Limited impact of weathered residues from the Deepwater Horizon oil spill on the gut-microbiome and foraging behavior of sheepshead minnows (Cyprinodon variegatus).

The Deepwater Horizon disaster of April 2010 was the largest oil spill in U.S. history and exerted catastrophic effects on several ecologically important fish species in the Gulf of Mexico (GoM). Within fish, the microbiome plays a key symbiotic role in maintaining host health and aids in acquiring nutrients, supporting immune function, and modulating behavior. The aim of this study was to examine if exposure to weathered oil might produce significant shifts in fish gut-associated microbial communities as determined from taxa and genes known for hydrocarbon degradation, and whether foraging behavior was affected. The gut microbiome (16S rRNA and shotgun metagenomics) of sheepshead minnow (Cyprinodon variegatus) was characterized after fish were exposed to oil in High Energy Water Accommodated Fractions (HEWAF; tPAH = 81.1 ± 12.4 µg/L) for 7 days. A foraging behavioral assay was used to determine feeding efficiency before and after oil exposure. The fish gut microbiome was not significantly altered in alpha or beta diversity. None of the most abundant taxa produced any significant shifts as a result of oil exposure, with only rare taxa showing significant shifts in abundance between treatments. However, several bioindicator taxa known for hydrocarbon degradation were detected in the oil treatment, primarily Sphingomonas and Acinetobacter. Notably, the genus Stenotrophomonas was detected in high abundance in 16S data, which previously was not described as a core member of fish gut microbiomes. Data also demonstrated that behavior was not significantly affected by oil exposure. Potential low bioavailability of the oil may have been a factor in our observation of minor shifts in taxa and no behavioral effects. This study lays a foundation for understanding the microbiome of captive sheepshead minnows and indicates the need for further research to elucidate the responses of the fish gut-microbiome under oil spill conditions.

Jaw size variation is associated with a novel craniofacial function for galanin receptor 2 in an adaptive radiation of pupfishes.

Understanding the genetic basis of novel adaptations in new species is a fundamental question in biology. Here we demonstrate a new role for galr2 in vertebrate craniofacial development using an adaptive radiation of trophic specialist pupfishes endemic to San Salvador Island, Bahamas. We confirmed the loss of a putative Sry transcription factor binding site upstream of galr2 in scale-eating pupfish and found significant spatial differences in galr2 expression among pupfish species in Meckel's cartilage using in situ hybridization chain reaction (HCR). We then experimentally demonstrated a novel role for Galr2 in craniofacial development by exposing embryos to Garl2-inhibiting drugs. Galr2-inhibition reduced Meckel's cartilage length and increased chondrocyte density in both trophic specialists but not in the generalist genetic background. We propose a mechanism for jaw elongation in scale-eaters based on the reduced expression of galr2 due to the loss of a putative Sry binding site. Fewer Galr2 receptors in the scale-eater Meckel's cartilage may result in their enlarged jaw lengths as adults by limiting opportunities for a circulating Galr2 agonist to bind to these receptors during development. Our findings illustrate the growing utility of linking candidate adaptive SNPs in non-model systems with highly divergent phenotypes to novel vertebrate gene functions.

Sex chromosome differentiation via changes in the Y chromosome repeat landscape in African annual killifishes Nothobranchius furzeri and N. kadleci.

Homomorphic sex chromosomes and their turnover are common in teleosts. We investigated the evolution of nascent sex chromosomes in several populations of two sister species of African annual killifishes, Nothobranchius furzeri and N. kadleci, focusing on their under-studied repetitive landscape. We combined bioinformatic analyses of the repeatome with molecular cytogenetic techniques, including comparative genomic hybridization, fluorescence in situ hybridization with satellite sequences, ribosomal RNA genes (rDNA) and bacterial artificial chromosomes (BACs), and immunostaining of SYCP3 and MLH1 proteins to mark lateral elements of synaptonemal complexes and recombination sites, respectively. Both species share the same heteromorphic XY sex chromosome system, which thus evolved prior to their divergence. This was corroborated by sequence analysis of a putative master sex determining (MSD) gene gdf6Y in both species. Based on their divergence, differentiation of the XY sex chromosome pair started approximately 2 million years ago. In all populations, the gdf6Y gene mapped within a region rich in satellite DNA on the Y chromosome long arms. Despite their heteromorphism, X and Y chromosomes mostly pair regularly in meiosis, implying synaptic adjustment. In N. kadleci, Y-linked paracentric inversions like those previously reported in N. furzeri were detected. An inversion involving the MSD gene may suppress occasional recombination in the region, which we otherwise evidenced in the N. furzeri population MZCS-121 of the Limpopo clade lacking this inversion. Y chromosome centromeric repeats were reduced compared with the X chromosome and autosomes, which points to a role of relaxed meiotic drive in shaping the Y chromosome repeat landscape. We speculate that the recombination rate between sex chromosomes was reduced due to heterochiasmy. The observed differences between the repeat accumulations on the X and Y chromosomes probably result from high repeat turnover and may not relate closely to the divergence inferred from earlier SNP analyses.

