Mitochondrial phylogeography and molecular evolution of the rhodopsin visual pigment in troglobitic populations of Astyanax mexicanus (De Filippi, 1853).

Cave-adapted animals provide a unique opportunity to study the evolutionary mechanisms underlying phenotypic, metabolic, behavioral, and genetic evolution in response to cave environments. The Mexican tetra ( Astyanax mexicanus) is considered a unique model system as it shows both surface and cave-dwelling morphs. To date, at least 33 different cave populations have been identified, with phylogenetic studies suggesting an origin from at least two independent surface lineages, thereby providing a unique opportunity to study parallel evolution. In the present study, we carried out the most exhaustive phylogeographic study of A. mexicanus to date, including cave and surface localities, using two mitochondrial markers (cytochrome b (cyt b) and cytochrome c oxidase subunit I ( COI)) and nuclear rhodopsin visual pigment ( rho). Additionally, we inferred the molecular evolution of rho within the two contrasting environments (cave and surface) and across three geographic regions (Sierra de El Abra, Sierra de Guatemala, and Micos). In total, 267 individuals were sequenced for the two mitochondrial fragments and 268 individuals were sequenced for the rho visual pigment from 22 cave and 46 surface populations. Phylogeographic results based on the mitochondrial data supported the two-lineage hypothesis, except for the Pachón and Chica caves, whose introgression has been largely documented. The Sierra de El Abra region depicted the largest genetic diversity, followed by the Sierra de Guatemala region. Regarding the phylogeographic patterns of rho, we recovered exclusive haplogroups for the Sierra de El Abra (Haplogroup I) and Sierra de Guatemala regions (Haplogroup IV). Moreover, a 544 bp deletion in the rho gene was observed in the Escondido cave population from Sierra de Guatemala, reducing the protein from seven to three intramembrane domains. This change may produce a loss-of-function (LOF) but requires further investigation. Regarding nonsynonymous ( dN) and synonymous ( dS) substitution rates (omega values ω), our results revealed the prevailing influence of purifying selection upon the rho pigment for both cave and surface populations (ω<1), but relaxation at the El Abra region. Notably, in contrast to the other two regions, we observed an increase in the number of dN mutations for Sierra de El Abra. However, given that a LOF was exclusively identified in the Sierra de Guatemala region, we cannot dismiss the possibility of a pleiotropic effect on the Rho protein.

Proteorhodopsin Phototrophy in Antarctic Coastal Waters.

Microbial proton-pumping rhodopsins are considered the simplest strategy among phototrophs to conserve energy from light. Proteorhodopsins are the most studied rhodopsins thus far because of their ubiquitous presence in the ocean, except in Antarctica, where they remain understudied. We analyzed proteorhodopsin abundance and transcriptional activity in the Western Antarctic coastal seawaters. Combining quantitative PCR (qPCR) and metagenomics, the relative abundance of proteorhodopsin-bearing bacteria accounted on average for 17, 3.5, and 29.7% of the bacterial community in Chile Bay (South Shetland Islands) during 2014, 2016, and 2017 summer-autumn, respectively. The abundance of proteorhodopsin-bearing bacteria changed in relation to environmental conditions such as chlorophyll a and temperature. Alphaproteobacteria, Gammaproteobacteria, and Flavobacteriia were the main bacteria that transcribed the proteorhodopsin gene during day and night. Although green light-absorbing proteorhodopsin genes were more abundant than blue-absorbing ones, the latter were transcribed more intensely, resulting in >50% of the proteorhodopsin transcripts during the day and night. Flavobacteriia were the most abundant proteorhodopsin-bearing bacteria in the metagenomes; however, Alphaproteobacteria and Gammaproteobacteria were more represented in the metatranscriptomes, with qPCR quantification suggesting the dominance of the active SAR11 clade. Our results show that proteorhodopsin-bearing bacteria are prevalent in Antarctic coastal waters in late austral summer and early autumn, and their ecological relevance needs to be elucidated to better understand how sunlight energy is used in this marine ecosystem. IMPORTANCE Proteorhodopsin-bearing microorganisms in the Southern Ocean have been overlooked since their discovery in 2000. The present study identify taxonomy and quantify the relative abundance of proteorhodopsin-bearing bacteria and proteorhodopsin gene transcription in the West Antarctic Peninsula's coastal waters. This information is crucial to understand better how sunlight enters this marine environment through alternative ways unrelated to chlorophyll-based strategies. The relative abundance of proteorhodopsin-bearing bacteria seems to be related to environmental parameters (e.g., chlorophyll a, temperature) that change yearly at the coastal water of the West Antarctic Peninsula during the austral late summers and early autumns. Proteorhodopsin-bearing bacteria from Antarctic coastal waters are potentially able to exploit both the green and blue spectrum of sunlight and are a prevalent group during the summer in this polar environment.

