The co-evolution of a host and its microbes colonizing different parts of the body including the body surface, oral cavity, gastrointestinal tract, and genitourinary tract is determined by complex symbiotic relationships (Ley et al., 2008; Chevalier et al., 2015). Numerous internal and external factors potentially affect the microbial community composition and structure, but the effects of temperature are more important and pervasive compared to many other external factors (Kohl and Yahn, 2016; Fontaine et al., 2018; Moeller et al., 2020; Zhu et al., 2021; Du et al., 2022; Huang et al., 2022). For example, warming decreases the relative abundance of Firmicutes in the gut microbiome and increases the ratio of putative pathogens in the western fence lizard Sceloporus occidentalis (Moeller et al., 2020) and alters the composition of gut microbial communities in the red-backed salamander Plethodon cinereus (Fontaine et al., 2018). The reduced relative abundance of bacteria of the genera Cellvibrio and Stenotrophomonas may adversely influence digestive performance in P. cinereus (Fontaine et al., 2018). Other studies on the brown bear Ursus arctos (Sommer et al., 2016), the Mongolian gerbil Meriones unguiculatus (Khakisahneh et al., 2020), and the thirteen-lined ground squirrel Ictidomys tridecemlineatus (Regan et al., 2022) provide indirect and direct evidence for the role of the microbiota in host physiology and survival. For example, compared with germ-free mice colonized with the gut microbiota from hibernating brown bears, germ-free mice colonized with the gut microbiota from summer brown bears trigger obesity, suggesting that seasonal changes in the gut microbiota can affect host energy metabolism and thereby result in mass gains of the body and fat (Sommer et al., 2016). Another example from I. tridecemlineatus shows that gut ureolytic microbes convert urea nitrogen into metabolites that can be absorbed by the host to promote protein balance during hibernation (Regan et al., 2022).

Host animals used to examine the effect of temperature on gut microbiota are either from the field, or maintained under controlled conditions in the laboratory. Field studies have examined the effects of external factors including seasonal temperature fluctuations on gut microbiota in a diverse array of vertebrate taxa hibernating [e.g., the brown frog Rana dybowskii (Tong et al., 2020), the Chinese alligator Alligator sinensis (Tang K.-Y. et al., 2019), I. tridecemlineatus (Chiang et al., 2022), U. arctos (Sommer et al., 2016), the greater horseshoe bat Rhinolophus ferrumequinum (Xiao et al., 2019), and the least horseshoe bat Rhinolophus pusillus (Liu et al., 2023)], or not hibernating [e.g., the plateau pika Ochotona curzoniae and the yak Bos mutus (Fu et al., 2020)] in winter. These field studies have yielded many interesting findings. In A. sinensis, for example, the increased relative abundance of bacteria of the genus Bacteroides in winter promotes the degradation of host mucin glycans, which is beneficial for hibernating Chinese alligators to pass the winter fasting period (Tang K.-Y. et al., 2019). Studies conducted under controlled conditions in the laboratory have paid more attention to the effect of temperature on gut microbiota. These laboratory studies consistently show that temperature increase or decrease affects the microbial community composition and structure, but conclusions are often drawn based on single species from a single sampling locality (e.g., Khakisahneh et al., 2020; Moeller et al., 2020; Arango et al., 2021; Hassenrück et al., 2021; Zhu et al., 2021; see also Chen et al., 2022). Whether and how the responses of gut microbiota to temperature change differ between host species distributed in thermally different regions or using thermally different habitats remain a sparsely studied area, but data on this topic are of great value in elucidating the role of gut microbiota in thermal adaptation. For example, gut microbial communities in tadpoles are more plastic in the invasive American bullfrog Lithobates catesbeianus than in the non-invasive green frog Lithobates clamitans, making the former species better able to adapt to a changing thermal environment (Fontaine and Kohl, 2020). Further work could usefully conduct comparative studies on multiple species from thermally contrasting environments to determine the role of gut microbiota in thermal adaptation.

Here, we focused on two oviparous lizard species of the family Agamidae, the Reeves’ butterfly lizard Leiolepis reevesii and the Przewalski’s toad-headed lizard Phrynocephalus przewalskii, living in thermally contrasting environments. L. reevesii is a medium-sized (up to 166 mm snout vent length, SVL), warm-climate lizard occurring in South China (Guangdong, Guangxi, and Hainan) and Vietnam (Figure 1A; Du et al., 2011), where air temperatures vary from 7.0 to 35.5°C.¹ P. przewalskii is a small (up to 66 mm SVL), cold-climate lizard occurring in North and Northwest China (Gansu, Inner Mongolia, Ningxia, and Qinghai) (Figure 1A; Wang, 2011), where air temperatures vary from −24.8 to 38.9°C (see text footnote 1). We designed a 2 species × 3 temperatures experiment, whereby we acclimated adult males of the two lizard species to 20, 28, and 36°C for 2 weeks and then collected their fecal and small-intestinal samples to analyze and compare the microbiota using 16S rRNA gene amplicon sequencing technology. We tested the following two hypotheses. First, the gut microbiota should help lizards adapt to thermal environments in which they live and, as such, gut microbial genes related to metabolic function should differ between the cold- and warm-climate species. Second, gut microbial communities should be more sensitive to temperature change in the warm-climate species living in thermally more stable environments.

