The molecular origin and evolution of dim‐light vision in mammals

The nocturnal origin of mammals is a longstanding hypothesis that is considered instrumental for the evolution of endothermy, a potential key innovation in this successful clade. This hypothesis is primarily based on indirect anatomical inference from fossils. Here, we reconstruct the evolutionary history of rhodopsin—the vertebrate visual pigment mediating the first step in phototransduction at low‐light levels—via codon‐based model tests for selection, combined with gene resurrection methods that allow for the study of ancient proteins. Rhodopsin coding sequences were reconstructed for three key nodes: Amniota, Mammalia, and Theria. When expressed in vitro, all sequences generated stable visual pigments with λ_MAX values similar to the well‐studied bovine rhodopsin. Retinal release rates of mammalian and therian ancestral rhodopsins, measured via fluorescence spectroscopy, were significantly slower than those of the amniote ancestor, indicating altered molecular function possibly related to nocturnality. Positive selection along the therian branch suggests adaptive evolution in rhodopsin concurrent with therian ecological diversification events during the Mesozoic that allowed for an exploration of the environment at varying light levels.     

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.     

Molecular adaptation of ammonia monooxygenase during independent pH specialization in Thaumarchaeota

Microbes are abundant in nature and often highly adapted to local conditions. While great progress has been made in understanding the ecological factors driving their distribution in complex environments, the underpinning molecular‐evolutionary mechanisms are rarely dissected. Therefore, we scrutinized the coupling of environmental and molecular adaptation in Thaumarchaeota, an abundant archaeal phylum with a key role in ammonia oxidation. These microbes are adapted to a diverse spectrum of environmental conditions, with pH being a key factor shaping their contemporary distribution and evolutionary diversification. We integrated high‐throughput sequencing data spanning a broad representation of ammonia‐oxidizing terrestrial lineages with codon modelling analyses, testing the hypothesis that ammonia monooxygenase subunit A (AmoA) – a highly conserved membrane protein crucial for ammonia oxidation and classical marker in microbial ecology – underwent adaptation during specialization to extreme pH environments. While purifying selection has been an important factor limiting AmoA evolution, we identified episodic shifts in selective pressure at the base of two phylogenetically distant lineages that independently adapted to acidic conditions and subsequently gained lasting ecological success. This involved nonconvergent selective mechanisms (positive selection vs. selection acting on variants fixed during an episode of relaxed selection) leading to unique sets of amino acid substitutions that remained fixed across the radiation of both acidophilic lineages, highlighting persistent adaptive value in acidic environments. Our data demonstrates distinct trajectories of AmoA evolution despite convergent phenotypic adaptation, suggesting that microbial environmental specialization can be associated with diverse signals of molecular adaptation, even for marker genes employed routinely by microbial ecologists.     

Adaptation and conservation of physiological systems in the evolution of human hypoxia tolerance

Analysis of human responses to hypobaric hypoxia in different lineages (lowlanders, Andean natives, Himalayan natives, and East Africans) indicates 'conservative' and 'adaptable' physiological characters involved in human responses to hypoxia. Conservative characters, derived by common descent, dominate and indeed define human physiology, but in five hypoxia response systems analyzed, we also found evidence for 'adaptable' characters at all levels of organization in all three high altitude lineages. Since Andeans and Himalayans have not shared common ancestry with East Africans for most of our species history, we suggest that their similar hypoxia physiology may represent the 'ancestral' condition for humans--an interpretation consistent with recent evidence indicating that our species evolved under 'colder, drier, and higher' conditions in East Africa where the phenotype would be simultaneously advantageous for endurance performance and for high altitude hypoxia. It is presumed that the phenotype was retained in low capacity form in highlanders and in higher capacity form in most lowland lineages (where it would be recognized by most physiologists as an endurance performance phenotype). Interestingly, it is easier for modern molecular evolution theory to account for the origin of 'adaptable' characters through positive selection than for conserved traits. Many conserved physiological systems are composed of so many gene products that it seems difficult to account for their unchanging state (for unchanging structure and function of hundreds of proteins linked in sequence to form the physiological system) by simple models of stabilizing selection.     

