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Theory asserts larger brains facilitate behaviours that enhance fitness. Research has demonstrated that increased brain size improves cognition and survival. However, the majority of research has focused on cross‐species comparisons. Experiments that manipulate selection to investigate the connection between brain size, behaviour and fitness are needed. Trinidadian killifish (Anablepsoides hartii) live in communities with (high predation: HP) and without (killifish only: KO) predators. Predator absence is associated with high population densities, increased intraspecific competition and evolved larger brain sizes. We tested for evolutionary shifts in behaviour by subjecting second‐generation lab‐reared killifish to a mirror aggression assay. We also quantify selection on brain size and behaviour by transplanting wild HP killifish to KO sites and tracking individual fitness (growth rates) with a mark‐recapture design. Lab‐reared killifish from KO sites—specifically males—exhibited higher levels of aggression than HP killifish. In the transplant experiment, HP killifish exhibited strong increases in aggression following the introduction to KO sites. Increased brain size was correlated with increased growth in transplanted HP killifish, yet there was no association between brain size, aggression and growth. Our results indicate that declines in predation and increased competition favour increases in aggression but further research is needed to determine if and how brain size and behaviour are linked through natural selection. Read the free Plain Language Summary for this article on the Journal blog. [ABSTRACT FROM AUTHOR]

Widely distributed Species in different geological and ecological environments often exhibit great intraspecific diversity. Aphaniops stoliczkanus (Day, 1872) is an endemic species of Middle East occurring over a vast geographic range in different basins. This species has been observed in freshwater, brackish water and hot sulphur springs; therefore, its populations are known to exhibit considerable morphological variation. In the present study, we have investigated the poorly studied populations of A. stoliczkanus of the Hormuz and Makran basins of Iran in terms of otolith morphology and morphometrics. According to the results, these populations can be divided into two distinct groups including east and west of the Strait of Hormuz. Our results also provide evidence that there is a relationship between otolith morphology and the environment, serving as environmental factors that may influence the formation of specific regions in the otolith shape. Despite the remarkable morphological similarities among otoliths in geographically close populations of Charak and Chiruyeh, our data purposed that geographically these populations have different dorsal tip shapes in their otolith. In comparison to the data from other studies, the otoliths of Qeshm Island appear to resemble the overall shape of otolith found in Oman very closely. However, it is believed that Qeshm Island population was separated from the Hormuz basin populations during the Holocene Sea level rise. Also, the outcomes of this study provide insights into the potential role of environmental factors in shaping intraspecific diversity and the migration patterns of this species within the Persian Gulf. [ABSTRACT FROM AUTHOR]

We analysed phylogenetic relationships within a major clade of Cyprinodontiformes (Teleostei) that includes five families of North American killifishes. We used DNA sequences from five genes for 130 species, with four fossil calibrations and three secondary calibrations, to generate a time-calibrated phylogeny. We estimated diversification rates, ancestral areas, and ancestral habitats for each node. Findings were interpreted within a detailed biogeographical synthesis. The results indicate that the clade arose in the Eocene along the Gulf of México coast. The speciation rate was uniform through time, except for acceleration in Cyprinodontidae after ~10.9 Mya. In other families, neither viviparity nor marine-to-freshwater transition was associated with accelerated speciation. Sea-level fluctuations might have created a speciation pump by stimulating cycles of dispersal and vicariance along the coast. Diversification also included many cases of inland immigration from coastal ancestors. For upland lineages, ancient river drainages accord with lineage distributions, including enigmatic disjunctions in Goodeidae and Fundulus. Diversification in uplands occurred via barrier displacement within alluvial or tectonically active landscapes. Killifishes also display high environmental tolerance and persist within harsh, peripheral environments unsuitable for most other fishes. Hence, a combination of clade antiquity, adaptability, dynamic geography, and persistence can explain the living diversity of New World killifishes. [ABSTRACT FROM AUTHOR]

Hypoxia tolerance in aquatic ectotherms involves a suite of behavioral and physiological responses at the organismal, tissue, and cellular levels. The current study evaluated two closely related killifish species (Fundulus heteroclitus, Fundulus majalis) to evaluate responses to acute moderate and acute severe hypoxia. Routine metabolic rate and loss of equilibrium were assessed, followed by analysis in skeletal muscle of markers of oxidative damage to proteins (2,4-DNPH), lipids (4-HNE), and DNA (8-OHdG), hypoxia signaling (HIF1α, HIF2α), cellular energy state (p-AMPK: AMPK), and protein degradation (Ubiquitin, LC3B, Calpain 2, Hsp70). Both species had a similar reduction in metabolic rate at low PO2. However, F. heteroclitus was the more hypoxia-tolerant species based on a lower PO2 at which there was loss of equilibrium, perhaps due in part to a lower oxygen demand at all oxygen tensions. Despite the differences in hypoxia tolerance between the species, skeletal muscle molecular markers were largely insensitive to hypoxia, and there were few differences in responses between the species. Thus, the metabolic depression observed at the whole animal level appears to limit perturbations in skeletal muscle in both species during the hypoxia treatments. [ABSTRACT FROM AUTHOR]

The decision to escalate aggression in a contest is based on the assessment of oneself and the opponent's capabilities, previous experiences and the asymmetries between contenders. Interestingly, there is increasing evidence that color polymorphism may also influence the result in a contest during agonistic encounters. Therefore, this research aimed to analyze, for the first time, the intra-sexual competition of adult males of the Mexican Rivulus Millerichthys robustus, testing the influence of color polymorphic variations during agonistic contests. The results of this study suggest that the aggressiveness between morphotypes is of orange and red over yellow. The orange morph was related to winning the greatest number of encounters, whereas yellow lost the most. The red morpho registered the greatest number of ties. Interestingly, in contests between more aggressive morphs (orange and red), fish tended to modulate and lower aggressiveness levels, possibly to avoid injuries. In contrast, the least aggressive morphotype (yellow) tended to raise its aggressiveness against the most successful morphotype and lower it in matches against its own color, changing to a scheme between contenders with symmetrical aggression levels. These results suggest that differential aggression between morphotypes can influence the evolution of color polymorphism in M. robustus, differentially influencing the fitness of the individuals. [ABSTRACT FROM AUTHOR]

Understanding how sublethal impacts of toxicants affect population‐relevant outcomes for organisms is challenging. We tested the hypotheses that the well‐known sublethal impacts of methylmercury (MeHg) and a polychlorinated biphenyl (PCB126) would have meaningful impacts on cohort growth and survival in yellow perch (Perca flavescens) and Atlantic killifish (Fundulus heteroclitus) populations, that inclusion of model uncertainty is important for understanding the sublethal impacts of toxicants, and that a model organism (zebrafish Danio rerio) is an appropriate substitute for ecologically relevant species (yellow perch, killifish). Our simulations showed that MeHg did not have meaningful impacts on growth or survival in a simulated environment except to increase survival and growth in low mercury exposures in yellow perch and killifish. For PCB126, the high level of exposure resulted in lower survival for killifish only. Uncertainty analyses increased the variability and lowered average survival estimates across all species and toxicants, providing a more conservative estimate of risk. We demonstrate that using a model organism instead of the species of interest does not necessarily give the same results, suggesting that using zebrafish as a surrogate for yellow perch and killifish may not be appropriate for predicting contaminant impacts on larval cohort growth and survival in ecologically relevant species. Our analysis also reinforces the notion that uncertainty analyses are necessary in any modeling assessment of the impacts of toxicants on a population because it provides a more conservative, and arguably realistic, estimate of impact. Environ Toxicol Chem 2024;43:2122–2133. © 2024 SETAC [ABSTRACT FROM AUTHOR]

