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Simple Summary: Arylalkylamine N-acetyltransferase (AANAT) is an enzyme that regulates the production of melatonin, a hormone that is essential for organisms to maintain circadian and annual rhythmic behavior. The AANAT gene has undergone gene duplication and inactivation during evolution. Nevertheless, the majority of mammals retain a solitary copy of AANAT within the biological genome, and the mechanisms by which this gene responds to environmental stimuli remain poorly understood. To address this issue, we investigated the adaptive evolution of AANAT at both the gene and protein levels. Our study revealed the presence of multiple mutation sites within the gene encoding AANAT, with variation in the structure of the AANAT protein observed across different geographic populations. We identified a positive selection of AANAT in populations residing at higher latitudes. Furthermore, individuals exhibiting longer hibernation periods displayed significantly higher catalytic efficiency of the AANAT enzyme compared to those with minimal hibernation behavior, suggesting a potential association between AANAT and hibernation rhythms. This study adds to our understanding of the adaptive evolution of AANAT and may provide molecular evidence for hibernation rhythm adaptation in bats. Arylalkylamine N-acetyltransferase (AANAT) is a crucial rate-limiting enzyme in the synthesis of melatonin. AANAT has been confirmed to be independently duplicated and inactivated in different animal taxa in order to adapt to the environment. However, the evolutionary forces associated with having a single copy of AANAT remain unclear. The greater horseshoe bat has a single copy of AANAT but exhibits different hibernation rhythms in various populations. We analyzed the adaptive evolution at the gene and protein levels of AANAT from three distinct genetic lineages in China: northeast (NE), central east (CE), and southwest (SW). The results revealed greater genetic diversity in the AANAT loci of the NE and CE lineage populations that have longer hibernation times, and there were two positive selection loci. The catalytic capacity of AANAT in the Liaoning population that underwent positive selection was significantly higher than that of the Yunnan population (p < 0.05). This difference may be related to the lower proportion of α helix and the variation in two interface residues. The adaptive evolution of AANAT was significantly correlated with climate and environment (p < 0.05). After controlling for geographical factors (latitude and altitude), the evolution of AANAT by the negative temperature factor was represented by the monthly mean temperature (r = −0.6, p < 0.05). The results identified the gene level variation, functional adaptation, and evolutionary driving factors of AANAT, provide an important foundation for further understanding the adaptive evolution of the single copy of AANAT in pteropods, and may offer evidence for adaptive hibernation rhythms in bats. [ABSTRACT FROM AUTHOR]

Hibernation, a survival strategy in mammals for extreme climates, induces physiological phenomena such as ischemia–reperfusion and metabolic shifts that hold great potential for advancements in modern medicine. Despite this, the molecular mechanisms underpinning hibernation remain largely unclear. This study used RNA‐seq and Iso‐seq techniques to investigate the changes in liver transcriptome expression of Rhinolophus pusillus during hibernation and active periods, as well as under different microhabitat temperatures. We identified 11 457 differentially expressed genes during hibernation and active periods, of which 395 showed significant differential expression. Genes associated with fatty acid catabolism were significantly upregulated during hibernation, whereas genes related to carbohydrate metabolism and glycogen synthesis were downregulated. Conversely, immune‐related genes displayed differential expression patterns: genes tied to innate immunity were significantly upregulated, while those linked to adaptive immunity and inflammatory response were downregulated. The analysis of transcriptomic data obtained from different microhabitat temperatures revealed that R. pusillus exhibited an upregulation of genes associated with lipid metabolism in lower microhabitat temperature. This upregulation facilitated an enhanced utilization rate of triglyceride, ultimately resulting in increased energy provision for the organism. Additionally, R. pusillus upregulated gluconeogenesis‐related genes regardless of the microhabitat temperature, demonstrating the importance of maintaining blood glucose levels during hibernation. Our transcriptomic data reveal that these changes in liver gene expression optimize energy allocation during hibernation, suggesting that liver tissue adaptively responds to the inherent stress of its function during hibernation. This study sheds light on the role of differential gene expression in promoting more efficient energy allocation during hibernation. It contributes to our understanding of how liver tissue adapts to the stressors associated with this state. [ABSTRACT FROM AUTHOR]