Abstract: In this study, 44 representative species from the Glires were selected, including taxa distributed across diverse altitudinal gradients and habitat types, and then two kinds of main rate-limiting enzyme genes in tricarboxylic acid (TCA) cycle were used to analyze the adaptive evolutionary characteristics, namely,CS, IDH3A, IDH3B, andIDH3G. Evolutionary analyses were employed to evaluate the selection pressure patterns under different habitat backgrounds. The results indicated thatCS, IDH3A, IDH3G, andIDH3Bwere generally subject to strong purifying selection, but the evolutionary rate exhibited significant heterogeneity among specific high-altitude lineages. Furthermore, phylogenetic analysis revealed a significant positive correlation between the evolutionary rates ofCSandIDH3Band altitude, suggesting that rodents may facilitate high-altitude adaptation through rapid evolution. Positively selected sites were detected in theCSgene across multiple independent high-altitude lineages based on the branch-site model; the spatial distribution of these positive selection sites in the three-dimensional protein structure suggests that different taxa may cope with hypoxic stress through distinct pathways, such as regulating enzyme activity or structural stability. This study suggests that high-altitude environments may continuously shape the evolutionary trajectory of key TCA cycle enzymes at the molecular level, which provide new comparative genomics evidence for understanding the genetic basis of metabolism in high-altitude adaptation in mammals.

Key words: high-altitude adaptation, citrate synthase, isocitrate dehydrogenase 3, Glires, positive correlation, positive selection sites