A new species of Melanorivulus is described from the upper Taquari River drainage, upper Paraguay River basin, in the Western Brazilian Cerrado. This species is a member of the Melanorivulus pictus group, endemic to the central Brazilian plateau and adjacent areas. Melanorivulus terena, new species, is endemic to the Paraguay River basin and is possibly closely related to other species endemic to the upper Paraná River basin, such as M. linearis, M. interruptus and M. egens, by the absence of distinctive dark marks on the humeral region in males. The new species is distinguished from all other species of the M. pictus group by a distal margin of unpaired fins and pelvic fin that is orangish red, and by the number of scales in the transversal series. The possible close relationship between the new species and other species recorded in the Paraná River basin may indicate an ancestral interconnection of the headwaters of the Rio Taquari (Paraguay River basin) with the headwaters of the upper Rio Verde and Rio Pardo (Paraná River basin). [ABSTRACT FROM AUTHOR]

ABSTRACT Hatching dynamics is key to population structure of annual fishes in temporary wetlands. Given the risk of false starts due to unpredictable hydroperiods, annual fish may employ risk‐spreading strategies in their hatching patterns consistent with bet hedging (such as the partial hatching of their egg bank and variable number of hatching cues) after being exposed to floods. However, the extent to which annual fish resort to bet hedging (as well as their potential drivers) over a wet–dry cycle is poorly understood. We assessed the hatching patterns of a South American killifish (Matilebias cyaneus; Cyprinodontiformes: Rivulidae) collected at different hydrological phases of a temporary wetland to test for variation in bet hedging over a wet–dry cycle. We incubated wild eggs of M. cyaneus obtained from male–female pairings separately reared in tanks originating in the ‘early‐wet’, ‘drying’ and ‘late‐wet’ phases of a temporary wetland and examined hatching patterns across a sequence of hydration trials under common‐garden conditions. We found variation in (i) the number of hatched and unhatched eggs and (ii) the number of hatched eggs across hydration trials among clutches from each phase (while controlling for parentage). Our results show that M. cyaneus variably commit to bet‐hedging hatching strategies over a wet–dry cycle, and suggest that the environmental conditions at specific periods of the year and the parental age at which eggs are produced drive annual fish hatching patterns. [ABSTRACT FROM AUTHOR]

The killifish genus Aphaniops consists of nine species distributed in Eastern Africa and the Middle East. However, distinguishing these species from each other based on morphological traits is challenging. Here we investigate the utility of otoliths (sagittae) in distinguishing between A. dispar, A. ginaonis, A. hormuzensis, A. kruppi and A. stoliczkanus. Our approach is based on otoliths from 89 specimens and involves (1) otolith morphometrics, following prior recommendations, (2) shape analysis of otolith contours based on discrete wavelet transformation—a novel method in killifish otolith research—and comparative statistical analyses. Both methods reveal significant interspecific variation in the otolith regions of the rostrum, antirostrum and excisura. While method (1) effectively discriminates most species, method (2) struggles to differentiate A. hormuzensis, A. stoliczkanus and A. kruppi. Additionally, both methods encounter challenges in correctly classifying A. hormuzensis due to the high otolith variability of this species in our sample. Possible factors accounting for their variability are environmental fluctuations at the sampled hot sulphuric spring (Khurgo) and potential introgressive hybridization. We conclude that otolith morphometry is a valuable tool for Aphaniops species identification. Furthermore, we found that the distinctiveness of species‐specific otolith traits increases with the divergence age of the species. [ABSTRACT FROM AUTHOR]

Researchers, managers, and policymakers have historically neglected non-game fishes relative to game fishes, and this oversight has extended to invasive non-game fishes in the United States. One such fish—the Eastern Banded Killifish (Fundulus diaphanus diaphanus Lesueur 1817)—has established and rapidly spread in Lake Michigan and connected waters since 2000. Here, we assess potential drivers of the successful invasion of Eastern Banded Killifish, as well as their potential to disrupt native communities and food webs. Specifically, we compare the trophic niche breadth and diet composition between Eastern Banded Killifish and a native subspecies, Western Banded Killifish (Fundulus diaphanus menona Jordan and Copeland 1877), using stable isotope and gut content metabarcoding analyses. Stable isotope analysis showed that Eastern Banded Killifish had a higher variance in littoral dependence and trophic position than Western Banded Killifish, but both stable isotope and gut content metabarcoding analyses revealed an overlap in the diet composition and trophic position between the subspecies. Eastern Banded Killifish may successfully establish outside its native range due to higher feeding variability than Western Banded Killifish, including in habitats historically unused by the native subspecies, but the trophic niche between these two subspecies was similar overall. This study provides insights into the successful invasion of a potentially overlooked non-game fish—Eastern Banded Killifish—while also comparing stable isotope and gut content metabarcoding analyses for an invasive freshwater fish for the first time. [ABSTRACT FROM AUTHOR]

Intraspecific variation in vertebrate eye size is well known. Ecological factors such as light availability are often correlated with shifts in relative eye size. However, experimental tests of selection on eye size are lacking. Trinidadian killifish (Anablepsoides hartii) are found in sites that differ in predation intensity. Sites that lack predators are characterized by lower light, high killifish densities, low resource availability, and intense competition for food. We previously found that killifish in sites that lack predators have evolved a larger "relative" eye size (eye size corrected for body size) than fish from sites with predators. Here, we used transplant experiments to test how selection operates on eye size when fish that are adapted to sites with predators are translocated into sites where predators are absent. We observed a significant "population × relative eye size" interaction; the relationship between relative eye size and a proxy for fitness (rates of individual growth) was positive in the transplanted fish. The trend was the opposite for resident fish. Such results provide experimental support that larger eyes enhance fitness and are favoured in environments characterized by low light and high competition. Graphical Abstract [ABSTRACT FROM AUTHOR]

Individual predator and prey species exhibit coupled population dynamics in simple laboratory systems and simple natural communities. It is unclear how often such pairwise coupling occurs in more complex communities, in which an individual predator species might feed on several prey species and an individual prey species might be attacked by several predators. To examine this problem, we applied multivariate autoregressive state-space (MARSS) models to 5-year time-series of monthly surveys of a predatory fish, the eastern mosquitofish (Gambusia holbrooki), and its littoral zone prey species, the least killifish (Heterandria formosa), in three locations in north Florida. The MARSS models were consistent with coupled predator–prey dynamics at two of the three locations. In one of these two locations, the estimated densities of the two species displayed classic predator–prey oscillations. In the third location, there was a positive effect of killifish density on mosquitofish density but no detectable effect of mosquitofish density on killifish density. In all three locations, increased submergent vegetation cover was associated with increased prey density but not increased predator density. Eigenvalues analyses for the joint predator–prey dynamics indicated that one of the cyclic locations had more stable dynamics than the other locations. The three different patterns demonstrate that the dynamics of a pairwise predator–prey interaction emerge not only from the characteristics of the prey and the predator, but also those of the habitat and trophic web in which the predator and prey are embedded. [ABSTRACT FROM AUTHOR]

