Whole genomes of Amazonian uakari monkeys reveal complex connectivity and fast differentiation driven by high environmental dynamism.

Despite showing the greatest primate diversity on the planet, genomic studies on Amazonian primates show very little representation in the literature. With 48 geolocalized high coverage whole genomes from wild uakari monkeys, we present the first population-level study on platyrrhines using whole genome data. In a very restricted range of the Amazon rainforest, eight uakari species (Cacajao genus) have been described and categorized into the bald and black uakari groups, based on phenotypic and ecological differences. Despite a slight habitat overlap, we show that posterior to their split 0.92 Mya, bald and black uakaris have remained independent, without gene flow. Nowadays, these two groups present distinct genetic diversity and group-specific variation linked to pathogens. We propose differing hydrology patterns and effectiveness of geographic barriers have modulated the intra-group connectivity and structure of bald and black uakari populations. With this work we have explored the effects of the Amazon rainforest's dynamism on wild primates' genetics and increased the representation of platyrrhine genomes, thus opening the door to future research on the complexity and diversity of primate genomics.

Nascent transcription reveals regulatory changes in extremophile fishes inhabiting hydrogen sulfide-rich environments.

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.

Epigenetic inheritance of DNA methylation changes in fish living in hydrogen sulfide-rich springs.

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.

Expression analyses of cave mollies (Poecilia mexicana) reveal key genes involved in the early evolution of eye regression.

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.

Local ancestry analysis reveals genomic convergence in extremophile fishes.

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'.

Bacterial Diversity in Replicated Hydrogen Sulfide-Rich Streams.

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.

Concordant Changes in Gene Expression and Nucleotides Underlie Independent Adaptation to Hydrogen-Sulfide-Rich Environments.

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.

Extreme environments and the origins of biodiversity: Adaptation and speciation in sulphide spring fishes.

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.

Molecular evolution and expression of oxygen transport genes in livebearing fishes (Poeciliidae) from hydrogen sulfide rich springs.

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.

Genome-scale data reveal that endemic Poecilia populations from small sulphidic springs display no evidence of inbreeding.

Populations with limited ranges can be highly vulnerable to changes in their environment and are, thus, of high conservation concern. Populations that experience human-induced range reductions are often highly inbred and lack genetic diversity, but it is unknown whether this is also the case for populations with naturally small ranges. The fishes Poecilia sulphuraria (listed as critically endangered) and Poecilia thermalis, which are endemic to small hydrogen sulphide-rich springs in southern Mexico, are examples of such populations with inherently small habitats. We used geometric morphometrics and population genetics to quantify phenotypic and genetic variation within and among two populations of P. sulphuraria and one population of P. thermalis. Principal component analyses revealed phenotypic and genetic differences among the populations. Evidence for inbreeding was low compared to populations that have undergone habitat reduction. The genetic data were also used to infer the demographic history of these populations to obtain estimates for effective population sizes and migration rates. Effective population sizes were large given the small habitats of these populations. Our results imply that these three endemic extremophile populations should each be considered separately for conservation purposes. Additionally, this study suggests that populations in naturally small habitats may have lower rates of inbreeding and higher genetic diversity than expected, and therefore may be better equipped to handle environmental perturbations than anticipated. We caution, however, that the inferred lack of inbreeding and the large effective population sizes could potentially be a result of colonization by genetically diverse ancestors.

Complexities of gene expression patterns in natural populations of an extremophile fish (Poecilia mexicana, Poeciliidae).

Variation in gene expression can provide insights into organismal responses to environmental stress and physiological mechanisms mediating adaptation to habitats with contrasting environmental conditions. We performed an RNA-sequencing experiment to quantify gene expression patterns in fish adapted to habitats with different combinations of environmental stressors, including the presence of toxic hydrogen sulphide (H2 S) and the absence of light in caves. We specifically asked how gene expression varies among populations living in different habitats, whether population differences were consistent among organs, and whether there is evidence for shared expression responses in populations exposed to the same stressors. We analysed organ-specific transcriptome-wide data from four ecotypes of Poecilia mexicana (nonsulphidic surface, sulphidic surface, nonsulphidic cave and sulphidic cave). The majority of variation in gene expression was correlated with organ type, and the presence of specific environmental stressors elicited unique expression differences among organs. Shared patterns of gene expression between populations exposed to the same environmental stressors increased with levels of organismal organization (from transcript to gene to physiological pathway). In addition, shared patterns of gene expression were more common between populations from sulphidic than populations from cave habitats, potentially indicating that physiochemical stressors with clear biochemical consequences can constrain the diversity of adaptive solutions that mitigate their adverse effects. Overall, our analyses provided insights into transcriptional variation in a unique system, in which adaptation to H2 S and darkness coincide. Functional annotations of differentially expressed genes provide a springboard for investigating physiological mechanisms putatively underlying adaptation to extreme environments.

Genomic resources for a model in adaptation and speciation research: characterization of the Poecilia mexicana transcriptome.

BACKGROUND: Elucidating the genomic basis of adaptation and speciation is a major challenge in natural systems with large quantities of environmental and phenotypic data, mostly because of the scarcity of genomic resources for non-model organisms. The Atlantic molly (Poecilia mexicana, Poeciliidae) is a small livebearing fish that has been extensively studied for evolutionary ecology research, particularly because this species has repeatedly colonized extreme environments in the form of caves and toxic hydrogen sulfide containing springs. In such extreme environments, populations show strong patterns of adaptive trait divergence and the emergence of reproductive isolation. Here, we used RNA-sequencing to assemble and annotate the first transcriptome of P. mexicana to facilitate ecological genomics studies in the future and aid the identification of genes underlying adaptation and speciation in the system. DESCRIPTION: We provide the first annotated reference transcriptome of P. mexicana. Our transcriptome shows high congruence with other published fish transcriptomes, including that of the guppy, medaka, zebrafish, and stickleback. Transcriptome annotation uncovered the presence of candidate genes relevant in the study of adaptation to extreme environments. We describe general and oxidative stress response genes as well as genes involved in pathways induced by hypoxia or involved in sulfide metabolism. To facilitate future comparative analyses, we also conducted quantitative comparisons between P. mexicana from different river drainages. 106,524 single nucleotide polymorphisms were detected in our dataset, including potential markers that are putatively fixed across drainages. Furthermore, specimens from different drainages exhibited some consistent differences in gene regulation. CONCLUSIONS: Our study provides a valuable genomic resource to study the molecular underpinnings of adaptation to extreme environments in replicated sulfide spring and cave environments. In addition, this study adds to the increasing number of genomic resources in the family Poeciliidae, which are widely used in comparative analyses of behavior, ecology, evolution, and medical genetics.