Animal collection and maintenance

The P. erythrurus and P. przewalskii individuals were captured from the Hoh-xil National Nature Reserve and Minqin Integrated Desert Control Experiment Station, respectively. Both areas are protected and only used for scientific research, and the two authorities gave us permission to capture the animals used in this study. Meanwhile, our field studies did not involve endangered or protected species. The periods of the two lizards’ collection were from June to July 2012. The lizards were active in their habitat during this season. The mating have already completed in both P. erythrurus and P. przewalskii, and only male lizards were used in our present study, so we could exclude the influence of reproduction on their metabolism. Toad-headed lizards P. erythrurus were captured by hand in the wild at Tuotuo River (34°13'N, 92°13'E), Qinghai province, China, in August 2012 (N=35). The mean body mass was 5.26±0.21 g and mean snout-vent length (SVL) was 4.98±0.32 cm. P. przewalskii individuals were collected from Minqin (38°38'N, 103°05'E), Gansu province, China, in July 2012 (N=42). The mean body mass and SVL were 5.75±0.38 g and 5.61±0.44 cm, respectively. The elevations of the two collection sites (measured by GPS, Magellan Explorist 600) were 4543 m and 1482 m, respectively. Climatic data (atmospheric pressure, highest and lowest temperature, air temperature and mean sunshine duration) of the two sampling sites were provided by the Chinese Climatic Data Centre (CDC) for the years from 1959 to 2009. All these climatic data were recorded at meteorological stations, which were situated at or near (< 2 km) the collection sites.

All captured lizards from the collecting zone were brought to the laboratory at Lanzhou University (36°05'N, 103°86'E) within 24 hours of capture. These lizards were maintained in a room at constant temperature (16±0.5°C), which was controlled by an air-conditioning system. As the habitat environmental conditions of P. erythrurus are quite different from our laboratory environment, especially the difference in PO₂, we needed to limit the possible effect of changed PO₂ on the physiological and biochemical characteristics of the lizards. P. erythrurus individuals were therefore kept in a non-pressurized hypoxic chamber (100 cm length, 45 cm width and 45 cm height) to simulate a low PO₂ at an altitude of 4550 m. Nitrogen gas was continuously added to the chamber to dilute the oxygen concentration to 11.8% (PO₂^≈90 mmHg, equivalent altitude of 4550 m). After the required oxygen concentration reached, it was then monitored and maintained by an oxygen controller (HCD-2B, Mei Cheng Oxygen Analysis Instruments Plant) connected to an electromagnetic valve to control the nitrogen flow. A 60 W bulb was suspended above one end of the chamber and operated for 10 h (from 0830h to 1830h) to provide a thermal gradient from 35 to 16°C. The bottom of the chamber was covered with a 10-mm depth of silver sand, and several bricks were placed in the chamber to give the lizards the opportunity to choose their optimal temperature or basking place. Meanwhile, fluorescent lamps were used to simulate natural light on a constant 12h: 12h light on: off cycle for each chamber. The climatic conditions and altitude of Lanzhou is similar to the Minqin, so the P. przewalskii were simply maintained in the chamber with PO₂=137mmHg. The other conditions were the same as for P. erythrurus. All lizards were fed mealworms and water ad libitum and all experiments were finished within 10 days of the collection.

Tissue sampling

The liver and skeletal muscle were blotted with absorbent paper to remove excess liquid and then weighed (accuracy: 0.001 g). The fresh tissues were used for mitochondrial assay, and the remaining tissues were immediately frozen in liquid nitrogen and then transferred into a cryogenic refrigerator and frozen at −80° C for subsequent assays of enzyme activity and gene expression.

