Convergent elevation of mitochondrial proton leakage improves thermogenesis in honeybees

The independent range expansions of the honeybees Apis cerana and A. mellifera from tropical origins into temperate environments have had profound ecological and agricultural impacts. Both species have convergently evolved enhanced thermogenic capacity relative to closely related taxa restricted to tropical and subtropical regions. However, the intrinsic physiological and molecular mechanisms enabling enhanced thermogenesis remain unclear. Here, we first show that A. cerana and A. mellifera exhibit increased proton leak, as evidenced by mitochondrial oxygen consumption assays, directly contributing to elevated heat production. Using a comparative framework supported by newly assembled high-quality genomes, we further identify accelerated evolution in mitochondrial metabolic pathways in both wide-ranged species. At the gene level, we identify Ptcd1 as a key candidate, showing signatures of positive selection and convergent amino acid substitution in both A. cerana and A. mellifera. Functional validation in Drosophila demonstrates that Ptcd1 promotes insect thermogenesis by modulating mitochondrial metabolism and ultrastructure. Moreover, knock-in Drosophila lines expressing Ptcd1 isoforms derived from A. cerana, the tropical species A. dorsata, and a targeted reversion mutation reveal that the Q208K substitution, unique to A. cerana and A. mellifera, increases proton leak and enhances heat generation. Together, these findings demonstrate that the independent fixation of a single amino acid change in PTCD1 has contributed to convergent enhancement of thermogenesis in both A. cerana and A. mellifera. This molecular innovation likely facilitated their expansion into temperate climates and highlights mitochondrial regulation as a fundamental axis of thermal adaptation shared across diverse animal lineages.