Minimal overall divergence of the gut microbiome in an adaptive radiation of Cyprinodon pupfishes despite potential adaptive enrichment for scale-eating.

Adaptive radiations offer an excellent opportunity to understand the eco-evolutionary dynamics of gut microbiota and host niche specialization. In a laboratory common garden, we compared the gut microbiota of two novel derived trophic specialist pupfishes, a scale-eater and a molluscivore, to closely related and distant outgroup generalist populations, spanning both rapid trophic evolution within 10 kya and stable generalist diets persisting over 11 Mya. We predicted an adaptive and highly divergent microbiome composition in the trophic specialists reflecting their rapid rates of craniofacial and behavioral diversification. We sequenced 16S rRNA amplicons of gut microbiomes from lab-reared adult pupfishes raised under identical conditions and fed the same high protein diet. In contrast to our predictions, gut microbiota largely reflected phylogenetic distance among species, rather than generalist or specialist life history, in support of phylosymbiosis. However, we did find significant enrichment of Burkholderiaceae bacteria in replicated lab-reared scale-eater populations. These bacteria sometimes digest collagen, the major component of fish scales, supporting an adaptive shift. We also found some enrichment of Rhodobacteraceae and Planctomycetia in lab-reared molluscivore populations, but these bacteria target cellulose. Overall phylogenetic conservation of microbiome composition contrasts with predictions of adaptive radiation theory and observations of rapid diversification in all other trophic traits in these hosts, including craniofacial morphology, foraging behavior, aggression, and gene expression, suggesting that the functional role of these minor shifts in microbiota will be important for understanding the role of the microbiome in trophic diversification.

Hybridization alters the shape of the genotypic fitness landscape, increasing access to novel fitness peaks during adaptive radiation.

Estimating the complex relationship between fitness and genotype or phenotype (i.e. the adaptive landscape) is one of the central goals of evolutionary biology. However, adaptive walks connecting genotypes to organismal fitness, speciation, and novel ecological niches are still poorly understood and processes for surmounting fitness valleys remain controversial. One outstanding system for addressing these connections is a recent adaptive radiation of ecologically and morphologically novel pupfishes (a generalist, molluscivore, and scale-eater) endemic to San Salvador Island, Bahamas. We leveraged whole-genome sequencing of 139 hybrids from two independent field fitness experiments to identify the genomic basis of fitness, estimate genotypic fitness networks, and measure the accessibility of adaptive walks on the fitness landscape. We identified 132 single nucleotide polymorphisms (SNPs) that were significantly associated with fitness in field enclosures. Six out of the 13 regions most strongly associated with fitness contained differentially expressed genes and fixed SNPs between trophic specialists; one gene (mettl21e) was also misexpressed in lab-reared hybrids, suggesting a potential intrinsic genetic incompatibility. We then constructed genotypic fitness networks from adaptive alleles and show that scale-eating specialists are the most isolated of the three species on these networks. Intriguingly, introgressed and de novo variants reduced fitness landscape ruggedness as compared to standing variation, increasing the accessibility of genotypic fitness paths from generalist to specialists. Our results suggest that adaptive introgression and de novo mutations alter the shape of the fitness landscape, providing key connections in adaptive walks circumventing fitness valleys and triggering the evolution of novelty during adaptive radiation.

