Geographical Variation in Thermal Adaptation of the Asiatic Toad ( Bufo gargarizans )

Thermal adaptation in animals can manifest in multiple dimensions, yet few studies have integrated behavioral, physiological, and molecular perspectives to gain a comprehensive understanding. In this study, we examined the thermal adaptation of the widely distributed Asiatic toad (Bufo gargarizans) across different climatic regions by comparing populations from northern and southern China. Our behavioral experiments demonstrate that southern populations prefer warmer conditions, while northern populations prefer cooler conditions. At the same ambient temperature, southern populations exhibit relatively higher metabolic rates. Transcriptomic and gene sequencing analyses revealed that among several temperature-sensitive receptor genes, TRPM4 shows the most pronounced genetic differentiation between northern and southern populations. Electrophysiological assays of TRPM4 function further indicated that the southern population exhibits marked sensitivity to moderately high temperatures (23°C and 35°C), whereas the northern population appears to have lost sensitivity to these temperatures. Furthermore, our correlation analyses revealed that the evolutionary divergence in behavior, physiology, and molecular characteristics between the northern and southern populations is closely linked to their local climatic temperatures. Ancestral sequence reconstruction showed that the temperature sensitivity of the gene TRPM4 in the ancestral B. gargarizans is most similar to that of the Chengdu population, suggesting a possible southwestern origin of B. gargarizans, followed by northward range expansion. Together, our integrative findings reveal a clear evolutionary divergence in thermal adaptation between northern and southern populations. Additionally, our study proposes a molecular phyloecological method for reconstructing ancestral geographic distributions using temperature receptor genes as valuable molecular markers.