RESUMEN
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⺠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⺠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⺠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.
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Pez Eléctrico , Subunidad alfa del Factor 1 Inducible por Hipoxia , Oxígeno , Animales , Ecosistema , Pez Eléctrico/genética , Pez Eléctrico/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Anaerobiosis , Oxígeno/metabolismoRESUMEN
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.
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Evolución Molecular , Rodopsina , Animales , Rodopsina/genética , Rodopsina/metabolismo , Peces/genética , Peces/metabolismo , Visión Ocular , EcosistemaRESUMEN
Cetaceans are fully aquatic mammals that descended from terrestrial ancestors, an iconic evolutionary transition characterized by adaptations for underwater foraging via breath-hold diving. Although the evolutionary history of this specialized behavior is challenging to reconstruct, coevolving sensory systems may offer valuable clues. The dim-light visual pigment, rhodopsin, which initiates phototransduction in the rod photoreceptors of the eye, has provided insight into the visual ecology of depth in several aquatic vertebrate lineages. Here, we use ancestral sequence reconstruction and protein resurrection experiments to quantify light-activation metrics in rhodopsin pigments from ancestors bracketing the cetacean terrestrial-to-aquatic transition. By comparing multiple reconstruction methods on a broadly sampled cetartiodactyl species tree, we generated highly robust ancestral sequence estimates. Our experimental results provide direct support for a blue-shift in spectral sensitivity along the branch separating cetaceans from terrestrial relatives. This blue-shift was 14 nm, resulting in a deep-sea signature (λmax = 486 nm) similar to many mesopelagic-dwelling fish. We also discovered that the decay rates of light-activated rhodopsin increased in ancestral cetaceans, which may indicate an accelerated dark adaptation response typical of deeper-diving mammals. Because slow decay rates are thought to help sequester cytotoxic photoproducts, this surprising result could reflect an ecological trade-off between rod photoprotection and dark adaptation. Taken together, these ancestral shifts in rhodopsin function suggest that some of the first fully aquatic cetaceans could dive into the mesopelagic zone (>200 m). Moreover, our reconstructions indicate that this behavior arose before the divergence of toothed and baleen whales.
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Buceo , Visión Nocturna , Rodopsina , Ballenas , Animales , Evolución Biológica , Fósiles , Rodopsina/metabolismo , Ballenas/genética , Ballenas/fisiologíaRESUMEN
Extreme environments, such as Antarctic habitats, present major challenges for many biological processes. Antarctic icefishes (Crynotothenioidea) represent a compelling system to investigate the molecular basis of adaptation to cold temperatures. Here, we explore how the sub-zero habitats of Antarctic icefishes have impacted rhodopsin (RH1) function, the temperature-sensitive dim-light visual pigment found in rod photoreceptors. Using likelihood models and ancestral reconstruction, we find that accelerated evolutionary rates in icefish RH1 underlie unique amino acid mutations absent from other deep-dwelling fishes, introduced before (S160A) and during (V259M) the onset of modern polar conditions. Functional assays reveal that these mutations red-shift rhodopsin spectral absorbance, consistent with spectral irradiance under sea ice. These mutations also lower the activation energy associated with retinal release of the light-activated RH1, and accelerate its return to the dark state, likely compensating for a cold-induced decrease in kinetic rates. These are adaptations in key properties of rhodopsin that mediate rod sensitivity and visual performance in the cold dark seas of the Antarctic.