Repeated evolution of vertebrate pollination syndromes in a recently diverged Andean plant clade

Although specialized interactions, including those involving plants and their pollinators, are often invoked to explain high species diversity, they are rarely explored at macroevolutionary scales. We investigate the dynamic evolution of hummingbird and bat pollination syndromes in the centropogonid clade (Lobelioideae: Campanulaceae), an Andean‐centered group of ∼550 angiosperm species. We demonstrate that flowers hypothesized to be adapted to different pollinators based on flower color fall into distinct regions of morphospace, and this is validated by morphology of species with known pollinators. This supports the existence of pollination syndromes in the centropogonids, an idea corroborated by ecological studies. We further demonstrate that hummingbird pollination is ancestral, and that bat pollination has evolved ∼13 times independently, with ∼11 reversals. This convergence is associated with correlated evolution of floral traits within selective regimes corresponding to pollination syndrome. Collectively, our results suggest that floral morphological diversity is extremely labile, likely resulting from selection imposed by pollinators. Finally, even though this clade's rapid diversification is partially attributed to their association with vertebrate pollinators, we detect no difference in diversification rates between hummingbird‐ and bat‐pollinated lineages. Our study demonstrates the utility of pollination syndromes as a proxy for ecological relationships in macroevolutionary studies of certain species‐rich clades.     

THE COMPLEX EVOLUTIONARY HISTORY OF SEEING RED: MOLECULAR PHYLOGENY AND THE EVOLUTION OF AN ADAPTIVE VISUAL SYSTEM IN DEEP‐SEA DRAGONFISHES (STOMIIFORMES: STOMIIDAE)

The vast majority of deep‐sea fishes have retinas composed of only rod cells sensitive to only shortwave blue light, approximately 480–490 nm. A group of deep‐sea dragonfishes, the loosejaws (family Stomiidae), possesses far‐red emitting photophores and rhodopsins sensitive to long‐wave emissions greater than 650 nm. In this study, the rhodopsin diversity within the Stomiidae is surveyed based on an analysis of rod opsin‐coding sequences from representatives of 23 of the 28 genera. Using phylogenetic inference, fossil‐calibrated estimates of divergence times, and a comparative approach scanning the stomiid phylogeny for shared genotypes and substitution histories, we explore the evolution and timing of spectral tuning in the family. Our results challenge both the monophyly of the family Stomiidae and the loosejaws. Despite paraphyly of the loosejaws, we infer for the first time that far‐red visual systems have a single evolutionary origin within the family and that this shift in phenotype occurred at approximately 15.4 Ma. In addition, we found strong evidence that at approximately 11.2 Ma the most recent common ancestor of two dragonfish genera reverted to a primitive shortwave visual system during its evolution from a far‐red sensitive dragonfish. According to branch‐site tests for adaptive evolution, we hypothesize that positive selection may be driving spectral tuning in the Stomiidae. These results indicate that the evolutionary history of visual systems in deep‐sea species is complex and a more thorough understanding of this system requires an integrative comparative approach.     

Parallel evolution in the major haemoglobin genes of eight species of Andean waterfowl

Theory predicts that parallel evolution should be common when the number of beneficial mutations is limited by selective constraints on protein structure. However, confirmation is scarce in natural populations. Here we studied the major haemoglobin genes of eight Andean duck lineages and compared them to 115 other waterfowl species, including the bar-headed goose ( Anser indicus ) and Abyssinian blue-winged goose ( Cyanochen cyanopterus ), two additional species living at high altitude. One to five amino acid replacements were significantly overrepresented or derived in each highland population, and parallel substitutions were more common than in simulated sequences evolved under a neutral model. Two substitutions evolved in parallel in the αA subunit of two (Ala-α8) and five (Thr-α77) taxa, and five identical βA subunit substitutions were observed in two (Ser-β4, Glu-β94, Met-β133) or three (Ser-β13, Ser-β116) taxa. Substitutions at adjacent sites within the same functional protein region were also observed. Five such replacements were in exterior, solvent-accessible positions on the A helix and AB corner of the αA subunit. Five others were in close proximity to inositolpentaphosphate binding sites, and two pairs of independent replacements occurred at two different α¹β¹ intersubunit contacts. More than half of the substitutions in highland lineages resulted in the acquisition of serine or threonine (18 gains vs. 2 losses), both of which possess a hydroxyl group that can hydrogen bond to a variety of polar substrates. The patterns of parallel evolution observed in these waterfowl suggest that adaptation to high-altitude hypoxia has resulted from selection on unique but overlapping sets of one to five amino acid substitutions in each lineage.     

