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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 the Poecilia mexicana species 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 of P. mexicana from their non-sulfidic ancestors and suggest shared responses among evolutionarily independent lineages.

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Mexico is a megadiverse region with a complex geological history, but it remains unclear to what extent the distribution of freshwater fish has been influenced by geographic barriers. This study examines the population level genetic divergence and phylogenetic relationships of species in the shortfin group of the subgenus Mollienesia (genus Poecilia), a group of live-bearing fishes that are widely distributed across Mexico, with sampling at a small geographic scale. Samples from over 50 locations were analyzed for six species by using phylogenetic and haplotype network approaches to assess genetic diversity across geographic ranges and to refine the distributions of species in this group. The results indicate that Mexican species have diversified following multiple, independent invasions from Middle America. Two species found north of the Trans-Mexican Volcanic Belt (TMVB) and one transversal species exhibited weak phylogenetic structure, likely due to the lack of physiographic barriers, recent colonization, and high dispersal rates among regions. In contrast, three species found south of the TMVB exhibited strong phylogenetic structure, reflecting a longer presence in the area and multiple physiographic barriers that isolated populations. This study identified mechanisms driving divergence and speciation, expanded the known range of several species, and resolved taxonomic uncertainties of populations.(AU)

México es una región megadiversa con una historia geológica compleja, pero se desconoce el nivel de influencia de las barreras geográficas sobre las distribuciones de los peces dulceacuícolas. Este estudio examina las relaciones filogenéticas, a escala geográfica pequeña, de las especies del grupo de aletas cortas del subgénero Mollienesia (género Poecilia), un grupo de peces vivíparos ampliamente distribuidos en México. Se analizaron muestras de seis especies en más de 50 localidades, utilizando métodos filogenéticos y de redes de haplotipos, para evaluar la diversidad genética y precisar las distribuciones de especies en este grupo. Los resultados indican que las especies mexicanas se han diversificado a partir de múltiples invasiones independientes desde Mesoamérica. Se detectó estructura filogenética débil en dos especies distribuidas al norte del Eje Neovolcánico y una especie que atraviesa el Eje Neovolcánico, posiblemente debido a la ausencia de barreras fisiográficas, colonización reciente y altas tasas de dispersión entre regiones. En contraste, se detectaron niveles altos de estructura filogenética en tres especies distribuidas del Eje Neovolcánico, lo que refleja una presencia más prolongada en el área y la existencia de múltiples barreras fisiográficas que aislaron a las poblaciones. Este estudio identificó mecanismos que promueven la divergencia y la especiación, expandió el rango conocido de varias especies y resolvió incertidumbres taxonómicas de algunas poblaciones.(AU)

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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.

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Eye regression occurs across cave-dwelling populations of many species and is often coupled with a decrease or loss in eye function. Teleost fishes are among the few vertebrates to undergo widespread colonization of caves and often exhibit eye regression with blindness. Cave populations of the poeciliid fish Poecilia mexicana (cave molly) exhibit reduced-albeit functional-eyes, offering the opportunity to investigate partial eye regression. We sequenced eye transcriptomes of cave and surface populations of P. mexicana to identify differentially expressed genes that potentially underlie eye regression in cave mollies. We identified 28 significantly differentially expressed genes, 20 of which were directly related to light sensitivity, eye structure and visual signaling. Twenty-six of these genes were downregulated in cave compared to surface populations. Functional enrichment analysis revealed eye-related gene ontologies that were under-represented in cave mollies. In addition, a set of co-expressed genes related to vision and circadian rhythm was correlated with habitat type (cave versus surface). Our study suggests that differential gene expression plays a key role in the beginning evolutionary stages of eye regression in P. mexicana, shedding further light on regressive evolution in cavefish.

