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The Mexican cavefish, Astyanax mexicanus (Characidae), has become an important model in evolutionary physiology and developmental biology, providing insights into the evolution of sensory systems, pigmentation, and metabolism. In contrast, comparatively little is known about the natural history and trophic ecology of this elusive cave inhabitant. We investigated cavefish from three independently colonized cave systems (Pachon, Tinaja, and Sabinos), which are located in the Sierra de El Abra of northeastern Mexico. Samples were collected multiple times throughout the year to investigate variation in body size, sex ratios, proportions of individuals with empty guts, and diet composition. We found consistent differences in body size among caves, and sex ratios were generally female biased, although to varying degrees. Gut content analyses indicated that cavefish consume food throughout the year, and diets are dominated by detritus, plant materials, and aquatic invertebrates. Especially in the Pachon cave, where we had the densest sampling, there was evidence for seasonal changes in diet composition that coincided with the rainy and dry seasons. Our findings potentially suggest that the cave environments in this system are characterized by continual nutrient limitation, rather than intermittent periods of starvation.

Across the tree of life, species have repeatedly evolved similar phenotypes. While well-studied for ecological traits, there is also evidence for convergent evolution of sexually selected traits. Swordtail fish (Xiphophorus) are a classic model system for studying sexual selection, and femaleXiphophorusexhibit strong mate preferences for large male body size and a range of sexually dimorphic ornaments. However, sexually selected traits have been lost multiple times in the genus. Phylogenetic relationships between species in this group have historically been controversial, likely as a result of prevalent gene flow, resulting in uncertainty over the number of losses of ornamentation and large body size. Here, we use whole-genome sequencing approaches to re-examine phylogenetic relationships within aXiphophorusclade that varies in the presence and absence of sexually selected traits. Using wild-caught individuals, we determine the phylogenetic placement of a small, unornamented species,X. continens, confirming an additional loss of ornamentation and large body size in the clade. With these revised phylogenetic relationships, we analyze evidence for coevolution between body size and other sexually selected traits using a phylogenetically independent contrasts approach. These results provide insights into the evolutionary pressures driving the recurrent loss of suites of sexually selected traits.

Phenotypic variation is common along environmental gradients, but it is often unknown to what extent it results from genetic differentiation between populations or phenotypic plasticity. We studied populations of a livebearing fish that have colonized streams rich in toxic hydrogen sulfide (H2S). In nature, there is strong phenotypic differentiation between adjacent sulfidic and nonsulfidic populations. In this study, we varied food availability to pregnant mothers from different populations to induce maternal effects, a form of plasticity, and repeatedly measured life-history and behavioral traits throughout the offspring’s ontogeny. Genetic differentiation affected most of the traits we measured, as sulfidic offspring tended to be born larger, mature later, have lower burst swimming performance, be more exploratory, and feed less accurately. In contrast, maternal effects impacted few traits and at a smaller magnitude, even though offspring from poorly provisioned mothers tended to be born larger and be more exploratory. Population differences and maternal effects (when both were present) acted synergistically, and there was no evidence for population differences in plasticity. Overall, our study suggests that phenotypic divergence between populations in nature is primarily caused by genetic differentiation, and that plasticity mediated by maternal effects accentuates—but does not cause—differences between populations.

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.

Our understanding of the mechanisms mediating the resilience of organisms to environmental change remains lacking. Heavy metals negatively affect processes at all biological scales, yet organisms inhabiting contaminated environments must maintain homeostasis to survive. Tar Creek in Oklahoma, USA, contains high concentrations of heavy metals and an abundance of Western mosquitofish (Gambusia affinis), though several fish species persist at lower frequency. To test hypotheses about the mechanisms mediating the persistence and abundance of mosquitofish in Tar Creek, we integrated ionomic data from seven resident fish species and transcriptomic data from mosquitofish. We predicted that mosquitofish minimize uptake of heavy metals more than other Tar Creek fish inhabitants and induce transcriptional responses to detoxify metals that enter the body, allowing them to persist in Tar Creek at higher density than species that may lack these responses. Tar Creek populations of all seven fish species accumulated heavy metals, suggesting mosquitofish cannot block uptake more efficiently than other species. We found population-level gene expression changes between mosquitofish in Tar Creek and nearby unpolluted sites. Gene expression differences primarily occurred in the gill, where we found upregulation of genes involved with lowering transfer of metal ions from the blood into cells and mitigating free radicals. However, many differentially expressed genes were not in known metal response pathways, suggesting multifarious selective regimes and/or previously undocumented pathways could impact tolerance in mosquitofish. Our systems-level study identified well characterized and putatively new mechanisms that enable mosquitofish to inhabit heavy metal-contaminated environments.

