Fast satellite DNA evolution in Nothobranchius annual killifishes.

Satellite DNA (satDNA) is a rapidly evolving class of tandem repeats, with some monomers being involved in centromere organization and function. To identify repeats associated with (peri)centromeric regions, we investigated satDNA across Southern and Coastal clades of African annual killifishes of the genus Nothobranchius. Molecular cytogenetic and bioinformatic analyses revealed that two previously identified satellites, designated here as NkadSat01-77 and NfurSat01-348, are associated with (peri)centromeres only in one lineage of the Southern clade. NfurSat01-348 was, however, additionally detected outside centromeres in three members of the Coastal clade. We also identified a novel satDNA, NrubSat01-48, associated with (peri)centromeres in N. foerschi, N. guentheri, and N. rubripinnis. Our findings revealed fast turnover of satDNA associated with (peri)centromeres and different trends in their evolution in two clades of the genus Nothobranchius.

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.

Early-life exposure to methylmercury induces reversible behavioral impairments and gene expression modifications in one isogenic lineage of mangrove rivulus fish Kryptolebias marmoratus.

Methylmercury (MeHg) is a ubiquitous bioaccumulative neurotoxicant present in aquatic ecosystems. It is known to alter behaviors, sensory functions and learning abilities in fish and other vertebrates. Developmental and early-life stages exposure to MeHg can lead to brain damage with immediate consequences on larvae behavior, but may also induce long term effects in adults after a detoxification period. However, very little is known about developmental origin of behavioral impairment in adults due to early exposure to MeHg. The aim of this study is to assess whether early-life MeHg exposure induces immediate and/or delayed effects on behaviors, related genes expression and DNA methylation (one of epigenetic mechanisms). To reach this goal, newly hatched larvae of mangrove rivulus fish, Kryptolebias marmoratus, were exposed to two sub-lethal concentrations of MeHg (90 µg/L and 135 µg/L) for 7 days, and immediate and delayed effects were assessed respectively in 7 dph (days post-hatching) and 90 dph fish. This species naturally produces isogenic lineages due to its self-fertilizing reproduction system, which is unique among vertebrates. It allows to study how environment stressors can influence organism's phenotype while minimizing genetic variability. As results, both MeHg exposures are associated with a decreased foraging efficiency and thigmotaxis, and a dose-dependent reduction in larvae locomotor activity. Regarding molecular analysis in larvae whole bodies, both MeHg exposures induced significant decreased expression of DNMT3a, MAOA, MeCP2 and NIPBL, and significant increase of GSS, but none of those genes underwent methylation changes in targeted CpGs. None of significant behavioral and molecular impairments observed in 7-dph larvae were found in 90-dph adults, which highlight a distinction between immediate and delayed effects of developmental MeHg exposure. Our results suggest implications of aminergic system and its neurotransmitters, redox/methylation trade-off and possibly other epigenetic mechanisms in MeHg neurotoxicity underlying behavioral alterations in rivulus.

Complete mitochondrial genome of the Spanish toothcarp, Aphanius iberus (Valenciennes, 1846) (Actinopterygii, Aphaniidae) and its phylogenetic position within the Cyprinodontiformes order.

BACKGROUND: The Spanish toothcarp (Aphanius iberus Valenciennes, 1846) is a small fish endemic to the eastern coastline of the Iberian Peninsula and is currently listed as "Endangered" (category IUCN: EN). It mainly inhabits brackish waters which can exhibit large fluctuations in temperature and salinity throughout the year. The genetics of A. iberus are not well-known since most studies have only evaluated the genetic structure of the species under a conservation framework in order to identify its potential conservation units. Different phylogenetic relationships of Aphanius have been published based on some particular genes. In the present study, the entire mitochondrial genome of A. iberus was obtained for the first time in the context of an A. iberus reference genome and a hypothesis regarding its phylogenetic position was considered. METHODS AND RESULTS: The mitogenome (a circular doble-stranded DNA sequence of 16,708 bp) was reconstructed and aligned against 83 Cyprinodontiformes and two outgroup taxa to identify the phylogenetic position of A. iberus. PartitionFinder was first used to test for the best evolutionary model and the phylogenetic analyses were performed using two methods: Maximun-Likelihood Approximation (IQ-Tree) and Bayesian inference (MrBayes). Our results show that A. iberus forms a sister group with Orestias ascotanensis, a cyprinodontiform species native to South America. CONCLUSIONS: The results were congruent with the traditional morphometric reconstructed trees and with a geological vicariant hypothesis involving Cyprinodontiformes where Aphaniidae is shown as a monophyletic family separated from the family Cyprinodontidae. The information gathered from this study is not only valuable for improving our understanding of the evolutionary history of A. iberus, but for future genomic studies involving the species.