Interspecific differences in microbial community were more pronounced in the fecal microbiota than in the small-intestinal microbiota. Bacteroidetes and Firmicutes were the top two dominant phyla in the fecal microbiota and Firmicutes and Proteobacteria were the top two dominant phyla in the small-intestinal microbiota in both species. These findings are consistent with earlier studies on amphibians (Zhou et al., 2020; Zhu et al., 2021), lizards (Ren et al., 2016; Kohl et al., 2017; Zhang et al., 2018; Montoya-Ciriaco et al., 2020; Chen et al., 2022), snakes (Tang W.-J. et al., 2019; Smith et al., 2021; Zhong et al., 2022), and turtles (Qu et al., 2020) where Bacteroidetes, Firmicutes, and Proteobacteria are the top three dominant phyla in the fecal and/or gut microbiota. An earlier study on three sympatric lizard species (Liolaemus parvus, Liolaemus ruibali, and Phymaturus williamsi) shows the stomach, hindgut and fecal microbiota display more pronounced interspecific differences in microbial community than the small-intestinal microbiota (Kohl et al., 2017). We also found that the fecal microbial community was more dissimilar than the small-intestinal microbial community. For example, ASVs of the genera Akkermansia, Butyricicoccus, and Cronobacter were classed as species-specific taxa in the fecal microbial community, but as generalists in the small-intestinal microbial community. More species-specific (L. reevesii or P. przewalskii) taxa and fewer generalists led to more pronounced interspecific differences in the fecal microbial community than in the small-intestinal microbial community.

The fecal and small-intestinal microbiota could help lizards adapt to thermal environments in which they live, as revealed by the following two pieces of evidence. First, alpha diversity was significantly higher in the cold-climate species (P. przewalskii) than in the warm-climate species (L. reevesii). High species diversity and functional redundancy are indicators of microbial resilience to return to ecosystem stability after disruption (Sommer et al., 2017). For example, to satisfy the physiological or energetic demands of the host in high-altitude and cold habitats, alpha diversity of the gut microbiota increases with altitude in the plateau pika O. curzoniae (Li et al., 2019) and the domestic yak Bos grunniens (Fan et al., 2020). Second, functional genes associated with carbohydrate metabolism were enhanced in P. przewalskii, conforming to the metabolic cold adaption hypothesis which predicts an increase in the metabolic rate of ectotherms from cold environments compared with their more temperate counterparts (Clarke, 2003; Schaefer and Walters, 2010). In a recent study on two Phrynocephalus lizards occurring in thermally different regions, Chen et al. (2022) found that the gut microbiota had a higher abundance of functional categories associated with metabolism in a high-altitude species (Phrynocephalus erythrurus) occurring from 4,500 to 5,300 m than in a low-altitude species (P. przewalskii) occurring from 500 to 1,700 m. Here, we found that, as in cattle where Firmicutes assimilate nutrients more efficiently in warm tropical environments (Zhang et al., 2022), the relative abundance of Firmicutes in the small-intestinal microbiota was higher in the warm-climate species than in the cold-climate species. Firmicutes are more efficient in dietary caloric intake than Bacteroidetes (Turnbaugh et al., 2006; Krajmalnik-Brown et al., 2012; Semova et al., 2012), and short-chain fatty acids produced by Firmicutes can be directly absorbed by the host as an energy source to promote mass gain (den Besten et al., 2013). That Firmicutes play a more important role in the absorption of nutrients in the warm-climate species further proves the contribution of microbial community to thermal adaptation in lizards.