The annual killifish Austrolebias charrua is an endangered species, endemic to the southern region of South America, which inhabits temporary ponds that emerges in the rainy season. The main anthropogenic threat driving the extinction of A. charrua stems from extensive agriculture, primarily due to the widrespread use of glyphosate-based herbicides near their habitats. Annual killifishes have been used as models for ecotoxicological studies but, up to now, there are no studies about reference genes in any Austrolebias species. This represents an obstacle to the use of qPCR-based technologies, the standard method for gene expression quantification. The present study aimed to select and validate potential reference genes for qPCR normalization in the annual killifish Austrolebias charrua considering different tissues, gender and environmental conditions. The candidate reference genes 18 s, actb, gapdh, ef1a, shox, eif3g, and the control gene atp1a1 were evaluated in male and female individuals in three different tissues (brain, liver, and gills) under two experimental conditions (control and acute exposition to Roundup Transorb®). The collected tissues were submitted to RNA extraction, followed by cDNA synthesis, cloning, sequencing, and qPCR. Overall, 18 s was the most stable reference gene, and 18 s and ef1a were the most stable combination. Otherwise, considering all variables, gapdh and shox were the least stable candidate genes. Foremost, suitable reference genes were validated in A. charrua, facilitating accurate mRNA quantification in this species, which might be useful for developing molecular tools of ecotoxicological assessment based on gene expression analysis for environmental monitoring of annual killifish. [ABSTRACT FROM AUTHOR]

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.

Three nominal species of the killifish genus Aplocheilus are reported from the lowlands of Sri Lanka. Two of these, Aplocheilus dayi and Aplocheilus werneri, are considered endemic to the island, whereas Aplocheilus parvus is reported from both Sri Lanka and Peninsular India. Here, based on a collection from 28 locations in Sri Lanka, also including a dataset of Asian Aplocheilus downloaded from GenBank, we present a phylogeny constructed from the mitochondrial cytochrome b (cytb), mitochondrial cytochrome c oxidase subunit 1 (cox1), and nuclear recombination activating protein 1 (rag1), and investigate the interrelationships of the species of Aplocheilus in Sri Lanka. The endemic Sri Lankan aplocheilid clade comprising A. dayi and A. werneri is recovered as the sister group to the clade comprising A. parvus from Sri Lanka and Aplocheilus blockii from Peninsular India. The reciprocal monophyly of A. dayi and A. werneri is not supported in our molecular phylogeny. A. dayi and A. werneri display strong sexual dimorphism, but species‐level differences are subtle, explained mostly by pigmentation patterns. Their phenotypes exhibit a parapatric distribution and may represent locally adapted forms of a single species. Alternatively, the present study does not rule out the possibility that A. dayi and A. werneri may represent an incipient species pair or that they have undergone introgression or hybridization in their contact zones. We provide evidence that the Nilwala‐Gin region of southwestern Sri Lanka may have acted as a drought refugium for these fishes. [ABSTRACT FROM AUTHOR]

Otolith morphology in the toothcarps of the family Aphaniidae is an important source of taxonomic information. However, little is known about the ontogenetic variation in otolith morphology. In this study, the development of otolith morphology in different life stages of Aphaniops kruppi, an endemic toothcarp of the Arabian Peninsula, is described and discussed. The results reveal (i) a significant correlation between standard length and otolith size (length) in larval and early juvenile stages, (ii) clear differences in otolith morphology between larvae/early juveniles and adults, and (iii) a temporal link between the appearance of the sulcus on the otolith's inner face and lifestyles, that is demersal in larvae and early juveniles, and pelagic in adults. Moreover, our new data on otoliths of Ap. kruppi propose that otoliths of larvae or early juveniles of Ap. kruppi can be recognized by adults not only because of their small size but also based on their short and rounded rostrum and antirostrum and shallow, wide excisura. As the above-mentioned characteristics are also found in the otoliths of juveniles and larvae of several other studied aphaniids, it can be concluded that otoliths are not diagnostic at the species level in Ap. kruppi, and this holds for other closely related species. [ABSTRACT FROM AUTHOR]

Water turbidity alters prey detectability and prey selection by a predator. In dimorphic mesopredators, the effect of water turbidity on foraging success may be sex specific, primarily due to sex differences in reproductive allotment, body size and vulnerability to predation. To experimentally test the effect of turbidity on prey consumption with respect to sex, we used turquoise killifish (Nothobranchius furzeri), a small fish from ephemeral savanna pools in southeast Africa that vary widely in turbidity. Large males possess conspicuous nuptial coloration while females are smaller and drab. Vision is assumed to be a fundamental sense for turquoise killifish, despite often living in very turbid water. As mesopredators, killifish regulate the invertebrate community in ephemeral pools. We tested the consumption of bloodworms (benthic and red‐coloured) and glassworms (pelagic and transparent) under clear (<1NTU) and turbid (320 NTU) water conditions. We found that turquoise killifish maintained their overall foraging success irrespective of turbidity. In both the clear and turbid water, the females consumed three times more food than males relative to their body mass. This likely stems from the females' high nutritional demands due to daily reproduction. It also suggests that females are not risk‐aversive in clear water despite their smaller size. Water turbidity affected the type of prey consumed by turquoise killifish and demonstrated its potential to affect the community structure of invertebrate species in ephemeral pools. [ABSTRACT FROM AUTHOR]

Externally laid eggs are often responsive to environmental cues; however, it is unclear how such plasticity evolves. In Trinidad, the killifish (Anablepsoides hartii) is found in communities with and without predators. Here, killifish inhabit shallower, ephemeral habitats in sites with predators. Such shifts may increase the exposure of eggs to air and lead to possible desiccation. We compared egg-hatching plasticity between communities by rearing eggs terrestrially on peat moss or in water. The timing of hatching did not differ between communities when eggs were reared in water. Eggs from sites with predators responded to terrestrial incubation by hatching significantly earlier compared with water-reared eggs. These responses were weaker in sites with no predators. Such divergent trends show that the presence of predators is associated with evolutionary shifts in hatching plasticity. Our results provide evidence for local adaptation in embryonic plasticity at the population scale. [ABSTRACT FROM AUTHOR]

Axis formation in fish and amphibians typically begins with a prepattern of maternal gene products. Annual killifish embryogenesis, however, challenges prepatterning models as blastomeres disperse and then aggregate to form the germ layers and body axes. We show that huluwa, a prepatterning factor thought to break symmetry by stabilizing β-catenin, is truncated and inactive in Nothobranchius furzeri. Nuclear β-catenin is not selectively stabilized on one side of the blastula but accumulates in cells forming the aggregate. Blocking β-catenin activity or Nodal signaling disrupts aggregate formation and germ layer specification. Nodal signaling coordinates cell migration, establishing an early role for this signaling pathway. These results reveal a surprising departure from established mechanisms of axis formation: Huluwa-mediated prepatterning is dispensable, and β-catenin and Nodal regulate morphogenesis. [ABSTRACT FROM AUTHOR]

