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Supplementary materials for Mitra et al. (2022) Ocular lens morphology is influenced by ecology and metamorphosis in frogs and toads

Additional file 1: Figure S1. Principal components analysis (PCA) plot of rlog transformed counts from the A) transcriptome-wide and B) vision gene coding sequence analyses illustrating the outlier juvenile, dark exposed sample. Figure S2. Enrichment of Gene ontology (GO) terms. Figure S3. Principal components analysis plot of rlog transformed counts of visual gene coding sequences. Figure S4. Spectral sensitivity of photoreceptors detected through microspectrophotometry in (A) one tadpole and (B) one adult Lithobates sphenocephalus. Figure S5. Examples of raw microspectrophotometry data and raw Gaussian fits (coloured and transparent) and Govardovskii et al. [55] template fits (black and non-transparent) for each cell and pigment type found in the tadpole (A–C) and adult (D–G) Lithobates sphenocephalus. Figure S6. Examples of raw microspectrophotometry data and raw Gaussian fits (cyan) and Govardovskii et al. [55] template fits (black) for RH1 ‘red’ rods in Lithobates sphenocephalus adults where the A2 template (solid) was a better fit than the A1 template (dashed). Figure S7. Expression profiles of significantly differentially expressed cone and rod phototransduction genes between tadpoles and juveniles. Figure S8. Comparison of averaged cross-normalized expression levels (TMM) for each major crystallin type. Figure S9. Expression profiles of taxon-specific lens crystallin genes that have not specifically been identified in frogs and that differ substantially between tadpole and adults (adjusted P-value < 0.05; Additional file 6). Figure S10. Comparison of averaged refractive increment index (dn/dc) for each major crystallin type. Table S1. Sample information and read counts for the frogs used in the RNA-seq analyses. Table S2. Summary data from fits of individual photoreceptor scans from one Lithobates sphenocephalus tadpole (n = 41) and one adult (n = 33) to visual pigment templates from Govardovskii et al. [55]. Table S3. Cross-normalized expression values (TMM) and computationally estimated refractive index increment (dn/dc) values for the ubiquitous and known frog taxon-specific specific crystallins.

Pupil constriction has important functional consequences for animal vision, yet the evolutionary mechanisms underlying diverse pupil sizes and shapes are poorly understood. We aimed to quantify the diversity and evolution of pupil shapes among amphibians and test for potential correlations to ecology based on functional hypotheses. Using photographs, we surveyed pupil shape across adults of 1293 amphibian species, 72 families, and 3 orders, and additionally for larval life stages for all families of frogs and salamanders with a biphasic ontogeny. For amphibians with a biphasic life history, we found that pupils change in many species that occupy distinct habitats before and after metamorphosis. In addition, we found that non-elongated (round or diamond) constricted pupils were correlated with species inhabiting consistently dim light environments (burrowing and aquatic species) and that elongated pupils (vertical and horizontal) were more common in species with larger absolute eye sizes. We propose that amphibians provide a valuable group within which to explore the anatomical, physiological, optical, and ecological mechanisms underlying the evolution of pupil shape.

1. The spectral characteristics of vertebrate ocular lenses affect the image of the world that is projected onto the retina, and thus help shape diverse visual capabilities. Here, we tested whether amphibian lens transmission is driven by adaptation to diurnal activity (bright light) and/or scansorial habits (complex visual environments). 2. Spectral transmission through the lenses of 79 species of frogs and 6 species of salamanders was measured, and data for 29 additional frog species compiled from published literature. Phylogenetic comparative methods were used to test ecological explanations of variation in lens transmission and to test for selection across traits. 3. Lenses of diurnal (day-active) and scansorial (climbing) frogs transmitted significantly less shortwave light than those of non-diurnal or non-scansorial amphibians, and evolutionary modelling suggested that these differences have resulted from differential selection. 4. The presence of shortwave-transparent lenses was common among the sampled amphibians, which implies that many are sensitive to shortwave light to some degree even in the absence of visual pigments maximally sensitive in the UV. This suggests that shortwave light, including the UV, could play an important role in amphibian behaviour and ecology. 5. Shortwave-absorbing lens pigments likely provide higher visual acuity to diurnally active frogs of multiple ecologies and to nocturnally active scansorial frogs. This new mechanistic understanding of amphibian visual systems suggests that shortwave-filtering lenses are adaptive not only in daylight conditions but also in scotopic conditions where high acuity is advantageous.

Supplemental Methods, Results, Figures, Tables, and References

Colubridae represents themost phenotypically diverse and speciose family of snakes, yet nowell-assembled and annotated genome exists for this lineage. Here, we report and analyze the genome of the garter snake, Thamnophis sirtalis, a colubrid snake that is an important model species for research in evolutionary biology, physiology, genomics, behavior, and the evolution of toxin resistance. Using the garter snake genome, we show how snakes have evolved numerous adaptations for sensing and securing prey, and identify features of snake genomestructure that provide insight into the evolution of amniote genomes.Analyses of the garter snake andother squamate reptile genomes highlight shifts in repeat element abundance andexpansionwithin snakes, uncover evidence of genes under positive selection, and provide revised neutral substitution rate estimates for squamates. Our identification of Z andW sex chromosome-specific scaffolds provides evidence for multiple origins of sex chromosome systems in snakes and demonstrates the value of this genome for studying sex chromosome evolution. Analysis of gene duplication and loss in visual and olfactory gene families supports a dim-light ancestral condition in snakes and indicates that olfactory receptor repertoires underwent an expansion early in snake evolution. Additionally, we provide some of the first links between secreted venom proteins, the genes that encode them, and their evolutionaryorigins ina rear-fanged colubridsnake, togetherwith newgenomic insight into the coevolutionary arms race between garter snakes and highly toxic newt prey that led to toxin resistance in garter snakes. Published version

Colubridae represents the most phenotypically diverse and speciose family of snakes, yet no well-assembled and annotated genome exists for this lineage. Here we report and analyze the genome of the garter snake, Thamnophis sirtalis, a colubrid snake that is an important model species for research in evolutionary biology, physiology, genomics, behavior and the evolution of toxin resistance. Using the garter snake genome, we show how snakes have evolved numerous adaptations for sensing and securing prey, and identify features of snake genome structure that provide insight into the evolution of amniote genomes. Analyses of the garter snake and other squamate reptile genomes highlight shifts in repeat element abundance and expansion within snakes, uncover evidence of genes under positive selection, and provide revised neutral substitution rate estimates for squamates. Our identification of Z and W sex chromosome-specific scaffolds provides evidence for multiple origins of sex chromosome systems in snakes and demonstrates the value of this genome for studying sex chromosome evolution. Analysis of gene duplication and loss in visual and olfactory gene families supports a dim-light ancestral condition in snakes and indicates that olfactory receptor repertoires underwent an expansion early in snake evolution. Additionally, we provide some of the first links between secreted venom proteins, the genes that encode them, and their evolutionary origins in a rear-fanged colubrid snake, together with new genomic insight into the coevolutionary arms race between garter snakes and highly toxic newt prey that led to toxin resistance in garter snakes.