Convergent evolution, where independent lineages evolve similar traits when adapting to similar habitats, is a common phenomenon and testament to the repeatability of evolutionary processes. Still, non-convergence is also common, and a major question is whether apparently idiosyncratic, lineage-specific evolutionary changes are reflective of chance events inherent to evolutionary processes, or whether they are also influenced by deterministic genetic or ecological factors. To address this question, we quantified the degree of convergence in genome-wide patterns of gene expression across lineages of livebearing fishes (family Poeciliidae) that span 40 million years of evolution and have colonized extreme environments in the form of toxic, hydrogen-sulfide-rich springs. We specifically asked whether the degree of convergence across lineage pairs was related to their phylogenetic relatedness or the ecological similarity of the habitats they inhabit. Using phylogenetic comparative analyses, we showed that the degree of convergence was highly variable across lineage pairs residing in sulfide springs. While closely related lineages did not exhibit higher degrees of convergence than distantly related ones, we uncovered a strong relationship between degree of convergence and ecological similarity. Our results indicate that variation in the degree of convergence is not merely noise associated with evolutionary contingency. Rather, cryptic environmental variation that is frequently ignored when we employ reductionist approaches can significantly contribute to adaptive evolution. This study highlights the importance of multivariate approaches that capture the complexities of both selective regimes and organismal design when assessing the roles of determinism and contingency in evolution.Significance StatementWhen different species adapt to similar environmental conditions, we frequently observe a mix between shared (convergent) and lineage-specific (nonconvergent) evolutionary changes. Shared changes provide evidence for the repeatability and predictability of evolution. However, it remains unclear whether lineage-specific changes are caused by random forces that limit the predictability of evolution, or whether they reflect deterministic processes shaped by unidentified genetic and environmental factors. By analyzing patterns of gene expression across fishes in extreme environments, we show that the degree of convergence between lineages is related to ecology, indicating that lineage-specific evolutionary changes are not just noise caused by random processes. Thus, acknowledging the complexity of nature in empirical research is critical if we want to predict evolution.

Divergent selection along ecological gradients can lead to speciation, and replicated speciation events occur when populations of multiple lineages undergo divergence following colonization of similar environments. In such instances, it remains unclear to what extent reproductive isolation evolves via convergent mechanisms at the genomic level due to biases associated with shared ancestry and variation in selection associated with geographic replicates typically studied in this context. We used a unique system in which three species of poeciliid fishes occur in sympatry in a toxic, hydrogen sulfide (H2S)-rich spring and an adjacent nonsulfidic stream to examine shared patterns of adaptive divergence across multiple levels of biological organization. Despite small spatial scales, we found strong genetic differentiation between populations in sulfidic and nonsulfidic habitats mediated by strong selection against migrants between habitat types. High levels of reproductive isolation were accompanied by convergent patterns of adaptation in morphological and physiological traits, as well as genome-wide patterns of gene expression across all three species. Furthermore, the mitochondrial genomes of each species exhibit shared signatures of selection on key genes involved in H2S toxicity. However, contrary to predictions of speciation theory, analyses of divergence across the nuclear genomes neither revealed evidence for clear genomic islands of speciation nor substantial congruence of outlier regions across population pairs. Instead, heterogenous regions of divergence spread across the genome suggest that selection for polygenic physiological adaptations likely facilitated the rapid evolution of high levels of reproductive isolation. Overall, we demonstrate that substantial convergence across multiple levels of biological organization can be mediated by non-convergent modifications at the genomic level. By disentangling variation in natural selection from lineage-specific evolution in this system of highly divergent, yet sympatric lineages, our results emphasize the outsized role of the genomic substrate upon which selection acts in driving convergent evolution at the phenotypic level.Significance StatementDivergent lineages that coexist in sympatry and are exposed to the same sources of natural selection provide a unique opportunity to study convergent evolution across levels of organization because confounding factors associated with geographic replications are eliminated. Using three sympatric lineages of livebearing fishes inhabiting toxic and adjacent nontoxic habitats, we show that the convergent evolution of phenotypic adaptation and reproductive isolation can evolve in the absence of substantial convergence at the genomic level. Our results suggest that the nature of selection is less important in producing genomic convergence than the initial genomic substrates that are available for selection to act on.

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 studied Poecilia mexicana populations that have repeatedly adapted to extreme sulphidic (H2S containing) environments. We measured visual sensitivity using opsin gene expression, as well as body pigmentation and water transmission 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. The light spectrum was only subtly different between environments and overall, we found no significant correlations between the light environment and visual sensitivity or body pigmentation. Altogether we found that sulphidic habitats select for differences in visual sensitivity and pigmentation; our data suggest that this effect is unlikely to be driven purely by the water’s spectral properties and may instead be from other correlated ecological changes.