Asunto(s)
Adaptación Fisiológica , Rodopsina , Rodopsina/genética , Adaptación Fisiológica/genética , Evolución Biológica , Visión Ocular , Ambientes Extremos , Regiones AntárticasRESUMEN
Empirical studies of genotype-phenotype-fitness maps of proteins are fundamental to understanding the evolutionary process, in elucidating the space of possible genotypes accessible through mutations in a landscape of phenotypes and fitness effects. Yet, comprehensively mapping molecular fitness landscapes remains challenging since all possible combinations of amino acid substitutions for even a few protein sites are encoded by an enormous genotype space. High-throughput mapping of genotype space can be achieved using large-scale screening experiments known as multiplexed assays of variant effect (MAVEs). However, to accommodate such multi-mutational studies, the size of MAVEs has grown to the point where a priori determination of sampling requirements is needed. To address this problem, we propose calculations and simulation methods to approximate minimum sampling requirements for multi-mutational MAVEs, which we combine with a new library construction protocol to experimentally validate our approximation approaches. Analysis of our simulated data reveals how sampling trajectories differ between simulations of nucleotide versus amino acid variants and among mutagenesis schemes. For this, we show quantitatively that marginal gains in sampling efficiency demand increasingly greater sampling effort when sampling for nucleotide sequences over their encoded amino acid equivalents. We present a new library construction protocol that efficiently maximizes sequence variation, and demonstrate using ultradeep sequencing that the library encodes virtually all possible combinations of mutations within the experimental design. Insights learned from our analyses together with the methodological advances reported herein are immediately applicable toward pooled experimental screens of arbitrary design, enabling further assay upscaling and expanded testing of genotype space.
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Aptitud Genética , Genotipo , Mutación , Simulación por Computador , Modelos Genéticos , Fenotipo , Evolución Molecular , Biblioteca de Genes , Sustitución de AminoácidosRESUMEN
Eukaryotic cells use G protein-coupled receptors (GPCRs) to convert external stimuli into internal signals to elicit cellular responses. However, how mutations in GPCR-coding genes affect GPCR activation and downstream signaling pathways remain poorly understood. Approaches such as deep mutational scanning show promise in investigations of GPCRs, but a high-throughput method to measure rhodopsin activation has yet to be achieved. Here, we scale up a fluorescent reporter assay in budding yeast that we engineered to study rhodopsin's light-activated signal transduction. Using this approach, we measured the mutational effects of over 1200 individual human rhodopsin mutants, generated by low-frequency random mutagenesis of the GPCR rhodopsin (RHO) gene. Analysis of the data in the context of rhodopsin's three-dimensional structure reveals that transmembrane helices are generally less tolerant to mutations compared to flanking helices that face the lipid bilayer, which suggest that mutational tolerance is contingent on both the local environment surrounding specific residues and the specific position of these residues in the protein structure. Comparison of functional scores from our screen to clinically identified rhodopsin disease variants found many pathogenic mutants to be loss of function. Lastly, functional scores from our assay were consistent with a complex counterion mechanism involved in ligand-binding and rhodopsin activation. Our results demonstrate that deep mutational scanning is possible for rhodopsin activation and can be an effective method for revealing properties of mutational tolerance that may be generalizable to other transmembrane proteins.
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Receptores Acoplados a Proteínas G , Rodopsina , Humanos , Rodopsina/genética , Rodopsina/química , Rodopsina/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/química , Transducción de Señal , Estructura Secundaria de Proteína , MutaciónRESUMEN
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.
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Opsinas de los Conos , Animales , Opsinas de los Conos/genética , Filogenia , Opsinas/genética , Peces/genética , Evolución MolecularRESUMEN
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.
RESUMEN
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.
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Adaptación Biológica/genética , Perciformes/genética , Rodopsina/genética , Selección Genética , Visión Ocular/genética , Animales , Agua Dulce , Perciformes/metabolismo , Rodopsina/metabolismo , América del SurRESUMEN
Snakes are known to express a rod visual opsin and two cone opsins, only (SWS1, LWS), a reduced palette resulting from their supposedly fossorial origins. Dipsadid snakes in the genus Helicops are highly visual predators that successfully invaded freshwater habitats from ancestral terrestrial-only habitats. Here, we report the first case of multiple SWS1 visual pigments in a vertebrate, simultaneously expressed in different photoreceptors and conferring both UV and violet sensitivity to Helicops snakes. Molecular analysis and in vitro expression confirmed the presence of two functional SWS1 opsins, likely the result of recent gene duplication. Evolutionary analyses indicate that each sws1 variant has undergone different evolutionary paths with strong purifying selection acting on the UV-sensitive copy and dN/dS â¼1 on the violet-sensitive copy. Site-directed mutagenesis points to the functional role of a single amino acid substitution, Phe86Val, in the large spectral shift between UV and violet opsins. In addition, higher densities of photoreceptors and SWS1 cones in the ventral retina suggest improved acuity in the upper visual field possibly correlated with visually guided behaviors. The expanded visual opsin repertoire and specialized retinal architecture are likely to improve photon uptake in underwater and terrestrial environments, and provide the neural substrate for a gain in chromatic discrimination, potentially conferring unique color vision in the UV-violet range. Our findings highlight the innovative solutions undertaken by a highly specialized lineage to tackle the challenges imposed by the invasion of novel photic environments and the extraordinary diversity of evolutionary trajectories taken by visual opsin-based perception in vertebrates.
