Dissecting Cold Tolerance in Drosophila ananassae: A Multi-Phenotypic and Bulk Segregant Analysis

Temperature strongly shapes the geographic distribution of insects, making cold tolerance an important component of adaptation to different thermal environments. Previous work in Drosophila ananassae identified variation in chill coma recovery time both within an ancestral Bangkok population and between this population and a derived Kathmandu population. However, cold tolerance was characterized only by chill coma recovery time, leaving open whether variation in this phenotype reflects cold tolerance more broadly. Here, we further characterized cold tolerance in iso-female lines from the Bangkok and Kathmandu populations and in recombinant inbred lines generated from the ancestral population, using lethal time and cold shock mortality in addition to chill coma recovery time. We showed that cold tolerance phenotypes differ between sexes, the three phenotypes showed partially overlapping but distinct patterns, and some recombinant inbred lines had extreme phenotypes with higher tolerance than the tolerant founder or lower tolerance than the sensitive founder. We performed bulk segregant analysis using the recombinant inbred lines that exhibited extreme chill coma recovery time to identify genomic regions associated with variation in this phenotype. We identified 22 candidate regions associated with variation in chill coma recovery time and showed that the genes in the putative regions were enriched in muscle development, metabolic processes, and cytoskeletal protein binding. Our results highlight the importance of considering multiple cold tolerance phenotypes and provide insights into the genetic architecture of cold tolerance variation in natural populations of Drosophila.