Hybridisation can be an important driver of evolutionary change, but hybridisation with invasive species can have adverse effects on native biodiversity. While hybridisation has been documented across taxa, there is limited understanding of ecological factors promoting patterns of hybridisation and the spatial distribution of hybrid individuals. We combined the results of ecological niche modelling (ENM) and restriction site‐associated DNA sequencing to test theories of niche conservatism and biotic resistance on the success of invasion, admixture, and extent of introgression between native and non‐native fishes. We related Maxent predictions of habitat suitability based on the native ranges of invasive Eastern Banded Killifish (Fundulus diaphanus diaphanus Lesueur 1817) and native Western Banded Killifish (Fundulus diaphanus menona Jordan and Copeland 1877) to admixture indices of individual Banded Killifish. We found that Eastern Banded Killifish predominated at sites predicted as suitable from their ENM, consistent with niche conservatism. Admixed individuals were more common as Eastern Banded Killifish habitat suitability declined. We also found that Eastern Banded Killifish were most common at sites closest to the presumed source of this invasion, whereas the proportion of admixed individuals increased with distance from the source of invasion. Lastly, we found little evidence that habitat suitability for Western Banded Killifish provides biotic resistance from either displacement by, or admixture with, invasive Eastern Banded Killifish. Our study demonstrates that ENMs can inform conservation‐relevant outcomes between native and invasive taxa while emphasising the importance of protecting isolated Western Banded Killifish populations from invasive conspecifics. [ABSTRACT FROM AUTHOR]

Non-genetic sources of phenotypic variation, such as the epigenome and the microbiome, could be important contributors to adaptive variation for species with low genetic diversity. However, little is known about the complex interaction between these factors and the genetic diversity of the host, particularly in wild populations. Here, we examine the skin microbiome composition of two closely-related mangrove killifish species with different mating systems (self-fertilising and outcrossing) under sympatric and allopatric conditions. This allows us to partition the influence of the genotype and the environment on their microbiome and (previously described) epigenetic profiles. We find the diversity and community composition of the skin microbiome are strongly shaped by the environment and, to a lesser extent, by species-specific influences. Heterozygosity and microbiome alpha diversity, but not epigenetic variation, are associated with the fluctuating asymmetry of traits related to performance (vision) and behaviour (aggression). Our study identifies that a proportion of the epigenetic diversity and microbiome differentiation is unrelated to genetic variation, and we find evidence for an associative relationship between microbiome and epigenetic diversity in these wild populations. This suggests that both mechanisms could potentially contribute to variation in species with low genetic diversity. Species with low genetic diversity have limited capacity to adapt to environmental change. This study finds that the skin microbiome and non-genetic (epigenetic) modifications of the DNA represent additional sources of variation that could help low diversity fish survive environmental challenges. [ABSTRACT FROM AUTHOR]

One of the most unique examples of adaptation to extreme environments is exemplified by diapauses (I, II and III) in annual killifish embryos. We aimed to understand the molecular mechanisms involved in the regulation of these arrests. In this context, we first comprehensively analysed the expression of genes using a transcriptomic approach to distinguish between two developmental trajectories: diapause induction and non-diapause induction. We focused on the process of diapause I induction by comparing the mRNA profiles at the maternal stage with those of diapause I-induced embryos, non-diapause I-induced embryos and embryos that had exited diapause I. Our analysis revealed approximately seven hundred transcripts that were differentially and exclusively expressed upon the induction of diapause I. The Gene Ontology (GO) categories of the gene-specific transcriptional regulator group, RNA metabolism, cytoskeletal protein and scaffold/adaptor proteins exhibited the highest representation among differentially expressed genes (DEGs) and coexpression analysis. Remarkably, the identification of different homeodomain transcription factors, cell cycle regulators and RNA processing/regulators as DEGs suggests that these regulators play important roles in Garcialebias charrua diapause I induction. Consistent with the results of the DEG analysis, the results of the Clust coexpression analysis revealed 5 and 3 patterns of diapause I-induced gene upregulation and downregulation, corresponding to 3939 and 1250 genes, respectively. Comparative expression analysis of genes and signalling pathways reported to contribute to diapauses in other species revealed that insulin/IGF, vitamin D, Wnt, polycomb and heat shock proteins are also involved in diapause I induction in annual killifish. [ABSTRACT FROM AUTHOR]

The maximum rate at which fish can take up oxygen from their environment to fuel aerobic metabolism is an important feature of their physiology and ecology. Methods to quantify maximum oxygen uptake rate (ṀO2), therefore, should reliably and reproducibly estimate the highest possible ṀO2 by an individual or species under a given set of conditions (peak ṀO2). This study determined peak ṀO2 and its repeatability in Gulf killifish, Fundulus grandis, subjected to three methods to elevate metabolism: swimming at increasing water speeds, during recovery after an exhaustive chase, and after ingestion of a large meal. Estimates of peak ṀO2 during swimming and after an exhaustive chase were repeatable across two trials, whereas peak ṀO2 after feeding was not. Peak ṀO2 determined by the three methods was significantly different from one another, being highest during swimming, lowest after an exhaustive chase, and intermediate after feeding. In addition, peak ṀO2 during recovery from an exhaustive chase depended on the length of time of recovery: in nearly 60% of the trials, values within the first hour of the chase were lower than those measured later. A novel and important finding was that an individual's peak ṀO2 was not repeatable when compared across methods. Therefore, the peak ṀO2 estimated for a group of fish, as well as the ranking of individual ṀO2 within that group, depends on the method used to elevate aerobic metabolism. [ABSTRACT FROM AUTHOR]

Behavioural interactions between alien invasive fishes and native fishes is considered one of the drivers of native fish decline. However, there are few experimental studies on their behavioural interactions at the individual level. In this study, we investigated the behavioural interactions between the threatened native killifish Valencia letourneuxi, and the alien invasive Eastern mosquitofish Gambusia holbrooki, thought to induce the killifish's rapid population decline. The aim was to assess aggressive behaviours, disruption in activity, stress levels, boldness, forging efficiency and shelter use by the native fish in the presence of the mosquitofish. Interspecies interactions were assessed in a three-tiered experimental setup, i.e. in an empty arena, in the presence of an artificial cover, and during feeding, using two opponents at each trial. The behaviours recorded and assessed were nips, following, approach, moving/immobile, top/bottom, erratic movement, cover use, feeding and latency to exit. The results showed that the mosquitofish was bolder, followed the native species, spent significantly less time under the cover and consumed food, as opposed to no following by the native species, significantly higher cover use and time immobile, as avoidance behaviours, and zero feeding in the presence of the mosquitofish. More significantly, the native species received direct aggression by the mosquitofish, as opposed to no aggression at all exhibited by it. Future research needs, as well as the conservation implications of our findings are briefly discussed. [ABSTRACT FROM AUTHOR]