Reproductive isolating mechanisms contributing to asymmetric hybridization in Killifishes (Fundulus spp.).

When species hybridize, one F1 hybrid cross type often predominates. Such asymmetry can arise from differences in a variety of reproductive barriers, but the relative roles and concordance of pre-mating, post-mating prezygotic, and post-zygotic barriers in producing these biases in natural animal populations have not been widely investigated. Here, we study a population of predominantly F1 hybrids between two killifish species (Fundulus heteroclitus and F. diaphanus) in which >95% of F1 hybrids have F. diaphanus mothers and F. heteroclitus fathers (D♀ × H♂). To determine why F. heteroclitus × F. diaphanus F1 hybrids (H♀ × D♂) are so rare, we tested for asymmetry in pre-mating reproductive barriers (female preference and male aggression) at a common salinity (10 ppt) and post-mating, pre-zygotic (fertilization success) and post-zygotic (embryonic development time and hatching success) reproductive barriers at a range of ecologically relevant salinities (0, 5, 10, and 15 ppt). We found that F. heteroclitus females preferred conspecific males, whereas F. diaphanus females did not, matching the observed cross bias in the wild. Naturally rare H♀ × D♂ crosses also had lower fertilization success than all other cross types, and a lower hatching success than the prevalent D♀ × H♂ crosses at the salinity found in the hybrid zone centre (10 ppt). Furthermore, the naturally predominant D♀ × H♂ crosses had a higher hatching success than F. diaphanus crosses at 10 ppt, which may further increase their relative abundance. The present study suggests that a combination of incomplete mating, post-mating pre-zygotic and post-zygotic reproductive isolating mechanisms act in concert to produce hybrid asymmetry in this system.

Molecular delimitation of the seasonal killifishes of the Hypsolebias antenori species group (Cyprinodontiformes, Rivulidae), with description of two new species from the Caatinga coastal basins, northeastern Brazil.

Hypsolebia antenori is a seasonal killifish considered to be broadly distributed along the Caatingas basins of northeastern Brazil, with records in the coastal drainages of the rio Pacoti, rio Jaguaribe, crrego Virglio and rio Apodi-Mossor basins. Based on morphological characters and molecular phylogenetic reconstructions, it was possible to diagnose two new species and restrict H. antenori to the rio Jaguaribe basin. Hypsolebias gongobira new species is described from a temporary pool in the rio Pacoti basin in Cear State, in syntopy with the species threatened H. longignatus. Hypsolebias bonita new species occurs in the floodplains of the rio Apodi-Mossor basin and in the crrego Virglio microbasin in the Furna Feia National Park, in Rio Grande do Norte State. We also discuss the conservation status of these new species and how the So Francisco Interbasin Water transfer, and the agricultural and urban expansion in the semiarid may be adversely affecting these seasonal killifishes.

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.

Phylogenomics reveals extensive introgression and a case of mito-nuclear discordance in the killifish genus Kryptolebias.