Mitochondrial isolation

For both species, 10 adult male lizards were used in the mitochondrial assay. The mitochondrial isolation was conducted according to published protocols with the following modifications. For liver mitochondria, the fresh organ was weighed and immediately homogenized in four volumes of ice-cold isolation buffer (250 mmol l^-1 sucrose, 5 mmol l^-1 Tris/HCl, pH 7.4, and 2 mmol l^-1 EGTA). The homogenate was spun at 3000 rpm for 5 min at 4° C, and the supernatant centrifuged at 12000 rpm for 10 min at 4° C. Then, the supernatant was discarded and the mitochondria were resuspended in an 800-µl isolation medium. In order to isolate the mitochondria of the skeletal muscle, the gastrocnemius from the hind-leg was removed, weighed and cut with scissors and homogenized in the isolation buffer (140 mmol l^-1 KCl, 50 mmol l^-1 Tris/HCl, pH 7.4, 2 mmol l^-1 EGTA, 0.5% (w/v) BSA, 5 mmol l^-1 MgCl₂, 1 mmol l^-1 ATP and 2.45 units ml^-1 Protease Type VIII), using a glass homogenizer. The homogenate was centrifuged at 3000 rpm for 5 min at 4° C, and the resulting supernatant was subjected to a high-speed spin cycle (12000 rpm, 10 min and 4° C). The mitochondrial pellet was resuspended in 650 µl medium (140 mmol l^-1KCl, 50 mmol l^-1 Tris/HCl, pH 7.4, 2 mmol l^-1 EGTA, 0.5% (w/v) BSA).

Mitochondrial O₂ consumption and proton leak measurement

Mitochondrial respiration was monitored on a Chlorolab 2 system (Hansatech Instruments, Norfolk, England) and thermostatically controlled at constant temperatures (20 and 30°C) by a circulator bath. The resulting mitochondrial solution (140 µl pre-assay) was saturated by room air with steadfast stirring at 50 rpm. The respiratory rate of state3 was determined in the presence of 5 mmol l^-1 succinate and 5 µmol l^-1of rotenone (to inhibit complex I of the respiratory chain) after the addition of 1 mmol l^-1 ADP, and the respiratory rate of state4 was measured after all the ADP was consumed. Thermal sensitivities were expressed as Q₁₀ = (K2/K1)^{10 /T2–T1}, where K1 and K2 are the reaction rates at temperatures T1 and T2, respectively.

Evaluation of possible uncoupling mechanisms followed the methods given by Guderley et al. and Rey et al., with some modifications [29,30]. Liver and skeletal muscle mitochondria from the fresh organs were used and the assays proceeded at 30° C. The potential contribution of UCP to mitochondrial respiration was evaluated by measuring inhibition of state4 using the addition of 2 mmol l^-1 guanosine diphosphate (GDP). The impact of inhibiting the adenine nucleotide translocase (ANT) was measured in an independent set of experiments and assessed by the addition of a non-competitive inhibitor, carboxyatractyloside (CAT), to a final concentration of 5 µM.

Enzyme activity measurement

A total of 10 lizards from each species were used for the analyses of enzyme activity. Tissues (liver and skeletal muscle) for lactate dehydrogenase (LDH, EC 1.1.1.27), citrate synthase (CS, E.C. 2.3.3.1) and β-hydroxyacyl coenzyme A dehydrogenase (HOAD, EC 1.1.1.35) assays were collected after the lizards were brought to our laboratory and stored at −80° C until analysis. Our methods for LDH and CS measurements followed those of Seebacher et al. [31], and the HOAD assay was conducted according to the published protocols of John-Alder and Joos [32], with some modifications. Tissue samples (0.05–0.15 g) were homogenized in nine volumes of ice-cold extraction medium (100 mmol l^-1 potassium phosphate (KH₂PO₄/K₂PO₄), 2 mmol l^-1 MgCl₂, 5 mmol l^-1EDTA, 1 mmol l^-1 reduced glutathione and 1% Triton X-100), and enzyme activities were determined in a temperature-controlled spectrophotometer (UV2000, Unico Instrument Co., Ltd., Shanghai, China). All assays were performed in duplicate at 20 and 30°C and enzyme activity was expressed in units of g^-1 wet tissue.

RNA isolation and cDNA synthesis

Total RNA was extracted from 50–100mg of liver and muscle using RNAiso Plus reagent (Takara Bio, Japan) according to the manufacturer’s instructions. RNA concentration and purity was measured using the NanoDrop 2000 (Thermo Scientific, USA) and the integrity was confirmed using 1.2% agarose gel (Gene Tech, Shanghai, China). The residue of genomic DNA was removed in a volume of 10 µl at 42° C for 2 min, containing 1 µg of RNA samples, 2 µL 5×gDNA Eraser Buffer, 1 µL gDNA Eraser and RNase Free dH₂O. Immediately after this, first-strand cDNA synthesis was performed by adding 4 µL of 5× PrimeScript Buffer, 1 µL of PrimerScript RT Enzyme Mix, 1 µL of RT Enzyme and 4 µL RNase Free dH₂O at 37 °C for 15 min, 85 °C for 15 s.