One of the main drivers of evolution is natural selection, which is when organisms better adapted to their environment are more likely to survive and reproduce. A common metaphor to explain this process is a landscape covered in peaks and valleys: the peaks represent genetic combinations or traits with high evolutionary fitness, while the valleys represent those with low fitness. As a population evolves and its environment changes, it moves among these peaks taking small steps across the landscape. However, there is a limit to how far an organism can travel in one leap. So, what happens when they need to cross a valley of low fitness to get to the next peak? To address this question, Patton et al. studied three young species of pupfish that recently evolved from a common ancestor and co-habit the same environment in the Caribbean. Patton et al. sequenced whole genomes of each new species and used this to build a genotypic fitness landscape, a network linking neighboring genotypes which each have a unique fitness value that was measured during field experiments. This revealed that most of the paths connecting the different species passed through valleys of low fitness. But there were rare, narrow ridges connecting each species. Next, Patton et al. found that new mutations as well as genetic variations that arose from mating with pupfish on other Caribbean islands altered genetic interactions and changed the shape of the fitness landscape. Ultimately, this significantly increased the accessibility of fitness peaks by both adding more ridges and decreasing the lengths of paths, expanding the realm of possible evolutionary outcomes. Understanding how fitness landscapes change during evolution could help to explain where new species come from. Other researchers could apply the same approach to estimate the genotypic fitness landscapes of other species, from bacteria to vertebrates. These networks could be used to visualize the complex fitness landscape that connects all lifeforms on Earth.

We get by with a little help from our friends: shared adaptive variation provides a bridge to novel ecological specialists during adaptive radiation.

Adaptive radiations involve astounding bursts of phenotypic, ecological and species diversity. However, the microevolutionary processes that underlie the origins of these bursts are still poorly understood. We report the discovery of an intermediate C. sp. 'wide-mouth' scale-eating ecomorph in a sympatric radiation of Cyprinodon pupfishes, illuminating the transition from a widespread algae-eating generalist to a novel microendemic scale-eating specialist. We first show that this ecomorph occurs in sympatry with generalist C. variegatus and scale-eating specialist C. desquamator on San Salvador Island, Bahamas, but is genetically differentiated, morphologically distinct and often consumes scales. We then compared the timing of selective sweeps on shared and unique adaptive variants in trophic specialists to characterize their adaptive walk. Shared adaptive regions swept first in both the specialist desquamator and the intermediate 'wide-mouth' ecomorph, followed by unique sweeps of introgressed variation in 'wide-mouth' and de novo variation in desquamator. The two scale-eating populations additionally shared 9% of their hard selective sweeps with the molluscivore C. brontotheroides, despite no single common ancestor among specialists. Our work provides a new microevolutionary framework for investigating how major ecological transitions occur and illustrates how both shared and unique genetic variation can provide a bridge for multiple species to access novel ecological niches.

A vertebrate adaptive radiation is assembled from an ancient and disjunct spatiotemporal landscape.

To investigate the origins and stages of vertebrate adaptive radiation, we reconstructed the spatial and temporal histories of adaptive alleles underlying major phenotypic axes of diversification from the genomes of 202 Caribbean pupfishes. On a single Bahamian island, ancient standing variation from disjunct geographic sources was reassembled into new combinations under strong directional selection for adaptation to the novel trophic niches of scale-eating and molluscivory. We found evidence for two longstanding hypotheses of adaptive radiation: hybrid swarm origins and temporal stages of adaptation. Using a combination of population genomics, transcriptomics, and genome-wide association mapping, we demonstrate that this microendemic adaptive radiation of novel trophic specialists on San Salvador Island, Bahamas experienced twice as much adaptive introgression as generalist populations on neighboring islands and that adaptive divergence occurred in stages. First, standing regulatory variation in genes associated with feeding behavior (prlh, cfap20, and rmi1) were swept to fixation by selection, then standing regulatory variation in genes associated with craniofacial and muscular development (itga5, ext1, cyp26b1, and galr2) and finally the only de novo nonsynonymous substitution in an osteogenic transcription factor and oncogene (twist1) swept to fixation most recently. Our results demonstrate how ancient alleles maintained in distinct environmental refugia can be assembled into new adaptive combinations and provide a framework for reconstructing the spatiotemporal landscape of adaptation and speciation.

