. Lizards were supplied sufficient mealworms and Tianzhu area is located to the east of Qilian Mountains, the water every two days. average environment temperature in breeding season is only 7.1 1C and annual rainfall is 700–800 mm (Yan et al., 2011). 2.4. Offspring morphological traits and female reproductive Contrary to these cool environmental conditions, the Minqin characters population we used in previous study inhabits a dry and warm climatic condition (3814’N, 10311’E, altitude E1400 m). This The cages were checked twice a day (morning and afternoon) semi-desert area is situated at the southwest edge of the Tengeli for offspring as soon as the first female gave birth. Newborns were Desert, the annual rainfall and temperature in breeding season is collected within a few hours after birth, and the morphological 113.6 mm and 18.3 1C, respectively (Zhang et al., 2010). In order traits were measured (mass70.01 g; length70.02 mm), includ- to facilitate the comparison, we cited the corresponding statistical ing body mass (BM), snout-vent length (SVL), tail length (TL), head data of Minqin population from our previous study. These lizards length (HL), head width (HW), arm length (AL), leg length (LL), were captured during early May 2008, in Minqin (38138’N, eye length (EL) and tympanum length (TyL). The parturition date, 103105’E), and the experiments were carried out in the similar postpartum body mass and litter size for each pregnant female laboratory conditions (Zhang et al., 2010). were recorded. The gestation periods were calculated from the 440 X.-L. Tang et al. / Journal of Thermal Biology 37 (2012) 438–444 date when female lizards were caught in the wild. The change of pregnant female’s body mass is represent by % mass change/d, which calculate by¼100 (m2 m1)/(m1 gestation period), where ‘‘m1’’¼mass at start of the experiment and ‘‘m2’’¼mass at termination of the experiment (Beuchat, 1988).