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Visión de Colores , Opsinas , Animales , Agua Dulce , Opsinas/genética , Opsinas/metabolismo , Filogenia , Células Fotorreceptoras Retinianas Conos/metabolismo , Opsinas de Bastones/genética , Serpientes/genética , Serpientes/metabolismoRESUMEN
Natural variation in the number, expression and function of sensory genes in an organism's genome is often tightly linked to different ecological and evolutionary forces. Opsin genes, which code for the first step in visual transduction, are ideal models for testing how ecological factors such as light environment may influence visual system adaptation. Neotropical cichlid fishes are a highly ecologically diverse group that evolved in a variety of aquatic habitats, including black (stained), white (opaque) and clear waters. We used cross-species exon capture to sequence Neotropical cichlid short wavelength-sensitive (SWS) opsins, which mediate ultraviolet (UV) to blue visual sensitivity. Neotropical cichlid SWS1 opsin (UV-sensitive) underwent a relaxation of selective constraint during the early phases of cichlid diversification in South America, leading to pseudogenization and loss. Conversely, SWS2a (blue-sensitive) experienced a burst of episodic positive selection at the base of the South American cichlid radiation. This burst coincides with SWS1 relaxation and loss, and is consistent with findings in ecomorphological studies characterizing a period of extensive ecological divergence in Neotropical cichlids. We use ancestral sequence reconstruction and protein modelling to investigate mutations along this ancestral branch that probably modified SWS2a function. Together, our results suggest that variable light environments played a prominent early role in shaping SWS opsin diversity during the Neotropical cichlid radiation. Our results also illustrate that long-term evolution under light-limited conditions in South America may have reduced visual system plasticity; specifically, early losses of UV sensitivity may have constrained the evolutionary trajectory of Neotropical cichlid vision.
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Cíclidos , Animales , Cíclidos/genética , Evolución Molecular , Opsinas/genética , Filogenia , América del SurRESUMEN
Primate colour vision depends on a matrix of photoreceptors, a neuronal post receptoral structure and a combination of genes that culminate in different sensitivity through the visual spectrum. Along with a common cone opsin gene for short wavelengths (sws1), Neotropical primates (Platyrrhini) have only one cone opsin gene for medium-long wavelengths (mws/lws) per X chromosome while Paleotropical primates (Catarrhini), including humans, have two active genes. Therefore, while female platyrrhines may be trichromats, males are always dichromats. The genus Alouatta is inferred to be an exception to this rule, as electrophysiological, behavioural and molecular analyses indicated a potential for male trichromacy in this genus. However, it is very important to ascertain by a combination of genetic and behavioural analyses whether this potential translates in terms of colour discrimination capability. We evaluated two howler monkeys (Alouatta spp.), one male A. caraya and one female A. seniculus, using a combination of genetic analysis of the opsin gene sequences and a behavioral colour discrimination test not previously used in this genus. Both individuals completed the behavioural test with performances typical of trichromatic colour vision and the genetic analysis of the sws1, mws, and lws opsin genes revealed three different opsin sequences in both subjects. These results are consistent with uniform trichromacy in both male and female, with presumed spectral sensitivity peaks similar to Catarrhini, at ~ 430 nm, 532 nm, and 563 nm for S-, M- and L-cones, respectively.