Significance Environmental factors can promote phenotypic variation through alterations in the epigenome and mediate adaptation of an organism to the environment. Observations suggest the adaptation of Poecilia mexicana fish to toxic, hydrogen sulfide–rich environments in southern Mexico may, in part, be promoted through epigenetic DNA methylation alterations that became generationally stable and are inherited to subsequent generations independent of the environment. Environmental epigenetics may provide an important mechanism mediating adaptation in this species. This is an observation that the epigenome is stably inherited generationally through the germline after the removal of an environmental stressor (i.e., hydrogen sulfide) from a wild population., 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.

microRNAs (miRNAs) are post-transcriptional regulators of gene expression and can play an important role in modulating organismal development and physiology in response to environmental stress. However, the role of miRNAs in mediating adaptation to diverse environments in natural study systems remains largely unexplored. Here, we characterized miRNAs and their expression in Poecilia mexicana, a species of small fish that inhabits both normal streams and extreme environments in the form of springs rich in toxic hydrogen sulphide (H2 S). We found that P. mexicana has a similar number of miRNA genes as other teleosts. In addition, we identified a large population of mature miRNAs that were differentially expressed between locally adapted populations in contrasting habitats, indicating that miRNAs may contribute to P. mexicana adaptation to sulphidic environments. In silico identification of differentially expressed miRNA-mRNA pairs revealed, in the sulphidic environment, the downregulation of miRNAs predicted to target mRNAs involved in sulphide detoxification and cellular homeostasis, which are pathways essential for life in H2 S-rich springs. In addition, we found that predicted targets of upregulated miRNAs act in the mitochondria (16.6% of predicted annotated targets), which is the main site of H2 S toxicity and detoxification, possibly modulating mitochondrial function. Together, the differential regulation of miRNAs between these natural populations suggests that miRNAs may be involved in H2 S adaptation by promoting functions needed for survival and reducing functions affected by H2 S. This study lays the groundwork for further research to directly demonstrate the role of miRNAs in adaptation to H2 S. Overall, this study provides a critical stepping-stone towards a comprehensive understanding of the regulatory mechanisms underlying the adaptive variation in gene expression in a natural system.

Ecological speciation, which relies heavily on selection driving the emergence of new species, has become the primary paradigm through which rapid species radiations are understood. In this way, selection, particularly ecological selection, is assumed to be the driver of most species radiations. However, in many radiations, such as the radiation of Lake Malawi’s cichlids, the assumption of selection as the driver of speciation has rarely been explicitly tested, and drift, often, has completely been ignored as potential factor. In order to understand the forces driving the divergence of Lake Malawi cichlids at the microevolutionary level, we studied the genetic and phenotypic divergence of ten allopatric populations of Maylandia zebra. We estimated effective population sizes as proxy for drift. Further, we compared neutral genetic differentiation to divergence in three phenotypic traits: body size, body shape, and melanophore counts. We found small, yet significant, population differentiation in all the studied traits across most populations. Population sizes were small rendering the potential for drift to be high. However, phenotypic differentiation exceeded neutral expectations for all traits suggesting divergent local selection. Our data suggest that natural, and potentially also sexual, selection may be the dominant force driving population differentiation in Lake Malawi’s rock-dwelling cichlids, despite the potential for drift in small populations.

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.

The role of ecology in phenotypic and species diversification is widely documented. Nonetheless, numerous nonadaptive processes can shape realized niches and phenotypic variation in natural populations, complicating inferences about adaptive evolution at macroevolutionary scales. We tested for evolved differences in thermal tolerances and their association with the realized thermal niche (including metrics describing diurnal and seasonal patterns of temperature extremes and variability) across a genus of tropical freshwater fishes reared in a standardized environment. There was limited evolution along the thermal niche axis associated with variation in maximum temperature and in upper thermal limits. In contrast, there was considerable diversification along the first major axis of the thermal niche associated with minimum temperatures and in lower thermal limits. Across our adaptive landscape analyses, 70% of species exhibited evidence of divergence in thermal niches. Most importantly, the first two major axes of thermal niche variation were significantly correlated with variation in lower thermal limits. Our results indicate adaptation to divergent thermal niches and adaptive evolution of related functional traits, and highlight the importance of divergence in lower thermal limits for the evolution of tropical biodiversity.

