Lemei Zhan

As ectotherms highly sensitive to environmental temperature fluctuations, skinks (a small lizard) are increasingly vulnerable to population instability due to global heatwaves. A clade model analysis of four Chinese skink species (Plestiodon capito, Plestiodon chinensis, Sphenomorphus indicus, and Scincella modesta) revealed positive selection acting on the ND6 gene in Sp. indicus. This species exhibits codon alterations in ND6, shifts its expression pathway and potentially decouples ND6 from high-temperature stress response mechanisms. To validate these findings, transcriptomic profiling was conducted to assess mitochondrial protein-coding gene (PCG) expression patterns under thermal stress. Using RT-qPCR, liver mitochondrial PCG transcript levels were compared between high-temperature (34 °C) and control (25 °C) groups in skink populations from distinct latitudes. Low-latitude species (P. chinensis and Sc. modesta) exhibited metabolic downregulation, characterized by a significant suppression of mitochondrial gene expression. Specifically, P. chinensis showed the downregulation of six mitochondrial genes (COII, COIII, ATP6, ND2, ND4, ND6) while upregulating one (ND1). By contrast, Sc. modesta showed the downregulation of nine genes (COI, COII, COIII, ATP8, ND1, ND3, ND4, ND4L, CYTB) and upregulated two (ND5, ND6). By contrast, high-latitude species exhibited divergent patterns: P. capito downregulated four genes (COI, COII, COIII, ND4L) and upregulated four others (ND1, ND2, ND3, ND4), whereas Sp. indicus downregulated six genes (COI, COII, ND2, ND3, ND4, ND4L) and upregulated one (ND5). These regulatory disparities suggest that low-latitude skinks have a greater capacity for metabolic depression to cope with chronic stress, whereas their high-latitude counterparts exhibit different adaptations. The findings provide valuable insights into assessing the adaptive potential of species in warming environments, particularly for ectotherms with limited thermoregulatory capacities.

Continual climate change strongly influences temperature conditions worldwide, making ectothermic animals as suitable species for studying the potential impact of climate change on global biodiversity. However, the study of how lizards distributed at different latitudes respond to climate change at the transcriptome level is still insufficient. According to the Climatic Variability Hypothesis (CVH), the range of climate fluctuations experienced by terrestrial animals throughout the year increases with latitude, so individuals at higher latitudes should exhibit greater thermal plasticity to cope with fluctuating environments. Mitochondria, as the energy center of vertebrate cells, may indicate species’ plasticity through the sensitivity of gene expression. In this study, we focused on the changes in transcript levels of liver mitochondrial protein-coding genes (PCGs) in skinks from the genus Plestiodon (P. capito and P. elegans) and the genus Scincella (S. modesta and S. reevesii) under low-temperature conditions of 8 °C, compared to the control group at 25 °C. Species within the same genus of skinks exhibit different latitudinal distribution patterns. We found that the two Plestiodon species, P. elegans and P. capito, employ a metabolic depression strategy (decreased transcript levels) to cope with low temperatures. In contrast, the two Scincella species show markedly different patterns: S. modesta exhibits significant increases in the transcript levels of six genes (metabolic compensation), while in S. reevesii, only two mitochondrial genes are downregulated (metabolic depression) compared to the control group. We also found that P. capito and S. modesta, which live at mid-to-high latitudes, exhibit stronger adaptive responses and plasticity at the mitochondrial gene level compared to P. elegans and S. reevesii, which live at lower latitudes. We suggest that this enhanced adaptability corresponds to more significant changes in a greater number of genes (plasticity genes).