Carbon fixation and rhodopsin systems in microbial mats from hypersaline lakes Brava and Tebenquiche, Salar de Atacama, Chile.

In this work, molecular diversity of two hypersaline microbial mats was compared by Whole Genome Shotgun (WGS) sequencing of environmental DNA from the mats. Brava and Tebenquiche are lakes in the Salar de Atacama, Chile, where microbial communities are growing in extreme conditions, including high salinity, high solar irradiance, and high levels of toxic metals and metaloids. Evaporation creates hypersaline conditions in these lakes and mineral precipitation is a characteristic geomicrobiological feature of these benthic ecosystems. The mat from Brava was more rich and diverse, with a higher number of different taxa and with species more evenly distributed. At the phylum level, Proteobacteria, Cyanobacteria, Chloroflexi, Bacteroidetes and Firmicutes were the most abundant, including ~75% of total sequences. At the genus level, the most abundant sequences were affilitated to anoxygenic phototropic and cyanobacterial genera. In Tebenquiche mats, Proteobacteria and Bacteroidetes covered ~70% of the sequences, and 13% of the sequences were affiliated to Salinibacter genus, thus addressing the lower diversity. Regardless of the differences at the taxonomic level, functionally the two mats were similar. Thus, similar roles could be fulfilled by different organisms. Carbon fixation through the Wood-Ljungdahl pathway was well represented in these datasets, and also in other mats from Andean lakes. In spite of presenting less taxonomic diversity, Tebenquiche mats showed increased abundance and variety of rhodopsin genes. Comparison with other metagenomes allowed identifying xantorhodopsins as hallmark genes not only from Brava and Tebenquiche mats, but also for other mats developing at high altitudes in similar environmental conditions.

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.

Contrast-FEL-A Test for Differences in Selective Pressures at Individual Sites among Clades and Sets of Branches.

A number of evolutionary hypotheses can be tested by comparing selective pressures among sets of branches in a phylogenetic tree. When the question of interest is to identify specific sites within genes that may be evolving differently, a common approach is to perform separate analyses on subsets of sequences and compare parameter estimates in a post hoc fashion. This approach is statistically suboptimal and not always applicable. Here, we develop a simple extension of a popular fixed effects likelihood method in the context of codon-based evolutionary phylogenetic maximum likelihood testing, Contrast-FEL. It is suitable for identifying individual alignment sites where any among the K≥2 sets of branches in a phylogenetic tree have detectably different ω ratios, indicative of different selective regimes. Using extensive simulations, we show that Contrast-FEL delivers good power, exceeding 90% for sufficiently large differences, while maintaining tight control over false positive rates, when the model is correctly specified. We conclude by applying Contrast-FEL to data from five previously published studies spanning a diverse range of organisms and focusing on different evolutionary questions.

Multi-scale simulation reveals that an amino acid substitution increases photosensitizing reaction inputs in Rhodopsins.

Evaluating the availability of molecular oxygen (O2 ) and energy of excited states in the retinal binding site of rhodopsin is a crucial challenging first step to understand photosensitizing reactions in wild-type (WT) and mutant rhodopsins by absorbing visible light. In the present work, energies of the ground and excited states related to 11-cis-retinal and the O2 accessibility to the ß-ionone ring are evaluated inside WT and human M207R mutant rhodopsins. Putative O2 pathways within rhodopsins are identified by using molecular dynamics simulations, Voronoi-diagram analysis, and implicit ligand sampling while retinal energetic properties are investigated through density functional theory, and quantum mechanical/molecular mechanical methods. Here, the predictions reveal that an amino acid substitution can lead to enough energy and O2 accessibility in the core hosting retinal of mutant rhodopsins to favor the photosensitized singlet oxygen generation, which can be useful in understanding retinal degeneration mechanisms and in designing blue-lighting-absorbing proteic photosensitizers.