Natural selection is often assumed to drive parallel functional diversification of the sexes. But males and females exhibit fundamental differences in their biology, and it remains largely unknown how sex differences affect macroevolutionary patterns. On microevolutionary scales, we understand how natural and sexual selection interact to give rise to sex-specific evolution during phenotypic diversification and speciation. Here we show that ignoring sex-specific patterns of functional trait evolution misrepresents the macroevolutionary adaptive landscape and evolutionary rates for 112 species of live-bearing fishes (Poeciliidae). Males and females of the same species evolve in different adaptive landscapes. Major axes of female morphology were correlated with environmental variables but not reproductive investment, while male morphological variation was primarily associated with sexual selection. Despite the importance of both natural and sexual selection in shaping sex-specific phenotypic diversification, species diversification was overwhelmingly associated with ecological divergence. Hence, the inter-predictability of mechanisms of phenotypic and species diversification may be limited in many systems. These results underscore the importance of explicitly addressing sex-specific diversification in empirical and theoretical frameworks of evolutionary radiations to elucidate the roles of different sources of selection and constraint. It is unclear how sex differences affect macroevolutionary patterns. In live-bearing fishes, the environment influences female morphology, while male morphological variation is primarily associated with sexual selection.

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.

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.

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.

Mitochondria have been known to be involved in speciation through the generation of Dobzhansky–Muller incompatibilities, where functionally neutral co-evolution between mitochondrial and nuclear genomes can cause dysfunction when alleles are recombined in hybrids. We propose that adaptive mitochondrial divergence between populations can not only produce intrinsic (Dobzhansky–Muller) incompatibilities, but could also contribute to reproductive isolation through natural and sexual selection against migrants, post-mating prezygotic isolation, as well as by causing extrinsic reductions in hybrid fitness. We describe how these reproductive isolating barriers can potentially arise through adaptive divergence of mitochondrial function in the absence of mito-nuclear coevolution, a departure from more established views. While a role for mitochondria in the speciation process appears promising, we also highlight critical gaps of knowledge: (1) many systems with a potential for mitochondrially-mediated reproductive isolation lack crucial evidence directly linking reproductive isolation and mitochondrial function; (2) it often remains to be seen if mitochondrial barriers are a driver or a consequence of reproductive isolation; (3) the presence of substantial gene flow in the presence of mito-nuclear incompatibilities raises questions whether such incompatibilities are strong enough to drive speciation to completion; and (4) it remains to be tested how mitochondrial effects on reproductive isolation compare when multiple mechanisms of reproductive isolation coincide. We hope this perspective and the proposed research plans help to inform future studies of mitochondrial adaptation in a manner that links genotypic changes to phenotypic adaptations, fitness, and reproductive isolation in natural systems, helping to clarify the importance of mitochondria in the formation and maintenance of biological diversity.

The observed distributions of two grazing minnows differ along a stream-size gradient in grassland streams and may be linked to temperature. In laboratory experiments, we assayed a suite of physiological traits along a temperature gradient and found that species differed in critical thermal maxima, with subtle differences in other traits., Earth’s atmosphere has warmed by ~1°C over the past century and continues to warm at an increasing rate. Effects of atmospheric warming are already visible in most major ecosystems and are evident across all levels of biological organization. Linking functional responses of individuals to temperature is critical for predicting responses of populations and communities to global climate change. The southern redbelly dace Chrosomus erythrogaster and the central stoneroller Campostoma anomalum are two minnows (Cyprinidae) that commonly occur in the Flint Hills region of the USA but show different patterns of occurrence, with dace largely occupying headwater reaches and stonerollers persisting in both headwater and intermediate-sized streams. We tested for differences between species in critical thermal maximum, energy metabolism, sustained swimming and activity over an ecologically relevant temperature gradient of acclimation temperatures. Typically, metrics increased with acclimation temperature for both species, although stoneroller activity decreased with temperature. We observed a significant interaction between species and temperature for critical thermal maxima, where stonerollers only had higher critical thermal maxima at the coldest temperature and at warm temperatures compared to the dace. We did not find evidence suggesting differences in the energy metabolism of dace and stonerollers. We detected interspecific differences in sustained swimming performance, with dace having higher swimming speed than stonerollers regardless of acclimation temperature. Finally, there was a significant interaction between temperature and species for activity; dace activity was higher at intermediate and warm temperatures compared to stonerollers. We observed subtle interspecific differences in how performance metrics responded to temperature that did not always align with observed patterns of distribution for these species. Thus, other ecological factors likely are important drivers of distributional patterns in these species.