Anguimorpha, within the order Squamata, represents a group with distinct morphological and behavioral characteristics in different ecological niches among lizards. Within Anguimorpha, there is a group characterized by limb loss, occupying lower ecological niches, concentrated within the subfamily Anguinae. Lizards with limbs and those without exhibit distinct locomotor abilities when adapting to their habitats, which in turn necessitate varying degrees of energy expenditure. Mitochondria, known as the metabolic powerhouses of cells, play a crucial role in providing approximately 95% of an organism’s energy. Functionally, mitogenomes (mitochondrial genomes) can serve as a valuable tool for investigating potential adaptive evolutionary selection behind limb loss in reptiles. Due to the variation of mitogenome structures among each species, as well as its simple genetic structure, maternal inheritance, and high evolutionary rate, the mitogenome is increasingly utilized to reconstruct phylogenetic relationships of squamate animals. In this study, we sequenced the mitogenomes of two species within Anguimorpha as well as the mitogenomes of two species in Gekkota and four species in Scincoidea. We compared these data with the mitogenome content and evolutionary history of related species. Within Anguimorpha, between the mitogenomes of limbless and limbed lizards, a branch-site model analysis supported the presence of 10 positively selected sites: Cytb protein (at sites 183 and 187), ND2 protein (at sites 90, 155, and 198), ND3 protein (at site 21), ND5 protein (at sites 12 and 267), and ND6 protein (at sites 72 and 119). These findings suggested that positive selection of mitogenome in limbless lizards may be associated with the energy requirements for their locomotion. Additionally, we acquired data from 205 mitogenomes from the NCBI database. Bayesian inference (BI) and Maximum Likelihood (ML) trees were constructed using the 13 mitochondrial protein-coding genes (PCGs) and two rRNAs (12S rRNA and 16S rRNA) from 213 mitogenomes. Our phylogenetic tree and the divergence time estimates for Squamata based on mitogenome data are consistent with results from previous studies. Gekkota was placed at the root of Squamata in both BI and ML trees. However, within the Toxicofera clade, due to long-branch attraction, Anguimorpha and (Pleurodonta + (Serpentes + Acrodonta)) were closely related groupings, which might indicate errors and also demonstrate that mitogenome-based phylogenetic trees may not effectively resolve long-branch attraction issues. Additionally, we reviewed the origin and diversification of Squamata throughout the Mesozoic era, suggesting that Squamata originated in the Late Triassic (206.05 Mya), with the diversification of various superfamilies occurring during the Cretaceous period. Future improvements in constructing squamate phylogenetic relationships using mitogenomes will rely on identifying snake and acrodont species with slower evolutionary rates, ensuring comprehensive taxonomic coverage of squamate diversity, and increasing the number of genes analyzed.

In the context of global warming, the frequency of severe weather occurrences, such as unexpected cold spells and heat waves, will grow, as well as the intensity of these natural disasters. Lizards, as a large group of reptiles, are ectothermic. Their body temperatures are predominantly regulated by their environment and temperature variations directly impact their behavior and physiological activities. Frequent cold periods and heat waves can affect their biochemistry and physiology, and often their ability to maintain their body temperature. Mitochondria, as the center of energy metabolism, are crucial for maintaining body temperature, regulating metabolic rate, and preventing cellular oxidative damage. Here, we used RT-qPCR technology to investigate the expression patterns and their differences for the 13 mitochondrial PCGs in Sphenomorphus incognitus (Squamata:Scincidae), also known as the brown forest skink, under extreme temperature stress at 4 °C, 8 °C, 34 °C, and 38 °C for 24 h, compared to the control group at 25 °C. In southern China, for lizards, 4 °C is close to lethal, and 8 °C induces hibernation, while 34/38 °C is considered hot and environmentally realistic. Results showed that at a low temperature of 4 °C for 24 h, transcript levels of ATP8, ND1, ND4, COI, and ND4L significantly decreased, to values of 0.52 ± 0.08, 0.65 ± 0.04, 0.68 ± 0.10, 0.28 ± 0.02, and 0.35 ± 0.02, respectively, compared with controls. By contrast, transcript levels of COIII exhibited a significant increase, with a mean value of 1.86 ± 0.21. However, exposure to 8 °C for 24 h did not lead to an increase in transcript levels. Indeed, transcript levels of ATP6, ATP8, ND1, ND3, and ND4 were significantly downregulated, to 0.48 ± 0.11, 0.68 ± 0.07, 0.41 ± 0.08, 0.54 ± 0.10, and 0.52 ± 0.07, respectively, as compared with controls. Exposure to a hot environment of 34 °C for 24 h led to an increase in transcript levels of COI, COII, COIII, ND3, ND5, CYTB, and ATP6, with values that were 3.3 ± 0.24, 2.0 ± 0.2, 2.70 ± 1.06, 1.57 ± 0,08, 1.47 ± 0.13, 1.39 ± 0.56, and 1.86 ± 0.12, respectively, over controls. By contrast, ND4L exhibited a significant decrease (to 0.31 ± 0.01) compared with controls. When exposed to 38 °C, the transcript levels of the 13 PCGs significantly increased, ranging from a 2.04 ± 0.23 increase in ND1 to a 6.30 ± 0.96 rise in ND6. Under two different levels of cold and heat stress, the expression patterns of mitochondrial genes in S. incognitus vary, possibly associated with different strategies employed by this species in response to low and high temperatures, allowing for rapid compensatory adjustments in mitochondrial electron transport chain proteins in response to temperature changes. Furthermore, this underscores once again the significant role of mitochondrial function in determining thermal plasticity in reptiles.

