Vision represents an excellent model for studying adaptation, given the genotype-tophenotype-map that has been characterized in a number of taxa. Fish possess a diverse range of visual sensitivities and adaptations to underwater light making them an excellent group to study visual system evolution. In particular, some speciose but understudied lineages can provide a unique opportunity to better understand aspects of visual system evolution such as opsin gene duplication and neofunctionalization. In this study, we characterized the visual system of Neotropical Characiformes, which is the result of several spectral tuning mechanisms acting in concert including gene duplications and losses, gene conversion, opsin amino acid sequence and expression variation, and A 1 /A 2 -chromophore shifts. The Characiforms we studied utilize three cone opsin classes (SWS2, RH2, LWS) and a rod opsin (RH1). However, the characiform's entire opsin gene repertoire is a product of dynamic evolution by opsin gene loss (SWS1, RH2) and duplication (LWS, RH1). The LWS-and RH1-duplicates originated from a teleost specific whole-genome duplication as well as characiform-specific duplication events. Both LWS-opsins exhibit gene conversion and, through substitutions in key tuning sites, one of the LWS-paralogs has acquired spectral sensitivity to green light. These sequence changes suggest reversion and parallel evolution of key tuning sites. In addition, characiforms exhibited species-specific differences in opsin expression. Finally, we found interspecific and intraspecific variation in the use of A 1 /A 2chromophores correlating with the light environment. These multiple mechanisms may be a result of the highly diverse visual environments where Characiformes have evolved.

33Vision represents an excellent model for studying adaptation, given the genotype-to-34 phenotype-map that has been characterized in a number of taxa. Fish possess a diverse 35 range of visual sensitivities and adaptations to underwater light making them an 36 excellent group to study visual system evolution. In particular, some speciose but 37 understudied lineages can provide a unique opportunity to better understand aspects of 38 visual system evolution such as opsin gene duplication and neofunctionalization. In this 39 study, we characterized the visual system of Neotropical Characiformes, which is the 40 result of several spectral tuning mechanisms acting in concert including gene 41 duplications and losses, gene conversion, opsin amino acid sequence and expression 42 variation, and A 1 /A 2 -chromophore shifts. The Characiforms we studied utilize three cone 43 opsin classes (SWS2, RH2, LWS) and a rod opsin (RH1). However, the characiform's 44 entire opsin gene repertoire is a product of dynamic evolution by opsin gene loss 45 (SWS1, RH2) and duplication (LWS, RH1). The LWS-and RH1-duplicates originated 46 from a teleost specific whole-genome duplication as well as characiform-specific 47 duplication events. Both LWS-opsins exhibit gene conversion and, through substitutions 48 in key tuning sites, one of the LWS-paralogs has acquired spectral sensitivity to green 49 light. These sequence changes suggest reversion and parallel evolution of key tuning 50 sites. In addition, characiforms exhibited species-specific differences in opsin 51 expression. Finally, we found interspecific and intraspecific variation in the use of A 1 /A 2 -52 chromophores correlating with the light environment. These multiple mechanisms may 53 be a result of the highly diverse visual environments where Characiformes have evolved. 54 55 58 relates to the environment. Evolutionary studies of genes such as the ones involved in 59 the first steps of vision can provide valuable insights in the acquisition of new functions 60 and their adaptive significance. In vertebrates, vision starts when light reaches the retina61 and is detected by rod (night vision) or cone (diurnal vision) photoreceptors.62 Photoreceptors are packed with visual pigments that are composed of two components: 63 an opsin protein with seven α-helices enclosing a ligand-binding pocket, and a light-64 sensitive chromophore, 11-cis retinal (Bowmaker 2008; Yokoyama 2008). There can be 65 3 multiple cone types containing different visual pigments that absorb light maximally in 66 different parts of the wavelength spectrum. 67 68 There are four classes of cone pigments encoded by opsin genes among vertebrates: a 69 short-wave class (SWS1) sensitive to ultraviolet-violet light (350-400 nm), a second 70 short-wave class (SWS2) sensitive to violet-blue (410-490 nm), a middle-wave class 71 (RH2) sensitive to green (480-535 nm), and a middle-to long-wave class (LWS) 72 sensitive to the green and red spectral region (490-570 nm) (Bowmaker and Hunt 2006; 73 Bowmaker 2008). All four ...