Evolution of a novel regulatory mechanism of hypoxia inducible factor in hypoxia-tolerant electric fishes.

Hypoxia is a significant source of metabolic stress that activates many cellular pathways involved in cellular differentiation, proliferation, and cell death. Hypoxia is also a major component in many human diseases and a known driver of many cancers. Despite the challenges posed by hypoxia, there are animals that display impressive capacity to withstand lethal levels of hypoxia for prolonged periods of time and thus offer a gateway to a more comprehensive understanding of the hypoxic response in vertebrates. The weakly electric fish genus Brachyhypopomus inhabits some of the most challenging aquatic ecosystems in the world, with some species experiencing seasonal anoxia, thus providing a unique system to study the cellular and molecular mechanisms of hypoxia tolerance. In this study, we use closely related species of Brachyhypopomus that display a range of hypoxia tolerances to probe for the underlying molecular mechanisms via hypoxia inducible factors (HIFs)-transcription factors known to coordinate the cellular response to hypoxia in vertebrates. We find that HIF1⍺ from hypoxia tolerant Brachyhypopomus species displays higher transactivation in response to hypoxia than that of intolerant species, when overexpressed in live cells. Moreover, we identified two SUMO-interacting motifs near the oxygen-dependent degradation and transactivation domains of the HIF1⍺ protein that appear to boost transactivation of HIF1, regardless of the genetic background. Together with computational analyses of selection, this shows that evolution of HIF1⍺ are likely to underlie adaptations to hypoxia tolerance in Brachyhypopomus electric fishes, with changes in two SUMO-interacting motifs facilitating the mechanism of this tolerance.

Adaptive Evolution of Nearctic Deepwater Fish Vision: Implications for Assessing Functional Variation for Conservation.

Intraspecific functional variation is critical for adaptation to rapidly changing environments. For visual opsins, functional variation can be characterized in vitro and often reflects a species' ecological niche but is rarely considered in the context of intraspecific variation or the impact of recent environmental changes on species of cultural or commercial significance. Investigation of adaptation in postglacial lakes can provide key insight into how rapid environmental changes impact functional evolution. Here, we report evidence for molecular adaptation in vision in 2 lineages of Nearctic fishes that are deep lake specialists: ciscoes and deepwater sculpin. We found depth-related variation in the dim-light visual pigment rhodopsin that evolved convergently in these 2 lineages. In vitro characterization of spectral sensitivity of the convergent deepwater rhodopsin alleles revealed blue-shifts compared with other more widely distributed alleles. These blue-shifted rhodopsin alleles were only observed in deep clear postglacial lakes with underwater visual environments enriched in blue light. This provides evidence of remarkably rapid and convergent visual adaptation and intraspecific functional variation in rhodopsin. Intraspecific functional variation has important implications for conservation, and these fishes are of conservation concern and great cultural, commercial, and nutritional importance to Indigenous communities. We collaborated with the Saugeen Ojibway Nation to develop and test a metabarcoding approach that we show is efficient and accurate in recovering the ecological distribution of functionally relevant variation in rhodopsin. Our approach bridges experimental analyses of protein function and genetics-based tools used in large-scale surveys to better understand the ecological extent of adaptive functional variation.

Multiple Ecological Axes Drive Molecular Evolution of Cone Opsins in Beloniform Fishes.

Ecological and evolutionary transitions offer an excellent opportunity to examine the molecular basis of adaptation. Fishes of the order Beloniformes include needlefishes, flyingfishes, halfbeaks, and allies, and comprise over 200 species occupying a wide array of habitats-from the marine epipelagic zone to tropical rainforest rivers. These fishes also exhibit a diversity of diets, including piscivory, herbivory, and zooplanktivory. We investigated how diet and habitat affected the molecular evolution of cone opsins, which play a key role in bright light and colour vision and are tightly linked to ecology and life history. We analyzed a targeted-capture dataset to reconstruct the evolutionary history of beloniforms and assemble cone opsin sequences. We implemented codon-based clade models of evolution to examine how molecular evolution was affected by habitat and diet. We found high levels of positive selection in medium- and long-wavelength beloniform opsins, with piscivores showing increased positive selection in medium-wavelength opsins and zooplanktivores showing increased positive selection in long-wavelength opsins. In contrast, short-wavelength opsins showed purifying selection. While marine/freshwater habitat transitions have an effect on opsin molecular evolution, we found that diet plays a more important role. Our study suggests that evolutionary transitions along ecological axes produce complex adaptive interactions that affect patterns of selection on genes that underlie vision.

