Insects are the most abundant and diverse terrestrial animals on the planet, yet few are capable of surviving in Antarctica's inhospitable climate. Genetic evidence indicates that Antarctic insects, as well as other terrestrial arthropods, have persisted throughout the repeated glaciation events of the Pleistocene and earlier. Thus, these species are ideal test cases for modeling the biogeography of terrestrial Antarctica and evolutionary responses to changing environments. The midge Belgica antarctica is perhaps the best studied Antarctic terrestrial arthropod in terms of physiology and genetics. This species is the southernmost free-living insect, and we recently participated in sequencing the genome and transcriptome of this species. However, a lack of information from closely related species has hindered our ability to pinpoint the precise evolutionary mechanisms that permit survival in Antarctica. In this proposal, we establish an international collaboration with scientists from the US, UK, France, and Chile to expand physiological and genomic research of Antarctic and sub-Antarctic midges. In addition to B. antarctica, our project focuses on Eretmoptera murphyi, a sub-Antarctic endemic that has invaded the maritime Antarctic, Halirytus magellanicus, a strictly Magellanic sub-Antarctic species endemic to Tierra del Fuego, and B. albipes, a sub-Antarctic species found on Crozet Island in the Indian Ocean. These four species are closely related and span an environmental gradient from sub-Antarctic to Antarctic habitats. Our central hypothesis is that shared mechanisms drive both population-level adaptation to local environmental conditions and macroevolutionary changes that permit a select few insects to tolerate Antarctic climates. Our Specific Aims are 1) Characterize conserved and species-specific adaptations to extreme environments through comparative physiology and transcriptomics, 2) Comparative genomics of Antarctic and sub-Antarctic midges to identify macroevolutionary signatures of Antarctic adaptation, and 3) Investigate patterns of diversification and location adaptation using population genomics. Our Broader Impacts include deploying an education professional with our research team to coordinate outreach and continuing our partnership with a Kentucky non-profit focused on K-12 STEM programming.