Cross-linking of bovine rhodopsin with sulfosuccinimidyl 4-(N maleimidomethyl)cyclohexane-1-carboxylate affects its functionality.

Rhodopsin is the photoreceptor protein involved in visual excitation in retinal rods. The functionality of bovine rhodopsin was determined following treatment with sulfosuccinimidyl 4-(N maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), a bifunctional reagent capable of forming covalent cross-links between suitable placed lysines and cysteines. Denaturing polyacrylamide gel electrophoresis showed that rhodopsin incubated with sulfo-SMCC generated intermolecular dimers, trimers, and higher oligomers, although most of the sulfo-SMCC-treated protein remained as a monomer. Minor alterations on the absorption spectrum of light-activated sulfo-SMCC-treated rhodopsin were observed. However, only ∼2% stimulation of the guanine nucleotide binding activity of transducin was measured in the presence of sulfo-SMCC-cross-linked photolyzed rhodopsin. Moreover, rhodopsin kinase was not able of phosphorylating sulfo-SMCC-cross-linked rhodopsin after illumination. Rhodopsin was purified in the presence of either 0.1% or 1% n-dodecyl ß-d-maltoside, to obtain dimeric and monomeric forms of the protein, respectively. Interestingly, no generation of the regular F1 and F2 thermolytic fragments was perceived with sulfo-SMCC-cross-linked rhodopsin either in the dimeric or monomeric state, implying the formation of intramolecular connections in the protein that might thwart the light-induced conformational changes required for interaction with transducin and rhodopsin kinase. Structural analysis of the rhodopsin three-dimensional structure suggested that the following lysine and cysteine pairs: Lys66/Lys67 and Cys316, Cys140 and Lys141, Cys140 and Lys248, Lys311 and Cys316, and/or Cys316 and Lys325 are potential candidates to generate intramolecular cross-links in the protein. Yet, the lack of fragmentation of sulfo-SMCC-treated Rho with thermolysin is consistent with the formation of cross-linking bridges between Lys66/Lys67 and Cys316, and/or Cys140 and Lys248.

Evidences for genetic differentiation within the highly endemic and endangered annual fish Austrolebias nigrofasciatus (Cyprinodontiformes: Rivulidae).

Samples of Austrolebias nigrofasciatus (n = 103), an endangered species of annual fish endemic to a small area of the Patos-Mirim lagoon system encompassing the São Gonçalo Channel lowlands, were collected from eight isolated temporary ponds, four located at the known distribution range of the species and four located along the Piratini River lowlands, where morphologically different individuals were found. In the laboratory, fragments of the mitochondrial cytochrome c oxidase I (coI), cytochrome b (cytb) and nuclear rhodopsin (rho) genes were amplified, purified and sequenced for 100, 99 and 58 of these individuals, respectively. Samples were further analysed using phylogenetic and phylogeographic methods to evaluate the patterns of genetic diversity and differentiation presented within and between populations, while assessing their evolutionary history, in order to guide the application of further conservation strategies. We found that the four new populations from the Piratini River lowlands encompass a different lineage of A. nigrofasciatus that diverged from that encountered in the São Gonçalo Channel at approximately 0.165 M years before present, during a population expansion and did not yet attain reciprocal monophyly. This divergence was associated with a glacial event that was preceded by an interglacial period putatively associated with the dispersal. Moreover, significant levels of genetic differentiation and a high number of exclusive haplotypes could be encountered even in micro-geographical scales, as in the comparisons between populations located within the same major lineage, indicating each of them may encompass independent management units. Conservation actions are certainly urgent, especially in the face of signs of a recent bottleneck.

Glucocorticoid and progesterone mechanisms in photoreceptor survival.