Into the Andes: multiple independent colonizations drive montane diversity in the Neotropical clearwing butterflies Godyridina

Understanding why species richness peaks along the Andes is a fundamental question in the study of Neotropical biodiversity. Several biogeographic and diversification scenarios have been proposed in the literature, but there is confusion about the processes underlying each scenario, and assessing their relative contribution is not straightforward. Here, we propose to refine these scenarios into a framework which evaluates four evolutionary mechanisms: higher speciation rate in the Andes, lower extinction rates in the Andes, older colonization times and higher colonization rates of the Andes from adjacent areas. We apply this framework to a species‐rich subtribe of Neotropical butterflies whose diversity peaks in the Andes, the Godyridina (Nymphalidae: Ithomiini). We generated a time‐calibrated phylogeny of the Godyridina and fitted time‐dependent diversification models. Using trait‐dependent diversification models and ancestral state reconstruction methods we then compared different biogeographic scenarios. We found strong evidence that the rates of colonization into the Andes were higher than the other way round. Those colonizations and the subsequent local diversification at equal rates in the Andes and in non‐Andean regions mechanically increased the species richness of Andean regions compared to that of non‐Andean regions (‘species‐attractor’ hypothesis). We also found support for increasing speciation rates associated with Andean lineages. Our work highlights the importance of the Andean slopes in repeatedly attracting non‐Andean lineages, most likely as a result of the diversity of habitats and/or host plants. Applying this analytical framework to other clades will bring important insights into the evolutionary mechanisms underlying the most species‐rich biodiversity hotspot on the planet.     

EVIDENCE FOR REPEATED LOSS OF SELECTIVE CONSTRAINT IN RHODOPSIN OF AMBLYOPSID CAVEFISHES (TELEOSTEI: AMBLYOPSIDAE)

The genetic mechanisms underlying regressive evolution—the degeneration or loss of a derived trait—are largely unknown, particularly for complex structures such as eyes in cave organisms. In several eyeless animals, the visual photoreceptor rhodopsin appears to have retained functional amino acid sequences. Hypotheses to explain apparent maintenance of function include weak selection for retention of light‐sensing abilities and its pleiotropic roles in circadian rhythms and thermotaxis. In contrast, we show that there has been repeated loss of functional constraint of rhodopsin in amblyopsid cavefishes, as at least three cave lineages have independently accumulated unique loss‐of‐function mutations over the last 10.3 Mya. Although several cave lineages still possess functional rhodopsin, they exhibit increased rates of nonsynonymous mutations that have greater effect on the structure and function of rhodopsin compared to those in surface lineages. These results indicate that functionality of rhodopsin has been repeatedly lost in amblyopsid cavefishes. The presence of a functional copy of rhodopsin in some cave lineages is likely explained by stochastic accumulation of mutations following recent subterranean colonization.     

Many‐to‐one form‐to‐function mapping weakens parallel morphological evolution

Evolutionary ecologists aim to explain and predict evolutionary change under different selective regimes. Theory suggests that such evolutionary prediction should be more difficult for biomechanical systems in which different trait combinations generate the same functional output: “many‐to‐one mapping.” Many‐to‐one mapping of phenotype to function enables multiple morphological solutions to meet the same adaptive challenges. Therefore, many‐to‐one mapping should undermine parallel morphological evolution, and hence evolutionary predictability, even when selection pressures are shared among populations. Studying 16 replicate pairs of lake‐ and stream‐adapted threespine stickleback (Gasterosteus aculeatus), we quantified three parts of the teleost feeding apparatus and used biomechanical models to calculate their expected functional outputs. The three feeding structures differed in their form‐to‐function relationship from one‐to‐one (lower jaw lever ratio) to increasingly many‐to‐one (buccal suction index, opercular 4‐bar linkage). We tested for (1) weaker linear correlations between phenotype and calculated function, and (2) less parallel evolution across lake‐stream pairs, in the many‐to‐one systems relative to the one‐to‐one system. We confirm both predictions, thus supporting the theoretical expectation that increasing many‐to‐one mapping undermines parallel evolution. Therefore, sole consideration of morphological variation within and among populations might not serve as a proxy for functional variation when multiple adaptive trait combinations exist.     

Positive selection and convergent evolution shape molecular phenotypic traits of innate immunity receptors in tits (Paridae)

Despite widespread variability and redundancy abounding animal immunity, little is currently known about the rate of evolutionary convergence (functionally analogous traits not inherited from a common ancestor) in host molecular adaptations to parasite selective pressures. Toll‐like receptors (TLRs) provide the molecular interface allowing hosts to recognize pathogenic structures and trigger early danger signals initiating an immune response. Using a novel combination of bioinformatic approaches, here we explore genetic variation in ligand‐binding regions of bacteria‐sensing TLR4 and TLR5 in 29 species belonging to the tit family of passerine birds (Aves: Paridae). Three out of the four consensual positively selected sites in TLR4 and six out of 14 positively selected positions in TLR5 were located on the receptor surface near the functionally important sites, and based on the phylogenetic pattern evolved in a convergent (parallel) manner. This type of evolution was also seen at one N‐glycosylation site and two positively selected phosphorylation sites, providing the first evidence of convergence in post‐translational modifications in evolutionary immunology. Finally, the overall mismatch between phylogeny and the clustering of surface charge distribution demonstrates that convergence is common in overall TLR4 and TLR5 molecular phenotypes involved in ligand binding. Our analysis did not reveal any broad ecological traits explaining the convergence observed in electrostatic potentials, suggesting that information on microbial symbionts may be needed to explain TLR evolution. Adopting state‐of‐the‐art predictive structural bionformatics, we have outlined a new broadly applicable methodological approach to estimate the functional significance of positively selected variation and test for the adaptive molecular convergence in protein‐coding polymorphisms.     