Ecologically mediated differences in electric organ discharge drive evolution in a sodium channel gene in South American electric fishes.

Active electroreception-the ability to detect objects and communicate with conspecifics via the detection and generation of electric organ discharges (EODs)-has evolved convergently in several fish lineages. South American electric fishes (Gymnotiformes) are a highly species-rich group, possibly in part due to evolution of an electric organ (EO) that can produce diverse EODs. Neofunctionalization of a voltage-gated sodium channel gene accompanied the evolution of electrogenic tissue from muscle and resulted in a novel gene (scn4aa) uniquely expressed in the EO. Here, we investigate the link between variation in scn4aa and differences in EOD waveform. We combine gymnotiform scn4aa sequences encoding the C-terminus of the Nav1.4a protein, with biogeographic data and EOD recordings to test whether physiological transitions among EOD types accompany differential selection pressures on scn4aa. We found positive selection on scn4aa coincided with shifts in EOD types. Species that evolved in the absence of predators, which likely selected for reduced EOD complexity, exhibited increased scn4aa evolutionary rates. We model mutations in the protein that may underlie changes in protein function and discuss our findings in the context of gymnotiform signalling ecology. Together, this work sheds light on the selective forces underpinning major evolutionary transitions in electric signal production.

Evolution of rhodopsin in flatfishes (Pleuronectiformes) is associated with depth and migratory behavior.

Visual signals are involved in many fitness-related tasks and are therefore essential for survival in many species. Aquatic organisms are ideal systems to study visual evolution, as the high diversity of spectral properties in aquatic environments generates great potential for adaptation to different light conditions. Flatfishes are an economically important group, with over 800 described species distributed globally, including halibut, flounder, sole, and turbot. The diversity of flatfish species and wide array of environments they occupy provides an excellent opportunity to understand how this variation translates to molecular adaptation of vision genes. Using models of molecular evolution, we investigated how the light environments inhabited by different flatfish lineages have shaped evolution in the rhodopsin gene, which is responsible for mediating dim-light visual transduction. We found strong evidence for positive selection in rhodopsin, and this was correlated with both migratory behavior and several fundamental aspects of habitat, including depth and freshwater/marine evolutionary transitions. We also identified several mutations that likely affect the wavelength of peak absorbance of rhodopsin, and outline how these shifts in absorbance correlate with the response to the light spectrum present in different habitats. This is the first study of rhodopsin evolution in flatfishes that considers their extensive diversity, and our results highlight how ecologically-driven molecular adaptation has occurred across this group in response to transitions to novel light environments.

Recreated Ancestral Opsin Associated with Marine to Freshwater Croaker Invasion Reveals Kinetic and Spectral Adaptation.

Rhodopsin, the light-sensitive visual pigment expressed in rod photoreceptors, is specialized for vision in dim-light environments. Aquatic environments are particularly challenging for vision due to the spectrally dependent attenuation of light, which can differ greatly in marine and freshwater systems. Among fish lineages that have successfully colonized freshwater habitats from ancestrally marine environments, croakers are known as highly visual benthic predators. In this study, we isolate rhodopsins from a diversity of freshwater and marine croakers and find that strong positive selection in rhodopsin is associated with a marine to freshwater transition in South American croakers. In order to determine if this is accompanied by significant shifts in visual abilities, we resurrected ancestral rhodopsin sequences and tested the experimental properties of ancestral pigments bracketing this transition using in vitro spectroscopic assays. We found the ancestral freshwater croaker rhodopsin is redshifted relative to its marine ancestor, with mutations that recapitulate ancestral amino acid changes along this transitional branch resulting in faster kinetics that are likely to be associated with more rapid dark adaptation. This could be advantageous in freshwater due to the redshifted spectrum and relatively narrow interface and frequent transitions between bright and dim-light environments. This study is the first to experimentally demonstrate that positively selected substitutions in ancestral visual pigments alter protein function to freshwater visual environments following a transition from an ancestrally marine state and provides insight into the molecular mechanisms underlying some of the physiological changes associated with this major habitat transition.

