The Molecular Evolution of Rhodopsin in Marine-derived and other Freshwater Fishes

Visual system evolution can be influenced by the spectral properties of light available in the environment. Variation in the dim-light specialized visual pigment rhodopsin is thought to result in functional shifts that optimize its sensitivity in relation to ambient spectral environments. Marine and freshwater environments have been shown to be characterized by different spectral properties and might be expected to place the spectral sensitivity of rhodopsin under different selection pressures. In Chapter two, I show that the rate ratio of non- synonymous to synonymous substitutions is significantly elevated in the rhodopsin gene of a South American clade of freshwater anchovies with marine ancestry. This signature of positive selection is not observed in the rhodopsin gene of the marine sister clade or in non-visual genes. In Chapter three I functionally characterize the effects of positively selected substitutions occurring on another independent invasion of freshwater made by ancestrally marine croakers. In vitro spectroscopic assays on ancestrally resurrected rhodopsin pigments reveal a red-shift in peak spectral sensitivity along the transitional branch, consistent with the wavelengths of light illuminating freshwater environments. Kinetics assays reveal that freshwater croaker rhodopsin might also possess more efficient dark adaptation. In Chapter four I use a comparative approach to show that substitutions with similar functional effects occur convergently during marine to freshwater transitions, but only in deeper-dwelling lineages. In Chapter five I investigate the molecular evolution of rhodopsin in Gymnotiformes, a clade of freshwater fishes with an alternative sensory modality specialized for dim-light environments. Rhodopsin is highly conserved in this clade, but bouts of positive selection are observed in association with ecological transitions, indicating that dim-light vision remains an important sensory modality in these freshwater fishes. Altogether, these studies show that shifts in selection pressures and substitutions that alter the functional properties of rhodopsin are frequently observed during ecological transitions into and within freshwater environments, as long as species inhabit depths where the attenuation of light is non-negligible. Furthermore, this thesis expands our understanding of the effects of ecology on visual evolution and its influence on the structural and functional properties of rhodopsin.Ph.D.2021-07-21 00:00:0

Metabarcoding to Detect Invasive Fishes data

Data associated with a DNA metabarcoding study of fish (invasive and native) biodiversity in four rivers in Ontario Sub directories 00_sample_map: Map of sampling locations and abundance of larval fishes collected at each site 01_database_build: Reference genetic database and taxonomic information for classification of metabarcoding sequencing reads 02_qiime2_analysis: De-noising and dereplicating DNA metabarcoding data 03_read_correction: Correction for contamination using no-template controls and single fish samples 04_sampling_stats: General summary statistics of sequencing 05_river_richness: Summaries and analyses of species richness across rivers 06_gear_richness: Comparison of gear types on species detected 07_primer_consistency: Comparison of primer target gene on species detected 08_sampling_depth: Comparison of number of samples collected to read depth per sample 09_database_comparison: Comparison of the reference databases for COI and 12S 10_amplicon_distance: Comparison of the genetic distance for COI and 12S metabarcoding primers</p

Transcriptome Assemblies

De novo Trinity assemblies of the colubrid snake eye transcriptomes

Sequence Alignments

Multiple sequence alignments for each gene

Draft Genome CDS

Sequences extracted from various draft genomes used in the selection analyses

Data from: Shifts in selective pressures on snake phototransduction genes associated with photoreceptor transmutation and dim-light ancestry

The visual systems of snakes are heavily modified relative to other squamates, a condition often thought to reflect their fossorial origins. Further modifications are seen in caenophidian snakes, where evolutionary transitions between rod and cone photoreceptors, termed photoreceptor transmutations, have occurred in many lineages. Little previous work, however, has focused on the molecular evolutionary underpinnings of these morphological changes. To address this, we sequenced seven snake eye transcriptomes and utilized new whole genome and targeted capture sequencing data. We used this data to analyze gene loss and shifts in selection pressures in phototransduction genes that may be associated with snake evolutionary origins and photoreceptor transmutation. We identified the surprising loss of rhodopsin kinase (GRK1), despite a low degree of gene loss overall and a lack of relaxed selection early during snake evolution. These results provide some of the first evolutionary genomic corroboration for a dim-light ancestor that lacks strong fossorial adaptations. Our results also indicate that snakes with photoreceptor transmutation experienced significantly different selection pressures from other reptiles. Significant positive selection was found primarily in cone-specific genes, but not rod-specific genes, contrary to our expectations. These results reveal potential molecular adaptations associated with photoreceptor transmutation, and also highlight unappreciated functional differences between rod- and cone-specific phototransduction proteins. This intriguing example of snake visual system evolution illustrates how the underlying molecular components of a complex system can be reshaped in response to changing selection pressures

Comparative sequence analyses of rhodopsin and RPE65 reveal patterns of selective constraint across hereditary retinal disease mutations

Retinitis pigmentosa (RP) comprises several heritable diseases that involve photoreceptor, and ultimately retinal, degeneration. Currently, mutations in over 50 genes have known links to RP. Despite advances in clinical characterization, molecular characterization of RP remains challenging due to the heterogeneous nature of causal genes, mutations, and clinical phenotypes. In this study, we compiled large datasets of two important visual genes associated with RP: rhodopsin, which initiates the phototransduction cascade, and the retinoid isomerase RPE65, which regenerates the visual cycle. We used a comparative evolutionary approach to investigate the relationship between interspecific sequence variation and pathogenic mutations that lead to degenerative retinal disease. Using codon-based likelihood methods, we estimated evolutionary rates (d N/d S) across both genes in a phylogenetic context to investigate differences between pathogenic and nonpathogenic amino acid sites. In both genes, disease-associated sites showed significantly lower evolutionary rates compared to nondisease sites, and were more likely to occur in functionally critical areas of the proteins. The nature of the dataset (e.g., vertebrate or mammalian sequences), as well as selection of pathogenic sites, affected the differences observed between pathogenic and nonpathogenic sites. Our results illustrate that these methods can serve as an intermediate step in understanding protein structure and function in a clinical context, particularly in predicting the relative pathogenicity (i.e., functional impact) of point mutations and their downstream phenotypic effects. Extensions of this approach may also contribute to current methods for predicting the deleterious effects of candidate mutations and to the identification of protein regions under strong constraint where we expect pathogenic mutations to occur.This work was supported by an NSERC Discovery Grant and a Foundation for Fighting Blindness seed grant to BSWC, Vision Science Research Fellowships to FEH, RKS, GMC, and AVN, and Ontario Graduate Fellowships to FEH and RKS. Amir Sabouhanian provided valuable feedback on the statistical analyses

PAML Results Tables

Detailed results and summary tables of each of the PAML analyses

PAML Files

Alignment and tree files needed to run the PAML analyses

FDR Control Results

Results of analyses to control the false discovery rate (FDR)