Duplication and adaptive evolution of the COR15 genes within the highly cold-tolerant Draba lineage (Brassicaceae)

Plants have evolved diverse adaptive mechanisms that enable them to tolerate abiotic stresses, to varying degrees, and such stresses may have strongly influenced evolutionary changes at levels ranging from molecular to morphological. Previous studies on these phenomena have focused on the adaptive evolution of stress-related orthologous genes in specific lineages. However, heterogenetic evolution of the paralogous genes following duplication has only been examined in a very limited number of stress-response gene families. The COR15 gene encodes a low molecular weight protein that plays an important role in protecting plants from cold stresses. Although two different copies of this gene have been found in the model species, Arabidopsis thaliana, evolutionary patterns of this small gene family in plants have not been previously explored. In this study, we cloned COR15-like sequences and performed evolutionary analyses of these sequences (including those previously reported) in the highly cold-tolerant Draba lineage and related lineages of Brassicaceae. Our phylogenetic analyses indicate that all COR15-like sequences clustered into four clades that corresponded well to the morphological lineages. Gene conversions were found to have probably occurred before/during the divergence of Brassica and Draba lineage. However, repeated, independent duplications of this gene have occurred in different lineages of Brassicaceae. Further comparisons of all sequences suggest that there have been significant inter-lineage differences in evolutionary rates between the duplicated and original genes. We assessed the likelihood that the differences between two well-supported gene subfamilies that appear to have originated from a single duplication, COR15a and COR15b, within the Draba lineage have been driven by adaptive evolution. Comparisons of their non-synonymous/synonymous substitution ratios and rates of predicted amino acid changes indicate that these two gene groups are evolving under different selective pressures and may be functionally divergent. This functional divergence was confirmed by comparing site-specific shifts in evolution indexes of the two groups of predicted proteins. The evidence of differential selection and possible functional divergence suggests that the duplication may be of adaptive significance, with possible implications for the explosive diversification of the Draba lineage during the cooling Quaternary stages and the following worldwide colonization of arid alpine and artic regions.     

Convergent Phenotypic Evolution of Rhodopsin for Dim-Light Sensing across Deep-Diving Vertebrates

Rhodopsin comprises an opsin attached to a retinal chromophore and is the only visual pigment conferring dim-light vision in vertebrates. On activation by photons, the retinal group becomes detached from the opsin, which is then inactive until it is recharged. Of all vertebrate species, those that dive face unique visual challenges, experiencing rapid decreases in light level and hunting in near darkness. Here, we combine sequence analyses with functional assays to show that the rhodopsin pigments of four divergent lineages of deep-diving vertebrates have undergone convergent increases in their retinal release rate. We compare gene sequences and detect parallel amino acids between penguins and diving mammals and perform mutagenesis to show that a single critical residue fully explains the observed increases in retinal release rate in both the emperor penguin and beaked whale. At the same time, we find that other shared sites have no significant effect on retinal release, implying that convergence does not always signify adaptive significance. We propose that accelerated retinal release confers rapid rhodopsin recharging, enabling the visual systems of diving species to adjust quickly to changing light levels as they descend through the water column. This contrasts with nocturnal species, where adaptation to darkness has been attributed to slower retinal release rates.     

Adaptive molecular evolution of a defence gene in sexual but not functionally asexual evening primroses

Theory predicts that sexual reproduction provides evolutionary advantages over asexual reproduction by reducing mutational load and increasing adaptive potential. Here, we test the latter prediction in the context of plant defences against pathogens because pathogens frequently reduce plant fitness and drive the evolution of plant defences. Specifically, we ask whether sexual evening primrose plant lineages (Onagraceae) have faster rates of adaptive molecular evolution and altered gene expression of a class I chitinase, a gene implicated in defence against pathogens, than functionally asexual evening primrose lineages. We found that the ratio of amino acid to silent substitutions (K(a) /K(s) = 0.19 vs. 0.11 for sexual and asexual lineages, respectively), the number of sites identified to be under positive selection (four vs. zero for sexual and asexual lineages, respectively) and the expression of chitinase were all higher in sexual than in asexual lineages. Our results are congruent with the conclusion that a loss of sexual recombination and segregation in the Onagraceae negatively affects adaptive structural and potentially regulatory evolution of a plant defence protein.     

