Organisms living at high altitudes must overcome three major environmental challenges: hypoxia, cold, and intense UV radiation. The molecular mechanisms that enable these challenges to be overcome have mainly been studied in endothermic organisms; relatively little attention has been paid to poikilothermic species. Here, we present deep transcriptome sequencing in two closely related lizards, the high altitude-dwelling Phrynocephalus erythrurus and the lowland-dwelling P. putjatia, to identify candidate genes under positive selection and to explore the convergent evolutionary adaptation of poikilothermic animals to high altitude life. More than 70 million sequence reads were generated for each species via Illumina sequencing. De novo assembly produced 56,845 and 63,140 transcripts for P. erythrurus and P. putjatia, respectively. P. erythrurus had higher Ka/Ks ratios than P. putjatia, implying an accelerated evolutionary rate in the high altitude lizard lineage. 206 gene ontology (GO) categories with accelerated evolutionary rates and 43 candidate positively selected genes were detected along the P. erythrurus lineage. Some of these GO categories have functions associated with responses to hypoxia, energy metabolism and responses to UV damage. We also found that the high-altitude ranid frog R. kukunoris had higher Ka/Ks ratios than the closely related low-altitude frog R. chensinensis, and that the functional categories with accelerated evolutionary rates in R. kukunoris overlapped extensively with those detected along the P. erythrurus lineage. The mechanisms of high altitude adaptation in P. erythrurus were tentatively inferred. By comparing two pairs of low- and high-altitude poikilothermic species, we found that similar functional categories had undergone positive selection in high altitude-dwelling Phrynocephalus and Rana lineages, indicating that similar mechanisms of adaptation to high altitude might have evolved in both genera. Our findings provide important guidance for future functional studies on high altitude adaptation in poikilothermic animals.

Investigating how highland amphibians respond to changes in ambient temperature may be of great significance for their fate prediction and effective conservation in the background of global warming. Here, using field individuals as the control group, we investigated the influence of high temperatures (20.5 and 25.5°C) and heat wave (15–26.6°C) on the thermal preference, critical thermal limits, locomotor performance, oxidative stress, and antioxidant enzyme activities in high-altitude frog Nanorana pleskei (3,490 m) endemic to the Qinghai-Tibet Plateau (QTP). After 2 weeks of acclimation to high temperatures and heat wave, the thermal preference (T_pref), critical thermal maximum (CT_max), and range of tolerable temperature significantly increased, while the critical thermal minimum (CT_min) was significantly decreased. The total time of jump to exhaustion significantly decreased, and burst swimming speed significantly increased in frogs acclimated in the high temperature and heat wave groups compared with the field group. In the high temperature group, the level of H₂O₂ and lipid peroxide (malondialdehyde, MDA), as well as the activities of glutathione peroxidase (GPX), glutathione reductase (GR), catalase (CAT), superoxide dismutase (SOD), and total antioxidant capacity (T-AOC) significantly increased in the liver or muscle. However, in the heat wave group, the MDA content significantly decreased in the liver, and antioxidants activities decreased in the liver and muscle except for CAT activities that were significantly increased in the liver. These results indicated that N. pleskei could respond to the oxidative stress caused by high temperatures by enhancing the activity of antioxidant enzymes. The heat wave did not appear to cause oxidative damage in N. pleskei, which may be attributed to the fact that they have successfully adapted to the dramatic temperature fluctuations on the QTP.

Ultraviolet-B radiation (UVBR), intensified by ozone depletion and climate change, poses a growing ecological threat to amphibians, particularly in high-elevation regions such as the Qinghai-Tibet Plateau. Endemic to this region, Rana kukunoris spans a wide range of elevations, where distinct populations may have evolved unique strategies and regulatory mechanisms to cope with UVBR. However, specific adaptive responses in adult frogs remain underexplored. This study compared the physiological responses of high- and low-altitude Rana kukunoris populations to UVBR exposure, focusing on dorsal color, immune function, antioxidant capacity, and DNA repair gene expression. High-altitude populations exhibited stable, dark pigmentation-potentially reducing the need for rapid melanin synthesis-alongside a robust immune profile and enhanced antioxidant enzyme activity, collectively conferring resilience against oxidative and immune stress under chronic UVBR exposure. Conversely, low-altitude populations exhibited pronounced UVBR-induced responses, including significant skin darkening, heightened immune activation evidenced by increased white blood cell counts, and increased oxidative damage marked by higher malondialdehyde (MDA) levels, coupled with reduced superoxide dismutase (SOD) and catalase (CAT) activities. Furthermore, tissue-specific upregulation of DNA repair genes in high-altitude populations suggested a stable DNA repair capacity adapted to high-UVBR environments. These findings reveal distinct physiological strategies within the same species for coping with UVBR across altitudinal gradients. Amid global increases in UVBR, this study offers novel insights into amphibian resilience in high-UVBR habitats and informs conservation strategies for populations across varying elevations.

In the context of climate change, understanding how indigenous amphibians of the Qinghai-Tibet plateau react to stresses and their coping mechanisms could be crucial for predicting their fate and successful conservation. A liver transcriptome for Nanorana pleskei was constructed using high-throughput RNA sequencing, and its gene expression was compared with frogs acclimated under either room temperature or high temperature and also heat wave exposed ones. A total of 126,465 unigenes were produced, with 66,924 (52.92%) of them being annotated. Up to 694 genes were found to be differently regulated as a result of abnormal temperature acclimatization. Notably, genes belonging to the heat shock protein (HSP) family were down-regulated in both treated groups. Long-term exposure to high-temperature stress may impair the metabolic rate of the frog and trigger the body to maintain a hypometabolic state in an effort to survive challenging times. During heat waves, unlike the high-temperature group, mitochondrial function was not impaired, and the energy supply was largely normal to support the highly energy-consuming metabolic processes. Genes were more transcriptionally suppressed when treated with high temperatures than heat waves, and the body stayed in low-energy states for combating these long-term adverse environments to survive. It might be strategic to preserve initiation to executive protein activity under heat wave stress. Under both stress conditions, compromising the protection of HSP and sluggish steroid activity occurred in frogs. Frogs were more affected by high temperatures than by heat waves.

Both the warming climate and ultraviolet-B radiation (UVBR) are considered to be notable environmental factors affecting amphibian population decline, with particular effects on tadpole development. However, the phenotypes of tadpoles living at high altitudes may be improved by moderately warming temperatures, reducing or eliminating the negative effects of oxidative damage caused by cool temperatures or strong UVBR at high altitudes. To verify this hypothesis, Rana kukunoris tadpoles, which live at high altitudes, were used to test the effect of the interaction of temperature and UVBR on their development and antioxidant systems in a fully factorial design. The tadpoles were exposed to UVBR and UVBR-free environments at 14 (cool temperature) and 22 °C (warm temperature), respectively. UVBR and a warm temperature had opposite influences on several traits of the tadpoles, including their survival, developmental rate, individual size, preferred body temperature, thermal tolerance temperature, oxidative damage, and enzymatic and nonenzymatic antioxidant systems. The moderate temperature could compensate for or override the negative effects of UVBR by increasing the tadpoles’ preferred body temperature and critical tolerance temperature, thus enhancing the locomotion ability and thermal sensitivity of their antioxidant systems. Furthermore, the dark skin coloration and aggregation behavior of R. kukunoris tadpoles may also be effective strategies for allowing them to resist UVBR and helping them to better adapt to a warming environment with stronger UVBR. Thus, it is possible that a moderate degree of warming may increase the capacity of living organisms to adapt to environmental changes and thus have positive effects on the development of tadpoles living at high altitudes.