Multi-locus phylogeny with dense Guiana Shield sampling supports new suprageneric classification of the neotropical three-barbeled catfishes (Siluriformes: Heptapteridae).

The catfish family Heptapteridae is ubiquitous across a range of freshwater habitats from southern Mexico to northern Argentina and contains 23 genera and 228 valid species. After a century of mostly morphology-based systematic analyses of these fishes, we provide the first molecular phylogenetic hypothesis spanning most valid Heptapteridae genera (16 of 23). We examined eight of 14 valid genera in the Nemuroglanis-subclade (Heptapterini), all valid genera in the Brachyglanis-subclade (Brachyglaniini) and most valid Brachyglaniini species (11 of 15). Maximum likelihood and Bayesian analyses of a 4156-base alignment of five gene regions (three mitochondrial: COI, Cyt b, and ND2; two nuclear: RAG2, Glyt) yielded thoroughly resolved and statistically robust phylogenies that were largely congruent with each other and with previous morphology-based hypotheses. We propose a revised phylogenetic classification consisting of two subfamilies (Rhamdiinae, Heptapterinae) each with two tribes. Dense taxonomic sampling of Brachyglaniini, including type species of Brachyglanis, Gladioglanis, Leptorhamdia, and Myoglanis, revealed widespread paraphyly. Newly recovered clades within Brachyglaniini are closely associated with either the upper Orinoco or the Essequibo suggesting repeated dispersals and/or range expansions/contractions across the western Guiana Shield highlands and from there to the upper Amazon and Brazilian Shield. These biogeographical processes appear to have been an important driver of allopatric diversification in the clade.

Is Niagara Falls a barrier to gene flow in riverine fishes? A test using genome-wide SNP data from seven native species.

Since the early Holocene, fish population genetics in the Laurentian Great Lakes have been shaped by the dual influences of habitat structure and post-glacial dispersal. Riverscape genetics theory predicts that longitudinal habitat corridors and unidirectional downstream water-flow drive the downstream accumulation of genetic diversity, whereas post-glacial dispersal theory predicts that fish genetic diversity should decrease with increasing distance from glacial refugia. This study examines populations of seven native fish species codistributed above and below the 58 m high Niagara Falls - a hypothesized barrier to gene flow in aquatic species. A better understanding of Niagara Falls' role as a barrier to gene flow and dispersal is needed to identify drivers of Great Lakes genetic diversity and guide strategies to limit exotic species invasions. We used genome-wide SNPs and coalescent models to test whether populations are: (a) genetically distinct, consistent with the Niagara Falls barrier hypothesis; (b) more genetically diverse upstream, consistent with post-glacial expansion theory, or downstream, consistent with the riverscape habitat theory; and (c) have migrated either upstream or downstream past Niagara Falls. We found that genetic diversity is consistently greater below Niagara Falls and the falls are an effective barrier to migration, but two species have probably dispersed upstream past the falls after glacial retreat yet before opening of the Welland Canal. Models restricting migration to after opening of the Welland Canal were generally rejected. These results help explain how river habitat features affect aquatic species' genetic diversity and highlight the need to better understand post-glacial dispersal pathways.

To see or not to see: molecular evolution of the rhodopsin visual pigment in neotropical electric fishes.