Rapid evolution of elaborate male coloration is driven by visual system in Australian fairy‐wrens (Maluridae)

The interplay between colour vision and animal signalling is of keen interest to behavioural ecologists and evolutionary biologists alike, but is difficult to address in terrestrial animals. Unlike most avian lineages, in which colour vision is relatively invariant among species, the fairy‐wrens and allies (Maluridae) show a recent gain of ultraviolet sensitivity (UVS). Here, we compare the rates of colour evolution on 11 patches for males and females across Maluridae in the context of their visual system. We measured reflectance spectra for 24 species, estimating five vision‐independent colour metrics as well as metrics of colour contrast among patches and sexual dichromatism in a receiver‐neutral colour space. We fit Brownian motion (BM) and Ornstein–Uhlenbeck (OU) models to estimate evolutionary rates for these metrics and to test whether male coloration, female coloration or dichromatism was driven by selective regimes defined by visual system or geography. We found that in general male coloration evolved rapidly in comparison with females. Male colour contrast was strongly correlated with visual system and expanded greatly in UVS lineages, whereas female coloration was weakly associated with geography (Australia vs. Papua New Guinea). These results suggest that dichromatism has evolved in Maluridae as males and females evolve at different rates, and are driven by different selection pressures.     

METABOLIC FLUX IS A DETERMINANT OF THE EVOLUTIONARY RATES OF ENZYME‐ENCODING GENES

Relationships between evolutionary rates and gene properties on a genomic, functional, pathway, or system level are being explored to unravel the principles of the evolutionary process. In particular, functional network properties have been analyzed to recognize the constraints they may impose on the evolutionary fate of genes. Here we took as a case study the core metabolic network in human erythrocytes and we analyzed the relationship between the evolutionary rates of its genes and the metabolic flux distribution throughout it. We found that metabolic flux correlates with the ratio of nonsynonymous to synonymous substitution rates. Genes encoding enzymes that carry high fluxes have been more constrained in their evolution, while purifying selection is more relaxed in genes encoding enzymes carrying low metabolic fluxes. These results demonstrate the importance of considering the dynamical functioning of gene networks when assessing the action of selection on system‐level properties.     

Adaptive evolution of G-protein coupled receptor genes

The phylogeny and patterns of nucleotide substitutions in the visual pigment genes, adrenergic receptor genes, muscarinic receptor genes, and in the human mas oncogene were studied by comparing their DNA sequences. The evolutionary tree obtained shows that the visual pigment genes and mas oncogene form one cluster and that the receptor genes form another. In the evolution of rhodopsin genes, synonymous substitutions outnumber nonsynonymous substitutions. This is consistent with the neutral theory of molecular evolution. However, the early evolutionary stages of alpha- and beta-adrenergic and muscarinic receptors are notable for significantly more nonsynonymous substitutions than synonymous substitutions, suggesting the acquisition of novel functional adaptations. Variable rates of nonsynonymous changes in different domains of these proteins reveal DNA segments that might have been important in their functional adaptations.      

Molecular dynamics simulation of dark-adapted rhodopsin in an explicit membrane bilayer: coupling between local retinal and larger scale conformational change

The light-driven photocycle of rhodopsin begins the photoreceptor cascade that underlies visual response. In a sequence of events, the retinal covalently attached to the rhodopsin protein undergoes a conformational change that communicates local changes to a global conformational change throughout the whole protein. In turn, the large-scale protein change then activates G-proteins and signal amplification throughout the cell. The nature of this change, involving a coupling between a local process and larger changes throughout the protein, may be important for many membrane proteins. In addition, functional work has shown that this coupling occurs with different efficiency in different lipid settings. To begin to understand the nature of the efficiency of this coupling in different lipid settings, we present a molecular dynamics study of rhodopsin in an explicit dioleoyl-phosphatidylcholine bilayer. Our system was simulated for 40 ns and provides insights into the very early events of the visual cascade, before the full transition and activation have occurred. In particular, we see an event near 10 ns that begins with a change in hydrogen bonding near the retinal and that leads through a series of coupled changes to a shift in helical tilt. This type of event, though rare on the molecular dynamics time-scale, could be an important clue to the types of coupling that occur between local and large-scale conformational change in many membrane proteins.