Natural Variation in Drosophila melanogaster Clock PolyQ Length: Geographic Gradients and Functional Properties

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, behavioural, 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 and find that their frequencies are geographically structured, most consistently along an east-west gradient: the Q25 and Q27 alleles show robust clines in longitude and in a bioclimatic axis of continentality, whereas latitudinal and altitudinal trends are weaker. Behavioural 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 showed the strongest, though modest, temperature sensitivity. 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 is geographically structured in patterns consistent with underlying climatic variation, highlighting the potential for low-complexity regions to modulate clock function in a context-dependent manner across environmental gradients.