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Regulating transcription allows organisms to respond to their environment, both within a single generation (plasticity) and across generations (adaptation). We examined transcriptional differences in gill tissues of fishes in thePoecilia mexicanaspecies complex (family Poeciliidae), which have colonized toxic springs rich in hydrogen sulfide (H₂S) in southern Mexico. There are gene expression differences between sulfidic and non-sulfidic populations, yet regulatory mechanisms mediating this gene expression variation remain poorly studied. We combined capped-small RNA sequencing (csRNA-seq), which captures actively transcribed (i.e. nascent) transcripts, and messenger RNA sequencing (mRNA-seq) to examine how variation in transcription, enhancer activity, and associated transcription factor binding sites may facilitate adaptation to extreme environments. csRNA-seq revealed thousands of differentially initiated transcripts between sulfidic and non-sulfidic populations, many of which are involved in H₂S detoxification and response. Analyses of transcription factor binding sites in promoter and putative enhancer csRNA-seq peaks identified a suite of transcription factors likely involved in regulating H₂S-specific shifts in gene expression, including several key transcription factors known to respond to hypoxia. Our findings uncover a complex interplay of regulatory processes that reflect the divergence of extremophile populations ofP. mexicanafrom their non-sulfidic ancestors and suggest shared responses among evolutionarily independent lineages. 

Abstract Adaptation to extreme environments often involves the evolution of dramatic physiological changes. To better understand how organisms evolve these complex phenotypic changes, the repeatability and predictability of evolution, and possible constraints on adapting to an extreme environment, it is important to understand how adaptive variation has evolved. Poeciliid fishes represent a particularly fruitful study system for investigations of adaptation to extreme environments due to their repeated colonization of toxic hydrogen sulfide–rich springs across multiple species within the clade. Previous investigations have highlighted changes in the physiology and gene expression in specific species that are thought to facilitate adaptation to hydrogen sulfide–rich springs. However, the presence of adaptive nucleotide variation in coding and regulatory regions and the degree to which convergent evolution has shaped the genomic regions underpinning sulfide tolerance across taxa are unknown. By sampling across seven independent lineages in which nonsulfidic lineages have colonized and adapted to sulfide springs, we reveal signatures of shared evolutionary rate shifts across the genome. We found evidence of genes, promoters, and putative enhancer regions associated with both increased and decreased convergent evolutionary rate shifts in hydrogen sulfide–adapted lineages. Our analysis highlights convergent evolutionary rate shifts in sulfidic lineages associated with the modulation of endogenous hydrogen sulfide production and hydrogen sulfide detoxification. We also found that regions with shifted evolutionary rates in sulfide spring fishes more often exhibited convergent shifts in either the coding region or the regulatory sequence of a given gene, rather than both. 

Environmental factors can promote phenotypic variation through alterations in the epigenome and facilitate adaptation of an organism to the environment. Although hydrogen sulfide is toxic to most organisms, the fish Poecilia mexicana has adapted to survive in environments with high levels that exceed toxicity thresholds by orders of magnitude. Epigenetic changes in response to this environmental stressor were examined by assessing DNA methylation alterations in red blood cells, which are nucleated in fish. Males and females were sampled from sulfidic and nonsulfidic natural environments; individuals were also propagated for two generations in a nonsulfidic laboratory environment. We compared epimutations between the sexes as well as field and laboratory populations. For both the wild-caught (F0) and the laboratory-reared (F2) fish, comparing the sulfidic and nonsulfidic populations revealed evidence for significant differential DNA methylation regions (DMRs). More importantly, there was over 80% overlap in DMRs across generations, suggesting that the DMRs have stable generational inheritance in the absence of the sulfidic environment. This is an example of epigenetic generational stability after the removal of an environmental stressor. The DMR-associated genes were related to sulfur toxicity and metabolic processes. These findings suggest that adaptation of P. mexicana to sulfidic environments in southern Mexico may, in part, be promoted through epigenetic DNA methylation alterations that become stable and are inherited by subsequent generations independent of the environment. 

Abstract Hydrogen sulfide is a toxic gas that disrupts numerous biological processes, including energy production in the mitochondria, yet fish in thePoecilia mexicanaspecies complex have independently evolved sulfide tolerance several times. Despite clear evidence for convergence at the phenotypic level in these fishes, it is unclear if the repeated evolution of hydrogen sulfide tolerance is the result of similar genomic changes. To address this gap, we used a targeted capture approach to sequence genes associated with sulfide processes and toxicity from five sulfidic and five nonsulfidic populations in the species complex. By comparing sequence variation in candidate genes to a reference set, we identified similar population structure and differentiation, suggesting that patterns of variation in most genes associated with sulfide processes and toxicity are due to demographic history and not selection. But the presence of tree discordance for a subset of genes suggests that several loci are evolving divergently between ecotypes. We identified two differentiation outlier genes that are associated with sulfide detoxification in the mitochondria that have signatures of selection in all five sulfidic populations. Further investigation into these regions identified long, shared haplotypes among sulfidic populations. Together, these results reveal that selection on standing genetic variation in putatively adaptive genes may be driving phenotypic convergence in this species complex. 

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 (H 2 S)—a toxicant that impairs mitochondrial function—across evolutionarily independent lineages of a fish ( Poecilia mexicana , Poeciliidae) from H 2 S-rich springs. Using comparative biochemical and physiological analyses, we found that mitochondrial function is maintained in the presence of H 2 S 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 H 2 S tolerance in different populations is likely caused by a combination of selection on standing genetic variation and de novo mutations. On a macroevolutionary scale, H 2 S 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 H 2 S 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 H 2 S adaptation in lineages that span 40 million years of evolution. 

Abstract Natural selection drives the evolution of traits to optimize organismal performance, but optimization of one aspect of performance can influence other aspects of performance. Here, we asked how phenotypic variation between locally adapted fish populations affects locomotion and ventilation, testing for functional trade‐offs and trait–performance correlations. Specifically, we investigated two populations of livebearing fish (Poecilia mexicana) that inhabit distinct habitat types (hydrogen‐sulphide‐rich springs and adjacent nonsulphidic streams). For each individual, we quantified different metrics of burst swimming during simulated predator attacks, steady swimming and gill ventilation. Coinciding with predictions, we documented significant population differences in all aspects of performance, with fish from sulphidic habitats exhibiting higher steady swimming performance and higher ventilation capacity, but slower burst swimming. There was a significant functional trade‐off between steady and burst swimming, but not between different aspects of locomotion and ventilation. Although our findings about population differences in locomotion performance largely parallel the results from previous studies, we provide novel insights about how morphological variation might impact ventilation and ultimately oxygen acquisition. Overall, our analyses provided insights into the functional consequences of previously documented phenotypic variation, which will help to disentangle the effects of different sources of selection that may coincide along complex environmental gradients. 

Abstract Visual sensitivity and body pigmentation are often shaped by both natural selection from the environment and sexual selection from mate choice. One way of quantifying the impact of the environment is by measuring how traits have changed after colonization of a novel habitat. To do this, we studiedPoecilia mexicanapopulations that have repeatedly adapted to extreme sulphidic (H₂S‐containing) environments. We measured visual sensitivity using opsin gene expression, as well as body pigmentation, for populations in four independent drainages. Both visual sensitivity and body pigmentation showed significant parallel shifts towards greater medium‐wavelength sensitivity and reflectance in sulphidic populations. Altogether we found that sulphidic habitats select for differences in visual sensitivity and pigmentation. Shifts between habitats may be due to both differences in the water's spectral properties and correlated ecological changes.