Potamanthidae belongs to the superfamily Ephemeroidea but has no complete mt genome released in the NCBI (except for two unchecked and one partial mt genome). Since the sister clade to Potamanthidae has always been controversial, we sequenced seven mt genomes of Potamanthidae (two species from Rhoenanthus and five species from Potamanthus) in order to rebuild the phylogenetic relationships of Potamanthidae in this study. The divergence time of Potamanthidae was also investigated by utilizing five fossil calibration points because of the indeterminate origin time. In addition, because Rhoenanthus coreanus and Potamanthus luteus are always in low-temperature environments, we aimed to explore whether these two species were under positive selection at the mt genome level. Amongst the 13 PCGs, CGA was used as the start codon in COX1, whereas other genes conformed to initiating with an ATN start codon. From this analysis, UUA (L), AUU (I), and UUU (F) had the highest usage. Furthermore, the DHU arm was absent in the secondary structure of S1 in all species. By combining the 13 PCGs and 2 rRNAs, we reconstructed the phylogenetic relationship of Potamanthidae within Ephemeroptera. The monophyly of Potamanthidae and the monophyly of Rhoenanthus and Potamanthus were supported in the results. The phylogenetic relationship of Potamanthidae + (Ephemeridae + Polymitarcyidae) was also recovered with a high prior probability. The divergence times of Potamanthidae were traced to be 90.44 Mya (95% HPD, 62.80–121.74 Mya), and the divergence times of Rhoenanthus and Potamanthus originated at approximately 64.77 Mya (95% HPD, 43.82–88.68 Mya), thus belonging to the late Pliocene Epoch or early Miocene Epoch. In addition, the data indicated that R. coreanus was under negative selection and that ATP8 and ND2 in Potamanthidae had a high evolutionary rate.

Ecological factors related to climate extremes have a significant influence on the adaptability of organisms, especially for ectotherms such as reptiles that are sensitive to temperature change. Climate extremes can seriously affect the survival and internal physiology of lizards, sometimes even resulting in the loss of local populations or even complete extinction. Indeed, studies have shown that the expression levels of the nuclear genes and mitochondrial genomes of reptiles change under low-temperature stress. At present, the temperature adaptability of reptiles has rarely been studied at the mitochondrial genome level. In the present study, the mitochondrial genomes of three species of lizards, Calotes versicolor, Ateuchosaurus chinensis, and Hemidactylus bowringii, which live in regions of sympatry, were sequenced. We used RT-qPCR to explore the level of mitochondrial gene expression under low-temperature stress, as compared to a control temperature. Among the 13 protein-coding genes (PCGs), the steady-state transcript levels of ND4L, ND1, ATP6, and COII were reduced to levels of 0.61 ± 0.06, 0.50 ± 0.08, 0.44 ± 0.16, and 0.41 ± 0.09 in C. versicolor, respectively, compared with controls. The transcript levels of the ND3 and ND6 genes fell to levels of just 0.72 ± 0.05 and 0.67 ± 0.05 in H. bowringii, compared with controls. However, the transcript levels of ND3, ND5, ND6, ATP6, ATP8, Cytb, and COIII genes increased to 1.97 ± 0.15, 2.94 ± 0.43, 1.66 ± 0.07, 1.59 ± 0.17, 1.46 ± 0.04, 1.70 ± 0.16, and 1.83 ± 0.07 in A. chinensis. Therefore, the differences in mitochondrial gene expression may be internally related to the adaptative strategy of the three species under low-temperature stress, indicating that low-temperature environments have a greater impact on A. chinensis, with a small distribution area. In extreme environments, the regulatory trend of mitochondrial gene expression in reptiles is associated with their ability to adapt to extreme climates, which means differential mitochondrial genome expression can be used to explore the response of different lizards in the same region to low temperatures. Our experiment aims to provide one new research method to evaluate the potential extinction of reptile species in warm winter climates.