RT-PCR amplification and quantitative real-time PCR

We designed new primers for the partial sequence of PGC-1α and PPAR-γaccording to the sequences of Anolis carolinensis and Eublepharis macularius obtained from GenBank; all primer sequences are described in Table 2. The polymerase chain reaction (PCR) amplification was conducted in a total volume of 20 µL containing 10×PCR buffer, 3 mM Mg²⁺, 0.50 µM of each primer, 0.50 mM of dNTPs, 0.50 units of Taq DNA polymerase (Sangon, Shanghai, China) and about 100 ng cDNA templates. Touchdown PCR protocols were used: 95° C for 5 min, 20 cycles of 95° C for 30 s, 65° C for 30 s, reducing by 2° C every two cycles to 51° C and 72° C for 1 min; 15 cycles at 55° C and 72° C for 10 min. PCR products were visualized on 2% low melting agarose gels. Bands were cut, purified with an EZ spin column DNA gel extraction kit (Sangon, China) and cloned into PMD18-T Vector (Takara Bio, Japan), and subsequently sequenced.

Expression patterns of these target genes were analysed using quantitative real-time PCR and β-actin was selected as an internal control for gene expression. We designed the specific primers for target or reference genes using the Primer Premier 5 (Table 3). The amplifications were performed in a 96-well plate in a 20 µL reaction volume containing 10 µl of 2×SYBR Master Mix Takara Bio, Japan), 0.4 µM of each primer, 2 µL of cDNA template, and DEPC-water. The PCR cycles were 95° C for 30s, 39 cycles of 95° C for 5s and Tm for 30s. All assays were performed in triplicate on 96-well plates, and for each of these we also conducted negative (RNA samples) and blank (water) controls using an CFX-96 real-time PCR machine (Bio-Rad Laboratories, Inc., CA, USA). The relative expression was calculated as 2^–ΔΔCt [33].

Statistical analyses

All data were tested for normality and homogeneity of variances to meet the assumptions of parametric testing prior to analysis, and no significant deviations from these assumptions were evident in the data. Data on morphological traits, values of Q₁₀ and gene expression were analysed using an ANOVA followed by a post-hoc Tukey’s test, and means values of mitochondrial uncoupling were compared using a paired t-test. Potential interactions between activity states (mitochondria and enzymes) and assay temperature were analyzed by two-way ANOVA. The values were reported as Means ± Standard error (s.e.m.) and were performed using SPSS release 16.0.0 (SPSS, Inc., Chicago, Illinois, USA).

Respiratory rate of liver and skeletal muscle mitochondria

For both liver and skeletal muscle mitochondria, the respiration rates of state3 and state4 increased with assay temperatures in the two species (Figure 1). Although the mean Q₁₀ was not significantly different between the two species, the values of Q₁₀ for state 3 and state 4 of both the liver and skeletal muscle in P. erythrurus were higher than those in P. przewalskii. The only exception was the mean Q₁₀ value of state 4 in P. erythrurus, which was lower than that in P. przewalskii (Table 4).

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Figure 1. Mitochondrial respiratory rate and the uncoupling of liver and skeletal muscle in Phrynocephalus erythrurus and Phrynocephalus przewalskii, respectively.

Values are means ± s.e.m.

https://doi.org/10.1371/journal.pone.0071976.g001

We also compared the possible interspecies variations in state3 and state4 for liver and skeletal muscle mitochondria, respectively. For liver mitochondria, the rate of state3 in P. erythrurus was much lower than that in P. przewalskii at the two assay temperatures (20° C:F_{1, 11} = 43.358, P<0.001; 30° C:F_{1, 12} = 25.250, P<0.001). Rates of state4 were also lower in P. erythrurus than in P. przewalskii at both 20 (F_1,11=29.940, P<0.001) and 30° C (F_1,13=113.065, P<0.001). At both assay temperatures, the maximal rates of liver mitochondria in P. przewalskii were approximately five- to eight-fold higher than in P. erythrurus; similarly, the oligomycin-inhibited rates of P. przewalskii were almost seven- to eight-fold higher than for P. erythrurus (Figure 1). On the other hand, skeletal muscle mitochondria in P. przewalskii had significantly higher maximal rates of succinate oxidation than the mitochondria of P. erythrurus at both assay temperatures (20° C:F_{1, 9} = 26.042, P<0.001; 30° C:F_{1, 14} = 8.963, P=0.010). There were also significant differences in the rate of state4 between the two species; the oligomycin-inhibited mitochondria from P. erythrurus had a lower respiratory rate than those from P. przewalskii. The rates of state3 and state4 at both assay temperatures were approximately twice as fast in P. przewalskii as in P. erythrurus (Figure 1).