2.5. Statistical analysis

The data were tested for normality and homogeneity of variances to meet the assumptions of parametric testing prior to analysis. Sex ratioswereexpressedbythenumberofmalesdividedbythetotal number of the offspring, and tested against the null expectation of a 1:1 sex ratio. The logistic regression was used to model litter sex ratios among four treatments (Wapstra et al., 2004). One-way ANOVA was used to evaluate the effect of gestation temperature on mean litter size, gestation period, the change of female body mass, offspring SVL and head length. One-way analysis of covariance (ANCOVA) with snout-vent length (SVL) as the covariate was used to determine whether gestation temperature had influence on other morphological Fig. 1. Comparison of offspring sex ratio between Tianzhu and Minqin popula- traits except for offspring SVL and head length. Furthermore, gesta- tions in Eremias multiocellata. The value in the column is the sample size of tion temperatures and populations in two-factor ANCOVAs (with SVL offspring. as covariate) were analyzed for their effects and possible interactions in explaining variation in female reproductive characters and off- spring morphological traits. A principal component analysis (varimax Tianzhu population had shorter gestation periods than Minqin rotation) was used to investigate the possible existence of morpho- for the same thermal condition. All female lizards lost weight metric characteristic of offspring in Tianzhu from different gestation dramatically during pregnancy, the least loss of body mass was temperatures. The Tukey’s test was used for all multiple comparisons. observed at 31 1C. The average litter size elevated with the All hypotheses were tested for statistical significance at the po0.05 increasing gestation temperatures and declined at 35 1C. The level. Data were analyzed using SPSS (Release 16.0.0, SPSS, Inc., females maintained at 31 1C had a maximum litter size of 4.09, Chicago, IL). and in 25, 29 and 35 1C group the litter size was 3.63, 3.83 and 3.36, respectively. These results are different from Minqin popu- lation, which the optimal temperature for mean litter size was at 3. Results 29 1C(Zhang et al., 2010). Additionally, the mean litter size of Tianzhu population was bigger than Minqin for the same thermal 3.1. Effect of gestation temperature on offspring sex ratio condition (Fig. 3). By the end of this experiment, we dissected the females that Logistic modeling of litter sex ratios revealed a predominant could not give birth, and undeveloped eggs were found in relationship between gestation temperatures and litter sex ratio oviducts and only the yolk material was detected in the eggs. 2 2 (rmodel¼4.466, Wald w ¼4.332, p¼0.037). The mothers produced These female lizards were excluded from data analyses. female biased offspring (n¼29, 41.38% were male, binomial test p¼0.458 against a null of 50% male) in 25 1C group but male biased 3.3. Effect of gestation temperature on offspring morphological traits offspring (n¼37, 67.57% were male, binomial test p¼0.047 against a null of 50% male) at 35 1C, while those reared at 29 and 31 1Cgave Gestation temperature had a significant effect on some morpho- birth to offspring with a balanced sex ratio (n¼42, 52.38% were male, logical traits of offspring, including SVL, TL, HL, HW, AL, LL and EL. binomial test p¼0.878 against a null of 50% male and n¼49, 53.06% However, there was no significant difference in BM and TyL among were male, binomial test p¼0.775 against a null of 50% male, four gestation temperature groups (Table 2). A principal component respectively). Compared with our previous study on the Minqin analysis resolved three components (with eigenvalues41) from eight population, the sex ratio of offspring in Tianzhu population was size-free morphological traits, accounting for 61.8% of variation in the skewed slightly. In the control group, females undergoing most of original data (Table 3). The first component (31.8% of variance their gestation in the field also gave birth to offspring in a balanced explained) had high positive loading for size-free values of body ratio (n¼34, 52.94% were male, binomial test p¼0.864 against a null mass and tail length. The second axis (18.3%) largely represented the of 50% male) (Fig. 1). Additionally, based on the regression analysis, it length of both the arm and the leg. The third component (11.8%) is indicated that 28.3 1C was the pivotal temperature, at which gravid represented the head width and tympanum length. Hatchlings from females should give 50% of each sex. four gestation temperatures had highly significant differences in their scores on all three components (All Po0.001). 3.2. Effect of gestation temperature on female reproductive We also compared offspring morphological traits of the two characters populations; include body mass, SVL, HW and HL. The results showed that born from the same gestation temperature, the Gestation period and change of mother’s body mass were neonate body size and body mass of Tianzhu population is smaller affected significantly by gestation temperatures, whereas average than those in Minqin (Fig. 5). litter size was not (Table 1).The gestation period increased with the declined ambient temperature non-linearly. An increase in ambient temperature from 25 to 29 1C reduced the gestation 4. Discussion period by 15 days. When the temperature rose from 31 1Cto 35 1C, the gestation period decreased 6 days (Table 1), which is Different gestation temperatures had significant effects on sex similar to the Minqin population (Fig. 4). Furthermore, the ratio of offspring in E. multiocellatas (Fig. 1). Gravid females X.-L. Tang et al. / Journal of Thermal Biology 37 (2012) 438–444 441

Table 1 Effect of gestation temperature on maternal reproduction in the viviparous lizard Eremias multiocellata.

Thermal treatments Laboratory Field Statistical analyses and (n¼9) result of the comparison 25 1C(n¼8) 29 1C(n¼12) 31 1C(n¼12) 35 1C(n¼11)

Body mass at start (g) 5.5870.13 5.8670.15 5.6170.10 5.5070.15 5.9770.34 1.097 ns Snout-vent length (mm) 60.1570.07 62.4270.08 61.3770.05 60.5170.08 61.3570.11 1.329 ns Tail length (mm) 77.770.35 69.370.54 69.270.47 77.370.34 67.470.38 1.337 ns Postpartum body mass (g) 4.4070.17 5.1770.22 5.2270.16 4.7870.09 4.4370.23 4.655nn Litter size 3.6370.32 3.8270.30 4.0970.25 3.6470.28 3.4070.31 0.815 ns Gestation period (day) 73.3871.69 58.0972.42 54.0971.25 48.5071.68 53.6271.07 25.500nnn (64–79) (46–69) (44–59) (38–56) (50–57) % mass change (d1) 0.2870.04 0.2370.05 0.1370.06 0.2570.07 0.1370.01 3.489n

Values are expressed as mean7SE and range. F ratios correspond to single effects and factor interactions in ANOVA (temperature as fixed factors) or ANCOVAs (with SVL as covariate, for all other traits). Tukey’s test was used in all multiple comparisons. Symbols immediately after F-values represent significant levels: ns, P40.05. n Po0.05 nn Po0.01 nnn Po0.001.