Hydrogen sulfide (H2S) is a respiratory toxicant that creates extreme environments tolerated by few organisms. H2S is also produced endogenously by metazoans and plays a role in cell signaling. The mechanisms of H2S toxicity and its physiological functions serve as a basis to discuss the multifarious strategies that allow animals to survive in H2S-rich environments. Despite their toxicity, H2S-rich environments also provide ecological opportunities, and complex selective regimes of covarying abiotic and biotic factors drive trait evolution in organisms inhabiting H2S-rich environments. Furthermore, adaptation to H2S-rich environments can drive speciation, giving rise to biodiversity hot spots with high levels of endemism in deep-sea hydrothermal vents, cold seeps, and freshwater sulfide springs. The diversity of H2S-rich environments and their inhabitants provides ideal systems for comparative studies of the effects of a clear-cut source of selection across vast geographic and phylogenetic scales, ultimately informing our understanding of how environmental stressors affect ecological and evolutionary processes.

We describe three new species of forest robin in the genus Stiphrornis; two from West Africa and one from the Congo Basin. Each species represents a distinct phylogenetic lineage based on genetic analysis. In addition to genetic differentiation, each new species is diagnosable from other Stiphrornis lineages by morphology, and by plumage. One of the new species appears to be restricted to the Central and Brong-Ahafo Regions of Ghana, and another is restricted to Benin and the Central Region of Ghana. In Ghana, these two new species presumably come into contact with Stiphrornis erythrothorax (Western Region of Ghana and westward), and there is evidence that one of the new species has a distinguishably different song from erythrothorax. The distribution of the third new species is primarily on the south bank of the Congo River, near the city of Kisangani. Recognition of these species provides additional evidence that Afrotropical forests are harbouring substantial cryptic diversity, and that our knowledge o...

The teleost brain is an energetically costly organ, which raises the question of how brain anatomy is shaped by divergent ecological factors in contrasting (extreme/resource-limited vs. benign) environments. A previous study compared different ecotypes of the teleost Poecilia mexicana in the Tacotalpa drainage system and found that cave fish had a smaller eye diameter, a smaller optic tectum and larger telencephalic lobes relative to ancestral surface-dwelling fish. Smaller eyes and a smaller optic tectum but larger telencephalic lobes were also found in fish from a sulphidic surface habitat near one of the caves, which the authors hypothesized to result from limited vision in turbid sulphide waters. In this study, we tested if repeated transitions along a replicated, natural toxicity gradient result in repeated (‘convergent’) anatomical changes of the teleost brain. We compared ecotypes in the P. mexicana species complex that have independently evolved increased tolerance to hydrogen sulphide (H2S) in three river drainages in southern Mexico, including a phylogenetically old H2S-adapted form (P. sulphuraria) and two P. mexicana ecotypes that represent earlier stages of adaptation to H2S. All H2S-adapted ecotypes exhibited smaller eyes, a smaller optic tectum volume and a smaller brain volume, but larger corpora cerebelli and hypothalamic volume than fish from non-sulphidic habitats. Drainage-specific effects were found for the telencephalic lobes, the total brain and eye size, as sexes responded differently to the presence of H2S depending on the drainage of origin. Turbidity and toxicity in sulphidic habitats may explain patterns of brain size divergence similar in direction (but not degree) to those observed in cave ecotypes. Hence, variation in brain anatomy reflects major ecological differences, and repeated ecological gradients can result in convergent differences in brain anatomy. Nonetheless, some unique patterns of brain differentiation suggest as yet unidentified differences in selection regimes between different sulphidic springs.

Effective conservation measures for endangered species require basic knowledge of habitat use and critical environmental variables influencing the species' occurrence. However, setting priorities may be difficult when multiple endangered species inhabit the same area and differ in habitat use. This study characterized the physical and chemical environment at 30 sites along a 2.5 km stretch of stream associated with the Banos del Azufre hydrogen sulphide spring complex, home to the only population of the widemouth gambusia (Gambusia eurystoma) in existence. Also present in the spring is Poecilia sulphuraria, a narrowly endemic and endangered species known from only a few populations. This study provides the most detailed report to date for physical and chemical drivers of species density in this small, extreme environment. Gambusia eurystoma were generally rare throughout the stream, and stream flow and substrate size were the best predictors of G. eurystoma densities. Conversely, P. sulphuraria were considerably more abundant, but no physical or chemical variables predicted their density among sites. Size distributions of adult P. sulphuraria were significantly influenced by stream flow and water chemistry, indicating a cost to living in greater proximity to toxic springs with high hydrogen sulphide concentrations. Overall, the results provide the first extensive report of environmental variation and factors associated with G. eurystoma densities, and indicate that conservation measures prioritizing environmental conditions for G. eurystoma will also benefit P. sulphuraria. Fish in sulphide springs provide prime examples of narrowly endemic species in desperate need of conservation, and their habitats face conservation challenges similar to those found in other springs with sympatric, endemic species in the world's arid regions. Copyright © 2016 John Wiley & Sons, Ltd.

