Hydrogen sulfide is a highly toxic gas that is lethal for most animals, even at low concentrations. In southern Mexico, small livebearing fish of the genus Poecilia have independently colonized multiple sulfidic springs, where hydrogen sulfide naturally occurs in high concentrations. These fish provide an opportunity to study how vertebrates have managed to survive in conditions that are lethal for most other organisms. Adaptation in sulfide springs also leads to reproductive isolation between sulfidic and non-sulfidic ecotypes, providing insights about how new species evolve. The objectives of this proposal are to identify convergent patterns of phenotypic and genetic differentiation in replicated pairs of sulfidic and non-sulfidic habitats. This comparison will reveal both traits and the underlying genetic changes that mediate sulfide tolerance. The research addresses three basic questions: (1) How do complex phenotypes evolve along replicated environmental gradients? Quantifying phenotypic variation (including behavioral, physiological, and morphological traits) in wild-caught individuals will identify convergent patterns of adaptive trait divergence in replicated pairs of sulfidic and non-sulfidic fish. Analyzing the same phenotypic traits in common garden-raised individuals will shed light on how heritable differentiation and phenotypic plasticity interact to express phenotypes in nature. (2) What are the genomic changes underlying trait divergence? Next generation sequencing technologies (RNA-Seq) will be used to identify genes that show signatures of divergent selection or differential expression, which will be used to identify candidate genes underlying adaptation to the extreme environments. (3) What are the functional consequences of the divergent traits expressed under different environmental conditions for reproductive isolation? Comparing growth rates of laboratory-reared fish exposed to either sulfidic or non-sulfidic conditions will provide a direct link between adaptation to sulfide and reproductive isolation in the form of immigrant inviability. Understanding organisms? responses to naturally occurring stressful environments can ultimately be used to better understand causes and consequences of organismal responses to anthropogenically altered environments. The project fosters collaboration in research and education between scientists and students from the United States and Mexico. This project establishes a public outreach platform for fish biologists to interact with fish hobbyist organizations as a means to disseminate research findings to the lay public.