Death of retinal photoreceptors is the basis of prevalent blinding diseases. Since steroids might have a therapeutic role in retinal degenerations, we compared the protective effects of dexamethasone and progesterone on photoreceptor death induced by mifepristone and light exposure. Therefore, we studied the effective protection doses for each steroid in the two models. In addition, we analyzed changes in the levels of pro- and antiapoptotic molecules, glucocorticoid receptors α and ß (GRα and GRß), and rhodopsin under conditions of successful protection and photoreceptor survival. Mifepristone and light exposure selectively damaged photoreceptors. In light exposed retinas, photoreceptors mainly disappeared in the dorsotemporal region, while mifepristone produced a uniform damage. Dexamethasone and progesterone, at the same dose of 4 mg/kg/day for 2 days, preserved over 88% photoreceptor nuclei in both models. Assessment of cell death regulators showed that, in control retinas, both steroids activated BCL-XL, a prosurvival molecule, and decreased BID, a proapoptotic regulator. After steroid treatment of damaged retinas, BCL-XL, BCL2 and BAX showed characteristic patterns depending on the use of dexamethasone or progesterone on mifepristone or light exposed retinas. By contrast, BID decreased with any injury-steroid combination. Changes in GRα or GRß levels did not correlate with survival but were consistent with a mechanism of ligand induced downregulation of receptor expression. GRß might be upregulated by progesterone. Both dexamethasone and progesterone increased retinal rhodopsin stores, suggesting a link between photoreceptor protection and transduction pathways. Results show that dexamethasone and progesterone induced comparable but not identical protection responses in each model.

Photoreceptors morphology and genetics of the visual pigments of Bothrops jararaca and Crotalus durissus terrificus (Serpentes, Viperidae).

Snakes inhabit a great variety of habitats, whose spectral quality of light may vary a lot and influence specific adaptations of their visual system. In this study, we investigated the genetics of the visual opsins and the morphology of retinal photoreceptors, of two nocturnal snakes from the Viperidae family, Bothrops jararaca and Crotalus durissus terrificus, which inhabit preferentially the Atlantic Rain Forest and the Brazilian Savannah, respectively. Total RNA was extracted from homogenized retinas and converted to cDNA. The opsin genes expressed in snake retinas, LWS, RH1, and SWS1, were amplified by polymerase chain reactions (PCRs) and sequenced. The absorption peak (λmax) of the opsins were estimated based on amino acids located at specific spectral tuning sites. Photoreceptor cell populations were analyzed using immunohistochemistry with anti-opsin antibodies. Results showed the same morphological cell populations and same opsins absorption peaks, in both viperid species: double and single cones with LWS photopigment and λmax at ∼555 nm; single cones with SWS1 photopigment and λmax at ∼360 nm; and rods with the rhodopsin RH1 photopigment and λmax at ∼500 nm. The results indicate adaptations to nocturnal habit in both species despite the differences in habitat, and the possibility of a dichromatic color vision at photopic conditions.

Melanopsin and rhodopsin mediate UVA-induced immediate pigment darkening: Unravelling the photosensitive system of the skin.

The mammalian skin has a photosensitive system comprised by several opsins, including rhodopsin (OPN2) and melanopsin (OPN4). Recently, our group showed that UVA (4.4 kJ/m2) leads to immediate pigment darkening (IPD) in murine normal and malignant melanocytes. We show the role of OPN2 and OPN4 as UVA sensors: UVA-induced IPD was fully abolished when OPN4 was pharmacologically inhibited by AA9253 or when OPN2 and OPN4 were knocked down by siRNA in both cell lines. Our data, however, demonstrate that phospholipase C/protein kinase C pathway, a classical OPN4 pathway, is not involved in UVA-induced IPD in either cell line. Nonetheless, in both cell types we have shown that: a) intracellular calcium signal is necessary for UVA-induced IPD; b) the involvement of CaMK II, whose inhibition, abolished the UVA-induced IPD; c) the role of CAMK II/NOS/sGC/cGMP pathway in the process since inhibition of either NOS or sGC abolished the UVA-induced IPD. Taken altogether, we show that OPN2 and OPN4 participate in IPD induced by UVA in murine normal and malignant melanocytes through a conserved common pathway. Interestingly, upon knockdown of OPN2 or OPN4, the UVA-driven IPD is completely lost, which suggests that both opsins are required and cooperatively signal in murine both cell lines. The participation of OPN2 and OPN4 system in UVA radiation-induced response, if proven to take place in human skin, may represent an interesting pharmacological target for the treatment of depigmentary disorders and skin-related cancer.

Genomic comparison between members of the Salinibacteraceae family, and description of a new species of Salinibacter (Salinibacter altiplanensis sp. nov.) isolated from high altitude hypersaline environments of the Argentinian Altiplano.