Table 2 Morphological traits of hatchling lizards (Eremias multiocellata) according to gestation temperature (Mean7SD).

Morphological traits Laboratory Field Statistical analyses and (n¼34) result of the comparison 251C(n¼29) 291C(n¼42) 311C(n¼49) 351C(n¼37)

Body mass (g) 0.51470.01 0.50570.01 0.48070.01 0.49670.01 0.52070.01 2.05 ns Snout-vent length (mm) 26.91870.02 27.20270.02 26.96570.02 26.62870.02 27.85870.04 2.43n Tail length (mm) 3.93670.04 3.89970.04 3.71770.08 3.67970.07 3.89770.04 4.26nn Head length (mm) 7.19370.06 7.38470.08 7.01770.07 7.18970.06 7.31970.03 5.77nnn Head width (mm) 4.29670.06 4.04970.05 3.97870.05 4.05670.06 3.91070.05 6.83nnn Fore-limb length (mm) 6.74170.08 6.20370.06 6.00070.08 6.33170.06 6.60070.05 21.05nnn Hind-limb length (mm) 9.25170.07 8.55570.10 8.23770.12 8.35170.08 8.67670.09 15.21nnn Eye length (mm) 2.66570.08 2.93270.06 2.93470.06 3.04570.07 2.89370.06 4.36nn Tympanum length (mm) 1.90270.04 1.81470.04 1.76870.04 1.77870.04 1.80370.05 1.70 ns

F ratios correspond to single effects and factor interactions in two-factor ANOVA (temperature and sex as fixed factors) or ANCOVAs (with SVL as covariate, for all other traits). Symbols immediately after F-values represent significant levels: ns, P40.05. n Po0.05 nn Po0.01 nnn Po0.001