Early-life exposure to weathered, unweathered and dispersed oil has persisting effects on ecologically relevant behaviors in sheepshead minnow.

The Deepwater Horizon oil spill released 3.19 million barrels of crude oil into the Gulf of Mexico, making it the largest oil spill in U.S. history. Weathering and the application of dispersants can alter the solubility of compounds within crude oil, thus modifying the acute toxicity of the crude oil to aquatic life. The primary aim of our study was to determine the lasting impact of early-life stage sheepshead minnow (Cyprinodon variegatus variegatus) exposure to weathered, unweathered and dispersed crude oil on prey capture, male aggression, novel object interaction and global DNA methylation. Embryos were exposed from 1 to 10 dpf to water accommodations of crude oil and were raised to adulthood in artificial seawater. Our results suggest exposure to crude oil did not result in lasting impairment of complex behavioral responses of male sheepshead minnow. Exposure to dispersed weathered oil, however, decreased border dwelling in response to a novel object (i.e. decreased anxiety). Principal component analysis revealed that exposure to weathered oil had no overarching effect, but that unweathered crude oil increased variability in exploratory behaviors but decreased variability in anxiety-associated behaviors. Further work is needed to understand the effects of oil exposure on fish behavior and the potential ecological impact of subtle behavioral changes in fishes.

Changes in regeneration-responsive enhancers shape regenerative capacities in vertebrates.

Vertebrates vary in their ability to regenerate, and the genetic mechanisms underlying such disparity remain elusive. Comparative epigenomic profiling and single-cell sequencing of two related teleost fish uncovered species-specific and evolutionarily conserved genomic responses to regeneration. The conserved response revealed several regeneration-responsive enhancers (RREs), including an element upstream to inhibin beta A (inhba), a known effector of vertebrate regeneration. This element activated expression in regenerating transgenic fish, and its genomic deletion perturbed caudal fin regeneration and abrogated cardiac regeneration altogether. The enhancer is present in mammals, shares functionally essential activator protein 1 (AP-1)-binding motifs, and responds to injury, but it cannot rescue regeneration in fish. This work suggests that changes in AP-1-enriched RREs are likely a crucial source of loss of regenerative capacities in vertebrates.

The combined effects of salinity, hypoxia, and oil exposure on survival and gene expression in developing sheepshead minnows, Cyprinodon variegatus.

The 2010 Deepwater Horizon oil spill released approximately 780 million liters of crude oil contaminating coastal habitats from Texas to Florida which are important habitats for many fish species during early life stages. These diverse habitats are also prone to rapid fluctuations in water quality, such as dissolved oxygen concentration and salinity. The consequence of combined exposure to crude oil and suboptimal environmental conditions during early life stage development of fish is still largely unknown. The objective of this project was to investigate the impacts of exposure to crude oil in combination with varying environmental stressors on developing Cyprinodon variegatus survival, growth, and gene expression. Three life stages (embryonic, post-hatch, and post-larval) were exposed to four nominal concentrations (6.25%, 12.5%, 50% and 100% with actual polycyclic aromatic hydrocarbon (PAH) concentrations ranging from 0 to 512 µg/L) of high energy water accommodated fractions (HEWAF) under different oxic (2.0 or >5.0 mg/L) and salinity (10 or 30 ppt) regimes at 30 °C for 48 h. We found that the post-larval developmental stage was the most sensitive to oil toxicity. Median lethal concentrations during the post-larval exposures followed a treatment-dependent pattern with the highest mortality observed under hypoxic-high salinity conditions (64.55 µg/L). Real-time PCR analysis identified down regulation of target genes, encoding cytochrome P450-1α (cyp1a1), erythropoietin (epo), and the aryl hydrocarbon receptor nuclear translocator (arnt1) only when oil exposure occurred under hypoxic-high salinity conditions in treatments with PAH concentrations greater than 226 µg/L. The target genes measured in this experiment are involved in the aryl hydrocarbon receptor signaling pathway which modulates metabolism of PAHs (a major component of crude oil), and the hypoxia inducible 1-α signaling pathway which is responsible for resilience to hypoxic stress, and it is known that disruption of these pathways can lead to an array of acute and chronic effects. Our results indicated that sheepshead minnow are most sensitive to oil exposure during the post-larval developmental stage. Survival data from this age-stage also indicate that oil toxicity response is exacerbated in hypoxic and high salinity environments. The increased mortality observed during the post-larval developmental stage might be attributed to the suppression of the aryl hydrocarbon receptor signaling and the hypoxia inducible 1-α signaling pathways which is evident in by the down-regulated expression of cyp1a1, epo, and arnt1. These findings provide more information about interactions between oil and abiotic factors which enable us to make better assumptions of the ecological impacts of DWH on coastal estuaries.