Background: Annual or seasonal killifishes (Cyprinodontiformes: Nothobranchiidae) are unique among fish in their ability to enter into developmental arrests (diapauses: DI, DII, and DIII). They have a short lifespan and their embryos are exceptionally tolerant to a variety of environmental stresses. These traits make them a popular model for studying vertebrate diapause, aging, stress tolerance, genome adaptation, and evolution. In such issues, in a comparative evolutionary framework, Fundulopanchax gardneri, a popular aquarium fish from Africa, is commonly used as a representative non‐annual model though its development is not studied in detail and whether it includes diapauses remains uncertain. Results: We described in detail for the first time embryonic development of F. gardneri and revealed it to resemble that in the undoubtedly annual Austrofundulus limnaeus killifish in displaying two developmental depressions. However, if compared with A. limnaeus, these developmental states look like "less intense" versions of DII and DIII rather than true diapauses. Conclusions: To determine whether developmental depressions in F. gardneri represent "true" diapauses or only their functional equivalents, detailed studies of embryonic development of different killifish both annual and non‐annual are needed. Before that, acceptance of F. gardneri as a representative non‐annual fish seems premature. Key Findings: Aplocheloid annual killifish (Cyprinodontiformes) are unique among fish in the ability to arrest development at different embryonic stages and prolong it for months and years. These and other traits make them a popular model in studying of aging, stress tolerance etc. In these studies, in a comparative framework, aplocheloid Fundulopanchax gardneri is usually used as a representative non‐annual fish though its developmental life‐history remains obscure.This study provides detailed description of F. gardneri development from early embryonic stages till sexual maturation with focus on developmental arrests, divergent developmental trajectories, duration of embryonic period, etc.The data provided in this study indicate F. gardneri to display developmental traits characteristic for annual killifish but in the "less expressed" form. Acceptance of F. gardneri as annual vs non‐annual is premature until the detailed study of embryonic development in other killifish both annual and non‐annual. [ABSTRACT FROM AUTHOR]

Kryptolebias marmoratus (Kmar), a teleost fish of the order Cyprinodontiformes, has a suite of unique phenotypes and behaviors not observed in other fishes. Many of these phenotypes are discrete and highly plastic—varying over time within an individual, and in some cases reversible. Kmar and its interfertile sister species, K. hermaphroditus, are the only known self‐fertile vertebrates. This unusual sexual mode has the potential to provide unique insights into the regulation of vertebrate sexual development, and also lends itself to genetics. Kmar is easily adapted to the lab and requires little maintenance. However, its internal fertilization and small clutch size limits its experimental use. To support Kmar as a genetic model, we compared alternative husbandry techniques to maximize recovery of early cleavage‐stage embryos. We find that frequent egg collection enhances yield, and that protease treatment promotes the greatest hatching success. We completed a forward mutagenesis screen and recovered several mutant lines that serve as important tools for genetics in this model. Several will serve as useful viable recessive markers for marking crosses. Importantly, the mutant kissylips lays embryos at twice the rate of wild‐type. Combining frequent egg collection with the kissylips mutant background allows for a substantial enhancement of early embryo yield. These improvements were sufficient to allow experimental analysis of early development and the successful mono‐ and bi‐allelic targeted knockout of an endogenous tyrosinase gene with CRISPR/Cas9 nucleases. Collectively, these tools will facilitate modern developmental genetics in this fascinating fish, leading to future insights into the regulation of plasticity. Research Highlights: A forward genetic screen in the self‐fertile fish, Kryptolebias marmoratus, identified useful mutants for use as markers and to enhance egg output.Optimization of husbandry further improved egg yield for embryo manipulation.New resources enabled targeted gene knockout in K. marmoratus using CRISPR/Cas9. [ABSTRACT FROM AUTHOR]

Small populations with limited range are often threatened by inbreeding and reduced genetic diversity, which can reduce fitness and exacerbate population decline. One of the most extreme natural examples is the Devils Hole pupfish (Cyprinodon diabolis), an iconic and critically endangered species with the smallest known range of any vertebrate. This species has experienced severe declines in population size over the last 30 years and suffered major bottlenecks in 2007 and 2013, when the population shrunk to 38 and 35 individuals, respectively. Here, we analysed 30 resequenced genomes of desert pupfishes from Death Valley, Ash Meadows and surrounding areas to examine the genomic consequences of small population size. We found extremely high levels of inbreeding (FROH = 0.34-0.81) and an increased amount of potentially deleterious genetic variation in the Devils Hole pupfish as compared to other species, including unique, fixed loss-of-function alleles and deletions in genes associated with sperm motility and hypoxia. Additionally, we successfully resequenced a formalin-fixed museum specimen from 1980 and found that the population was already highly inbred prior to recent known bottlenecks. We thus document severe inbreeding and increased mutation load in the Devils Hole pupfish and identify candidate deleterious variants to inform management of this conservation icon.

Simple Summary: The old world killifish Aphanius fasciatus is a typical inhabitant of transition waters along the coasts of the central and eastern Mediterranean Sea, where it lives in environments (salt ponds, estuaries, and coastal lagoons) with large variations in salt, oxygen, and temperature. Therefore, A. fasciatus could be an interesting non-model species to be studied to evaluate the response of the mitochondrial oxidative phosphorylation (OXPHOS) genes to the variations and extremes of environmental factors. In this study, the sequences of three OXPHOS genes were analyzed in six populations of A. fasciatus to detect mutations and sites subject to selection. The results indicate that mutations were detected in two genes of the Greek population. Moreover, positively selected sites were also found. The information we obtained from the mitochondrial DNA sequences of A. fasciatus adds to the growing data on selective pressure acting on mitochondrial DNA. These results should be explored from the perspective of the local adaptation of a species highly tolerant to wide fluctuations of environmental parameters and should be supported using experimental evidence to better understand the interplay between historical climatic events and local adaptation and how each of them contributes to shaping the genetic structure of this species. Mitochondrial oxidative phosphorylation (OXPHOS) genes are a system subject to selection under determined environmental constraints despite a neutral evolution model that has long been hypothesized for the mitochondrial genome. In this study, the sequences of ND1, Cytb, and COI OXPHOS genes were analyzed in six populations of the eurythermal and euryhaline killifish A. fasciatus, to detect non-synonymous mutations leading to amino acid changes and to check whether selection acted on them using tests of recombination and selection. The results indicate a high COI and Cytb gene diversity and a high percentage of private haplotypes in all populations. In the Greek population, non-synonymous nucleotide substitutions were observed in the N-terminal region of COI and Cytb. Positively selected sites were also found. The information we obtained from the mitochondrial DNA sequences of A. fasciatus adds to the growing data on selective pressure acting on mitochondrial DNA in non-model species. These results should be explored from the perspective of the local adaptation of eurythermal and euryhaline species and supported using experimental evidence to better understand the interplay between historical climatic events and local adaptation and how each of them contributes to shaping the genetic structure of this species. [ABSTRACT FROM AUTHOR]

Divergent ecological character displacement (ECD) is the competition-driven divergence in resource use-related phenotypic traits between coexisting species. It is considered one of the primary drivers of ecological diversification and adaptive radiation. We analyzed phenotypic and ecological variation in 2 African annual killifish species of the genus Nothobranchius : N. eggersi and N. melanospilus in sympatry and N. melanospilus in allopatry. Our aim was to test whether allopatric and sympatric populations of N. melanospilus differ morphologically from each other and from N. eggersi and examine whether these differences are consistent with the predictions of ECD. We find that sympatric N. melanospilus differ from allopatric N. melanospilus and differ from N. eggersi more strongly than the latter. Our data satisfy four criteria for demonstrating ECD: Differences in phenotypes between allopatric and sympatric N. melanospilus are greater than expected by chance; the divergence pattern between allopatric and sympatric N. melanospilus results from an evolutionary shift rather than from ecological sorting; morphological differences observed reflect differences in resource use; and, lastly, sites of allopatry and sympatry do not differ in food resource availability or other ecological conditions. Our results suggest that competition is the main driver of the observed divergence between two N. melanospilus populations. [ABSTRACT FROM AUTHOR]