Introgression is a widespread evolutionary process leading to phylogenetic inconsistencies among distinct parts of the genomes, particularly between mitochondrial and nuclear-based phylogenetic reconstructions (e.g., mito-nuclear discordances). Here, we used mtDNA and genome-wide nuclear sites to provide the first phylogenomic-based hypothesis on the evolutionary relationships within the killifish genus Kryptolebias. In addition, we tested for evidence of past introgression in the genus given the multiple reports of undergoing hybridization between its members. Our mtDNA phylogeny generally agreed with the relationships previously proposed for the genus. However, our reconstruction based on nuclear DNA revealed an unknown lineage - Kryptolebias sp. 'ESP' - as the sister group of the self-fertilizing mangrove killifishes, K. marmoratus and K. hermaphroditus. All individuals sequenced of Kryptolebias sp. 'ESP' had the same mtDNA haplotype commonly observed in K. hermaphroditus, demonstrating a clear case of mito-nuclear discordance. Our analysis further confirmed extensive history of introgression between Kryptolebias sp. 'ESP' and K. hermaphroditus. Population genomics analyses indicate no current gene flow between the two lineages, despite their current sympatry and history of introgression. We also confirmed introgression between other species pairs in the genus that have been recently reported to form hybrid zones. Overall, our study provides a phylogenomic reconstruction covering most of the Kryptolebias species, reveals a new lineage hidden in a case of mito-nuclear discordance, and provides evidence of multiple events of ancestral introgression in the genus. These findings underscore the importance of investigating different genomic information in a phylogenetic framework, particularly in taxa where introgression is common as in the sexually diverse mangrove killifishes.

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.

Investigating the utility of Anchored Hybrid Enrichment data to investigate the relationships among the Killifishes (Actinopterygii: Cyprinodontiformes), a globally distributed group of fishes.

The Killifishes (Cyprinodontiformes) are a diverse and well-known group of fishes that contains sixteen families inclusive of Anablepidae, Aphaniidae Aplocheilidae, Cubanichthyidae, Cyprinodontidae, Fluviphylacidae, Fundulidae, Goodeidae, Nothobranchiidae, Orestiidae, Pantanodontidae, Poeciliidae, Procatopodidae, Profundulidae, Rivulidae, and Valenciidae and more than 1,200 species that are globally distributed in tropical and temperate, freshwater and estuarine habitats. The evolutionary relationships among the families within the group, based on different molecular and morphological data sets, have remained uncertain. Therefore, the objective of this study was to use a targeted approach, anchored hybrid enrichment, to investigate the phylogenetic relationships among the families within the Cyprindontiformes. This study included more than 100 individuals, representing all sixteen families within the Cyprinodontiformes, including many recently diagnosed families. We recovered an average of 244 loci per individual. These data were submitted to phylogenetic analyses (RaxML and ASTRAL) and although we recovered many of the same relationships as in previous studies of the group, several novel sets of relationships for other families also were recovered. In addition, two well-established clades (Suborders Cyprinodontoidei and Aplocheilodei) were recovered as monophyletic and are in agreement with most previous studies. We also assessed the degree of gene tree discordance in our dataset to evaluate support for alternative topological hypotheses for interfamilial relationships within the Cyprinodontiformes using a variety of different analyses. The results from this study will provide a robust, historical framework needed to investigate a plethora of biogeographic, taxonomic, ecological, and physiological questions for this group of fishes.

Genome sequencing and transcriptomic analysis of the Andean killifish Orestias ascotanensis reveals adaptation to high-altitude aquatic life.

Orestias ascotanensis (Cyprinodontidae) is a teleost pupfish endemic to springs feeding into the Ascotan saltpan in the Chilean Altiplano (3,700 m.a.s.l.) and represents an opportunity to study adaptations to high-altitude aquatic environments. We have de novo assembled the genome of O. ascotanensis at high coverage. Comparative analysis of the O. ascotanensis genome showed an overall process of contraction, including loss of genes related to G-protein signaling, chemotaxis and signal transduction, while there was expansion of gene families associated with microtubule-based movement and protein ubiquitination. We identified 818 genes under positive selection, many of which are involved in DNA repair. Additionally, we identified novel and conserved microRNAs expressed in O. ascotanensis and its closely-related species, Orestias gloriae. Our analysis suggests that positive selection and expansion of genes that preserve genome stability are a potential adaptive mechanism to cope with the increased solar UV radiation to which high-altitude animals are exposed to.

The endangered White Sands pupfish (Cyprinodon tularosa) genome reveals low diversity and heterogenous patterns of differentiation.