RESUMEN
High-altitude environments present a range of biochemical and physiological challenges for organisms through decreases in oxygen, pressure, and temperature relative to lowland habitats. Protein-level adaptations to hypoxic high-altitude conditions have been identified in multiple terrestrial endotherms; however, comparable adaptations in aquatic ectotherms, such as fishes, have not been as extensively characterized. In enzyme proteins, cold adaptation is attained through functional trade-offs between stability and activity, often mediated by substitutions outside the active site. Little is known whether signaling proteins [e.g., G protein-coupled receptors (GPCRs)] exhibit natural variation in response to cold temperatures. Rhodopsin (RH1), the temperature-sensitive visual pigment mediating dim-light vision, offers an opportunity to enhance our understanding of thermal adaptation in a model GPCR. Here, we investigate the evolution of rhodopsin function in an Andean mountain catfish system spanning a range of elevations. Using molecular evolutionary analyses and site-directed mutagenesis experiments, we provide evidence for cold adaptation in RH1. We find that unique amino acid substitutions occur at sites under positive selection in high-altitude catfishes, located at opposite ends of the RH1 intramolecular hydrogen-bonding network. Natural high-altitude variants introduced into these sites via mutagenesis have limited effects on spectral tuning, yet decrease the stability of dark-state and light-activated rhodopsin, accelerating the decay of ligand-bound forms. As found in cold-adapted enzymes, this phenotype likely compensates for a cold-induced decrease in kinetic rates-properties of rhodopsin that mediate rod sensitivity and visual performance. Our results support a role for natural variation in enhancing the performance of GPCRs in response to cold temperatures.
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Altitud , Rodopsina/química , Animales , Teorema de Bayes , Evolución Biológica , Bolivia , Bagres , Proteínas y Péptidos de Choque por Frío/química , Frío , Cristalografía por Rayos X , Ecuador , Evolución Molecular , Geografía , Células HEK293 , Humanos , Cinética , Mutación , Perú , FilogeniaRESUMEN
Bats are excellent models for studying the molecular basis of sensory adaptation. In Chiroptera, a sensory trade-off has been proposed between the visual and auditory systems, though the extent of this association has yet to be fully examined. To investigate whether variation in visual performance is associated with echolocation, we experimentally assayed the dim-light visual pigment rhodopsin from bat species with differing echolocation abilities. While spectral tuning properties were similar among bats, we found that the rate of decay of their light-activated state was significantly slower in a nonecholocating bat relative to species that use distinct echolocation strategies, consistent with a sensory trade-off hypothesis. We also found that these rates of decay were remarkably slower compared with those of other mammals, likely indicating an adaptation to dim light. To examine whether functional changes in rhodopsin are associated with shifts in selection intensity upon bat Rh1 sequences, we implemented selection analyses using codon-based likelihood clade models. While no shifts in selection were identified in response to diverse echolocation abilities of bats, we detected a significant increase in the intensity of evolutionary constraint accompanying the diversification of Chiroptera. Taken together, this suggests that substitutions that modulate the stability of the light-activated rhodopsin state were likely maintained through intensified constraint after bats diversified, being finely tuned in response to novel sensory specializations. Our study demonstrates the power of combining experimental and computational approaches for investigating functional mechanisms underlying the evolution of complex sensory adaptations.
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Adaptación Biológica , Quirópteros/fisiología , Ecolocación , Evolución Molecular , Rodopsina/fisiología , Animales , Adaptación a la Oscuridad , Cinética , Visión OcularRESUMEN
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.