Mechanisms of mitochondrial uncoupling

In liver, GDP significantly reduced the oxygen uptake of oligomycin-inhibiting state4 respiration in P. przewalskii (N=6, paired t-test, t=7.211, P=0.001) and P. erythrurus (N=5, paired t-test, t=4.311, P=0.013), respectively. However, the addition of CAT reduced oxygen uptake, but only in P. przewalskii (N=7, paired t-test, t=4.103, P=0.006), not in P. erythrurus (N=6, paired t-test, t=2.447, P=0.058). For skeletal muscle mitochondria, neither GDP (N=4, paired t-test, t=2.390, P=0.097) nor CAT (N=4, paired t-test, t=2.593, P=0.122) significantly reduced the oxygen uptake in P. przewalskii. On the other hand, the reverse results were found in the skeletal muscle mitochondria of P. erythrurus; both GDP (N=5, paired t-test, t=5.434, P=0.006) and CAT (N=5, paired t-test, t=3.895, P=0.018) dramatically reduced oxygen uptake at 30° C (Figure 1).

We also analysed possible interspecies differences related to the effect of GDP and CAT on the oxygen uptake of liver or skeletal muscle mitochondria (Figure 1). In liver, P. przewalskii mitochondria were inhibited by GDP (F_1,15=8.879, P=0.009) and CAT (F_1,17=16.765, P=0.001), and had a significantly higher respiratory rate than those from P. erythrurus. Similar to the results for liver mitochondria, CAT-inhibited skeletal muscle mitochondria from P. przewalskii consumed oxygen faster than those from P. erythrurus (F_1,11=5.110, P=0.045). However, inhibition by GDP did not significantly affect the respiratory rate between the two species (F_1,11=3.758, P=0.079).

Metabolic enzyme activity

In both the liver and skeletal muscle of the two species, LDH activity increased significantly with the assay temperatures (all F>5.982, P<0.026). The differences in LDH activity between the liver and skeletal muscle of the two species were also analysed. Liver LDH activity in P. przewalskii was notably higher than in P. erythrurus at 20° C (F_1,18=6.092, P=0.024) and 30° C (F_1,18=4.999, P=0.038) (Figure 2), but no significant difference was observed in the skeletal muscle of either of the two species at both assay temperatures (20° C:F_{1, 17} = 0.300, P=0.591; 30° C:F_{1, 17} = 0.230, P=0.638) (Figure 3).

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Figure 2. Activities of lactate dehydrogenase (LDH), citrate synthase (CS) and β-hydroxyacyl coenzyme A dehydrogenase (HOAD) at 20 and 30°C in the liver of Phrynocephalus erythrurus (Open columns) and Phrynocephalus przewalskii (Twill columns).

Values are means ± s.e.m.

https://doi.org/10.1371/journal.pone.0071976.g002

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Figure 3. Activities of lactate dehydrogenase (LDH), citrate synthase (CS) and β-hydroxyacyl coenzyme A dehydrogenase (HOAD) at 20 and 30°C in the skeletal muscle of Phrynocephalus erythrurus (Open columns) and Phrynocephalus przewalskii (Twill columns).

Values are means ± s.e.m.

https://doi.org/10.1371/journal.pone.0071976.g003

The assay temperatures had a significant effect on the CS activity in all tissues and both species (all F>4.743, P<0.050). However, there was no significant difference in CS activity for either liver or skeletal muscle between the two species at 20 and 30°C, respectively (all F<1.003, P>0.300) (Figures 2, 3).

The HOAD activity changed significantly with assay temperature only in the skeletal muscle of the two species (P. erythrurus: F_1,18=15.604, P=0.001; P. przewalskii: F_1,17=5.622, P=0.033) (Figure 3), and not in liver (P. erythrurus: F_1,18=2.328, P=0.144; P. przewalskii: F_1,16=0.394, P=0.539) (Figure 2). The HOAD activity of P. erythrurus in skeletal muscle was significantly higher than in P. przewalskii at 20 (F_1,16=7.965, P=0.012) and 30° C (F_1,16=7.642, P=0.014). However, there was no significant variation between livers of the two species at 20 or 30°C (all F<1.117, P>0.305). The thermal sensitivity of four enzyme activities, expressed as Q₁₀ values calculated between 20 and 30°C, were compared between the two species for liver and skeletal muscle. Q₁₀ values of each enzyme activities were similar between the two assay temperatures (all P>0.05) (Table 4).