The White Sands pupfish (Cyprinodon tularosa), endemic to New Mexico in Southwestern North America, is of conservation concern due in part to invasive species, chemical pollution, and groundwater withdrawal. Herein, we developed a draft reference genome and use it to provide biological insights into the evolution and conservation of C. tularosa. We used our assembly to localize microsatellite markers previously used to demarcate evolutionary significant units (ESU), quantified genomic divergence and transposable element profiles between species, and compared C. tularosa genomic diversity related species. Our de novo assembly of PacBio Sequel II error-corrected reads resulted in a 1.08 Gb draft genome with a contig N50 of 1.4 Mb and 25,260 annotated protein coding genes, including 95% of the expected Actinopterygii conserved complete single-copy orthologues. Many of the C. tularosa microsatellite markers used for conservation assessments fell within, or near, genes and exhibited a pattern of increased heterozygosity near genic areas compared to those in intergenic regions. Nuclear alignments between these two species revealed 193 genes contained in rapidly diverging tracts; transposable element profiles were largely concordant and suggest a shared, rapid expansion of LINE and Gypsy elements. Genome-wide heterozygosity was markedly lower in C. tularosa compared to estimates from other related species, probably because of smaller long-term effective population sizes constrained by their isolated and limited habitat. Overall, these inferences provide new insights into C. tularosa that should help inform future management efforts.

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.

Molecular phylogeny of the Austrolebias/ adloffi/ group (Cyprinodontiformes, Rivulidae), with description of two new endangered and highly endemic species of annual killifishes from the Laguna dos Patos system, southern Brazil.

The Austrolebias adloffi species group encompasses a diverse lineage of annual killifishes that occurs along the Laguna dos Patos/Lagoa Mirim system, in both Brazilian and Uruguayan territories. We herein employ an integrative taxonomy approach to describe two new species of the group, inferring their phylogenetic relationships and evaluating their conservation status. Austrolebias cheffei sp. nov. and Austrolebias lourenciano sp. nov. are herein described from the western portion of the Laguna dos Patos system. Austrolebias cheffei is distinguished from the remaining species of the A. adloffi species group by presenting a yellowish green or yellowish blue dorsal fin, with wide black to dark brown bars extending from the base to the middle portion of the dorsal and anal fins in the males. Austrolebias lourenciano is distinguished from the remaining species of the A. adloffi species group by presenting a yellowish green dorsal fin, with light yellow or light bluish bars forming small triangles, interspersed with small dark brown rows of blotches in the dorsal fin base, and greenish blue anal fin, sometimes with lighter elongated yellowish iridescent blotches, limited to the basal region. According to mitochondrial cytb sequences, both species are reciprocally monophyletic relative to other species of the A. adloffi species group, and present positive barcoding gap values. Interestingly, both new species form a grade that is closely related to Austrolebias aff. minuano 1, an undescribed species that occurs at the opposite margin of the Laguna dos Patos. Among the other evaluated species, A. bagual, A. aff. minuano 1, A. nigrofasciatus, A. pelotapes, A. pongondo, A. arachan, and A. viarius also revealed to be reciprocally monophyletic, whereas A. minuano and A. adloffi revealed to be paraphyletic in regard to A. charrua and A. aff. minuano 2, respectively, and A. nachtigalli is subdivided in two clades, one of which including A. reicherti, which points to the need of a taxonomic review of the group. In addition, we discussed the conservation status of the new species, corrected the type locality of A. pongondo, and provided a dichotomous identification key of the A. adloffi species group.

The influence of hypoxia on the cardiac transcriptomes of two estuarine species - C. variegatus and F. grandis.