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The molecular basis of convergent phenotypes is often unknown. However, convergence at a genomic level is predicted when there are large population sizes, gene flow among diverging lineages or strong genetic constraints. We used whole-genome resequencing to investigate genomic convergence in fishes ( Poecilia spp.) that have repeatedly colonized hydrogen sulfide (H2S)-rich environments in Mexico. We identified genomic similarities in both single nucleotide polymorphisms (SNPs) and structural variants (SVs) among independently derived sulfide spring populations, with approximately 1.2% of the genome being shared among sulfidic ecotypes. We compared these convergent genomic regions to candidate genes for H2S adaptation identified from transcriptomic analyses and found that a significant proportion of these candidate genes (8%) were also in regions where sulfidic individuals had similar SNPs, while only 1.7% were in regions where sulfidic individuals had similar SVs. Those candidate genes included genes involved in sulfide detoxification, the electron transport chain (the main toxicity target of H2S) and other processes putatively important for adaptation to sulfidic environments. Regional genomic similarity across independent populations exposed to the same source of selection is consistent with selection on standing variation or introgression of adaptive alleles across divergent lineages. However, combined with previous analyses, our data also support that adaptive changes in mitochondrially encoded subunits arose independently via selection on de novo mutations. Pressing questions remain on what conditions ultimately facilitate the independent rise of adaptive alleles at the same loci in separate populations, and thus, the degree to which evolution is repeatable or predictable. This article is part of the theme issue 'Convergent evolution in the genomics era: new insights and directions'.

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In this paper, we outline the use of a mitochondria-targeted ratiometric mass spectrometry probe, MitoA, to detect in vivo changes in mitochondrial hydrogen sulfide (H2S) in Poecilia mexicana (family Poeciliidae). MitoA is introduced via intraperitoneal injection into the animal and is taken up by mitochondria, where it reacts with H2S to form the product MitoN. The MitoN/MitoA ratio can be used to assess relative changes in the amounts of mitochondrial H2S produced over time. We describe the use of MitoA in the fish species P. mexicana to illustrate the steps for adopting the use of MitoA in a new organism, including extraction and purification of MitoA and MitoN from tissues followed by tandem mass spectrometry. In this proof-of-concept study we exposed H2S tolerant P. mexicana to 59 µM free H2S for 5 h, which resulted in increased MitoN/MitoA in brain and gills, but not in liver or muscle, demonstrating increased mitochondrial H2S levels in select tissues following whole-animal H2S exposure. This is the first time that accumulation of H2S has been observed in vivo during whole-animal exposure to free H2S using MitoA. This article has an associated First Person interview with the first author of the paper.

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Extreme environments typically require costly adaptations for survival, an attribute that often translates to an elevated influence of habitat conditions on biotic communities. Microbes, primarily bacteria, are successful colonizers of extreme environments worldwide, yet in many instances, the interplay between harsh conditions, dispersal, and microbial biogeography remains unclear. This lack of clarity is particularly true for habitats where extreme temperature is not the overarching stressor, highlighting a need for studies that focus on the role other primary stressors (e.g., toxicants) play in shaping biogeographic patterns. In this study, we leveraged a naturally paired stream system in southern Mexico to explore how elevated hydrogen sulfide (H2S) influences microbial diversity. We sequenced a portion of the 16S rRNA gene using bacterial primers for water sampled from three geographically proximate pairings of streams with high (> 20 µM) or low (~ 0 µM) H2S concentrations. After exploring bacterial diversity within and among sites, we compared our results to a previous study of macroinvertebrates and fish for the same sites. By spanning multiple organismal groups, we were able to illuminate how H2S may differentially affect biodiversity. The presence of elevated H2S had no effect on overall bacterial diversity (p = 0.21), a large effect on community composition (25.8% of variation explained, p < 0.0001), and variable influence depending upon the group-whether fish, macroinvertebrates, or bacteria-being considered. For bacterial diversity, we recovered nine abundant operational taxonomic units (OTUs) that comprised a core H2S-rich stream microbiome in the region. Many H2S-associated OTUs were members of the Epsilonproteobacteria and Gammaproteobacteria, which both have been implicated in endosymbiotic relationships between sulfur-oxidizing bacteria and eukaryotes, suggesting the potential for symbioses that remain to be discovered in these habitats.

