Natural Variation in Clock polyQ Length Is Associated with Circadian Function to Climatic Gradients in Drosophila melanogaster

Natural variation in circadian clock genes provides a powerful framework for understanding how organisms respond to environmental heterogeneity. The Clock (Clk) gene encodes a core transcriptional regulator of circadian rhythms and contains a polymorphic polyglutamine (polyQ) tract whose evolutionary significance remains unclear. Here, we integrate population genomic, behavioral, and molecular analyses to investigate the functional and geographic patterns of Clk polyQ variation in Drosophila melanogaster. Using data from 127 European populations, we identify 11 Clk polyQ alleles whose frequencies show significant associations with latitude, longitude, and principal components derived from bioclimatic variables, indicating strong geographic structure. Behavioral assays of near-isogenic lines revealed that polyQ length modulates circadian function under thermal challenge: most alleles maintained stable free-running periods across temperatures, whereas the intermediate-length Q25 allele exhibited reduced temperature compensation. Circadian phase showed pronounced allele-specific sensitivity to elevated temperature in laboratory assays, although phase variation did not display a consistent relationship with geographic variables. At the molecular level, luciferase reporter assays showed that longer polyQ alleles exhibited higher transcriptional activity, linking polyQ length to CLK-mediated gene expression. Together, these results demonstrate that natural variation in Clk polyQ length has measurable functional consequences for circadian regulation and exhibits strong geographic structuring, highlighting the potential for low-complexity regions to modulate clock function in a context-dependent manner across environmental gradients.