The application of tandem MALDI-TOF MS screening with 16S rRNA gene sequencing of selected isolates has been demonstrated to be an excellent approach for retrieving novelty from large-scale culturing. The application of such methodologies in different hypersaline samples allowed the isolation of the culture-recalcitrant Salinibacter ruber second phylotype (EHB-2) for the first time, as well as a new species recently isolated from the Argentinian Altiplano hypersaline lakes. In this study, the genome sequences of the different species of the phylum Rhodothermaeota were compared and the genetic repertoire along the evolutionary gradient was analyzed together with each intraspecific variability. Altogether, the results indicated an open pan-genome for the family Salinibacteraceae, as well as the codification of relevant traits such as diverse rhodopsin genes, CRISPR-Cas systems and spacers, and one T6SS secretion system that could give ecological advantages to an EHB-2 isolate. For the new Salinibacter species, we propose the name Salinibacter altiplanensis sp. nov. (the designated type strain is AN15T=CECT 9105T=IBRC-M 11031T).

Accelerated Evolution and Functional Divergence of the Dim Light Visual Pigment Accompanies Cichlid Colonization of Central America.

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.

Molecular phylogeny and timing of diversification in South American Cynolebiini seasonal killifishes.

The rich biological diversity of South America has motivated a series of studies associating evolution of endemic taxa with the dramatic geologic and climatic changes that occurred during the Cainozoic. The organism here studied is the killifish tribe Cynolebiini, a group of seasonal fishes uniquely inhabiting temporary pools formed during the rainy seasons. The Cynolebiini are found in open vegetation areas inserted in the main tropical and subtropical South American phytogeographical regions east of the Andes. Here, we present the first molecular phylogeny sampling all the eight genera of the Cynolebiini, using fragments of two mitochondrial and four nuclear genes for 35 species of Cynolebiini plus 19 species as outgroups. The dataset, 4448bp, was analysed under Bayesian and maximum likelihood approaches, providing a relatively well solved tree, which retrieves high support values for the Cynolebiini and most included clades. The resulting tree was used to estimate the time of divergence in included lineages using two cyprinodontiform fossils to calibrate the tree. We further investigated historical biogeography through the likelihood-based DEC model. Our estimates indicate that divergence between the clades comprising New World and Old World aplocheiloids occurred during the Eocene, about 50Mya, much more recent than the Gondwanan fragmentation scenario assumed in previous studies. This estimation is nearly synchronous to estimated splits involving other South American and African vertebrate clades, which have been explained by transoceanic dispersal through an ancient Atlantic island chain during the Palaeogene. We estimate that Cynolebiini split from its sister group Cynopoecilini in the Oligocene, about 25Mya and that Cynolebiini started to diversify giving origin to the present genera during the Miocene, about 20-14Mya. The Cynolebiini had an ancestral origin in the Atlantic Forest and probably were not present in the open vegetation formations of central and northeastern South America until the Middle Miocene, when expansion of dry open vegetation was favoured by cool temperatures and strike seasonality. Initial splitting between the genera Cynolebias and Simpsonichthys during the Miocene (about 14Mya) is attributed to the uplift of the Central Brazilian Plateau.

Evolution of nonspectral rhodopsin function at high altitudes.

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.

Microbial and viral-like rhodopsins present in coastal marine sediments from four polar and subpolar regions.

Rhodopsins are broadly distributed. In this work, we analyzed 23 metagenomes corresponding to marine sediment samples from four regions that share cold climate conditions (Norway; Sweden; Argentina and Antarctica). In order to investigate the genes evolution of viral rhodopsins, an initial set of 6224 bacterial rhodopsin sequences according to COG5524 were retrieved from the 23 metagenomes. After selection by the presence of transmembrane domains and alignment, 123 viral (51) and non-viral (72) sequences (>50 amino acids) were finally included in further analysis. Viral rhodopsin genes were homologs of Phaeocystis globosa virus and Organic lake Phycodnavirus. Non-viral microbial rhodopsin genes were ascribed to Bacteroidetes, Planctomycetes, Firmicutes, Actinobacteria, Cyanobacteria, Proteobacteria, Deinococcus-Thermus and Cryptophyta and Fungi. A rescreening using Blastp, using as queries the viral sequences previously described, retrieved 30 sequences (>100 amino acids). Phylogeographic analysis revealed a geographical clustering of the sequences affiliated to the viral group. This clustering was not observed for the microbial non-viral sequences. The phylogenetic reconstruction allowed us to propose the existence of a putative ancestor of viral rhodopsin genes related to Actinobacteria and Chloroflexi. This is the first report about the existence of a phylogeographic association of the viral rhodopsin sequences from marine sediments.