The expression of PGC-1α and PPAR-γ mRNA

In liver, the expression of PGC-1α (F_1,12=5.361, P<0.001) and PPAR-γ (F_1,12=3.479, P<0.001) mRNAs was significantly higher in P. przewalskii, with 2.9-fold more PGC-1α and 1.6-fold more PPAR-γ in P. przewalskii compared to P. erythrurus, respectively (Figure 4). The expression of these two genes in skeletal muscle followed a similar pattern, with a lower expression level of the PGC-1α (F_1,11=2.669, P<0.01) and PPAR-γ (F_1,11=1.284, P<0.01) mRNAs in P. erythrurus (Figure 4).

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Figure 4. Real-time PCR analysis of PGC-1α and PPAR-γ mRNA levels in liver and skeletal muscle from Phrynocephalus erythrurus and Phrynocephalus przewalskii.

Values are means ± s.e.m. in each group.

https://doi.org/10.1371/journal.pone.0071976.g004

Low respiratory rate and high ATP production efficiency of mitochondria in P. erythrurus

The protein-specific maximal oxidative capacities of skeletal muscle mitochondria in P. erythrurus were only about 50% of the capacities of the mitochondria from P. przewalskii. Mitochondria from P. erythrurus liver present an even lower maximal respiratory rate, being 12~18% of those from the liver of P. przewalskii. The mitochondrial respiratory rate in P. erythrurus was also lower than some other reptiles in hibernation or cold seasons [34,35]. In general, the mitochondrial respiratory rate is obviously different among vertebrates, with the exact rate depending on the phylogeny and environmental conditions [10,36,37]. The mitochondrial respiratory rate of many ectothermic animals living in cold environments is lower than that of ectothermic animals living in warm environments [30]. Our results were consistent with these. Additionally, mitochondria are also believed to be an oxygen-sensitive element in cells, and mitochondrial respiratory rate and efficiency could decline at low PO₂ [1]. Therefore, it is likely that the reduced respiratory rate of mitochondria in P. erythrurus could be a result of the synergistic effects of long-term living under cold temperature and low PO₂ at high altitude. Furthermore, such variation at mitochondrial level may be directly related to the effect of temperature on mitochondrial membrane structure or the activity of the electron transport chain [34,38]. However, we do not know the structure of the mitochondrial membrane, and membrane potential can be affected by low temperature or low oxygen, thus causing depression of mitochondrial metabolism. These aspects will be the subject of particular attention in our future work.

Although the mitochondrial respiratory rate of P. erythrurus was much lower than many ectothermic animals, our results for proton leak indicated that liver mitochondrial efficiency was significantly higher in this species. The proton leak of liver mitochondria was estimated to account for 7~8% and 24~26% of state3 in P. erythrurus and P. przewalskii, respectively. Furthermore, the proton leak of P. erythrurus liver mitochondria accounted for a greater proportion of state4 than in P. przewalskii (74~79% VS. 43~48%). These results on one hand are associated with a higher respiratory control rate (RCR) in P. erythrurus than that in P. przewalskii; on the other hand, may also directly relate to the biological function of proton leak. Numerous studies suggested that the main functions of proton leak are heat generation and reducing the production of ROS [39]. So a higher proton leak of P. erythrurus in the resting state (state4) should contribute to produce more heat, therefore benefitting P. erythrurus by maintaining a suitable core temperature to withstand the low cave temperature (measured by iButton, with an average temperature below 6° C, which close to its lethal temperature). However, a higher proton leak may accelerate the utilization of nutrient and speed up the depletion time of intrinsic body energy stores, a greater fat utilization mentioned below could be an effective strategy to compensate such high-energy consumption during resting state. Furthermore, a proton leak that is reduced to less than 10% during the active state (state3) of P. erythrurus will improve the utilization of oxygen and ATP production. On the other hand, the proton leak of the two lizards accounts for almost equal proportions in either state4 or state3 in skeletal muscle. In accordance with our results, similar results were also predominantly found in some assays on mammalian skeletal muscles [14,40]. The possible explanation could be related with the constant average value of mitochondrial ATP turnover during the rest-to-exercise transition, while the rate of proton leak across the mitochondrial inner membrane (which is controlled by ATP turnover) also remains constant [14].

