Geographic Divergence and the Genomic Basis of Reproductive Diapause in <i>Drosophila triauraria</i>

Adjusting reproduction timing to environmental cues is essential for lifetime fitness. In many insects, reproductive diapause shows clinal variation along environmental gradients such as photoperiod and temperature. How such continuous trait variation may be encoded at the molecular level and maintained in the presence of gene flow remains largely elusive. The fruit fly Drosophila triauraria is distributed across a wide latitudinal range of the Japanese archipelago. Northern strains exhibit a strong photoperiodic reproductive diapause in females, whereas southern strains fail to arrest ovarian development even under short-day conditions at low temperatures. These distinct phenotypes and the presumable clinal variation in between provide an ideal opportunity to examine the molecular basis of latitudinal divergence. We first investigated diapause induction in both females and males from previously reported and newly tested strains collected from the regions spanning ~26° N-43° N along the Japanese archipelago. The assessment revealed continuous geographic variation in sensitivity to photoperiod and temperature. We then analysed the whole-genome sequences of 21 strains, including 14 newly sequenced, to identify genomic regions underlying this divergence. In addition to the conventional F_ST analysis, we applied a "monophyletic window" approach suitable for limited sample sizes. The analysis identified a candidate region containing putative E-box and TER-box sequence motifs of the timeless (tim) gene, which has been previously implicated in diapause regulation in multiple insect species. The quantitative PCR analysis further supported a partial association between the tim expression and the incidence of female diapause. These findings reinforce the growing evidence for a role of circadian clock genes in the adaptive regulation of reproductive diapause and demonstrate the utility of tree-based approaches for detecting genomic regions of geographic divergence.