Mechanisms Underlying Adaptation to Life in Hydrogen Sulfide–Rich Environments Joanna L. Kelley,* ,1 Lenin Arias-Rodriguez, 2 Dorrelyn Patacsil Martin, 3 Muh-Ching Yee, 4 Carlos D. Bustamante, 3 and Michael Tobler* ,5 1 School of Biological Sciences, Washington State University 2 Divisi on Acade ´mica de Ciencias Biol ogicas, Universidad Ju arez Aut onoma de Tabasco, Villahermosa, Tabasco, Me ´xico 3 Department of Genetics, Stanford University 4 Department of Plant Biology, Carnegie Institution for Science, Stanford, CA 5 Division of Biology, Kansas State University *Corresponding author: E-mail: tobler@ksu.edu;joanna.l.kelley@wsu.edu. Associate editor: Yuseob Kim Abstract Hydrogen sulfide (H 2 S) is a potent toxicant interfering with oxidative phosphorylation in mitochondria and creating extreme environmental conditions in aquatic ecosystems. The mechanistic basis of adaptation to perpetual exposure to H 2 S remains poorly understood. We investigated evolutionarily independent lineages of livebearing fishes that have colonized and adapted to springs rich in H 2 S and compared their genome-wide gene expression patterns with closely related lineages from adjacent, nonsulfidic streams. Significant differences in gene expression were uncovered between all sulfidic and nonsulfidic population pairs. Variation in the number of differentially expressed genes among population pairs corresponded to differences in divergence times and rates of gene flow, which is consistent with neutral drift driving a substantial portion of gene expression variation among populations. Accordingly, there was little evidence for conver- gent evolution shaping large-scale gene expression patterns among independent sulfide spring populations. Nonetheless, we identified a small number of genes that was consistently differentially expressed in the same direction in all sulfidic and nonsulfidic population pairs. Functional annotation of shared differentially expressed genes indicated upregulation of genes associated with enzymatic H 2 S detoxification and transport of oxidized sulfur species, oxidative phosphorylation, energy metabolism, and pathways involved in responses to oxidative stress. Overall, our results suggest that modification of processes associated with H 2 S detoxification and toxicity likely complement each other to mediate elevated H 2 S tolerance in sulfide spring fishes. Our analyses allow for the development of novel hypotheses about biochemical and physiological mechanisms of adaptation to extreme environments. Key words: ecological physiology, evolution, extreme environments, gene expression, H 2 S, Poecilia mexicana (Poeciliidae), RNA-sequencing. Introduction Extreme environments are characterized by physiochemical stressors lethal to most organisms, and their clearly defined and replicated selective regimes enable hypothesis-driven tests of organismal responses at all levels of biological organi- zation (Waterman 1999; Bell 2012). Extremophiles can with- stand such stressful conditions and provide ideal systems to study mechanisms underlying physiological adaptation (Storey KB and Storey JM 2005; Nevo 2011). In addition, they shed light into life’s capacities and limitations to adapt to novel environmental conditions, and understanding the ways organisms function in the context of natural stressors provides basic insights into the biochemical, physiological, and developmental processes that govern life (Waterman 1999; Tobler et al. 2015). Hydrogen sulfide (H 2 S) is a physi- ochemical stressor that is produced in many aquatic environ- ments through geochemical or biological processes (Muyzer and Stams 2008). H 2 S has been hypothesized to have played a critical role in the origin of life and caused mass extinctions; accordingly, it has influenced basic physiological properties of organisms and shaped evolutionary diversification on Earth (Kump et al. 2005; Olson and Straub 2015). H 2 S has a wide variety of cytotoxic effects (Beauchamp et al. 1984; Reiffenstein et al. 1992), but its toxicity primarily unfolds through the inhibition of cytochrome c oxidase (COX) in the mitochondrial respiratory chain, which halts ATP produc- tion (Cooper and Brown 2008). Exposure to environmental H 2 S can consequently limit an organism’s ability to survive and reproduce (Bagarinao 1992). Although environmental concentrations of H 2 S are typically low or highly transient, high and sustained concentrations create extreme environ- ments that are lethal for most life (Tobler and Plath 2011; Riesch et al. 2015). Few metazoans have colonized H 2 S-rich habitats and evolved strategies to cope with the continuous exposure to this respiratory toxicant, giving rise to unique ecological communities in deep-sea hydrothermal vents, Article ß The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Open Access Mol. Biol. Evol. 33(6):1419–1434 doi:10.1093/molbev/msw020 Advance Access publication February 9, 2016 1419