the mother’s body temperature was very close to the ambient Table 3 temperature (Yan et al., 2011). These results suggest that the Loading of the first three axes of a principal component analysis on offspring different sex ratios among groups were caused by gestation morphological traits in Eremias multiocellata. temperatures. We could exclude some impacts which also Morphological traits Factor loading affected the sex ratio of offspring, such as embryonic sex reversal, differential fertilization, or different embryonic mortality. First, in PC1 PC2 PC3 terms of function, there is no difference between sex reversal and TSD during embryonic development. If sex is determined, and Body mass 0.827n 0.207 0.060 Tail length 0.794n 0.165 0.101 then reversed due to temperature, the ecological result is the Head width 0.152 0.199 0.698n same (Thompson et al., 2003). Second, mating competition could Head length 0.596 0.228 0.251 be excluded, for the adult females were caught just after mating Arm length 0.212 0.691n 0.160 n at late May (evident by fresh mating scars). These results are Leg length 0.284 0.753 0.198 similar to our previous study on another population of the same Eye length 0.300 0.715 0.052 Tympanum length 0.095 0.026 0.829n species living at low altitude in Minqin (Zhang et al., 2010) and Variance explained (%) 31.8 18.3 11.8 provide additional evidence that this viviparous lizard is a TSD species. Size effects are removed in all cases by using residuals from the regressions on Robert and Thompson (2001) first discovered the TSD was not snout-vent length of neonates. All data were log-transformed. exclusive to oviparous lizards, but also occurred in viviparous lizard n Variables with the main contribution to each factor. Eulamprus tympanum. Up to now only four separate species had been confirmed as TSD viviparous lizards (Ji et al., 2006; Wapstra et al., 2004; Zhang et al., 2010) and they live in substantially varied environmental conditions and exhibit different TSD patterns. It is maintained at 35 1C produced more male offspring than those interesting that these species exhibit cold mountainous distributions. maintained at 25 1C. Each of the laboratory groups was provided However, very few studies have been concerned with how the sex with the same condition except for the ambient temperature, and ratios of offspring in the same species respond to different 442 X.-L. Tang et al. / Journal of Thermal Biology 37 (2012) 438–444 environmental conditions. Pen et al. (2010) first investigated the because their available body volumes were highly packed by effect of ambient temperatures on offspring sex ratios in two developing embryos (Ji et al., 1997, 2001; Lourdais et al., 2002; populations of a viviparous species with TSD, and found that Lin et al., 2008). Furthermore, mothers may reduce their activity N. ocellatus inhabiting different climatic extremes differed in sex- levels and forage less to avoid higher risks of predation and determining mechanisms, with TSD in lowlands and GSD in high- energy costs of moving around (Cooper et al. 1990, Schwarzkopf lands. Another analysis on the American crocodile Crocodylus acutus and Shine 1992). Thus, total intake energy may be insufficient for indicated that nearby populations presented very different sex ratios mother’s thermoregulation, maintenance and reproduction, and depending upon local conditions (Escobedo-Galva´netal.,2011). From the pregnant females had to consume the self-stored energy in the results of present and our previous studies, we can find the order to provide a proper condition for embryos development offspring sex ratios in Tianzhu population were less skewed than that (Shine, 2004). Our previous work has proved that mother’s body of Minqin population (Fig. 1). Both studies were carried out under the temperature was slightly higher at 25 1C but lower at 35 1C than same condition but on different populations. The Tianzhu population the ambient temperature. It indicated pregnant E. multiocellata was located at a higher altitude and the monthly mean temperature consumed more energy and adapt themselves to the extreme was significant lower than the Minqin area (Fig. 2). From the previous studies mentioned above, we speculated that the different skewed sex ratio between two populations may be mainly caused by the local thermal environment. In addition, we found E. multiocellatas has sex chromosome and belongs to female heterogamety (ZZ/ZW) (unpub- lished work). At present it is difficult to know whether the genetic factor or the thermal condition both affected sex ratio between Minqin and Tianzhu. Recent work on the oviparous skink Bassiana duperreyi illustrated sex ratio of offspring could be influenced by combinations of genes, temperature, and hormone (Radder et al., 2007; 2009). This exemplifies the complexity of sex determining mechanisms in reptiles and the distinct possibility that multiple factors act simultaneously and interactively. The length of the gestation period is usually shortened in warmer environments in reptiles (Du et al., 2007; Shine and Harlow, 1996; Xu and Ji, 2007). In this study, when the gestation temperature increased from 25 to 29 1C, the gestation period shortened by about 15 days. But the gestation period was only reduced by approximately 6 days when the temperature increased from 31 to 35 1C. This result is similar to that of some viviparous lizards (Ji et al., 2006; Shine and Harlow, 1993), as well as the Minqin population of E. multiocellatas (Fig. 4). It indicates that the relationship between gestation temperature and length Fig. 3. Comparison of mean litter size between Tianzhu and Minqin populations in Eremias multiocellata. The gestation temperatures population interaction of gestation period is non-linear, and pregnant females may affected mean litter size of female significantly (F3,77 ¼3.08, P¼0.007). The benefit less from selecting higher ambient temperatures in terms Tianzhu population had bigger mean litter size than Minqin at the same thermal of the reduced gestation length (Li et al., 2009). condition. Although sufficient food and water were provided, all female lizards lost weight during the pregnancy. However, females from 25 1Cto351C treatment groups lost more weight than other groups. The least loss of body weight was occurred at 31 1C. The daily food intake of pregnant females may significantly reduce,

Fig. 4. Comparison of gestation period between Tianzhu and Minqin populations in Eremias multiocellata. The length of gestation period shortened with the higher thermal environment non-linearly in both populations, and gestation tempera- tures population interaction had notable effect on gestation period

Fig. 2. Monthly mean temperature of Tianzhu and Minqin from 1953 to 2009. (F3,77 ¼109.90, Po0.001). X.-L. Tang et al. / Journal of Thermal Biology 37 (2012) 438–444 443