Environmental temperature alters alpha and beta diversities of the gut microbiota in a diverse array of animal taxa (e.g., Bestion et al., 2017; Fontaine et al., 2018; Zhu et al., 2019, 2021; Cao et al., 2022). Our results showed that the cold-climate species responded to temperature increase or decrease by adjusting alpha diversity of microbial community, whereas the warm-climate species did so by adjusting beta diversity of microbial community. Reduced alpha diversity has been reported for P. cinereus (Fontaine et al., 2018), the common lizard Zootoca vivipara (Bestion et al., 2017), and the domestic chicken Gallus domesticus (Zhu et al., 2019) exposed to warm thermal conditions. Reduced alpha diversity may result in a decrease in genetic diversity and thereby inhibit the host’s ability to respond to environmental stress and resist infection (Pollock et al., 2019). In this study, we found that, as in mice exposed to a high temperature of 36°C (Cao et al., 2022), alpha diversity (observed ASVs and Shannon indices) of the fecal microbiota increased with increasing temperatures in P. przewalskii. Such a temperature-dependent change in alpha diversity may be due to the relatively high proportions of Parabacteroides and Citrobacter in P. przewalskii acclimated to 20°C (Figure 1D). The increased relative abundance of some bacterial genera such as Citrobacter and Pseudomonas of the phylum Proteobacteria reduces microbial diversity in the gut of the brown tree frog Polypedates megacephalus artificially hibernating in the laboratory (Weng et al., 2016). In L. reevesii, however, we found that neither in the fecal microbiota nor in the small-intestinal microbiota did alpha diversity differ significantly among the three temperature treatments. This finding is consistent with previous studies on the northern leopard frog Rana pipiens (Kohl and Yahn, 2016) and the tropical clawed frog Xenopus tropicalis (Li et al., 2020). Temperature increase or decrease led to higher microbial heterogeneity in L. reevesii than in P. przewalskii (Figure 3), indicating that microbial communities were more changeable in the warm-climate species. Plasticity in the gut microbial community potentially helps host animals adapt to harsh environments with extreme climate events, limited food resources, or toxic exposure (Alberdi et al., 2016). The responses of microbial community to temperature increase or decrease differed between the two agamid lizards, and this difference presumably results from the fact that they use thermally different habitats and differ in thermal tolerance (Lin, 2008; Qu et al., 2011; Wang, 2013).

The microbial composition was thermally more sensitive in L. reevesii than in P. przewalskii. Fecal or small-intestinal bacteria of the phyla Bacteroidetes, Firmicutes, and Proteobacteria and 12 genera belonging to the phyla Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria were affected by acclimation temperature in L. reevesii. However, in P. przewalskii, only fecal bacteria of the phyla Bacteroidetes and Verrucomicrobia and four genera belonging to the phyla Bacteroidetes, Firmicutes, and Verrucomicrobia were affected by acclimation temperature. The trends of variation in the relative abundance of Bacteroidetes and Verrucomicrobia differed between the two lizard species. More specifically, bacteria of the phylum Bacteroidetes enriched in the 20 and 36°C treatments in P. przewalskii, bacteria of the phylum Bacteroidetes enriched in the 28°C treatment in L. reevesii, and bacteria of the phylum Verrucomicrobia enriched in the 28°C treatment in P. przewalskii. Bacteria of the phyla Bacteroidetes and Verrucomicrobia can use host-derived mucin glycans in the absence of dietary substrates (Sonnenburg et al., 2005; Carey et al., 2013). It has been reported for R. dybowskii (Tong et al., 2020), I. tridecemlineatus (Dill-McFarland et al., 2014; Stevenson et al., 2014), and U. arctos (Sommer et al., 2016) that the increased relative abundance of Bacteroidetes or Verrucomicrobia and the decreased relative abundance of Firmicutes during hibernation help hosts adapt to long-term fasting in winter. The increased relative abundance of Verrucomicrobia was observed in L. reevesii acclimated to 20°C, though statistically not significant. Additionally, as in Andrias davidianus (Zhu et al., 2021), the relative abundance of Firmicutes and Proteobacteria increased at low or high acclimation temperatures in L. reevesii. In Drosophila subobscura, decisive effects of heat stress on the gut microbiota composition differ between the sexes, with warming reducing the relative abundances of Firmicutes in females, increasing the relative abundances of Firmicutes, and reducing the relative abundances of Proteobacteria in males (Jaramillo and Castañeda, 2021). The result for male D. subobscura is consistent with this study based on male adults of L. reevesii. High Firmicutes abundance (or Firmicutes/Bacteroidetes ratio) is related to obesity in humans (Clemente et al., 2012), and to weight gain and immune function in birds (Zhang et al., 2015) and mammals (Ley et al., 2005). The Firmicutes/Bacteroidetes ratio increased with elevated temperature in L. reevesii, indicating an increased capacity for energy harvest. However, in cold-climate vertebrates such as the Brandt’s vole Lasiopodomys brandtii (Bo et al., 2019) and the Tibetan sheep Ovis aries (Fan et al., 2021), cold stress increases the Firmicutes/Bacteroidetes ratio to maintain the host’s metabolic balance. Compared with active counterparts, hibernating animals often have a higher relative abundance of Proteobacteria in their gut microbiota (Weng et al., 2016; Song et al., 2020; Tong et al., 2020; Cao et al., 2023). Such a difference is also evident in animals not hibernating in winter (Sun et al., 2016; Ferguson et al., 2018), indicating that the bacterial phylum Proteobacteria may have an important role in helping hosts adapt to fasting (starvation) or cold conditions. It is worth noting that most taxa of the phylum Proteobacteria have pathogenic or antipathogenic functions (Wiggins et al., 2011; Ramsey et al., 2015). The relative abundances of putative pathogens did not differ significantly among three temperature treatments in L. reevesii, but were enriched in P. przewalskii acclimated to 20°C. The accelerated colonization of pathogenic bacteria in the gut at low temperatures has been observed in other cold-climate vertebrates (Liu et al., 2022; Tong et al., 2023). Bacteria of the genus Akkermansia can enhance metabolic homeostasis and anti-inflammatory properties (Everard et al., 2013; Shin et al., 2014). The relative abundance of Akkermansia bacteria decreased in P. przewalskii acclimated to 20°C, suggesting that, as in X. tropicalis (Li et al., 2020), low-temperatures exposure may lead to a decrease in immunity in this cold-climate lizard. Temperature-dependent changes in the microbial composition differed between L. reevesii and P. przewalskii, and this difference can be explained by the following two reasons. First, host genetic background is a key determinant of the microbial composition and structure (Kokou et al., 2018; Weinstein et al., 2021). Second, host thermal tolerance modulates the microbial response to temperature change (Kokou et al., 2018). Overall, our data showed that the microbial community was thermally more sensitive in the warm-climate lizard than in the cold-climate lizard, and that lizards can adapt to temperature increase or decrease by adjusting the gut microbiota.