Low anaerobic metabolism and possibly greater fat utilization in P. erythrurus

It is assumed that animals exposed to a cold or hypoxic environment over an evolutionary time frame have, through natural selection, developed and maintained characters that are beneficial to maintaining ATP synthesis despite continually limited oxygen availability [41]. Numerous papers suggest that anaerobic metabolism is not extensively used for long time ATP supplement in terrestrial vertebrates. In our present study, anaerobic metabolism in the liver of P. erythrurus is significantly lower than that in P. przewalskii, which is implied by lower LDH activity as well as a lower LDH/CS ratio in liver. These results do not conform to the expectation that LDH activity would increase in cold temperatures, as well as low PO₂ conditions (Pasteur Effect) [31,35]. However, for better living in cold and hypoxia conditions, endemic animals inhabiting high altitudes have revealed a number of strategies to adjust the anaerobic metabolism level, including isozyme spectrum and specific expression of LDH-M and LDH-H [42–44]. Furthermore, in accordance with our results, similar results were also predominantly found in some native animals at high altitude and hibernating animals [17,45]. Therefore, low LDH activity in P. erythrurus might the result of selectively evolved under the cold and hypoxia pressure of the Qinghai–Tibet Plateau. Such low anaerobic metabolism is also thought to be related to a more efficient coupling between ATP demand and ATP supply, allowing for a more effective integration between glycolysis and oxidative metabolism [45]. In contrast, there is no significant variation in LDH activities in the skeletal muscle of the two lizards, in accordance with the similar thermal sensitivities of LDH. Both P. erythrurus and P. przewalskii are mainly dependent on short burst locomotors in the wild. So the non-sustained contraction and relaxation of skeletal muscle in P. erythrurus can be achieved by anaerobic energy production, and a similar phenomenon has also been reported in some other species [41].

Fatty acid oxidation in reptiles is expected to be less important than in mammals, because HOAD activity and the HOAD/CS ratio is lower in reptilian species than in other vertebrates [46,47]. However, a higher free fat acid (FFA) (data not shown) and HOAD activity, accompanied by a higher HOAD/CS ratio, were found in P. erythrurus, suggesting a substantial proportion of the energy supply may derive from lipid oxidation in this species. The similar results were also detected in Tupinambis merianae and some other reptiles [37,48]. HOAD activity indicates the relative oxidation capacity of fatty acid, although, unlike CS, it does not give an indication of total aerobic capacity, and only measures the portion of fat oxidation. Increased fatty acid metabolism in liver acts to diminish the use of glycogen reserves, and leads to the accumulation of more glycogen in the P. erythrurus liver (proved by liver transmission electron microscopy, data not shown). In addition, the increase of capacity for fatty acid oxidation in skeletal muscle would limit the use of modest glycogen reserves, and this pattern, together with the constancy of CS in both liver and skeletal muscle examined, emphasises the nature of aerobic metabolism in P. erythrurus.

Low expression of metabolism-related genes in P. erythrurus

Studies on mammals have indicated that both low temperature and hypoxia could induce PGC-1α and PPAR-γ expression in various tissues. However, P. erythrurus living in cold temperature and low PO₂ conditions showed no increased expression of PGC-1α and PPAR-γ. Such downregulation of gene expression could be related to its effective adjustment at proton leak and enzyme level, and high body temperature and enough oxygen availability in its habitat could also eliminate the influence of cold and low PO₂ on the expression of PGC-1α and PPAR-γ in P. erythrurus. Firstly, we found that P. erythrurus could achieve a body temperature of over 30° C during activity in their habitat, even when the ambient temperature was only about 10~15°C (unpublished data). Secondly, studies on Phrynocephalus lizards indicate that some physiological traits were significantly changed only when the PO₂ was as low as 60 mmHg [19]. Previous studies on the oxygen-dependence of respiration also indicate that changing atmospheric PO₂ mainly has a significant impact on the PO₂ of alveolar air and arterial blood rather than on the PO₂ of cell or mitochondria, while mitochondria can achieve a maximum respiratory rate even when PO₂ is at a level of 15 mmHg [11]. Therefore, these characteristics, which are indicative of physiological, behavioural and structural aspects of P. erythrurus, provide a considerable contribution to living at cold temperatures and low PO₂ environmental conditions, rather than metabolism-related genes. However, the function and mechanism of these genes in animals endemic to high altitudes remains unclear.