Fig. 5. Comparison of offspring morphological traits between Tianzhu and Minqin populations in Eremias multiocellata. Body mass (F3,261¼2.04, P¼0.037), head length (F3,261¼5.25, Po0.001) and head width (F3,261¼4.61, Po0.001) are all significantly different, but the SVL was not (F3,261¼0.28, P¼0.98). environments by thermoregulation (Yan et al., 2011). Moreover, which was rarely testified and was criticized as artificial or unpro- body mass of female lizards decreased least at 31 1C, suggestion ven (Ashton, 2001; Gaston et al., 1998). This morphology related that the mothers consumed less energy and nutrient at optimal rule suggested that in cooler climatic conditions usually temperature for pregnancy. have shorter arms or legs than the animals in warmer climates Neonate size can influence offspring survival (Ferguson and Fox, (Mayr, 1956), thus the smaller morphological traits in Tianzhu 1984; Henrich, 1988). The evolution of offspring number and size is population seemed as an adaptation strategy to cooler temperature. presumably mediated by the balance of the fecundity advantage of Firstly, the smaller has a larger body surface relative to its producing small offspring against the survival advantage of large mass. Smaller-sized individuals in cooler environments may be offspring. In present study, the mean litter size at 31 1C was higher able to regulate their body temperature more rapidly and precisely than those at other temperatures, which was different from the (Ashton and Feldman, 2003). Furthermore, ectotherms metabolic results in Minqin population (optimal temperature for mean litter rate generally decreases with hypothermia (Patterson and Davies, size was at 29 1C) (Zhang et al., 2010). Moreover, the mean litter size 1984), but the rate of mass-specific energy expenditure may of Tianzhu population was bigger than those maintained at the increase with decreased body size (Peters, 1986). Therefore, the same thermal condition in Minqin (Fig. 3). We also analyzed offspring with the smaller body size in Tianzhu could benefit more offspring morphology of both populations, and found the offspring with an effective energy strategy in cooler environments. in Tianzhu population had smaller body size (Fig. 5). These results In conclusion, our data provide evidence and support the fact predicted that the female lizards in Tianzhu may exhibit the ‘‘bigger that geographic variations affect the phenotypes and reproduction of litter size with smaller neonates’’ reproductive strategy. This kind of reptilian species. Recent work showed female heterogamety (ZZ/ZW) female reproductive strategy is unusual but similar to the Sceloporus exists in E. multiocellata, and it is not clear whether the different occidental which lives at high elevation site (Sinervo, 1990). On one skewed sex ratios between populations is influenced by genetic hand, smaller offspring incubated faster (Fig. 4), which may posi- factors simultaneously. A previous study has already testified live- tively influence hatching success in environments with very short bearing lizards at different climatic extremes of the species distribu- seasons (Porter and Tracy, 1983). On the other hand, larger clutches tion differ in their sex-determining mechanisms (Pen et al., 2010). may be able to satiate predators at hatching, thereby enhancing Heretofore, it is still unknown whether E. multiocellata have the survival probability per offspring and maintain the population at a similar characteristics. Future work in our laboratory will address this stable level (McGinley, 1989; Post, 1998). Furthermore, the direction issue in E. multiocellata. of this trade-off may also reflect the particular environmental conditions at these mountain sites or lineage-specific responses to these conditions, or both (Mathies and Andrews, 1995; Sinervo, Acknowledgments 1990; Sinervo and Licht, 1991). Gestation temperatures significantly affected morphological We thank F. Yue, D. J. Zhang, X. Meng, X. F. Yan and Y. Xin for traits of offspring other than body mass and tympanum length in help in various ways. Research funding was provided by the E. multiocellata, and offspring developed at 25 1Cand291Chad National Natural Science Foundation of China (No. 30670263). relative larger size (SVL, TL, AL and LL) than at 31 1Cand351C. This result is consistent with studies on many other reptile species that References ectotherm at cooler temperatures exhibit temperature-induced plasticity which produces larger hatchlings (Deeming and Angilletta, M., Oufiero, C.E., Leache, A.D., 2006. Direct and indirect effects of Ferguson 2004; Booth, 2006). But more importantly, the body size environmental temperature on the evolution of reproductive strategies: an information-theoretic approach. Am. Nat. 168, 123–135. and body mass of Tianzhu population is smaller than those in Angilletta, M.J., Niewiarowski, P.H., Navas, C.A., 2002. The evolution of thermal Minqin (Fig. 5),itisavaluableevidencetoproveAllen’srule physiology in ectotherms. J. Therm. Biol. 27, 249–268. 444 X.-L. Tang et al. / Journal of Thermal Biology 37 (2012) 438–444