Turquoise killifish (Nothobranchius furzeri) evolved a naturally short lifespan of about six months and exhibit aging hallmarks that affect multiple organs. These hallmarks include protein aggregation, telomere shortening, cellular senescence, and systemic inflammation. Turquoise killifish possess the full spectrum of vertebrate-specific innate and adaptive immune system. However, during their recent evolutionary history, they lost subsets of mucosal-specific antibody isoforms that are present in other teleosts. As they age, the immune system of turquoise killifish undergoes dramatic cellular and systemic changes. These changes involve increased inflammation, reduced antibody diversity, an increased prevalence of pathogenic microbes in the intestine, and extensive DNA damage in immune progenitor cell clusters. Collectively, the wide array of age-related changes occurring in turquoise killifish suggest that, despite an evolutionary separation spanning hundreds of millions of years, teleosts and mammals share common features of immune system aging. Hence, the spontaneous aging observed in the killifish immune system offers an excellent opportunity for discovering fundamental and conserved aspects associated with immune system aging across vertebrates. Additionally, the species' naturally short lifespan of only a few months, along with its experimental accessibility, offers a robust platform for testing interventions to improve age-related dysfunctions in the whole organism and potentially inform the development of immune-based therapies for human aging-related diseases. [ABSTRACT FROM AUTHOR]

Nonnative species may display unique life history traits when established in habitats with distinctive environmental and biotic contexts compared with their native ecosystems. Gulf killifish (Fundulus grandis), native to coastal habitats of the Gulf of Mexico, are established in several inland river systems where they pose a potential threat to native fishes. In the Pecos River, Texas, nonnative Gulf killifish have shown a high rate of piscivory compared with native coastal populations; otherwise, little is known about the ecology of the species in inland systems. We examined reproductive characteristics, size, and age of Gulf killifish in the Pecos River. We found that reproduction takes place approximately between late March and September, with the gonadosomatic index of females showing a large and extended peak in spring and a second minor peak in late August–September. Our age estimations indicate that this population consists mostly of fish <2 years old, similarly to that reported for coastal populations. Total length ranged 11.9–143.4 mm for males and 32.3–162.5 mm for females, indicating a sexually dimorphic size structure with Pecos River individuals reaching larger sizes compared with coastal populations. The relatively large size and piscivorous nature of Gulf killifish, along with tolerance for a wide range of environmental conditions, are attributes in nonnative species hypothesized to promote invasion success and replacement of native species. Las especies no nativas pueden mostrar características ecológicas únicas cuando se establecen en hábitats con contextos ambientales y bióticos distintivos en comparación con sus ecosistemas nativos. Los Gulf killifish (Fundulus grandis), nativos de los hábitats costeros del Golfo de México, se establecen en varios sistemas fluviales interiores donde representan una amenaza potencial para los peces nativos. En el río Pecos, Texas, los Gulf killifish no nativos han mostrado un alto nivel de piscivoría en comparación con las poblaciones costeras nativas; de lo contrario, se sabe poco sobre la ecología de la especie en los sistemas interiores. Examinamos las características reproductivas, el tamaño y la edad de los Gulf killifish en el río Pecos. Encontramos que la reproducción ocurre aproximadamente entre finales de marzo y septiembre, con un índice gonadosomático de hembras que muestra un pico grande y prolongado en primavera y un segundo pico menor a finales de agosto-septiembre. Nuestras estimaciones de edad sugieren que esta población se compone principalmente de peces de menos de dos años, como se ha informado para las poblaciones costeras. La longitud total osciló entre 11,9 y 143,4 mm para los machos y entre 32,3 y 162,5 mm para las hembras, lo que sugiere una estructura de tamaño sexualmente dimórfica con individuos del río Pecos que alcanzan tamaños más grandes en comparación con las poblaciones costeras. El tamaño relativamente grande y el comportamiento piscívoro de los Gulf killifish, junto con la tolerancia a una amplia gama de condiciones ambientales, son atributos de las especies no nativas que se supone que promueven el éxito de la invasión y el reemplazo de las especies nativas. [ABSTRACT FROM AUTHOR]

We describe Argolebias adrianae, a new species of killifish from a small temporary wetland in the Paraná Forest ecoregion with no regular or predictable temporal pattern of water availability. This habitat is in the Lower Iguazú River Basin, known for its high fish endemism, but until now, only two species of Rivulidae were reported from it, but from the Araucarian Forest ecoregion. The genus Argolebias was previously only known from the lower portions of the Paraguay, Paraná, and Uruguay basins and middle Paraná. The new species is distinguished from all congeners by its unique coloration, which includes a conspicuously dark grey anterior third portion of the dorsal fin and the absence of iridescent spots on the basal half of the pectoral fin in live adult males, as well as dark grey spots on the anterocentral portion of the flanks of females. Our phylogenetic analysis shows A. adrianae to be closely related to Argolebias guarani from the adjacent Middle Paraná basin. We also provide data on the ecology, ontogeny of coloration, and chorion ornamentation of this species. Our findings have important implications for understanding the biogeography, ecology, and evolution of mechanisms that enable organisms to thrive in highly stochastic environments like this one. [ABSTRACT FROM AUTHOR]

Specific subpopulations of neurons in nerve and sensory systems must be developed and maintained, and this is accomplished in significant part by neurotrophins (NTs) and the signaling receptors on which they act, called tyrosine protein kinase receptors (Trks). The neurotrophins–tyrosine protein kinase receptors (NTs/Trks) system is involved in sensory organ regulation, including the visual system. An NTs/Trks system alteration is associated with neurodegeneration related to aging and diseases, including retinal pathologies. An emergent model in the field of translational medicine, for instance, in aging study, is the annual killifish belonging to the Nothobranchius genus, thanks to its short lifespan. Members of this genus, such as Nothobranchius guentheri, and humans share a similar retinal stratigraphy. Nevertheless, according to the authors' knowledge, the occurrence and distribution of the NTs/Trks system in the retina of N. guentheri has never been investigated before. Therefore, the present study aimed to localize neurotrophin BDNF, NGF, and NT-3 and TrkA, TrkB, and TrkC receptors in the N. guentheri retina using the immunofluorescence method. The present investigation demonstrates, for the first time, the occurrence of the NTs/Trks system in N. guentheri retina and, consequently, the potential key role of these proteins in the biology and survival of the retinal cells. [ABSTRACT FROM AUTHOR]

Topminnows of the Teleost genus Fundulus serve as model organisms in ecotoxicology because of their broad physiological tolerance and propensity to breed in captivity. This research has been primarily limited to intraspecific comparisons, due to incomplete understanding of the evolutionary history of the genus, which is necessary for use of phylogenetic comparative methods. Interspecific relationships of topminnows remain unresolved, despite recent advances in mitochondrial and nuclear genome sequencing. Specifically, interrelationships of a group containing the starhead topminnows (Fundulus blairae, F. dispar, F. escambiae, F. lineolatus, and F. nottii) typically yield low node support values. Here, we present the first annotated mitochondrial genome of the Starhead Topminnow (F. dispar) and provide a phylogenetic hypothesis for starhead topminnows within the genus Fundulus. DNA was isolated from a specimen of F. dispar collected in Kentucky, USA. The circular genome is 16,564 bp long and contains 13 protein-coding genes, two ribosomal RNAs (rRNA), 22 transfer RNAs (tRNA), and one control region (D-loop). Our phylogenetic analysis supports a sister relationship between F. dispar and a group containing F. notatus and F. olivaceus. This data helps to resolve the phylogenetic placement of starhead topminnows. [ABSTRACT FROM AUTHOR]