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Evolución Molecular , Peces/genética , Rodopsina/genética , Secuencia de Aminoácidos , Animales , Ecosistema , Luz , Filogenia , Rodopsina/química , Visión OcularRESUMEN
Vertebrate retinas are generally composed of rod (dim-light) and cone (bright-light) photoreceptors with distinct morphologies that evolved as adaptations to nocturnal/crepuscular and diurnal light environments. Over 70 years ago, the "transmutation" theory was proposed to explain some of the rare exceptions in which a photoreceptor type is missing, suggesting that photoreceptors could evolutionarily transition between cell types. Although studies have shown support for this theory in nocturnal geckos, the origins of all-cone retinas, such as those found in diurnal colubrid snakes, remain a mystery. Here we investigate the evolutionary fate of the rods in a diurnal garter snake and test two competing hypotheses: (i) that the rods, and their corresponding molecular machinery, were lost or (ii) that the rods were evolutionarily modified to resemble, and function, as cones. Using multiple approaches, we find evidence for a functional and unusually blue-shifted rhodopsin that is expressed in small single "cones." Moreover, these cones express rod transducin and have rod ultrastructural features, providing strong support for the hypothesis that they are not true cones, as previously thought, but rather are modified rods. Several intriguing features of garter snake rhodopsin are suggestive of a more cone-like function. We propose that these cone-like rods may have evolved to regain spectral sensitivity and chromatic discrimination as a result of ancestral losses of middle-wavelength cone opsins in early snake evolution. This study illustrates how sensory evolution can be shaped not only by environmental constraints but also by historical contingency in forming new cell types with convergent functionality.
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Evolución Biológica , Ritmo Circadiano , Colubridae/fisiología , Células Fotorreceptoras Retinianas Conos/citología , Animales , Inmunohistoquímica , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Células Fotorreceptoras Retinianas Conos/ultraestructura , Pigmentos Retinianos/metabolismo , Células Fotorreceptoras Retinianas Bastones/citología , Células Fotorreceptoras Retinianas Bastones/ultraestructura , Rodopsina/metabolismo , Transducina/metabolismoRESUMEN
Cichlids encompass one of the most diverse groups of fishes in South and Central America, and show extensive variation in life history, morphology, and colouration. While studies of visual system evolution in cichlids have focussed largely on the African rift lake species flocks, Neotropical cichlids offer a unique opportunity to investigate visual system evolution at broader temporal and geographic scales. South American cichlid colonization of Central America has likely promoted accelerated rates of morphological evolution in Central American lineages as they encountered reduced competition, renewed ecological opportunity, and novel aquatic habitats. To investigate whether such transitions have influenced molecular evolution of vision in Central American cichlids, we sequenced the dim-light rhodopsin gene in 101 Neotropical cichlid species, spanning the diversity of the clade. We find strong evidence for increased rates of evolution in Central American cichlid rhodopsin relative to South American lineages, and identify several sites under positive selection in rhodopsin that likely contribute to adaptation to different photic environments. We expressed a Neotropical cichlid rhodopsin protein invitro for the first time, and found that while its spectral tuning properties were characteristic of typical vertebrate rhodopsin pigments, the rate of decay of its active signalling form was much slower, consistent with dim light adaptation in other vertebrate rhodopsins. Using site-directed mutagenesis combined with spectroscopic assays, we found that a key amino acid substitution present in some Central American cichlids accelerates the rate of decay of active rhodopsin, which may mediate adaptation to clear water habitats.
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Cíclidos/genética , Adaptación a la Oscuridad/genética , Rodopsina/genética , Animales , Evolución Biológica , América Central , Ecosistema , Evolución Molecular , Proteínas del Ojo/genética , Variación Genética/genética , Lagos , Luz , Mutagénesis Sitio-Dirigida , FilogeniaRESUMEN
Bats represent one of the largest and most striking nocturnal mammalian radiations, exhibiting many visual system specializations for performance in light-limited environments. Despite representing the greatest ecological diversity and species richness in Chiroptera, Neotropical lineages have been undersampled in molecular studies, limiting the potential for identifying signatures of selection on visual genes associated with differences in bat ecology. Here, we investigated how diverse ecological pressures mediate long-term shifts in selection upon long-wavelength (Lws) and short-wavelength (Sws1) opsins, photosensitive cone pigments that form the basis of colour vision in most mammals, including bats. We used codon-based likelihood clade models to test whether ecological variables associated with reliance on visual information (e.g. echolocation ability and diet) or exposure to varying light environments (e.g. roosting behaviour and foraging habitat) mediated shifts in evolutionary rates in bat cone opsin genes. Using additional cone opsin sequences from newly sequenced eye transcriptomes of six Neotropical bat species, we found significant evidence for different ecological pressures influencing the evolution of the cone opsins. While Lws is evolving under significantly lower constraint in highly specialized high-duty cycle echolocating lineages, which have enhanced sonar ability to detect and track targets, variation in Sws1 constraint was significantly associated with foraging habitat, exhibiting elevated rates of evolution in species that forage among vegetation. This suggests that increased reliance on echolocation as well as the spectral environment experienced by foraging bats may differentially influence the evolution of different cone opsins. Our study demonstrates that different ecological variables may underlie contrasting evolutionary patterns in bat visual opsins, and highlights the suitability of clade models for testing ecological hypotheses of visual evolution.