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The colonization of novel environments often involves changes in gene expression, protein coding sequence, or both. Studies of how populations adapt to novel conditions, however, often focus on only one of these two processes, potentially missing out on the relative importance of different parts of the evolutionary process. In this study, our objectives were 1) to better understand the qualitative concordance between conclusions drawn from analyses of differential expression and changes in genic sequence and 2) to quantitatively test whether differentially expressed genes were enriched for sites putatively under positive selection within gene regions. To achieve this, we compared populations of fish (Poecilia mexicana) that have independently adapted to hydrogen-sulfide-rich environments in southern Mexico to adjacent populations residing in nonsulfidic waters. Specifically, we used RNA-sequencing data to compare both gene expression and DNA sequence differences between populations. Analyzing these two different data types led to similar conclusions about which biochemical pathways (sulfide detoxification and cellular respiration) were involved in adaptation to sulfidic environments. Additionally, we found a greater overlap between genes putatively under selection and differentially expressed genes than expected by chance. We conclude that considering both differential expression and changes in DNA sequence led to a more comprehensive understanding of how these populations adapted to extreme environmental conditions. Our results imply that changes in both gene expression and DNA sequence-sometimes at the same loci-may be involved in adaptation.

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Organisms adapted to physiochemical stressors provide ideal systems to study evolutionary mechanisms that drive adaptation and speciation. This review study focuses on livebearing fishes of the Poecilia mexicana species complex (Poeciliidae), members of which have repeatedly colonized hydrogen sulphide (H2 S)-rich springs. H2 S is a potent respiratory toxicant that creates extreme environmental conditions in aquatic ecosystems. There is also a rich history of research on H2 S in toxicology and biomedicine, which has facilitated the generation of a priori hypotheses about the proximate mechanisms of adaptation. Testing these hypotheses through the application of high-throughput genomic and transcriptomic analyses has led to the identification of the physiological underpinnings mediating adaptation to H2 S-rich environments. In addition, systematic natural history studies have provided a nuanced understanding of how the presence of a physiochemical stressor interacts with other sources of selection to drive evolutionary change in a variety of organismal traits, including physiology, morphology, behaviour and life history. Adaptation to extreme environments in P. mexicana also coincides with ecological speciation, and evolutionarily independent lineages span almost the full range of the speciation continuum from panmixia to complete reproductive isolation. Multiple mechanisms of reproductive isolation are involved in reducing gene flow between adjacent populations that are adapted to contrasting environmental conditions. Comparative studies among evolutionarily independent lineages within the P. mexicana species complex and, more recently, other members of the family Poeciliidae that have colonized H2 S-rich environments will provide insights into the factors facilitating or impeding convergent evolution, providing tangible links between micro-evolutionary processes and macro-evolutionary patterns.

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Hydrogen sulfide (H2S) is a natural toxicant in some aquatic environments that has diverse molecular targets. It binds to oxygen transport proteins, rendering them non-functional by reducing oxygen-binding affinity. Hence, organisms permanently inhabiting H2S-rich environments are predicted to exhibit adaptive modifications to compensate for the reduced capacity to transport oxygen. We investigated 10 lineages of fish of the family Poeciliidae that have colonized freshwater springs rich in H2S-along with related lineages from non-sulfidic environments-to test hypotheses about the expression and evolution of oxygen transport genes in a phylogenetic context. We predicted shifts in the expression of and signatures of positive selection on oxygen transport genes upon colonization of H2S-rich habitats. Our analyses indicated significant shifts in gene expression for multiple hemoglobin genes in lineages that have colonized H2S-rich environments, and three hemoglobin genes exhibited relaxed selection in sulfidic compared to non-sulfidic lineages. However, neither changes in gene expression nor signatures of selection were consistent among all lineages in H2S-rich environments. Oxygen transport genes may consequently be predictable targets of selection during adaptation to sulfidic environments, but changes in gene expression and molecular evolution of oxygen transport genes in H2S-rich environments are not necessarily repeatable across replicated lineages.

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