Adaptations at the genetic level ultimately lead to the resilience of species, allowing them to colonize new habitats, to recover following environmental changes, and to diversify. This project examines the way organisms colonize and adapt to extreme environments, namely freshwater caves. Caves are hostile habitats where adaptation at the genetic level is essential for survival. Cave organisms commonly have a special set of traits collectively known as 'troglomorphy,' which include changes in both form (reduced dependence on eyes, longer legs and antennae) and function (tolerance to low oxygen, better sense of smell). These traits in combination with the geographical isolation of caves make them a perfect study system to answer long-standing questions on adaptation, and species diversification. This project uses genetic methods to study the geographic distribution of two crustacean species (Asellus aquaticus and Niphargus hrabei), which have populations that can be found in surface waters and freshwater caves throughout Europe. In order to better understand how these species colonized and adapted to life in darkness, this project will identify the important genes that play a role in troglomorphy, and examine how they are controlled (switched on or off). The identification of genes that are switched on or off when exposed to different conditions (surface vs. caves) will help to clarify how genetics and environment ultimately come together to drive the form and function of living organisms. Additionally, cave ecosystems often contain rare, or new, species that are left undiscovered due to the difficultly of accessing and studying these habitats. Unfortunately, many cave species are endangered (pollution, habitat destruction, overexploitation of aquifers, etc.), and the opportunity to gain knowledge from these ideal study systems is quickly vanishing. In addition to the scientific knowledge generated during this project, it will also result in the training of graduate and undergraduate students in state-of-the-art molecular laboratory techniques and computational analyses. The computer software developed for these analyses will be made available online, providing other researchers with the opportunity to use these new resources. Exploration of these caves will likely result in the discovery and description of new species. Photo and video footage gathered during these expeditions will be made public as a documentary highlighting cave exploration and research. Results from this project will be offered in a series of public seminars and outreach activities. Impacts from this research will aid in the understanding of these important but very threatened ecosystems and will help us better understand conservation needs for the organisms in these systems. The unique characteristics of aquatic caves and of their predominantly crustacean biodiversity nominate them as particularly interesting study subjects for evolutionary biologists. The present study capitalizes on a perfect natural experiment, the Molnar Janos thermal cave system in Budapest, Hungary. This intricate freshwater cave system and the immediately adjacent Malom Lake present the ideal opportunity to address questions of colonization, adaptation, and evolution. Despite marked environmental differences between the cave and surface waters, both localities are inhabited by natural populations of two emerging model cave species, the isopod Asellus aquaticus and the amphipod Niphargus hrabei. This project aims to employ these populations' phylogeographic histories as robust frameworks on which to evaluate the transcriptional and epigenetic basis behind the adaptive divergence of traits involved in troglomorphy, namely vision and chemoreception. This investigation will be undertaken using comparative DNA methylation (BsRADseq) and RNA sequencing (RNAseq) approaches. The identification and evaluation of differentially expressed/methylated genes and pathways will provide a solid bridge between genotype-phenotype, and aid in the understanding of patterns of molecular evolution in cave systems. The results will depict, in a phylogenetically informed context, a close to complete picture of the molecular basis behind vision and chemoreception in A. aquaticus and N. hrabei, of the role these traits play in cave adaptation, and of the evolution of troglomorphy in the subphylum Crustacea. With these, the present study will contribute to the discovery of evolutionarily significant molecular mechanisms that permit the survival and evolution of life in caves and other extreme environments. These findings will undoubtedly yield valuable insights into the molecular underpinnings of adaptation and their role in evolutionary processes across environments and across the tree of life.