Genetic variation is critical for adaptive evolution. Despite its importance, there is still limited evidence in support of some prominent theoretical models explaining the maintenance of genetic polymorphism within populations. We examined 84 populations of Xiphophorus variatus, a livebearing fish with a genetic polymorphism associated with physiological performance, to test: (1) whether niche differentiation explains broad-scale maintenance of polymorphism, (2) whether polymorphism is maintained among populations by local adaptation and migration, or (3) whether heterogeneity in explicit environmental variables could be linked to levels of polymorphism within populations. We found no evidence of climatic niche differentiation that could generate or maintain broad geographic variation in polymorphism. Subsequently, hierarchical partitioning of genetic richness and partial mantel tests revealed that 76% of the observed genetic richness was partitioned within populations with no effect of geographic distance on polymorphism. These results strongly suggest a lack of migration-selection balance in the maintenance of polymorphism, and model selection confirmed a significant relationship between environmental heterogeneity and genetic richness within populations. Few studies have demonstrated such effects at this scale, and additional studies in other taxa should examine the generality of gene-by-environment interactions across populations to better understand the dynamics and scale of balancing selection.

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 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

Summary Ecological sources of selection are key drivers of evolutionary change in populations. Information on the ecological relevance of such evolutionary shifts is comparatively sparse and has received renewed interest. The framework of ecological stoichiometry is useful to investigate the reciprocal effects between ecology and evolution, because data on somatic stoichiometry of ancestral and descendent populations can be used to predict ecological functions, such as nutrient recycling, using mass balance-based models. Here, we investigated whether divergent populations of livebearing fishes (genus Poecilia) have diverged in elemental composition. We tested whether adaptation to local environmental conditions is manifested in changes of somatic stoichiometry by measuring carbon (C), nitrogen (N), phosphorus (P) and sulphur (S) contents of wild-caught individuals inhabiting sulphidic (extreme) and non-sulphidic (benign) habitats. We also attempted to isolate the sources (i.e. genetic, environmental and their interaction) of intraspecific variation in stoichiometry. Finally, we tested whether shifts in somatic stoichiometry impinge on the rates at which key nutrients (N and P) are excreted. We found significant differentiation in somatic stoichiometry between fish from the two different habitat types in two of three river drainages, with fish from sulphidic habitats having lower C but higher P and S contents. Even though there was evidence for temporal variation and plasticity in elemental composition, differences between sulphidic and non-sulphidic populations in P and S contents were maintained in laboratory populations over multiple generations. Finally, some sulphidic and non-sulphidic population pairs differed in the rates of N and P excretions, although excretion rates were not related to somatic stoichiometry. Together, these results show that the elemental composition of organisms appears to have the characteristics typically observed in the evolution of biochemical, physiological and morphological traits. Studying taxa that have undergone replicated evolution illuminate some of the evolutionary mechanisms that drive variation in somatic elemental composition. Applying stoichiometric principles to such variation, as we have performed here, is a useful, yet underutilised approach to understand the ecological relevance of evolutionary change.

Environmental variation can shape phenotypic variation in organisms, but most evidence for trait differentiation comes from analyses of dichotomous habitat types that differ in only one or few key environmental factors. In reality, environmental variation is often more subtle, gradual and multifarious. Here, we investigated geographic variation in body shape of two darter species (Etheostoma spectabile and Etheostoma flabellare; Percidae) that occur along river gradients. This study addressed three specific questions: Is there intraspecific geographic variation in the two species across different sites in the Ozark Highlands of Oklahoma (USA)? Is phenotypic variation across sites correlated with abiotic environmental conditions? Do the two species share site-specific (i.e. convergent) phenotypic variation in areas where they occur together? Our results indicated significant body shape variation in both species. Population differences in body shape were particularly correlated with variation in substrate composition. The combined analysis of both species indicated a small but significant effect of convergence on body shape wherever they are sympatric; shared variation, however, was not related to any environmental variables included in the analysis. While it remains unclear whether phenotypic variation in these species is due to heritable differentiation or environmentally induced plasticity, our results indicate that even subtle and gradual environmental variation can induce substantial variation in phenotypes on a relatively small spatial scale.

Summary Divergent selection along environmental gradients connecting locally restricted extreme habitats and adjacent benign habitats can shape convergent evolution of traits involved in coping with physiochemical stressors and can drive speciation. At the same time, the presence of such stressors alters aspects of the biotic environment, including resource availability and competitive regimes. However, it remains unclear whether and how the ecology of populations occurring in both extreme and benign environments varies in a predictable fashion. We investigated the trophic ecology of live-bearing fishes of the genus Poecilia that have independently colonised multiple springs containing toxic hydrogen sulphide in southern Mexico. Sulphide spring fish are adapted to the unique environmental conditions and are reproductively isolated from ancestral populations in adjacent non-sulphidic habitats. We used gut content analyses to test whether colonisation of extreme habitats was accompanied by shifts of trophic resource use and expansions of trophic niche width. Furthermore, we tested whether dietary shifts were reflected in trophic morphology by comparing intestinal tract lengths among populations using both wild-caught and common garden-raised individuals. Gut content analyses revealed that fish inhabiting toxic springs expanded their trophic niche width and changed their dietary resource use from detritus and algae to sulphide bacteria and invertebrates. This dietary shift was paralleled by changes in intestinal tract morphology, whereby sulphide spring fish had shorter intestines than fish from adjacent non-sulphidic habitats. Analysis of common garden-raised fish indicated that morphological differences between sulphidic and non-sulphidic populations are at least in part due to genetic differentiation. Both patterns of trophic resource use and differentiation in trophic morphology were consistent across replicated pairs of sulphidic and non-sulphidic populations, although the magnitude of differentiation varied among river drainages. Our results suggest that colonisation of and adaptation to sulphide springs in southern Mexico was paralleled by convergent changes in trophic ecology. This highlights the complexity of environmental gradients and the necessity of considering multiple sources of selection when studying the evolution of complex phenotypes.

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.

The brain is a plastic organ, and so intraspecific studies that compare results obtained from wild individuals with those from common-garden experiments are crucial for studies aiming to understand brain evolution. We compared volumes of brain regions between reproductively isolated populations of a neotropical fish, Poecilia mexicana, that has locally adapted to perpetual darkness (Cueva Luna Azufre), toxic hydrogen sulphide in a surface stream (El Azufre) or a combination of both stressors (Cueva del Azufre). Wild fish showed habitat-dependent differences: enlarged telencephalic lobes and reduced optic tecta were found in fish living in darkness and sulphidic waters, in darkness without hydrogen sulphide or exposed to light and sulphide; fish from the sulphidic cave additionally showed enlarged cerebella. Comparison with common-garden reared fish detected a general decrease in brain size throughout populations in the lab, and little of the brain size divergence between lab-reared ecotypes that was seen in wild-caught fish. The pronounced differences in brain region volumes between ecotypes in the wild might be interpreted within the framework of mosaic evolution; however, the outcomes of common-garden experiments indicate a high amount of phenotypic plasticity. Our study thus highlights the importance of combining the investigation of brain size in wild populations with common-garden experiments for answering questions of brain evolution.

Extreme environments are characterised by the presence of physicochemical stressors and provide unique study systems to address problems in evolutionary ecology research. Sulphide springs provide an example of extreme freshwater environments; because hydrogen sulphide’s adverse physiological effects induce mortality in metazoans even at micromolar concentrations. Sulphide springs occur worldwide, but while microbial communities in sulphide springs have received broad attention, little is known about macroinvertebrates and fish inhabiting these toxic environments. We reviewed qualitative occurrence records of sulphide spring faunas on a global scale and present a quantitative case study comparing diversity patterns in sulphidic and adjacent non-sulphidic habitats across replicated river drainages in Southern Mexico. While detailed studies in most regions of the world remain scarce, available data suggests that sulphide spring faunas are characterised by low species richness. Dipterans (among macroinvertebrates) and cyprinodontiforms (among fishes) appear to dominate the communities in these habitats. At least in fish, there is evidence for the presence of highly endemic species and populations exclusively inhabiting sulphide springs. We provide a detailed discussion of traits that might predispose certain taxonomic groups to colonize sulphide springs, how colonizers subsequently adapt to cope with sulphide toxicity, and how adaptation may be linked to speciation processes.

The Pecos gambusia (Gambusia nobilis) is an endangered poeciliid native to western Texas and eastern New Mexico. The decline of G. nobilis in Texas is likely the result of habitat alteration and introduction of potentially invasive congeners (e.g., the introduced largespring gambusia, Gambusia geiseri). In the East Sandia Cienega, observers have noted mating between G. geiseri and G. nobilis. Because potential hybridization between native and introduced species could have detrimental effects on reproductive fitness, we compare mean brood size, gonadosomatic indices, and standard length of both G. geiseri and G. nobilis as a first step in assessing the impacts of G. geiseri introduction in this area.