Although the influence of acclimation temperature on the microbial composition and structure differed between L. reevesii and P. przewalskii, the two species exhibited similar microbial functions enriched at different acclimation temperatures. Pathways related to environmental information processing (i.e., ABC transporters and two-component system) were enriched in lizards acclimated to 20 or 36°C, confirming the existence of a functional response of the gut microbial community to temperature (Nachin et al., 2005). ABC transporters pathway uses ATP hydrolysis to transport a wide variety of substrates (i.e., sugars, amino acids, glycans, and phospholipids) (Gottesman and Ambudkar, 2001). This pathway might promote food absorption to help the host survive in the season or environment that is cold and/or short of food (Xia et al., 2021). Two-component system pathway is involved in the information communication between microbes and their environment, allowing bacteria to sense and respond to environmental change. Consistent with a study on goitred gazelles (Gazella subgutturosa) exposed to low temperatures in winter (Qin et al., 2022), our study showed that the relative abundant of two-component system was significantly higher in P. przewalskii acclimated to the lowest temperature (20°C). This finding suggests the role of two-component system in cold adaptation. Additionally, the temperature-dependence of pathways associated with amino acid metabolism, lipid metabolism, and metabolism of cofactors and vitamins seen in L. reevesii suggests that metabolic functions of bacteria are thermally more sensitive in warm-climate lizards. Consistent with studies on the Wistar rat Rattus norvegicus (Qu et al., 2021) and the rice frog Fejervarya limnocharis (Huang and Liao, 2021), amino acid metabolism and lipid metabolism were enriched in L. reevesii acclimated to 36°C. Overall, both species of lizards studied herein, L. reevesii in particular, could cope with temperature changes by adjusting the relative abundance of functional categories associated with metabolism and environmental information processing.

With the findings of this study, we can draw the following conclusions. First, the fecal microbiota displays more pronounced interspecific differences in microbial community than the small-intestinal microbiota in both species of lizards occurring in thermally different regions. Second, the response of fecal and small-intestinal microbiota to temperature increase or decrease differs between the warm-climate (L. reevesii) and cold-climate (P. przewalskii) species, with more bacterial taxa affected by acclimation temperature in L. reevesii than in P. przewalskii. Third, both species, the warm-climate species in particular, can cope with temperature changes by adjusting the relative abundance of functional categories associated with metabolism and environmental information processing. Fourth, the fecal microbiota contributes to cold-climate adaptation in P. przewalskii, as revealed by the fact that functional genes associated with carbohydrate metabolism were enhanced in this species. Taken together, our results validate the hypotheses tested, of which one suggests that the gut microbiota should help lizards adapt to thermal environments in which they live and one suggests that microbial communities should be thermally more sensitive in warm-climate lizards than in cold-climate lizards.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm.nih.gov/, PRJNA901369.

The animal study was approved by the Laboratory Animal Care and Animal Ethics Committee of Nanjing Normal University and Hainan Tropical Ocean University. The study was conducted in accordance with the local legislation and institutional requirements.

X-MZ: Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. J-QC: Investigation, Writing – review & editing. YD: Funding acquisition, Investigation, Writing – review & editing. C-XL: Investigation, Resources, Writing – review & editing. Y-FQ: Data curation, Formal analysis, Writing – review & editing. L-HL: Conceptualization, Funding acquisition, Investigation, Supervision, Writing – original draft, Writing – review & editing. XJ: Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing.