Kryptolebias Marmoratus Research Articles

Skin ionocyte density of amphibious killifishes is shaped by phenotypic plasticity and constitutive interspecific differences.

When amphibious fishes are on land, gill function is reduced or eliminated and the skin is hypothesized to act as a surrogate site of ionoregulation. Skin ionocytes are present in many fishes, particularly those with amphibious life histories. We used nine closely related killifishes spanning a range of amphibiousness to first test the hypothesis that amphibious killifishes have evolved constitutively increased skin ionocyte density to promote ionoregulation on land. We found that skin ionocyte densities were constitutively higher in five of seven amphibious species examined relative to exclusively water-breathing species when fish were prevented from leaving water, strongly supporting our hypothesis. Next, to examine the scope for plasticity, we tested the hypothesis that skin ionocyte density in amphibious fishes would respond plastically to air-exposure to promote ionoregulation in terrestrial environments. We found that air-exposure induced plasticity in skin ionocyte density only in the two species classified as highly amphibious, but not in moderately amphibious species. Specifically, skin ionocyte density significantly increased in Anablepsoides hartii (168%) and Kryptolebias marmoratus (37%) following a continuous air-exposure, and only in K. marmoratus (43%) following fluctuating air-exposure. Collectively, our data suggest that highly amphibious killifishes have evolved both increased skin ionocyte density as well as skin that is more responsive to air-exposure compared to exclusively water-breathing and less amphibious species. Our findings are consistent with the idea that gaining the capacity for cutaneous ionoregulation is a key evolutionary step that enables amphibious fishes to survive on land.

Out of water in the dark: Plasticity in visual structures and function in an amphibious fish

Many fishes encounter periods of prolonged darkness within their lifetime, yet the consequences for the visual system are poorly understood. We used an amphibious fish (Kryptolebias marmoratus) that occupies dark terrestrial environments during seasonal droughts to test whether exposure to prolonged darkness diminishes visual performance owing to reduced optic tectum (OT) size and/or neurogenesis. We performed a 3-week acclimation with a 2 2 factorial design, in which fish were either acclimated to a 12 h:12 h or 0 h:24 h light:dark photoperiod in water or in air. We found that water-exposed fish had poorer visual acuity when acclimated to the dark, while air-acclimated fish had poorer visual acuity regardless of photoperiod. The ability of K. marmoratus to capture aerial prey from water followed a similar trend, suggesting that good vision is important for hunting effectively. Changes in visual acuity did not result from changes in OT size, but air-acclimated fish had 37% fewer proliferating cells in the OT than water-acclimated fish. AsK. marmoratus are unable to eat on land, reducing cell proliferation in the OT may serve as a mechanism to reduce maintenance costs associated with the visual system. Overall, we suggest that prolonged darkness and air exposure can impair vision in K. marmoratus, and that changes in visual performance may be mediated, in part, by OT neurogenesis. More broadly, we show that plastic changes to the visual system of fishes can have potential consequences for organismal performance and fitness.

Retention of larval skin traits in adult amphibious killifishes: a cross-species investigation.

The gills are the primary site of exchange in fishes. However, during early life-stages or in amphibious fishes, ionoregulation and gas-exchange may be primarily cutaneous. Given the similarities between larval and amphibious fishes, we hypothesized that cutaneous larval traits are continuously expressed in amphibious fishes across all life-stages to enable the skin to be a major site of exchange on land. Alternatively, we hypothesized that cutaneous larval traits disappear in juvenile stages and are re-expressed in amphibious species in later life-stages. We surveyed six species spanning a range of amphibiousness and characterized cutaneous ionocytes and neuroepithelial cells (NECs) as representative larval skin traits at up to five stages of development. We found that skin ionocyte density remained lower and constant in exclusivelywater-breathing, relative to amphibious species across development, whereas in amphibious species ionocyte density generally increased. Additionally, adults of the most amphibious species had the highest cutaneous ionocyte densities. Surprisingly, cutaneous NECs were only identified in the skin of one amphibious species (Kryptolebias marmoratus), suggesting that cutaneous NECs are not a ubiquitous larval or amphibious skin trait, at least among the species we studied. Our data broadly supports the continuous-expression hypothesis, as three of four amphibious experimental species expressed cutaneous ionocytes in all examined life-stages. Further, the increasing density of cutaneous ionocytes across development in amphibious species probably facilitates the prolonged occupation of terrestrial habitats.

Context-dependent relationships between swimming, terrestrial jumping and body composition in the amphibious fish Kryptolebias marmoratus.

Understanding the mechanisms that create phenotypic variation within and among populations is a major goal of physiological ecology. Variation may be a consequence of functional trade-offs (i.e. improvement in one trait comes at the expense of another trait) or alternatively may reflect the intrinsic quality of an organism (i.e. some individuals are simply better overall performers than others). There is evidence for both ideas in the literature, suggesting that environmental context may mediate whether variation results from trade-offs or differences in individual quality. We tested this overarching 'context dependence' hypothesis by comparing the aquatic and terrestrial athletic performance of the amphibious fish Kryptolebias marmoratus captured from two contrasting habitats, a large pond and small burrows. Overall, pond fish were superior terrestrial athletes but burrow fish were better burst swimmers, suggestive of a performance trade-off at the population level. Within each population, however, there was no evidence of a performance trade-off. In burrow fish, athletic performance was positively correlated with muscle content and body condition, consistent with the individual quality hypothesis. In pond fish, there was only a relationship between glycolytic white muscle and aquatic burst performance. Notably, pond fish were in better body condition, which may mask relationships between condition and athletic performance. Overall, our data highlight that population-level trends are insufficient evidence for the existence of phenotypic trade-offs in the absence of similar within-population patterns. Furthermore, we only found evidence for the individual quality hypothesis in one population, suggesting that patterns of phenotypic covariance are context dependent.

Genetic Structure of the Mangrove Killifish Kryptolebias hermaphroditus Costa, 2011 (Cyprinodontiformes: Aplocheiloidei) Supports A Wide Connection among its Populations.

The Kryptolebias marmoratus species group is composed of the only three vertebrate species that lack females. These species present only males and simultaneously hermaphroditic individuals; that are able to reproduce by allogamy, with males, or by autogamy, performing self-fertilization and generating clones of themselves. The proportion of males is variable among those species and even among their populations. Kryptolebias hermaphroditus has the smallest proportion of males. Indeed, no males have been recorded in most known populations. This is a mainly autogamous species, with small populations having a disjunct distribution along the eastern and northern coast of Brazil. Species presenting such adaptations would be expected to have an elevated rate of genetic population structure, reflecting any barriers that obstruct gene flow between populations. Partial sequences of the mitochondrial cytochrome c oxidase I (COI) gene from 335 individuals were sampled to perform a population analysis. Only a single haplotype of COI, widely distributed throughout all the sampled populations, was recovered for K. hermaphroditus. Here we hypothesize that the high degree of communication within populations is probably the main biological feature leading to this pattern.

Novel spikey ionocytes are regulated by cortisol in the skin of an amphibious fish.

Cortisol is a major osmoregulatory hormone in fishes. Cortisol acts upon the gills, the primary site of ionoregulation, through modifications to specialized ion-transporting cells called ionocytes. We tested the hypothesis that cortisol also acts as a major regulator of skin ionocyte remodelling in the amphibious mangrove rivulus (Kryptolebias marmoratus) when gill function ceases during the water-to-land transition. When out of water, K. marmoratus demonstrated a robust cortisol response, which was linked with the remodelling of skin ionocytes to increase cell cross-sectional area and Na+-K+-ATPase (NKA) content, but not when cortisol synthesis was chemically inhibited by metyrapone. Additionally, we discovered a novel morphology of skin-specific ionocyte that are spikey with multiple cell processes. Spikey ionocytes increased in density, cell cross-sectional area and NKA content during air exposure, but not in metyrapone-treated fish. Our findings demonstrate that skin ionocyte remodelling during the water-to-land transition in amphibious fish is regulated by cortisol, the same hormone that regulates gill ionocyte remodelling in salinity-challenged teleosts, suggesting conserved hormonal function across diverse environmental disturbances and organs in fishes.

Molecular mechanisms of embryonic tail development in the self-fertilizing mangrove killifish Kryptolebias marmoratus.

ABSTRACTUsing the self-fertilizing mangrove killifish, we characterized two mutants, shorttail (stl) and balltail (btl). These mutants showed abnormalities in the posterior notochord and muscle development. Taking advantage of a highly inbred isogenic strain of the species, we rapidly identified the mutated genes, noto and msgn1 in the stl and btl mutants, respectively, using a single lane of RNA sequencing without the need of a reference genome or genetic mapping techniques. Next, we confirmed a conserved morphant phenotype in medaka and demonstrate a crucial role of noto and msgn1 in cell sorting between the axial and paraxial part of the tail mesoderm. This novel system could substantially accelerate future small-scale forward-genetic screening and identification of mutations. Therefore, the mangrove killifish could be used as a complementary system alongside existing models for future molecular genetic studies.

Predicting the in-between: Present and future habitat suitability of an intertidal euryhaline fish

New World mangrove trees are foundation species, and their range is predicted to expand northward with climate change. Foundation species are commonly prioritized for conservation, with the goal of preserving the entire community that depends on them. However, no studies have explicitly investigated whether mangrove-dependent species' ranges will track the northward expansion of New World mangrove forests. We use the mangrove rivulus fish, Kryptolebias marmoratus, to investigate shifts in habitat suitability in response to various climate change scenarios (Representative Concentration Pathways 2.6, 4.5, 6.0, and 8.5). Niche models for coastal species focus on traditional climatic variables (e.g., precipitation, temperature) even though coastal habitats also are directly influenced by marine variables (e.g., sea surface salinity). We employ a novel data integration method that combines marine and climatic variables, and that accounts for model selection uncertainty using model averaging to provide robust estimates of habitat suitability. Contrary to expectation, suitability of rivulus habitat is predicted to increase in the south and decrease or remain unchanged in the north across all climate change scenarios. Thus, rivulus might experience range contraction, not expansion. Habitat became more suitable with increased salinity of the saltiest month and precipitation of the driest quarter. In laboratory settings, rivulus have higher survival, reproductive success, and growth rates in low salinities. This discrepancy suggests that some combination of the responses of rivulus and its competitors to environmental change will restrict rivulus to habitats that laboratory experiments consider suboptimal. Our models suggest that focusing conservation decisions on foundation species could overestimate habitat availability and resilience of affiliated communities while simultaneously underestimating species declines and extinction risks.

Effects of crowding stress on the hypothalamo-pituitary-interrenal axis of the self-fertilizing fish, Kryptolebias marmoratus

We tested whether crowding stress affects the hypothalamo-pituitary-interrenal (HPI) axis of the self-fertilizing fish, Kryptolebias marmoratus, which is known to be aggressive in the laboratory conditions but sometimes found as a group from a single land crab burrow in the wild. The projection of corticotropin-releasing hormone (CRH) neurons to the adrenocorticotropic hormone (ACTH) cells in the pituitary was confirmed by dual-label immunohistochemistry; CRH-immunoreactive (ir) fibers originating from cell bodies located in the lateral tuberal nucleus (NLT) of the hypothalamus were observed to project to ACTH-ir cells in the rostral pars distalis of the pituitary. Then, fish were reared solitary or in pairs for 14 days, and the number of CRH-ir cell bodies in the NLT of the hypothalamus and cortisol levels in the body without head region were compared. The number of CRH-ir cell bodies and cortisol levels were significantly higher in paired fish. These results indicate that crowding stress affects the HPI axis in K. marmoratus which thrive in small burrows with limited water volume.

Against the Odds: Hybrid Zones between Mangrove Killifish Species with Different Mating Systems.

Different mating systems are expected to affect the extent and direction of hybridization. Due to the different levels of sexual conflict, the weak inbreeder/strong outbreeder (WISO) hypothesis predicts that gametes from self-incompatible (SI) species should outcompete gametes from self-compatible (SC) ones. However, other factors such as timing of selfing and unilateral incompatibilities may also play a role on the direction of hybridization. In addition, differential mating opportunities provided by different mating systems are also expected to affect the direction of introgression in hybrid zones involving outcrossers and selfers. Here, we explored these hypotheses with a unique case of recent hybridization between two mangrove killifish species with different mating systems, Kryptolebias ocellatus (obligately outcrossing) and K. hermaphroditus (predominantly self-fertilizing) in two hybrid zones in southeast Brazil. Hybridization rates were relatively high (~20%), representing the first example of natural hybridization between species with different mating systems in vertebrates. All F1 individuals were sired by the selfing species. Backcrossing was small, but mostly asymmetrical with the SI parental species, suggesting pattern commonly observed in plant hybrid zones with different mating systems. Our findings shed light on how contrasting mating systems may affect the direction and extent of gene flow between sympatric species, ultimately affecting the evolution and maintenance of hybrid zones.

Seeing in the swamp: hydrogen sulfide inhibits eye metabolism and visual acuity in a sulfide-tolerant fish.

In fish, vision may be impaired when eye tissue is in direct contact with environmental conditions that limit aerobic ATP production. We hypothesized that the visual acuity of fishes exposed to hydrogen sulfide (H2S)-rich water would be altered owing to changes in cytochrome c oxidase (COX) activity. Using the H2S-tolerant mangrove rivulus (Kryptolebias marmoratus), we showed that a 10 min exposure to greater than or equal to 200 µM of H2S impaired visual acuity and COX activity in the eye. Visual acuity and COX activity were restored in fish allowed to recover in H2S-free water for up to 1 h. Since K. marmoratus are found in mangrove pools with H2S concentrations exceeding 1000 µM, visual impairment may impact predator avoidance, navigation and foraging behaviour in the wild.

Filling the gaps: phylogeography of the self-fertilizing Kryptolebias species (Cyprinodontiformes: Rivulidae) along South American mangroves.

Mangrove killifishes of the genus Kryptolebias have been historically classified as rare because of their small size and cryptic nature. Major gaps in distribution knowledge across mangrove areas, particularly in South America, challenge the understanding of the taxonomic status, biogeographical patterns and genetic structuring of the lineages composing the self-fertilizing "Kryptolebias marmoratus species complex." In this study, the authors combined a literature survey, fieldwork and molecular data to fill major gaps of information about the distribution of mangrove killifishes across western Atlantic mangroves. They found that selfing mangrove killifishes are ubiquitously distributed across the Caribbean, Central and South American mangroves and report 14 new locations in South America, extending the range of both the "Central clade" and "Southern clade" lineages which overlap in the Amazon. Although substantial genetic differences were found between clades, the authors also found further genetic structuring within clades, with populations in Central America, north and northeast Brazil generally showing higher levels of genetic diversity compared to the clonal ones in southeast Brazil. The authors discuss the taxonomic status and update the geographical distribution of the Central and Southern clades, as well as potential dispersal routes and biogeographical barriers influencing the distribution of the selfing mangrove killifishes in the western Atlantic mangroves.

Does leaving water make fish smarter? Terrestrial exposure and exercise improve spatial learning in an amphibious fish.

Amphibious fishes transition between aquatic and terrestrial habitats, and must therefore learn to navigate two dramatically different environments. We used the amphibious killifish Kryptolebias marmoratus to test the hypothesis that the spatial learning ability of amphibious fishes would be altered by exposure to terrestrial environments because of neural plasticity in the brain region linked to spatial cognition (dorsolateral pallium). We subjected fish to eight weeks of fluctuating air-water conditions or terrestrial exercise before assessing spatial learning using a bifurcating T-maze, and neurogenesis in the dorsolateral pallium by immunostaining for proliferating cell nuclear antigen. In support of our hypothesis, we found that air-water fluctuations and terrestrial exercise improved some markers of spatial learning. Moreover, air-water and exercised fish had 39% and 46% more proliferating cells in their dorsolateral pallium relative to control fish, respectively. Overall, our findings suggest that fish with more terrestrial tendencies may have a cognitive advantage over those that remain in water, which ultimately may influence their fitness in both aquatic and terrestrial settings. More broadly, understanding the factors that promote neural and behavioural plasticity in extant amphibious fishes may provide insights into how ancestral fishes successfully colonized novel terrestrial environments before giving rise to land-dwelling tetrapods.

Genome-wide DNA methylation of the liver reveals delayed effects of early-life exposure to 17-α-ethinylestradiol in the self-fertilizing mangrove rivulus

ABSTRACT Organisms exposed to endocrine disruptors in early life can show altered phenotype later in adulthood. Although the mechanisms underlying these long-term effects remain poorly understood, an increasing body of evidence points towards the potential role of epigenetic processes. In the present study, we exposed hatchlings of an isogenic lineage of the self-fertilizing fish mangrove rivulus for 28 days to 4 and 120 ng/L of 17-α-ethinylestradiol. After a recovery period of 140 days, reduced representation bisulphite sequencing (RRBS) was performed on the liver in order to assess the hepatic genome-wide methylation landscape. Across all treatment comparisons, a total of 146 differentially methylated fragments (DMFs) were reported, mostly for the group exposed to 4 ng/L, suggesting a non-monotonic effect of EE2 exposure. Gene ontology analysis revealed networks involved in lipid metabolism, cellular processes, connective tissue function, molecular transport and inflammation. The highest effect was reported for nipped-B-like protein B (NIPBL) promoter region after exposure to 4 ng/L EE2 (+ 21.9%), suggesting that NIPBL could be an important regulator for long-term effects of EE2. Our results also suggest a significant role of DNA methylation in intergenic regions and potentially in transposable elements. These results support the ability of early exposure to endocrine disruptors of inducing epigenetic alterations during adulthood, providing plausible mechanistic explanations for long-term phenotypic alteration. Additionally, this work demonstrates the usefulness of isogenic lineages of the self-fertilizing mangrove rivulus to better understand the biological significance of long-term alterations of DNA methylation by diminishing the confounding factor of genetic variability.

Mitochondrial physiology and responses to elevated hydrogen sulphide in two isogenic lineages of an amphibious mangrove fish

Hydrogen sulphide (H2S) is toxic and can act as a selective pressure on aquatic organisms, facilitating a wide range of adaptations for life in sulphidic environments. Mangrove rivulus (Kryptolebias marmoratus) inhabit mangrove swamps and have developed high tolerance to environmental H2S. They are hermaphroditic and can self-fertilize, producing distinct isogenic lineages with different sensitivity to H2S. Here, we tested the hypothesis that observed differences in responses to H2S are the result of differences in mitochondrial functions. For this purpose, we performed two experimental series, testing (1) the overall mitochondrial oxidizing capacities and (2) the kinetics of apparent H2S mitochondrial oxidation and inhibition in two distinct lineages of mangrove rivulus, originally collected from Belize and Honduras. We used permeabilized livers from both lineages, measured mitochondrial oxidation, and monitored changes during gradual increases of sulphide. Ultimately, we determined that each lineage has a distinct strategy for coping with elevated H2S, indicating divergences in mitochondrial function and metabolism. The Honduras lineage has higher anaerobic capacity substantiated by higher lactate dehydrogenase activity and higher apparent H2S oxidation rates, likely enabling them to tolerate H2S by escaping aquatic H2S in a terrestrial environment. However, Belize fish have increased cytochrome c oxidase and citrate synthase activities as well as increased succinate contribution to mitochondrial respiration, allowing them to tolerate higher levels of aquatic H2S without inhibition of mitochondrial oxygen consumption. Our study reveals distinct physiological strategies in genetic lineages of a single species, indicating possible genetic and/or functional adaptations to sulphidic environments at the mitochondrial level.

Miraculous skin metamorphosis for beached amphibious fish

Taking those first steps on land after emerging from the primordial swamp required a lot more than simply changing the way the earliest pioneers moved and breathed. The ancient innovators must also have sealed their skins to prevent water loss in the relatively dry surroundings. They may even have repurposed their external layer to absorb oxygen when their gills collapsed. Modern amphibious mangrove rivulus (Kryptolebias marmoratus) probably reprise this epic transformation each time they flip in the fin prints of their ancestors on land. So how do these modern-day descendants of the earliest land colonists pull off the feat each time they emerge? An international team of researchers from China, Canada, Germany and the USA, led by Andrew Whitehead from the University of California Davis (UCD), USA, and Patricia Wright from the University of Guelph, Canada, investigated the skin of beached mangrove rivulus to find out how the extraordinary creatures carry off this miraculous metamorphosis.As mangrove rivulus thrive on the eastern shore of America, from Florida to Brazil, the team decided to compare the skins of a population from Honduras, which makes its home in brackish mangrove swamps, with another population that frequents freshwater ponds in Belize. The intrepid creatures routinely make their homes above the waterline, so Tessa Blanchard, from the University of Guelph, Canada, made sure that the fish were comfortably ensconced in individual moist containers for 1, 6, 24, 72 and 168 h before collecting portions of their skin. Scott Kelly, from York University, Canada, then scrutinised the samples and the team was amazed to see dramatic changes – even within an hour of leaving the water. The fish restructured the gaps between the skin cells that solvents, ions and other compounds usually pass through to make the skin more robust, and Kelly saw large numbers of blood vessels near the surface of the beached fish's skin, which pick up oxygen from the air and transport it quickly to the cells that balance the salts in the fish's bodies.Yunwei Dong, from Ocean University of China, also analysed the gene expression patterns in the fish's skin during a sabbatical in the Whitehead lab, and discovered that the animals reduced the expression of genes that control the amount of collagen in their skin, which could help them to absorb oxygen and water, in addition to allowing them to get around on land more easily. The beached fish also altered the expression of genes that help them to absorb oxygen and maintain their delicate internal salt balance. Meanwhile, the skins of the high and dry fish began producing a group of proteins, Rhesus proteins, which are essential for disposing of toxic ammonia – the fish equivalent of urea waste produced by humans – which they usually eject through the gills in water. In addition, the team noticed that the fish increased the gene expression of aquaporins – specialised channel proteins to absorb water from their surroundings – to prevent themselves from drying out. Meanwhile, at the University of Münster, Germany, Angela Noll and Juergen Schmitz compared the genes of the mangrove rivulus with those of non-amphibious fish, and found that several of the key genes that allow the amphibious fish to emerge from water have changed significantly, underlining their importance for life on land.‘When K. marmoratus transitions from water to air, the skin quickly alters its barrier and exchange functions to delicately balance these dual purposes on land’, says Whitehead, who is keen to understand the genetic mutations that could explain why some mangrove rivulus populations adjust better to life on land than others.

Evolutionary game in an androdioecious population: Coupling of outcrossing and male production

Androdioecy, the coexistence of hermaphrodites and males, is very rare in vertebrates but occurs in mangrove killifish living in ephemeral or unstable habitats. Hermaphrodites reproduce both by outcrossing with males and by selfing. Outbreeding is advantageous because of inbreeding depression, but it requires encounters with males. The advantages of a propensity for outcrossing among hermaphrodites and the production of males affect each other very strongly. To study the evolutionary coupling of these two aspects, we here analyze a simple evolutionary game for a population composed of three phenotypes: outcrossing-oriented hermaphrodites, selfing-oriented hermaphrodites, and males. Outcrossing-oriented hermaphrodites first attempt to search for males and perform outcrossing if they encounter males. If they fail to encounter males, they reproduce via selfing. Selfing-oriented hermaphrodites simply reproduce by selfing. The replicator dynamics may show bistability, in which both the androdioecious population (with outcrossing-oriented hermaphrodites and males) and the pure hermaphroditic population are locally stable. The model shows the fraction of males is either zero or relatively high (more than 25%), which is not consistent with the observed low fraction of males (less than 5%). To explain this discrepancy, we studied several models including immigration and enforced copulation. We concluded that the observed pattern can be most likely explained by a population dominated by selfing-oriented hermaphrodites receiving immigration of males.

Acclimation to prolonged aquatic hypercarbia or air enhances hemoglobin‑oxygen affinity in an amphibious fish

When the amphibious mangrove rivulus (Kryptolebias marmoratus) leaves water for extended periods, hemoglobin-O2 binding affinity increases. We tested the hypothesis that the change in affinity was a consequence of hemoglobin isoform switching driven by exposure to environments associated with increased internal CO2 levels. We exposed K. marmoratus to either water (control, pH 8.1), air, aquatic hypercarbia (5.1 kPa CO2, pH 6.6–6.8), or aquatic acid (isocarbic control, pH 6.6–6.8), for 7 days, and measured hemoglobin-O2 affinity spectrophotometrically. We found that mangrove rivulus compensated for elevated CO2 and aquatic acid exposure by shifting hemoglobin-O2 affinity back to aquatic (control) levels when measured at an ecologically-relevant high CO2 level that would be experienced in vivo. Using proteomics, we found that the hemoglobin subunits present in the blood did not change between treatments, but air and aquatic acid exposure altered the abundance of cathodic hemoglobin subunits. We therefore conclude that hemoglobin isoform switching is not a primary strategy used by mangrove rivulus to adjust P50 under these conditions. Abundances of other RBC proteins also differed between treatment groups relative to control fish (e.g. Rhesus protein type A, band 3 anion exchanger). Overall, our data indicate that both aquatic hypercarbia and aquatic acidosis create similar changes in hemoglobin-O2 affinity as air exposure. However, the protein-level consequences differ between these groups, indicating that the red blood cell response of mangrove rivulus can be modulated depending on the environmental cue received.

More than meets the eye: syntopic and morphologically similar mangrove killifish species show different mating systems and patterns of genetic structure along the Brazilian coast.

Different mating systems can strongly affect the extent of genetic diversity and population structure among species. Given the increased effects of genetic drift on reduced population size, theory predicts that species undergoing self-fertilisation should have greater population structure than outcrossed species; however, demographic dynamics may affect this scenario. The mangrove killifish clade is composed of the two only known examples of self-fertilising species among vertebrates (Kryptolebias marmoratus and Kryptolebias hermaphroditus). A third species in this clade, Kryptolebias ocellatus, inhabits mangrove forests in southeast Brazil; however, its mating system and patterns of genetic structure have been rarely explored. Here, we examined the genetic structure and phylogeographic patterns of K. ocellatus along its distribution, using mitochondrial DNA and microsatellites to compare its patterns of genetic structure with the predominantly selfing and often-syntopic, K. hermaphroditus. Our results indicate that K. ocellatus reproduces mainly by outcrossing, with no current evidence of selfing, despite being an androdioecious species. Our results also reveal a stronger population subdivision in K. ocellatus compared to K. hermaphroditus, contrary to the theoretical predictions based on reproductive biology of the two species. Our findings indicate that, although morphologically similar, K. ocellatus and K. hermaphroditus had remarkably different evolutionary histories when colonising the same mangrove areas in southeastern Brazil, with other factors (e.g., time of colonisation, dispersal/establishment capacity) having more profound effects on the current population structuring of those species than differences in mating systems.

Hydrogen sulphide sensitivity and tolerance in genetically distinct lineages of a selfing mangrove fish (Kryptolebias marmoratus).

Mangroves are critical marine habitats. High hydrogen sulphide (H2S) is a feature of these important ecosystems and its toxicity creates a challenge for mangrove inhabitants. The mangrove rivulus (Kryptolebias marmoratus) is a selfing, hermaphroditic, amphibious fish that can survive exposure to 1116μM H2S in the wild. These fish rely on cutaneous respiration for gas and ion exchange when emerged. We hypothesized that the skin surface is fundamentally important in H2S tolerance in these mangrove fish by limiting H2S permeability. To test our hypothesis, we first disrupted the skin surface in one isogenic lineage and measured H2S tolerance and sensitivity. We increased water H2S concentration until emersion as a measure of the ability to sense and react to H2S, which we refer to as sensitivity. We then determined H2S tolerance by preventing emersion and increasing H2S until loss of equilibrium (LOE). The H2S concentration at emersion and LOE were significantly affected by disrupting the skin surface, providing support that the skin is involved in limiting H2S permeability. Capitalizing on their unique reproductive strategy, we used three distinct isogenic lineages to test the hypothesis that there would be genetic differences in H2S sensitivity and tolerance. We found significant differences in emersion concentration only among lineages, suggesting a genetic component to H2S sensitivity but not tolerance. Our study also demonstrated that external skin modifications and avoidance behaviours are two distinct strategies used to tolerate ecologically relevant H2S concentrations and likely facilitate survival in challenging mangrove habitats.