Hybrid gene misregulation in multiple developing tissues within a recent adaptive radiation of Cyprinodon pupfishes.

Genetic incompatibilities constitute the final stages of reproductive isolation and speciation, but little is known about incompatibilities that occur within recent adaptive radiations among closely related diverging populations. Crossing divergent species to form hybrids can break up coadapted variation, resulting in genetic incompatibilities within developmental networks shaping divergent adaptive traits. We crossed two closely related sympatric Cyprinodon pupfish species-a dietary generalist and a specialized molluscivore-and measured expression levels in their F1 hybrids to identify regulatory variation underlying the novel craniofacial morphology found in this recent microendemic adaptive radiation. We extracted mRNA from eight day old whole-larvae tissue and from craniofacial tissues dissected from 17-20 day old larvae to compare gene expression between a total of seven F1 hybrids and 24 individuals from parental species populations. We found 3.9% of genes differentially expressed between generalists and molluscivores in whole-larvae tissues and 0.6% of genes differentially expressed in craniofacial tissue. We found that 2.1% of genes were misregulated in whole-larvae hybrids whereas 19.1% of genes were misregulated in hybrid craniofacial tissues, after correcting for sequencing biases. We also measured allele specific expression across 15,429 heterozygous sites to identify putative compensatory regulatory mechanisms underlying differential expression between generalists and molluscivores. Together, our results highlight the importance of considering misregulation as an early indicator of genetic incompatibilities in the context of rapidly diverging adaptive radiations and suggests that compensatory regulatory divergence drives hybrid gene misregulation in developing tissues that give rise to novel craniofacial traits.

Evidence for a role of arginine vasotocin receptors in the gill during salinity acclimation by a euryhaline teleost fish.

The nonapeptide arginine vasotocin (AVT) regulates osmotic balance in teleost fishes, but its mechanisms of action are not fully understood. Recently, it was discovered that nonapeptide receptors in teleost fishes are differentiated into two V1a-type, several V2-type, and two isotocin (IT) receptors, but it remains unclear which receptors mediate AVT's effects on gill osmoregulation. Here, we examined the role of nonapeptide receptors in the gill of the euryhaline Amargosa pupfish (Cyprinodon nevadensis amargosae) during osmotic acclimation. Transcripts for the teleost V1a-type receptor v1a2 were upregulated over fourfold in gill 24 h after transferring pupfish from 7.5 ppt to seawater (35 ppt) or hypersaline (55 ppt) conditions and downregulated after transfer to freshwater (0.3 ppt). Gill transcripts for the nonapeptide degradation enzyme leucyl-cystinyl aminopeptidase (LNPEP) also increased in fish acclimating to 35 ppt. To test whether the effects of AVT on the gill might be mediated by a V1a-type receptor, we administered AVT or a V1-type receptor antagonist (Manning compound) intraperitoneally to pupfish before transfer to 0.4 ppt or 35 ppt. Pupfish transferred to 35 ppt exhibited elevated gill mRNA abundance for cystic fibrosis transmembrane conductance regulator (cftr), but that upregulation diminished under V1-receptor inhibition. AVT inhibited the increase in gill Na+/Cl- cotransporter 2 (ncc2) transcript abundance that occurs following transfer to hypoosmotic environments, whereas V1-type receptor antagonism increased ncc2 mRNAs even without a change in salinity. These findings indicate that AVT acts via a V1-type receptor to regulate gill Cl- transport by inhibiting Cl- uptake and facilitating Cl- secretion during seawater acclimation.

Dynamics of Gene Expression Responses for Ion Transport Proteins and Aquaporins in the Gill of a Euryhaline Pupfish during Freshwater and High-Salinity Acclimation.