Arago´ n, P., Lobo, J.M., Olalla-Ta´rraga, M.A´ ., Rodrı´guez, M.A´ ., 2010. The Contribution Møller, A.P., 2004. Protandry, sexual selection and climate change. Global Change of Contemporary Climate to Ectothermic and Endothermic Vertebrate Dis- Biol. 10, 2028–2035. tributions in a Glacial Refuge. Blackwell Publishing Ltd, pp. 40–49.. Mathies, T., Andrews, R.M., 1995. Thermal and reproductive biology of high and Ashton, K.G., 2001. Are ecological and evolutionary rules being dismissed prema- low elevation populations of the lizard Sceloporus scalaris: implications for the turely? Diversity Distrib. 7, 289–295. evolution of viviparity. Oecologia 104, 101–111. Ashton, K.G., Feldman, C.R., 2003. Bergman’s Rule in Nonavian Reptiles Mayr, E., 1956. Geographical character gradients and climatic adaptation. Evolu- Turtles Follow it, Lizard and Snakes Reverse it. Blackwell Publishing Ltd tion 10, 105–108. pp. 1151–1163.. Parmesan, C., Yohe, G., 2003. A globally coherent fingerprint of climate change Beuchat, C.A., 1988. Temperature effects during gestation in a viviparous lizard. J. impacts across natural systems. Nature 421, 37–42. Therm. Biol. 13, 135–142. McGinley, M.A., 1989. The influence of a positive correlation between clutch size Blanckenhorn, W.U., Stillwell, R.C., Young, K.A., Fox, C.W., Ashton, K.G., 2006. When and offspring fitness on the optimal offspring size. Evol. Ecol. 3, 150–156. rensch meets bergmann: does sexual size dimorphism change systematically Patterson, J., Davies, P.M.C., 1984. The influence of temperature, sexual condition, with latitude? Evolution 60, 2004–2011. and season on the metabolic rate of the lizard Psammodromus hispanicus. Booth, D.T., 2006. Influence of incubation temperature on hatchling phenotype in J. Comp. Physiol. B: Biochem., Syst., Environ. Physiol. 154, 311–316. reptiles. Physiol. Biochem. Zool. 79 (2), 274–281. Pen, I., Uller, T., Feldmeyer, B., Harts, A., While, G.M., Wapstra, E., 2010. Climate- Bull, J.J., 1983. Evolution of Sex Determining Mechanisms. The Benjamin/Cum- driven population divergence in sex-determining systems. Nature 468, mings Publishing Company, Inc. 436–438. ChamaillE´ -Jammes, S., Massot, M., AragO´ N, P., Clobert, J., 2006. Global Warming Peters, R.H., 1986. The Ecological Implications of Body Size. Cambridge University and Positive Fitness Response in Mountain Populations of Common Lizards Press. Lacerta vivipara. Blackwell Science Ltd, pp. 392–402.. Pulido, F., Berthold, P., 2004. Microevolutionary response to climatic change. Adv. Cooper Jr, W.E., Vitt, L.J., Hedges, R., Huey, R.B., 1990. Locomotor impairment and Ecol. Res. 35, 151–183. defense in gravid lizards (Eumecesl aticeps): behaviourals hift in activitym ay Porter, W.P., Tracy, C.R., 1983. Biophysical analyses of energetics, time-space offset costs of reproductionin an active forager. Behav. Ecol. Sociobiol. 27, utilization, and distributional limits. In: Huey, R.B., Pianka, E.R., Schoener, T.W. 153–157. (Eds.), Lizard Ecology: Studies of a Model Organism. Harvard University Press, Deeming, D.C., Ferguson, M.W.J., 2004. Egg Incubation: Its Effects on Embryonic Cambridge, MA, pp. 55–83. Development in Birds and Reptiles. Cambridge University Press. Post, W., 1998. Advantages of coloniality in female boat-tailed Grackles. Wilson Du, W.