Anthropogenic habitat degradation and alien invasive species have led to the rapid decline of freshwater fish biodiversity globally. The knowledge of threatened species' embryogenesis and larval development could be important for the design of appropriate conservation measures to reverse their decline. Here, we describe the embryonic and larval development of the globally threatened Peloponnese Valencia (Valencia robertae) to inform urgently needed ex situ and in situ conservation initiatives, such as safety stock creation, conservation translocation, and population monitoring. The development of V. robertae is described from the embryonic to the juvenile stage from in vivo imaging, for the first time in detail for this species and genus. Valencia robertae's fertilised eggs are large (approximately 2 mm), spherical, macrolecithal, translucent, with negative buoyancy, filaments at the outer surface, and several oil globules. They have a long incubation period (approximately 18 days at 20 ± 1 °C) and, in laboratory conditions, a high hatching rate (84%, n = 89). Various types of chromatophores are visible in the embryo, incl. melanophores, xanthophores, and iridophores at the dorsal area of the eye and at the iris. Embryos at hatching measure approximately 5.4 mm SL (6.5 mm TL) and have well-developed caudal and pectoral fins, large eyes, and well-developed mouth; exogenous feeding starts at 24–48 h post hatching. Sequential formation of fins continues with the development of the anal and dorsal fins and is completed by the formation of the pelvic fins, at approximately 11 mm SL (25–30 days post hatching). The ecological and conservation implications of our findings are discussed. [ABSTRACT FROM AUTHOR]

Understanding the mechanisms of parasite community assembly can be confounded by phylogenetic distance among host species. Addressing this requires focusing on parasite communities within closely related taxa. Thus, we took a macroecological approach to examining parasite community structure within Killifish species in the genus Fundulus to disentangle the effects of host phylogeny and ecological variables. We constructed a database of parasite communities within Fundulus species from 15 published and unpublished surveys covering the Atlantic coast of the US and Canada. The database was expanded by sampling sites in underrepresented provinces and states, totaling 10 Fundulus species from 57 unique geographic sites. Univariate analysis of observed parasite species richness among Fundulus populations in the dataset found that latitude, climate type, and salinity were the dominant factors determining parasite species richness. Multivariate analysis found that host species and landscape type were the most important factors in determining the similarity of parasite assemblages. Unexpectedly, parasite species richness decreased in low latitudes, and host phylogenetic distance was not found to be a significant factor in the similarity of parasite communities. These results indicate that commonly reported large-scale drivers of parasite community structure, such as latitude and phylogeny, could have diminished significance at the host genus level relative to host ecology, biogeography, and local landscape factors. [ABSTRACT FROM AUTHOR]

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.

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.

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.

The African turquoise killifish (Nothobranchius furzeri) has emerged as a popular model organism for neuroscience research in the last decade. One of the reasons for its popularity is its short lifespan for a vertebrate organism. However, little research has been carried out using killifish in behavioral tests, especially looking at changes in their behavior upon aging. Therefore, we used the open field and the novel tank diving test to unravel killifish locomotion, exploration-related behavior, and behavioral changes over their adult lifespan. The characterization of this behavioral baseline is important for future experiments involving pharmacology to improve the aging phenotype. In this study, two cohorts of fish were used, one cohort was tested in the open field test and one cohort was tested in the novel tank diving test. Each cohort was tested from the age of 6 weeks to the age of 24 weeks and measurements were performed every three weeks. In the open field test, we found an increase in the time spent in the center zone from 18 weeks onward, which could indicate altered exploration behavior. However, upon aging, the fish also showed an increased immobility frequency and duration. In addition, after the age of 15 weeks, their locomotion decreased. In the novel tank diving test, we did not observe this aging effect on locomotion or exploration. Killifish spent around 80% of their time in the bottom half of the tank, and we could not observe habituation effects, indicating slow habituation to novel environments. Moreover, we observed that killifish showed homebase behavior in both tests. These homebases are mostly located near the edges of the open field test and at the bottom of the novel tank diving test. Altogether, in the open field test, the largest impact of aging on locomotion and exploration was observed beyond the age of 15 weeks. In the novel tank diving test, no effect of age was found. Therefore, to test the effects of pharmacology on innate behavior, the novel tank diving test is ideally suited because there is no confounding effect of aging. [ABSTRACT FROM AUTHOR]

Sarcopenia, the age‐related decline in muscle function, places a considerable burden on health‐care systems. While the stereotypic hallmarks of sarcopenia are well characterized, their contribution to muscle wasting remains elusive, which is partly due to the limited availability of animal models. Here, we have performed cellular and molecular characterization of skeletal muscle from the African killifish—an extremely short‐lived vertebrate—revealing that while many characteristics deteriorate with increasing age, supporting the use of killifish as a model for sarcopenia research, some features surprisingly reverse to an "early‐life" state in the extremely old stages. This suggests that in extremely old animals, there may be mechanisms that prevent further deterioration of skeletal muscle, contributing to an extension of life span. In line with this, we report a reduction in mortality rates in extremely old killifish. To identify mechanisms for this phenomenon, we used a systems metabolomics approach, which revealed that during aging there is a striking depletion of triglycerides, mimicking a state of calorie restriction. This results in the activation of mitohormesis, increasing Sirt1 levels, which improves lipid metabolism and maintains nutrient homeostasis in extremely old animals. Pharmacological induction of Sirt1 in aged animals was sufficient to induce a late life‐like metabolic profile, supporting its role in life span extension in vertebrate populations that are naturally long‐lived. Collectively, our results demonstrate that killifish are not only a novel model to study the biological processes that govern sarcopenia, but they also provide a unique vertebrate system to dissect the regulation of longevity. [ABSTRACT FROM AUTHOR]

The genetic architecture of phenotypic traits can affect the mode and tempo of trait evolution. Human‐altered environments can impose strong natural selection, where successful evolutionary adaptation requires swift and large phenotypic shifts. In these scenarios, theory predicts that adaptation is due to a few adaptive variants of large effect, but empirical studies that have revealed the genetic architecture of rapidly evolved phenotypes are rare, especially for populations inhabiting polluted environments. Fundulus killifish have repeatedly evolved adaptive resistance to extreme pollution in urban estuaries. Prior studies, including genome scans for signatures of natural selection, have revealed some of the genes and pathways important for evolved pollution resistance, and provide context for the genotype–phenotype association studies reported here. We created multiple quantitative trait locus (QTL) mapping families using progenitors from four different resistant populations, and using RAD‐seq genetically mapped variation in sensitivity (developmental perturbations) following embryonic exposure to a model toxicant PCB‐126. We found that one to two large‐effect QTL loci accounted for resistance to PCB‐mediated developmental toxicity. QTLs harbored candidate genes that govern the regulation of aryl hydrocarbon receptor (AHR) signaling. One QTL locus was shared across all populations and another was shared across three populations. One QTL locus showed strong signatures of recent natural selection in the corresponding wild population but another QTL locus did not. Some candidate genes for PCB resistance inferred from genome scans in wild populations were identified as QTL, but some key candidate genes were not. We conclude that rapidly evolved resistance to the developmental defects normally caused by PCB‐126 is governed by few genes of large effect. However, other aspects of resistance beyond developmental phenotypes may be governed by additional loci, such that comprehensive resistance to PCB‐126, and to the mixtures of chemicals that distinguish urban estuaries more broadly, may be more genetically complex. [ABSTRACT FROM AUTHOR]