Pupfishes (genus Cyprinodon) evolved some of the broadest salinity tolerances of teleost fishes, with some taxa surviving in conditions from freshwater to nearly 160 ppt. In this study, we examined transcriptional dynamics of ion transporters and aquaporins in the gill of the desert Amargosa pupfish (Cyprinodon nevadensis amargosae) during rapid salinity change. Pupfish acclimated to 7.5 ppt were exposed to freshwater (0.3 ppt), seawater (35 ppt), or hypersaline (55 ppt) conditions over 4 h and sampled at these salinities over 14 d. Plasma osmolality and Cl- concentration became elevated 8 h after the start of exposure to 35 or 55 ppt but returned to baseline levels after 14 d. Osmolality recovery was paralleled by increased gill Na+/K+-ATPase activity and higher relative levels of messenger RNAs (mRNAs) encoding cystic fibrosis transmembrane conductance regulator (cftr) and Na+/K+/2Cl- cotransporter-1 (nkcc1). Transcripts encoding one Na+-HCO3- cotransporter-1 isoform (nbce1.1) also increased in the gills at higher salinities, while a second isoform (nbce1.2) increased expression in freshwater. Pupfish in freshwater also had lower osmolality and elevated gill mRNAs for Na+/H+ exchanger isoform-2a (nhe2a) and V-type H+-ATPase within 8 h, followed by increases in Na+/H+ exchanger-3 (nhe3), carbonic anhydrase 2 (ca2), and aquaporin-3 (aqp3) within 1 d. Gill mRNAs for Na+/Cl- cotransporter-2 (ncc2) also were elevated 14 d after exposure to 0.3 ppt. These results offer insights into how coordinated transcriptional responses for ion transporters in the gill facilitate reestablishment of osmotic homeostasis after changes in environmental salinity and provide evidence that the teleost gill expresses two Na+-HCO3- cotransporter-1 isoforms with different roles in freshwater and seawater acclimation.

Morphological and taxonomic descriptions of a new genus and species of killifishes (Teleostei: Cyprinodontiformes) from the high Andes of northern Chile.

A new genus and species, Pseudorestias lirimensis, is described from the southern part of the Chilean Altiplano. While sharing several characters that clearly align the new species with Orestias, this new fish is characterized by numerous autapomorphies: the Meckel cartilage is a continuous cartilage that broadly expands posteriorly (in large specimens, it keeps its anterior part and is resorbed posteriorly), the basibranchials are fused into one long element, the second pharyngobranchial is not displaced dorsally over pharyngobranchial tooth plate 3+4, but they are aligned, the anterior and posterior ceratohyals are closely articulated keeping a scarce amount of cartilage between both bones and ventral to them, ossified middle and distal dorsal radials are present in females as well as ossified middle and distal anal radials. Pseudorestias lirimensis presents strong sexual dimorphism associated to size. Females are almost twice as large and long than males, neuromast lines are absent in males, a mesethmoid is present in males, squamation on head is reduced in males, and ossified middle and distal radial of dorsal fin are cartilaginous in males. Pseudorestias and Orestias are suggested as the sole members of the tribe Orestiini. A list of characters diagnosing the tribe is provided. The presence of the new genus is interpreted as a possible result of the ecosystem isolation where the fish is living from surrounding basins-as early as possibly from the Miocene-Pliocene times-and its physical and chemical characteristics. Small populations, living conditions, small habitat, and reduced distribution make this species a strong candidate to be considered critically endangered, a situation already established for all other Chilean species living in the Altiplano. There is high probability it will become extinct due to water demands and climate change in the region.

Molecular signatures of longevity: Insights from cross-species comparative studies.