Functional variation in rhodopsin, the dim-light-specialized visual pigment, frequently occurs in species inhabiting light-limited environments. Variation in visual function can arise through two processes: relaxation of selection or adaptive evolution improving photon detection in a given environment. Here, we investigate the molecular evolution of rhodopsin in Gymnotiformes, an order of mostly nocturnal South American fishes that evolved sophisticated electrosensory capabilities. Our initial sequencing revealed a mutation associated with visual disease in humans. As these fishes are thought to have poor vision, this would be consistent with a possible sensory trade-off between the visual system and a novel electrosensory system. To investigate this, we surveyed rhodopsin from 147 gymnotiform species, spanning the order, and analysed patterns of molecular evolution. In contrast with our expectation, we detected strong selective constraint in gymnotiform rhodopsin, with rates of non-synonymous to synonymous substitutions lower in gymnotiforms than in other vertebrate lineages. In addition, we found evidence for positive selection on the branch leading to gymnotiforms and on a branch leading to a clade of deep-channel specialized gymnotiform species. We also found evidence that deleterious effects of a human disease-associated substitution are likely to be masked by epistatic substitutions at nearby sites. Our results suggest that rhodopsin remains an important component of the gymnotiform sensory system alongside electrolocation, and that photosensitivity of rhodopsin is well adapted for vision in dim-light environments.

Genetic structure of endangered lake chubsucker Erimyzon sucetta in Canada reveals a differentiated population in a precarious habitat.

We used mitochondrial DNA to assess the genetic structure of endangered lake chubsucker Erimyzon sucetta across its Canadian range. We found unique mitochondrial haplotypes in Lyons Creek, a tributary of the Niagara River that faces a strong potential for habitat deterioration. Lyons Creek may therefore serve as a reservoir of unique genetic diversity. The sensitivity of Lyons Creek, combined with the genetic uniqueness of its E. sucetta population, call for further investigation into whether this population should be considered a separate designatable unit for conservation purposes.

Always chew your food: freshwater stingrays use mastication to process tough insect prey.

Chewing, characterized by shearing jaw motions and high-crowned molar teeth, is considered an evolutionary innovation that spurred dietary diversification and evolutionary radiation of mammals. Complex prey-processing behaviours have been thought to be lacking in fishes and other vertebrates, despite the fact that many of these animals feed on tough prey, like insects or even grasses. We investigated prey capture and processing in the insect-feeding freshwater stingray Potamotrygon motoro using high-speed videography. We find that Potamotrygon motoro uses asymmetrical motion of the jaws, effectively chewing, to dismantle insect prey. However, CT scanning suggests that this species has simple teeth. These findings suggest that in contrast to mammalian chewing, asymmetrical jaw action is sufficient for mastication in other vertebrates. We also determined that prey capture in these rays occurs through rapid uplift of the pectoral fins, sucking prey beneath the ray's body, thereby dissociating the jaws from a prey capture role. We suggest that the decoupling of prey capture and processing facilitated the evolution of a highly kinetic feeding apparatus in batoid fishes, giving these animals an ability to consume a wide variety of prey, including molluscs, fishes, aquatic insect larvae and crustaceans. We propose Potamotrygon as a model system for understanding evolutionary convergence of prey processing and chewing in vertebrates.

Multilocus molecular phylogeny of the suckermouth armored catfishes (Siluriformes: Loricariidae) with a focus on subfamily Hypostominae.

The Neotropical catfish family Loricariidae is the fifth most species-rich vertebrate family on Earth, with over 800 valid species. The Hypostominae is its most species-rich, geographically widespread, and ecomorphologically diverse subfamily. Here, we provide a comprehensive molecular phylogenetic reappraisal of genus-level relationships in the Hypostominae based on our sequencing and analysis of two mitochondrial and three nuclear loci (4293bp total). Our most striking large-scale systematic discovery was that the tribe Hypostomini, which has traditionally been recognized as sister to tribe Ancistrini based on morphological data, was nested within Ancistrini. This required recognition of seven additional tribe-level clades: the Chaetostoma Clade, the Pseudancistrus Clade, the Lithoxus Clade, the 'Pseudancistrus' Clade, the Acanthicus Clade, the Hemiancistrus Clade, and the Peckoltia Clade. Results of our analysis, which included type- and non-type species for every valid genus in Hypostominae, support the reevaluation and restriction of several historically problematic genera, including Baryancistrus, Cordylancistrus, Hemiancistrus, and Peckoltia. Much of the deep lineage diversity in Hypostominae is restricted to Guiana Shield and northern Andean drainages, with three tribe-level clades still largely restricted to the Guiana Shield. Of the six geographically widespread clades, a paraphyletic assemblage of three contain lineages restricted to drainages west of the Andes Mountains, suggesting that early diversification of the Hypostominae predated the late Miocene surge in Andean uplift. Our results also highlight examples of trophic ecological diversification and convergence in the Loricariidae, including support for three independent origins of highly similar and globally unique morphological specializations for eating wood.