-G., Hu, L.-J., Lu, J.-L., Zhu, L.-J., 2007. Effects of incubation temperature on Bulletin 110, 489–496. embryonic development rate, sex ratio and post-hatching growth in the Radder, R.S., Pike, D.A., Quinn, A.E., Shine, R., 2009. Offspring sex in a lizard Chinese three-keeled pond turtle, Chinemys reevesii. Aquaculture 272, depends on egg size. Curr. Biol. 19, 1102–1105. 747–753. Radder, R.S., Quinn, A.E., Georges, A., Sarre, S.D., Shine, R., 2007. Genetic evidence Du, W.-G., Shine, R., 2008. The influence of hydric environments during egg for co-occurrence of chromosomal and thermal sex-determining systems in a lizard. Biol. Lett. 4, 176–178. incubation on embryonic heart rates and offspring phenotypes in a scincid Robert, K.A., Thompson, M.B., 2001. Sex determination: viviparous lizard selects lizard (Lampropholis guichenoti). Comp. Biochem. Physiol. A: Mol. Integr. sex of embryos. Nature 412, 698–699. Physiol. 151, 102–107. Schwarzkopf, L., Shine, R., 1992. Costs of reproduction in lizards: escape tactics Du, W.G., Feng, J.H., 2008. Phenotypic effects of thermal mean and fluctuations on and susceptibility to predation. Behav. Ecol. Sociobiol. 31, 17–25. embryonic development and hatchling traits in a lacertid lizard, Takydromus Shine, R., 2004. Does viviparity evolve in cold climate reptiles because pregnant septentrionalis. J. Exp. Zool. A: Ecol. Genet. Physiol. 309A, 138–146. females maintain stable (not high) body temperatures? Evolution 58, Dunn, P., 2004. Breeding dates and reproductive performance. Adv. Ecol. Res. 35, 1809–1818. 69–87. Shine, R., Harlow, P., 1993. Maternal thermoregulation influences offspring Escobedo-Galva´n, A.H., Gonza´lez-Salazar, C., Lo´ pez-Alcaide, S., Arroyo-Pen˜a, V.B., viability in a viviparous lizard. Oecologia 96, 122–127. Martı´nez-Meyer, E., 2011. Will all species with temperature-dependent sex Shine, R., Harlow, P.S., 1996. Maternal manipulation of offspring phenotypes via determination respond the same way to climate change? A reply to Kallimanis nest-site selection in an oviparous lizard. Ecology 77, 1808–1817. (2010). Oikos 120, 795–799. Sinervo, B., 1990. The evolution of maternal investment in lizards: an experi- Ferguson, G.W., Fox, S.F., 1984. Annual variation and survival advantage of large mental and comparative analysis of egg size and its effects on offspring juvenile side-blotched lizards, Uta stansburiana: its causes and evolutionary performance. Evolution 44, 279–294. significance. Evolution 38, 342–349. Sinervo, B., Licht, P., 1991. Hormonal and physiological control of clutch size, egg Gaston, K.J., Blackburn, T.M., Spicer, J.I., 1998. Rapoport’s rule: time for an epitaph? size, and egg shape in side&\blotched lizards (Uta stansburiana): constraints on Trends Ecol. Evol. 13, 70–74. the evolution of lizard life histories. J. Exp. Zool. 257, 252–264. Henrich, S., 1988. Variation in offspring sizes of poeciliid fish Herandria formosa in Thompson, M.B., Seebacher, F., Robert, K.A., 2003. Facultative sex allocation in the relation to fitness. Oikos 51, 13–18. viviparous lizard Eulamprus tympanum, a species with temperature-dependent Huey, R.B., Tewksbury, J.J., 2009. Can behavior douse the fire of climate warming? sex determination. Aust. J. Zool. 51, 367–370. Proc. Natl. Acad. Sci. USA 106, 3647–3648. Valenzuela, N., Adams, C., Janzen, F.J., 2003. Pattern does not equal process: Janzen, J.F., 1994. Climate Change and Temperature-Dependent Sex Determination exactly when is sex environmentally determined? Am. Nat., 676–683. in Reptiles. Proceedings of the National Academy of Sciences 91, 7487–7490. Wapstra, Erik, Uller, Tobias, Sinn, L.D., Olsson, Mats, Mazurek, Katrina, Joss, Jean, Ji, X., Xie, Y.