Much of the current research on longevity focuses on the aging process within a single species. Several molecular players (e.g. IGF1 and MTOR), pharmacological compounds (e.g. rapamycin and metformin), and dietary approaches (e.g. calorie restriction and methionine restriction) have been shown to be important in regulating and modestly extending lifespan in model organisms. On the other hand, natural lifespan varies much more significantly across species. Within mammals alone, maximum lifespan differs more than 100 fold, but the underlying regulatory mechanisms remain poorly understood. Recent comparative studies are beginning to shed light on the molecular signatures associated with exceptional longevity. These include genome sequencing of microbats, naked mole rat, blind mole rat, bowhead whale and African turquoise killifish, and comparative analyses of gene expression, metabolites, lipids and ions across multiple mammalian species. Together, they point towards several putative strategies for lifespan regulation and cancer resistance, as well as the pathways and metabolites associated with longevity variation. In particular, longevity may be achieved by both lineage-specific adaptations and common mechanisms that apply across the species. Comparing the resulting cross-species molecular signatures with the within-species lifespan extension strategies will improve our understanding of mechanisms of longevity control and provide a starting point for novel and effective interventions.

Adaptive introgression from distant Caribbean islands contributed to the diversification of a microendemic adaptive radiation of trophic specialist pupfishes.

Rapid diversification often involves complex histories of gene flow that leave variable and conflicting signatures of evolutionary relatedness across the genome. Identifying the extent and source of variation in these evolutionary relationships can provide insight into the evolutionary mechanisms involved in rapid radiations. Here we compare the discordant evolutionary relationships associated with species phenotypes across 42 whole genomes from a sympatric adaptive radiation of Cyprinodon pupfishes endemic to San Salvador Island, Bahamas and several outgroup pupfish species in order to understand the rarity of these trophic specialists within the larger radiation of Cyprinodon. 82% of the genome depicts close evolutionary relationships among the San Salvador Island species reflecting their geographic proximity, but the vast majority of variants fixed between specialist species lie in regions with discordant topologies. Top candidate adaptive introgression regions include signatures of selective sweeps and adaptive introgression of genetic variation from a single population in the northwestern Bahamas into each of the specialist species. Hard selective sweeps of genetic variation on San Salvador Island contributed 5 times more to speciation of trophic specialists than adaptive introgression of Caribbean genetic variation; however, four of the 11 introgressed regions came from a single distant island and were associated with the primary axis of oral jaw divergence within the radiation. For example, standing variation in a proto-oncogene (ski) known to have effects on jaw size introgressed into one San Salvador Island specialist from an island 300 km away approximately 10 kya. The complex emerging picture of the origins of adaptive radiation on San Salvador Island indicates that multiple sources of genetic variation contributed to the adaptive phenotypes of novel trophic specialists on the island. Our findings suggest that a suite of factors, including rare adaptive introgression, may be necessary for adaptive radiation in addition to ecological opportunity.

MicroRNA miR-29 controls a compensatory response to limit neuronal iron accumulation during adult life and aging.

BACKGROUND: A widespread modulation of gene expression occurs in the aging brain, but little is known as to the upstream drivers of these changes. MicroRNAs emerged as fine regulators of gene expression in many biological contexts and they are modulated by age. MicroRNAs may therefore be part of the upstream drivers of the global gene expression modulation correlated with aging and aging-related phenotypes. RESULTS: Here, we show that microRNA-29 (miR-29) is induced during aging in short-lived turquoise killifish brain and genetic antagonism of its function induces a gene-expression signature typical of aging. Mechanicistically, we identified Ireb2 (a master gene for intracellular iron delivery that encodes for IRP2 protein), as a novel miR-29 target. MiR-29 is induced by iron loading and, in turn, it reduces IRP2 expression in vivo, therefore limiting intracellular iron delivery in neurons. Genetically modified fish with neuro-specific miR-29 deficiency exhibit increased levels of IRP2 and transferrin receptor, increased iron content, and oxidative stress. CONCLUSIONS: Our results demonstrate that age-dependent miR-29 upregulation is an adaptive mechanism that counteracts the expression of some aging-related phenotypes and its anti-aging activity is primarily exerted by regulating intracellular iron homeostasis limiting excessive iron-exposure in neurons.

Context dependence in complex adaptive landscapes: frequency and trait-dependent selection surfaces within an adaptive radiation of Caribbean pupfishes.