Morphology does not predict performance: jaw curvature and prey crushing in durophagous stingrays.

All stingrays in the family Myliobatidae are durophagous, consuming bivalves and gastropods, as well as decapod crustaceans. Durophagous rays have rigid jaws, flat teeth that interlock to form pavement-like tooth plates, and large muscles that generate bite forces capable of fracturing stiff biological composites (e.g. mollusk shell). The relative proportion of different prey types in the diet of durophagous rays varies between genera, with some stingray species specializing on particular mollusk taxa, while others are generalists. The tooth plate module provides a curved occlusal surface on which prey is crushed, and this curvature differs significantly among myliobatids. We measured the effect of jaw curvature on prey-crushing success in durophagous stingrays. We milled aluminum replica jaws rendered from computed tomography scans, and crushed live mollusks, three-dimensionally printed gastropod shells, and ceramic tubes with these fabricated jaws. Our analysis of prey items indicate that gastropods were consistently more difficult to crush than bivalves (i.e. were stiffer), but that mussels require the greatest work-to-fracture. We found that replica shells can provide an important proxy for investigations of failure mechanics. We also found little difference in crushing performance between jaw shapes, suggesting that disparate jaws are equally suited for processing different types of shelled prey. Thus, durophagous stingrays exhibit a many-to-one mapping of jaw morphology to mollusk crushing performance.

Out of the blue: adaptive visual pigment evolution accompanies Amazon invasion.

Incursions of marine water into South America during the Miocene prompted colonization of freshwater habitats by ancestrally marine species and present a unique opportunity to study the molecular evolution of adaptations to varying environments. Freshwater and marine environments are distinct in both spectra and average intensities of available light. Here, we investigate the molecular evolution of rhodopsin, the photosensitive pigment in the eye that activates in response to light, in a clade of South American freshwater anchovies derived from a marine ancestral lineage. Using likelihood-based comparative sequence analyses, we found evidence for positive selection in the rhodopsin of freshwater anchovy lineages at sites known to be important for aspects of rhodopsin function such as spectral tuning. No evidence was found for positive selection in marine lineages, nor in three other genes not involved in vision. Our results suggest that an increased rate of rhodopsin evolution was driven by diversification into freshwater habitats, thereby constituting a rare example of molecular evolution mirroring large-scale palaeogeographic events.

The evolutionary origins of diadromy inferred from a time-calibrated phylogeny for Clupeiformes (herring and allies).

One of the most remarkable types of migration found in animals is diadromy, a life-history behaviour in which individuals move between oceans and freshwater habitats for feeding and reproduction. Diadromous fishes include iconic species such as salmon, eels and shad, and have long fascinated biologists because they undergo extraordinary physiological and behavioural modifications to survive in very different habitats. However, the evolutionary origins of diadromy remain poorly understood. Here, we examine the widely accepted productivity hypothesis, which states that differences in productivity between marine and freshwater biomes determine the origins of the different modes of diadromy. Specifically, the productivity hypothesis predicts that anadromous lineages should evolve in temperate areas from freshwater ancestors and catadromous lineages should evolve in tropical areas from marine ancestors. To test this, we generated a time-calibrated phylogeny for Clupeiformes (herrings, anchovies, sardines and allies), an ecologically and economically important group that includes high diversity of diadromous species. Our results do not support the productivity hypothesis. Instead we find that the different modes of diadromy do not have predictable ancestry based on latitude, and that predation, competition and geological history may be at least as important as productivity in determining the origins of diadromy.

Paleo-drainage basin connectivity predicts evolutionary relationships across three Southeast Asian biodiversity hotspots.