-Y., Sun, P.-Y., Zheng, X.-Z., 1997. Sexual dimorphism and female Shine, Richard, 2009. Climate effects on offspring sex ratio in a viviparous reproduction in a viviparous snake, Elaphe rufodorsata. J. Herpetol. 31, lizard. J. Anim. Ecol. 78, 84–90. 420–422. Wapstra, E., Olsson, M., Shine, R., Edwards, A., Swain, R., Joss, J.M.P., 2004. Maternal Ji, X., Xu, X.-F., Du, W.-G., 2001. Female reproductive output and neonate basking behaviour determines offspring sex in a viviparous reptile. Proc. R. characteristics in a viviparous water snake (Sinonatrix annularis). Acta Zool. Soc. London. Ser. B 271, 230–232. Sinica 47, 231–234. Warner, D.A., Shine, R., 2007. Reproducing lizards modify sex allocation in Ji, X., Lin, L.H., Luo, L.G., Lu, H.L., Gao, J.F., Han, J., 2006. Gestation temperature response to operational sex ratios. Biol. Lett. 3, 47–50. affects sexual phenotype, morphology, locomotor performance, and growth of Witt, M.J., Hawkes, L.A., Godfrey, M.H., Godley, B.J., Broderick, A.C., 2010. Predict- neonatal brown forest skinks, Sphenomorphus indicus. Biol. J. Linn. Soc. 88, ing the impacts of climate change on a globally distributed species: the case of 453–463. the loggerhead turtle. J. Exp. Biol. 213, 901–911. Kallimanis, A.S., 2010. Temperature dependent sex determination and climate Xu, D.-D., Ji, X., 2007. Sexual dimorphism, female reproduction and egg incubation change. Oikos 119, 197–200. in the oriental leaf-toed gecko (Hemidactylus bowringii) from southern China. Li, H., Qu, Y.-F., Hu, R.-B., Ji, X., 2009. Evolution of viviparity in cold-climate lizards: Zoology 110, 20–27. testing the maternal manipulation hypothesis. Evol. Ecol. 23, 777–790. Yan, X.F., Tang, X.L., Yue, F., Zhang, D.J., Xin, Y., Wang, C., Chen, Q., 2011. Influence Li, H., Qu, Y.F., Ding, G.H., Ji, X., 2011. Life-history variation with respect to of ambient temperature on maternal thermoregulation and neonate pheno- experienced thermal environments in the lizard, Eremias multiocellata (Lacer- types in a viviparous lizard, Eremias multiocellata, during the gestation period. tidae). Zool. Sci. 28, 332–338. J. Therm. Biol. 36, 187–192. Lin, L.-H., Li, H., An, H., Ji, X., 2008. Do temperature fluctuations during incubation Zhang, D.-J., Tang, X.-L., Yue, F., Chen, Z., Li, R.-D., Chen, Q., 2010. Effect of gestation always play an important role in shaping the phenotype of hatchling reptiles? temperature on sexual and morphological phenotypes of offspring in a J. Therm. Biol. 33, 193–199. viviparous lizard, Eremias multiocellata. J. Therm. Biol. 35, 129–133. Lourdais, O., Bonnet, X., Doughty, P., 2002. Costs of anorexia during pregnancy in a Zhao, K.T., 1999. . In: Zhao, E.M., Zhao, K.T., Zhou, K.Y. (Eds.), Fauna viviparous snake (Vipera aspis). J. Exp. Zool. 292, 487–493. Sinica, Reptilia. Science Press, Beijing, pp. 219–242. 2 (: Lacertilia).