Atlantic killifish (Fundulus heteroclitus) is a valuable model in evolutionary toxicology to study how the interactions between genetic and environmental factors serve the adaptive ability of organisms to resist chemical pollution. Killifish populations inhabiting environmental toxicant-contaminated New Bedford Harbor (NBH) show phenotypes tolerant to polychlorinated biphenyls (PCBs) and differences at the transcriptional and genomic levels. However, limited research has explored epigenetic alterations and metabolic effects in NBH killifish. To identify the involvement of epigenetic and metabolic regulation in the adaptive response of killifish, we investigated tissue- and sex-specific differences in global DNA methylation and metabolomic profiles of NBH killifish populations, compared to sensitive populations from a non-polluted site, Scorton Creek (SC). The results revealed that liver-specific global DNA hypomethylation and differential metabolites were evident in fish from NBH compared with those from SC. The sex-specific differences were not greater than the tissue-specific differences. We demonstrated liver-specific enriched metabolic pathways (e.g., amino acid metabolic pathways converged into the urea cycle and glutathione metabolism), suggesting possible crosstalk between differential metabolites and DNA hypomethylation in the livers of NBH killifish. Additional investigation of methylated gene regions is necessary to understand the functional role of DNA hypomethylation in the regulation of enzyme-encoding genes associated with metabolic processes and physiological changes in NBH populations. [ABSTRACT FROM AUTHOR]

Using African annual killifishes of the genus Nothobranchius from temporary savannah pools with rapid karyotype and sex chromosome evolution, we analysed the chromosomal distribution of telomeric (TTAGGG)n repeat and Nfu‐SatC satellite DNA (satDNA; isolated from Nothobranchius furzeri) in 15 species across the Nothobranchius killifish phylogeny, and with Fundulosoma thierryi as an out‐group. Our fluorescence in situ hybridization experiments revealed that all analysed taxa share the presence of Nfu‐SatC repeat but with diverse organization and distribution on chromosomes. Nfu‐SatC landscape was similar in conspecific populations of Nothobranchius guentheri and Nothobranchius melanospilus but slightly‐to‐moderately differed between populations of Nothobranchius pienaari, and between closely related Nothobranchius kuhntae and Nothobranchius orthonotus. Inter‐individual variability in Nfu‐SatC patterns was found in N. orthonotus and Nothobranchius krysanovi. We revealed mostly no sex‐linked patterns of studied repetitive DNA distribution. Only in Nothobranchius brieni, possessing multiple sex chromosomes, Nfu‐SatC repeat occupied a substantial portion of the neo‐Y chromosome, similarly as formerly found in the XY sex chromosome system of turquoise killifish N. furzeri and its sister species Nothobranchius kadleci—representatives not closely related to N. brieni. All studied species further shared patterns of expected telomeric repeats at the ends of all chromosomes and no additional interstitial telomeric sites. In summary, we revealed (i) the presence of conserved satDNA class in Nothobranchius clades (a rare pattern among ray‐finned fishes); (ii) independent trajectories of Nothobranchius sex chromosome differentiation, with recurrent and convergent accumulation of Nfu‐SatC on the Y chromosome in some species; and (iii) genus‐wide shared tendency to loss of telomeric repeats during interchromosomal rearrangements. Collectively, our findings advance our understanding of genome structure, mechanisms of karyotype reshuffling, and sex chromosome differentiation in Nothobranchius killifishes from the genus‐wide perspective. [ABSTRACT FROM AUTHOR]

Melanorivulus rossoi has a restricted distribution and, due to land modifications for agricultural use, may be on the verge of extinction. So, understanding its gametic development and reproductive cycle can expand the knowledge of the species and possible reproductive and/or ecological interventions. Specimens were collected in swamps adjacent to crop areas in Campo Grande, Mato Grosso do Sul, Brazil. Individuals were fixed in Karnovsky's solution, gonadal samples were extracted and submitted to the histological preparation protocol. 221 individuals were analyzed, with the highest abundance of individuals collected from February to September. Larger fish were observed in the last months of the year, especially in November, while the smallest ones were collected in June. For females, all individuals presented ovarian follicles in different periods of maturation and gonadal differentiation. The same was observed for males, with constant proportions of spermatic cells. Thus, for M. rossoi, it has been found that the production of gametic cells in both male and female gonads is constant, characterizing a synchronous cell development in males and females. The data observed for Melanorivulus rossoi compared to M. punctatus, previously studied by our research group, showed that despite the species being related, the gametogenesis data have some differences. [ABSTRACT FROM AUTHOR]

Poeciliids are widely recognized as successful invaders, possessing traits associated with invasion success. Native to Central America and south‐eastern Mexico, the twospot livebearer (Pseudoxiphophorus bimaculatus) is a species recently recognized as invasive in both Central and northern Mexico. Despite its invasive status, limited research exists on its invasion process and the potential threats it poses to native species. In this study, we conducted a comprehensive review of the current knowledge on the twospot livebearer and mapped its current and potential distribution worldwide. The twospot livebearer shares similar traits with other successful invaders within the same family. Notably, it exhibits high fecundity throughout the year and demonstrates resilience to highly polluted and oxygen‐deprived water conditions. This fish serves as a host for several parasites, including generalists, and has been extensively translocated for commercial purposes. Recently, it has also been used for biocontrol within its native range. Apart from existing outside its native range, the twospot livebearer, under current climate conditions and if transported there, could readily colonize biodiversity hotspots in tropical zones worldwide, including the Caribbean Islands, the Horn of Africa, North of Madagascar Island, south‐eastern Brazil, and others located in southern and eastern Asia. Given that this fish is highly plastic and our Species Distribution Model, we consider that all areas with a habitat suitability >0.2 should prevent its arrival and establishment. Our findings underscore the urgent need to recognize this species as a threat to freshwater native topminnows and prevent its introduction and spread. [ABSTRACT FROM AUTHOR]

Channa argus (Northern Snakehead) was first discovered in Pennsylvania and the Delaware River watershed in 2004 in Franklin Delano Roosevelt Park (FDRP), Philadelphia, PA. We monitored this population by boat and/or backpack electrofishing from 2005 to 2021. Our objective was to summarize common population characteristics and compare these with other non-native Northern Snakehead populations. Length-at-age data indicated the FDRP population was slower growing and lacked larger individuals relative to other populations. Data from stomach dissection documented feeding on Anguilla rostrata (American Eel), Lepomis macrochirus (Bluegill), Lepomis gibbosus (Pumpkinseed), Fundulus diaphanus (Banded Killifish), and Micropterus salmoides (Largemouth Bass), suggesting potential impacts to these species. Backpack electrofishing in 2005 and 2008 showed significant decreases in the densities of American Eel and Banded Killifish which may be due to predation by Northern Snakehead. [ABSTRACT FROM AUTHOR]

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.

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.