Variation in pigmentation traits is an effective window to evolutionary processes due to their importance for survival and reproduction. In particular, one of the leading hypotheses for the maintenance of conspicuous pigmentation in natural populations is its signaling function in mate choice. Here, we demonstrate the occurrence of melanism in poeciliid fishes of the genus Poecilia that inhabits toxic, hydrogen sulfide springs in southern Mexico and the absence of melanism from closely related populations in reference habitats lacking hydrogen sulfide. Assays of female mate preference in both habitat types were used to examine whether divergence in female preference for melanism contributes to its maintenance in hydrogen sulfide springs. We found significant variation in female preferences for melanistic males. Specifically, melanistic females from the toxic spring exhibited a significant preference for melanistic males, while non-melanistic females from the same population exhibited no preference. Females from the non-sulfidic reference population discriminated strongly against melanistic males. Preferences of melanistic females appear to be a significant force in the maintenance of melanism in sulfidic habitats and suggest a change in preference as the divergence from non-sulfidic ancestors. Potential polymorphism in preference within the hydrogen sulfide spring indicates that preference for melanistic males may not be environmentally controlled. Thus, a change in preference following divergence can promote the maintenance of variation in pigmentation within populations and between divergent habitats.

The cichlid fishes of Lake Malawi represent one of the most diverse adaptive radiations of vertebrates known. Among the rock-dwelling cichlids (mbuna), closely related sympatric congeners possess similar trophic morphologies (i.e. cranial and jaw structures), defend overlapping or adjacent territories, but can be easily distinguished based on male nuptial coloration. The apparent morphological similarity of congeners, however, leads to an ecological conundrum: theory predicts that ecological competition should lead to competitive exclusion. Hence, we hypothesized that slight, yet significant, ecological differences accompanied the divergence in sexual signals and that the divergence of ecological and sexual traits is correlated. To evaluate this hypothesis, we quantified body shape, a trait of known ecological importance, in populations of Maylandia zebra, a barred, widespread mbuna, and several sympatric nonbarred congeners. We found that the barred populations differ in body shape from their nonbarred sympatric congeners and that the direction of shape differences was consistent across all barred vs. nonbarred comparisons. Barred populations are generally deeper bodied which may be an adaptation to the structurally complex habitat they prefer, whereas the nonbarred species have a more fusiform body shape, which may be adaptive in their more open microhabitat. Furthermore, M. zebra populations sympatric with nonbarred congeners differ from populations where the nonbarred phenotype is absent and occupy less morphospace, indicating potential ecological character displacement. Mitochondrial DNA as well as published AFLP data indicated that the nonbarred populations are not monophyletic and therefore may have evolved multiple times independently. Overall our data suggest that the evolution of coloration and body shape may be coupled as a result of correlational selection. We hypothesize that correlated evolution of sexually selected and ecological traits may have contributed to rapid speciation as well as the maintenance of diversity in one of the most diverse adaptive radiations known.

Understanding the ecological consequences of evolutionary change is a central challenge in contemporary biology. We propose a framework based on the ˜25 elements represented in biology, which can serve as a conduit for a general exploration of poorly understood evolution-to-ecology links. In this framework, known as ecological stoichiometry, the quantity of elements in the inorganic realm is a fundamental environment, while the flow of elements from the abiotic to the biotic realm is due to the action of genomes, with the unused elements excreted back into the inorganic realm affecting ecological processes at higher levels of organization. Ecological stoichiometry purposefully assumes distinct elemental composition of species, enabling powerful predictions about the ecological functions of species. However, this assumption results in a simplified view of the evolutionary mechanisms underlying diversification in the elemental composition of species. Recent research indicates substantial intraspecific variation in elemental composition and associated ecological functions such as nutrient excretion. We posit that attention to intraspecific variation in elemental composition will facilitate a synthesis of stoichiometric information in light of population genetics theory for a rigorous exploration of the ecological consequences of evolutionary change.

This study investigated a rapidly vanishing group of fishes in the genus Characodon (including Characodon lateralis and Characodon audax) from the upper Rio Mezquital drainage in Mexico. Using specimens from museum collections, morphological variation was assessed to quantify body shape differentiation among historic (i.e. extirpated and extant) collection sites. In both sexes, body shape (particularly head shape, the proportion of the caudal peduncle and the position and size of dorsal and anal fins) varied significantly among populations and species. Variation among collection sites could at least partially be attributed to geography, as the presence of distinct hydrographic units and a major waterfall coincided with major body shape differences. These results are discussed in the light of previously published molecular genetic analyses, as they have direct implications for taxonomic problems and the need for conservation measures for these endangered fishes.