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Quirópteros/genética , Opsinas de los Conos/genética , Ecosistema , Evolución Molecular , Animales , Conducta Apetitiva , Ecolocación/fisiología , Conducta Alimentaria , Luz , Filogenia , TranscriptomaRESUMEN
Cetaceans have undergone a remarkable evolutionary transition that was accompanied by many sensory adaptations, including modification of the visual system for underwater environments. Recent sequencing of cetacean genomes has made it possible to begin exploring the molecular basis of these adaptations. In this study we use in vitro expression methods to experimentally characterize the first step of the visual transduction cascade, the light activation of rhodopsin, for the killer whale. To investigate the spectral effects of amino acid substitutions thought to correspond with absorbance shifts relative to terrestrial mammals, we used the orca gene as a background for the first site-directed mutagenesis experiments in a cetacean rhodopsin. The S292A mutation had the largest effect, and was responsible for the majority of the spectral difference between killer whale and bovine (terrestrial) rhodopsin. Using codon-based likelihood models, we also found significant evidence for positive selection in cetacean rhodopsin sequences, including on spectral tuning sites we experimentally mutated. We then investigated patterns of ecological divergence that may be correlated with rhodopsin functional variation by using a series of clade models that partitioned the data set according to phylogeny, habitat, and foraging depth zone. Only the model partitioning according to depth was significant. This suggests that foraging dives might be a selective regime influencing cetacean rhodopsin divergence, and our experimental results indicate that spectral tuning may be playing an adaptive role in this process. Our study demonstrates that combining computational and experimental methods is crucial for gaining insight into the selection pressures underlying molecular evolution.
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Adaptación Biológica , Pigmentos Retinianos/genética , Rodopsina/genética , Selección Genética , Orca/genética , Animales , Evolución Biológica , Evolución Molecular , Expresión Génica , Luz , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Filogenia , Conformación Proteica , Pigmentos Retinianos/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Relación Estructura-Actividad , Orca/metabolismoRESUMEN
Like many aquatic vertebrates, whales have blue-shifting spectral tuning substitutions in the dim-light visual pigment, rhodopsin, that are thought to increase photosensitivity in underwater environments. We have discovered that known spectral tuning substitutions also have surprising epistatic effects on another function of rhodopsin, the kinetic rates associated with light-activated intermediates. By using absorbance spectroscopy and fluorescence-based retinal release assays on heterologously expressed rhodopsin, we assessed both spectral and kinetic differences between cetaceans (killer whale) and terrestrial outgroups (hippo, bovine). Mutation experiments revealed that killer whale rhodopsin is unusually resilient to pleiotropic effects on retinal release from key blue-shifting substitutions (D83N and A292S), largely due to a surprisingly specific epistatic interaction between D83N and the background residue, S299. Ancestral sequence reconstruction indicated that S299 is an ancestral residue that predates the evolution of blue-shifting substitutions at the origins of Cetacea. Based on these results, we hypothesize that intramolecular epistasis helped to conserve rhodopsin's kinetic properties while enabling blue-shifting spectral tuning substitutions as cetaceans adapted to aquatic environments. Trade-offs between different aspects of molecular function are rarely considered in protein evolution, but in cetacean and other vertebrate rhodopsins, may underlie multiple evolutionary scenarios for the selection of specific amino acid substitutions.