Increased nutrient loading has led to eutrophication of coastal shelf waters which has resulted in increased prevalence of persistent hypoxic zones - areas in which the dissolved oxygen content of the water drops below 2 mg/L. The northern Gulf of Mexico, fed primarily by the Mississippi River watershed, undergoes annual establishment of one of the largest hypoxic zones in the world. Exposure to hypoxia can induce physiological impacts in fish cardiac systems that include bradycardia, changes in stroke volume, and altered cardiovascular vessel development. While these impacts have been addressed at the functional level, there is little information regarding the molecular basis for these changes. This study used transcriptomic analysis techniques to interrogate the effects of hypoxia exposure on the developing cardiovascular system in newly hatched larvae of two estuarine species that occupy the same ecological niche - the sheepshead minnow (Cyprinodon variegatus) and the Gulf killifish (Fundulus grandis). Results suggest that while differential gene expression is largely distinct between the two species, downstream impacts on pathways and functional responses such as reduced cardiac hypertrophy, modulation of blood pressure, and increased incidence of apoptosis appear to be conserved. Further, differences in the magnitude of these conserved responses may suggest that the length of embryonic development could impart a level of resiliency to hypoxic perturbation in early life stage fish.

Genomic fingerprints of palaeogeographic history: The tempo and mode of rift tectonics across tropical Africa has shaped the diversification of the killifish genus Nothobranchius (Teleostei: Cyprinodontiformes).

This paper reports a phylogeny of the African killifishes (Genus Nothobranchius, Order Cyprinodontiformes) informed by five genetic markers (three nuclear, two mitochondrial) of 80 taxa (seven undescribed and 73 of the 92 recognized species). These short-lived annual fishes occupy seasonally wet habitats in central and eastern Africa, and their distribution coincides largely with the East African Rift System (EARS). The fossil dates of sister clades used to constrain a chronometric tree of all sampled Nothobranchius recovered the origin of the genus at ~13.27 Mya. It was followed by the radiations of six principal clades through the Neogene. An ancestral area estimation tested competing biogeographical hypotheses to constrain the ancestral origin of the genus to the Nilo-Sudan Ecoregion, which seeded a mid-Miocene dispersal event into the Coastal ecoregion, followed closely (~10 Mya) by dispersals southward across the Mozambique coastal plain into the Limpopo Ecoregion. Extending westwards across the Tanzanian plateau, a pulse of radiations through the Pliocene were associated with dispersals and fragmentation of wetlands across the Kalahari and Uganda Ecoregions. We interpret this congruence of drainage rearrangements with dispersals and cladogenic events of Nothobranchius to reflect congruent responses to recurrent uplift and rifting. The coevolution of these freshwater fishes and wetlands is attributed to ultimate control by tectonics, as the EARS extended southwards during the Neogene. Geobiological consilience of the combined evidence supports a tectonic hypothesis for the evolution of Nothobranchius.

Few Fixed Variants between Trophic Specialist Pupfish Species Reveal Candidate Cis-Regulatory Alleles Underlying Rapid Craniofacial Divergence.

Investigating closely related species that rapidly evolved divergent feeding morphology is a powerful approach to identify genetic variation underlying variation in complex traits. This can also lead to the discovery of novel candidate genes influencing natural and clinical variation in human craniofacial phenotypes. We combined whole-genome resequencing of 258 individuals with 50 transcriptomes to identify candidate cis-acting genetic variation underlying rapidly evolving craniofacial phenotypes within an adaptive radiation of Cyprinodon pupfishes. This radiation consists of a dietary generalist species and two derived trophic niche specialists-a molluscivore and a scale-eating species. Despite extensive morphological divergence, these species only diverged 10 kya and produce fertile hybrids in the laboratory. Out of 9.3 million genome-wide SNPs and 80,012 structural variants, we found very few alleles fixed between species-only 157 SNPs and 87 deletions. Comparing gene expression across 38 purebred F1 offspring sampled at three early developmental stages, we identified 17 fixed variants within 10 kb of 12 genes that were highly differentially expressed between species. By measuring allele-specific expression in F1 hybrids from multiple crosses, we found that the majority of expression divergence between species was explained by trans-regulatory mechanisms. We also found strong evidence for two cis-regulatory alleles affecting expression divergence of two genes with putative effects on skeletal development (dync2li1 and pycr3). These results suggest that SNPs and structural variants contribute to the evolution of novel traits and highlight the utility of the San Salvador Island pupfish system as an evolutionary model for craniofacial development.

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.