Understanding factors driving diversity across biodiversity hotspots is critical for formulating conservation priorities in the face of ongoing and escalating environmental deterioration. While biodiversity hotspots encompass a small fraction of Earth's land surface, more than half the world's plants and two-thirds of terrestrial vertebrate species are endemic to these hotspots. Tropical Southeast (SE) Asia displays extraordinary species richness, encompassing four biodiversity hotspots, though disentangling multiple potential drivers of species richness is confounded by the region's dynamic geological and climatic history. Here, we use multilocus molecular genetic data from dense multispecies sampling of freshwater fishes across three biodiversity hotspots, to test the effect of Quaternary climate change and resulting drainage rearrangements on aquatic faunal diversification. While Cenozoic geological processes have clearly shaped evolutionary history in SE Asian halfbeak fishes, we show that paleo-drainage re-arrangements resulting from Quaternary climate change played a significant role in the spatiotemporal evolution of lowland aquatic taxa, and provide priorities for conservation efforts.

Ichthyoplankton metabarcoding: An efficient tool for early detection of invasive species establishment.

Detection of invasive species is critical for management but is often limited by challenges associated with capture, processing and identification of early life stages. DNA metabarcoding facilitates large-scale monitoring projects to detect establishment early. Here, we test the use of DNA metabarcoding to monitor invasive species by sequencing over 5000 fishes in bulk ichthyoplankton samples (larvae and eggs) from four rivers of ecological and cultural importance in southern Canada. We were successful in detecting species known from each river and three invasive species in two of the four rivers. This includes the first detection of early life-stage rudd in the Credit River. We evaluated whether sampling gear affected the detection of invasive species and estimates of species richness, and found that light traps outperform bongo nets in both cases. We also found that the primers used for the amplification of target sequences and the number of sequencing reads generated per sample affect the consistency of species detections. However, these factors have less impact on detections and species richness estimates than the number of samples collected and analysed. Our analyses also show that incomplete reference databases can result in incorrectly attributing DNA sequences to invasive species. Overall, we conclude that DNA metabarcoding is an efficient tool for monitoring the early establishment of invasive species by detecting evidence of reproduction but requires careful consideration of sampling design and the primers used to amplify, sequence and classify the diversity of native and potentially invasive species.

It's a family matter: molecular phylogenetics of Atheriniformes and the polyphyly of the surf silversides (family: Notocheiridae).

Phylogenetic relationships among families of Atheriniformes have long been problematic. The affinities of one of the most enigmatic lineages, surf silversides (Notocheiridae), have proven particularly elusive due to this taxon's unique morphology and rarity in museum collections. In this study, we use mitochondrial and nuclear sequence data to generate a phylogeny for seven of the eight families of Atheriniformes. Our results reveal that four families within Atheriniformes (Atherinopsidae, Notocheiridae, Atherinidae, Melanotaeniidae) are not monophyletic. Most notably, Notocheiridae is polyphyletic, with Notocheirus hubbsi nested within New World silversides (Atherinopsidae), while members of Iso are sister to all other Old World Atheriniforms. These data suggest that the unique morphology of Notocheirus and Iso is a result of adaptive convergent evolution to the high-energy surf habitat where these species live.

Modulation of bioelectric cues in the evolution of flying fishes.

Changes to allometry, or the relative proportions of organs and tissues within organisms, is a common means for adaptive character change in evolution. However, little is understood about how relative size is specified during development and shaped during evolution. Here, through a phylogenomic analysis of genome-wide variation in 35 species of flying fishes and relatives, we identify genetic signatures in both coding and regulatory regions underlying the convergent evolution of increased paired fin size and aerial gliding behaviors. To refine our analysis, we intersected convergent phylogenomic signatures with mutants with altered fin size identified in distantly related zebrafish. Through these paired approaches, we identify a surprising role for an L-type amino acid transporter, lat4a, and the potassium channel, kcnh2a, in the regulation of fin proportion. We show that interaction between these genetic loci in zebrafish closely phenocopies the observed fin proportions of flying fishes. The congruence of experimental and phylogenomic findings point to conserved, non-canonical signaling integrating bioelectric cues and amino acid transport in the establishment of relative size in development and evolution.