Extreme environments test the limits of life; yet, some organisms thrive in harsh conditions. Extremophile lineages inspire questions about how organisms can tolerate physiochemical stressors and whether the repeated colonization of extreme environments is facilitated by predictable and repeatable evolutionary innovations. We identified the mechanistic basis underlying convergent evolution of tolerance to hydrogen sulfide (H2S)—a toxicant that impairs mitochondrial function—across evolutionarily independent lineages of a fish (Poecilia mexicana, Poeciliidae) from H2S-rich springs. Using comparative biochemical and physiological analyses, we found that mitochondrial function is maintained in the presence of H2S in sulfide spring P. mexicana but not ancestral lineages from nonsulfidic habitats due to convergent adaptations in the primary toxicity target and a major detoxification enzyme. Genome-wide local ancestry analyses indicated that convergent evolution of increased H2S tolerance in different populations is likely caused by a combination of selection on standing genetic variation and de novo mutations. On a macroevolutionary scale, H2S tolerance in 10 independent lineages of sulfide spring fishes across multiple genera of Poeciliidae is correlated with the convergent modification and expression changes in genes associated with H2S toxicity and detoxification. Our results demonstrate that the modification of highly conserved physiological pathways associated with essential mitochondrial processes mediates tolerance to physiochemical stress. In addition, the same pathways, genes, and—in some instances—codons are implicated in H2S adaptation in lineages that span 40 million years of evolution.

The notorious plasticity of gene expression responses and the complexity of environmental gradients complicate the identification of adaptive differences in gene regulation among populations. We combined transcriptome analyses in nature with common-garden and exposure experiments to establish cause-effect relationships between the presence of a physiochemical stressor and expression differences, as well as to test how evolutionary change and plasticity interact to shape gene expression variation in natural systems. We studied two evolutionarily independent population pairs of an extremophile fish (Poecilia mexicana) living in toxic, hydrogen sulphide (H S)-rich springs and adjacent nontoxic habitats and assessed genomewide expression patterns of wild-caught and common-garden-raised individuals exposed to different concentrations of H S. We found that 7.7% of genes that were differentially expressed between sulphidic and nonsulphidic ecotypes remained differentially expressed in the laboratory, indicating that sources of selection other than H S-or plastic responses to other environmental factors-contribute substantially to gene expression patterns observed in the wild. Concordantly differentially expressed genes in the wild and the laboratory were primarily associated with H S detoxification, sulphur processing and metabolic physiology. While shared, ancestral plasticity played a minor role in shaping gene expression variation observed in nature, we documented evidence for evolved population differences in the constitutive expression as well as the H S inducibility of candidate genes. Mechanisms underlying gene expression variation also varied substantially across the two ecotype pairs. These results provide a springboard for studying evolutionary modifications of gene regulatory mechanisms that underlie expression variation in locally adapted populations.

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22Extreme environments test the limits of life. Still, some organisms thrive in harsh conditions, 23 begging the question whether the repeated colonization of extreme environments is facilitated by 24 predictable and repeatable evolutionary innovations. We identified the mechanistic basis underlying 25 convergent evolution of tolerance to hydrogen sulfide (H2S)-a potent toxicant that impairs 26 mitochondrial function-across evolutionarily independent lineages of a fish (Poecilia mexicana, 27 Poeciliidae) from H2S-rich freshwater springs. We found that mitochondrial function is maintained 28 in the presence of H2S in sulfide spring P. mexicana, but not ancestral lineages in adjacent nonsulfidic 29 habitats, due to convergent adaptations in both the primary toxicity target and a major detoxification 30 enzyme. Additionally, we show that H2S tolerance in 10 independent lineages of sulfide spring fishes 31 across multiple genera of Poeciliidae is mediated by convergent modification and expression changes 32 of genes associated with H2S toxicity and detoxification. Our results demonstrate that the repeated 33 modification of highly conserved physiological pathways associated with essential mitochondrial 34 processes enabled the colonization of novel environments. 35 36 3 Stephen J. Gould was a fierce proponent of the importance of contingency in evolution, famously 37 quipping that replaying the "tape of life" would lead to different outcomes every time (1). 38Mitochondrial genomes were historically thought to be a prime example of such contingency 39 evolution, because alternative genetic variants were assumed to be selectively neutral (2). This 40 paradigm has been shifting, with mounting evidence that mitochondria-and genes encoded in the 41 mitochondrial genome-play an important role in adaptation, especially in the context of 42 physiochemical stress (3). However, it often remains unclear how genetic variation in mitochondrial 43 genomes and nuclear genes that contribute to mitochondrial function translates to variation in 44 physiological and organismal function. Furthermore, it is not known whether exposure to similar 45 selective regimes may cause convergent modifications of mitochondrial genomes and emergent 46 biochemical and physiological functions in evolutionarily independent lineages. Extreme 47 environments that represent novel ecological niches are natural experiments to address questions 48 about mechanisms underlying mitochondrial adaptations and illuminate the predictability of adaptive 49 evolution of mitochondria. Among the most extreme freshwater ecosystems are springs with high 50 levels of hydrogen sulfide (H2S), a potent respiratory toxicant lethal to metazoans due to its 51 inhibition of mitochondrial ATP production (4). Multiple lineages of livebearing fishes (Poeciliidae) 52 have colonized H2S-rich springs throughout the Americas and independently evolved tolerance to 53 sustained H2S concentrations orders of magnitudes higher than those encountered by ancestral 54 lineages in nonsulfid...

Hydrogen sulfide (H2S) is a toxic gas that inhibits the final step in the mitochondrial electron transport chain. This gas, however, is produced endogenously at low levels to promote vasodilation, angiogenesis, and reduce inflammation. Both a deficiency and an excess of H2S are associated with disease therefore, maintaining H2S homeostasis is critical for proper physiologic function. Despite the surge in biomedical interest in H2S as a therapeutic and a factor in the etiology of disease, the regulatory mechanisms remain largely unknown. Progress has been hampered by the lack of study systems that exhibit both sulfide susceptibility and sulfide tolerance within a single species that would permit comparative analyses toward uncovering mechanisms that regulate responses to H2S. Poecilia mexicana is a species of fish with populations that are naturally adapted to either non‐sulfidic or sulfidic environments. Offspring reared in the lab under non‐sulfidic conditions maintain their tolerance or non‐tolerance to H2S. We hypothesized that H2S‐tolerant fish differentially express genes and modify mitochondrial function to tolerate toxic levels of H2S relative to non‐tolerant fish. We have found that H2S‐tolerant fish differentially express SQR, VEGF, CSE, COX, and P450 (which are involved in H2S responses in other models) under control and H2S conditions relative to non‐tolerant fish. Furthermore, mitochondria isolated from H2S‐tolerant fish livers exhibit modifications in the coupling of electron transport and oxidative phosphorylation compared to non‐tolerant fish mitochondria. These data suggest that this is a valuable model to elucidate mechanisms involved in regulating H2S.