The adaptive landscape provides the foundational bridge between micro- and macroevolution. One well-known caveat to this perspective is that fitness surfaces depend on ecological context, including competitor frequency, traits measured, and resource abundance. However, this view is based largely on intraspecific studies. It is still unknown how context-dependence affects the larger features of peaks and valleys on the landscape which ultimately drive speciation and adaptive radiation. Here, I explore this question using one of the most complex fitness landscapes measured in the wild in a sympatric pupfish radiation endemic to San Salvador Island, Bahamas by tracking survival and growth of laboratory-reared F2 hybrids. I present new analyses of the effects of competitor frequency, dietary isotopes, and trait subsets on this fitness landscape. Contrary to expectations, decreasing competitor frequency increased survival only among very common phenotypes, whereas less common phenotypes rarely survived despite few competitors, suggesting that performance, not competitor frequency, shapes large-scale features of the fitness landscape. Dietary isotopes were weakly correlated with phenotype and growth, but did not explain additional survival variation. Nonlinear fitness surfaces varied substantially among trait subsets, revealing one-, two-, and three-peak landscapes, demonstrating the complexity of selection in the wild, even among similar functional traits.

The African Turquoise Killifish: A Model for Exploring Vertebrate Aging and Diseases in the Fast Lane.

Why and how organisms age remains a mystery, and it defines one of the biggest challenges in biology. Aging is also the primary risk factor for many human pathologies, such as cancer, diabetes, cardiovascular diseases, and neurodegenerative diseases. Thus, manipulating the aging rate and potentially postponing the onset of these devastating diseases could have a tremendous impact on human health. Recent studies, relying primarily on nonvertebrate short-lived model systems, have shown the importance of both genetic and environmental factors in modulating the aging rate. However, relatively little is known about aging in vertebrates or what processes may be unique and specific to these complex organisms. Here we discuss how advances in genomics and genome editing have significantly expanded our ability to probe the aging process in a vertebrate system. We highlight recent findings from a naturally short-lived vertebrate, the African turquoise killifish, which provides an attractive platform for exploring mechanisms underlying vertebrate aging and age-related diseases.

Cloning of circadian rhythmic pathway genes and perturbation of oscillation patterns in endocrine disrupting chemicals (EDCs)-exposed mangrove killifish Kryptolebias marmoratus.

To investigate the effect of endocrine disrupting chemicals (EDCs) on the circadian rhythm pathway, we cloned clock and circadian rhythmic pathway-associated genes (e.g. Per2, Cry1, Cry2, and BMAL1) in the self-fertilizing mangrove killifish Kryptolebias marmoratus. The promoter region of Km-clock had 1 aryl hydrocarbon receptor element (AhRE, GTGCGTGACA) and 8 estrogen receptor (ER) half-sites, indicating that the AhRE and ER half sites would likely be associated with regulation of clock protein activity during EDCs-induced cellular stress. The Km-clock protein domains (bHLH, PAS1, PAS2) were highly conserved in five additional fish species (zebrafish, Japanese medaka, Southern platyfish, Nile tilapia, and spotted green pufferfish), suggesting that the fish clock protein may play an important role in controlling endogenous circadian rhythms. The promoter regions of Km-BMAL1, -Cry1, -Cry2, and -Per2 were found to contain several xenobiotic response elements (XREs), indicating that EDCs may be able to alter the expression of these genes. To analyze the endogenous circadian rhythm in K. marmoratus, we measured expression of Km-clock and other circadian rhythmic genes (e.g. Per2, Cry1, Cry2, and BMAL1) in different tissues, and found ubiquitous expression, although there were different patterns of transcript amplification during different developmental stages. In an estrogen (E2)-exposed group, Km-clock expression was down-regulated, however, a hydroxytamoxifen (TMX, nonsteroid estrogen antagonist)-exposed group showed an upregulated pattern of Km-clock expression, suggesting that the expression of Km-clock is closely associated with exposure to EDCs. In response to the exposure of bisphenol A (BPA) and 4-tert-octyphenol (OP), Km-clock expression was down-regulated in the pituitary/brain, muscle, and skin in both gender types (hermaphrodite and secondary male). In juvenile K. marmoratus liver tissue, expression of Km-clock and other circadian rhythmic pathway-associated genes showed a regular oscillation pattern over a period of approximately 24h during a 12L:12D cycle. However, the circadian rhythm of BPA-exposed juvenile K. marmoratus liver tissue was perturbed over a 12L:12D period. This study will aid in our understanding of how EDCs perturb endogenous circadian rhythms, particularly in BPA-exposed fish liver tissue.