Retinal Pre-Conditioning by CD59a Knockout Protects against Light-Induced Photoreceptor Degeneration.

Complement dysregulation plays a key role in the pathogenesis of age-related macular degeneration (AMD), but the specific mechanisms are incompletely understood. Complement also potentiates retinal degeneration in the murine light damage model. To test the retinal function of CD59a, a complement inhibitor, CD59a knockout (KO) mice were used for light damage (LD) experiments. Retinal degeneration and function were compared in WT versus KO mice following light damage. Gene expression changes, endoplasmic reticulum (ER) stress, and glial cell activation were also compared. At baseline, the ERG responses and rhodopsin levels were lower in CD59aKO compared to wild-type (WT) mice. Following LD, the ERG responses were better preserved in CD59aKO compared to WT mice. Correspondingly, the number of photoreceptors was higher in CD59aKO retinas than WT controls after LD. Under normal light conditions, CD59aKO mice had higher levels than WT for GFAP immunostaining in Müller cells, mRNA and protein levels of two ER-stress markers, and neurotrophic factors. The reduction in photon capture, together with the neurotrophic factor upregulation, may explain the structural and functional protection against LD in the CD59aKO.

Population Structure of the Rockpool Blenny Entomacrodus vomerinus Shows Source-Sink Dynamics among Ecoregions in the Tropical Southwestern Atlantic.

The Tropical Southwestern Atlantic is characterized by prominent ecosystems with large-scale oceanographic complexity. Yet, the evolutionary processes underlying genetic differentiation and connectivity in this region remain largely unknown. Entomacrodus vomerinus (Valenciennes, 1836) is a demersal fish with planktonic larvae endemic to this marine province, inhabiting shallow tidal pools in continental and oceanic reef environments. We evaluated the population structure, genetic diversity and gene flow of E. vomerinus using mitochondrial data (CYTB and COI) and nuclear (rhodopsin, RHO) DNA sequences. We sampled a total of 85 individuals, comprising 46 from three oceanic archipelagos with varying distance from the coast (São Pedro and São Paulo-SS, Fernando de Noronha-FE and Rocas Atoll-RA) and 39 from two localities in northeastern Brazilian coast (Rio Grande do Norte-RN and Bahia-BA). Multilocus analysis revealed the presence of three Evolutionarily Significant Units-ESUs (SS, FE+RA, and RN+BA), which are in accordance with distinct marine ecoregions. Coalescent analyses showed that the central ESU has a larger effective population size than the other two, suggesting strong asymmetries in the genetic diversity across the species range. Moreover, they showed that gene flow is highly asymmetric, suggesting a source-sink dynamics from the central ESU into the remaining ones, in agreement with oceanic currents. Together, these results provide insights in the evolutionary mechanisms facilitating diversification in this marine province.

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.

Syndromic and non-syndromic forms of retinitis pigmentosa: a comprehensive Italian clinical and molecular study reveals new mutations.

Mutations in more than 60 different genes have been associated with non-syndromic and syndromic retinitis pigmentosa (RP), a heterogeneous group of inherited retinal dystrophies. To increase the understanding of the molecular epidemiology of the disease in Italy, we analyzed 56 patients with syndromic and non-syndromic forms of RP attending the Retinitis Pigmentosa Center of San Paolo Hospital (Milan, Italy). Patients underwent detailed clinical examination. Genomic DNA isolated from peripheral blood samples was screened for mutations in different genes according to RP form by direct sequencing analysis. The impact of novel missense mutations on protein functions was predicted by in silico analysis and protein sequence alignment. Cosegregation analysis was performed between available family members. Forty-one of the 56 probands analyzed had non-syndromic and 15 had syndromic RP forms. Putative disease-causing mutations were identified in 19 of 56 unrelated RP probands. Mutation screening identified a total of 22 different heterozygous variants. Notably, 12 of these putative pathogenic mutations have not been previously reported. New variants were found to be located on the USH2A, RPGR, EYS, and RHO genes. All 3 new variants detected in X-linked RP probands were confirmed in other affected family members. We found a positivity rate of 24.4% and 60% for probands with non-syndromic and syndromic RP, respectively. This is the first report of RPGR X-linked RP proband-ORF15 mutations in Italian patients with X-linked (XL)-RP. In addition, this is the first report of data regarding the association between EYS mutations and non-syndromic RP forms in the Italian population.