Color vision has evolved multiple times in both vertebrates and invertebrates and is largely determined by the number and variation in spectral sensitivities of distinct opsin subclasses. However, because of the difficulty of expressing long-wavelength (LW) invertebrate opsins in vitro, our understanding of the molecular basis of functional shifts in opsin spectral sensitivities has been biased toward research primarily in vertebrates. This has restricted our ability to address whether invertebrate Gq protein-coupled opsins function in a novel or convergent way compared to vertebrate Gt opsins. Here we develop a robust heterologous expression system to purify invertebrate rhodopsins, identify specific amino acid changes responsible for adaptive spectral tuning, and pinpoint how molecular variation in invertebrate opsins underlie wavelength sensitivity shifts that enhance visual perception. By combining functional and optophysiological approaches, we disentangle the relative contributions of lateral filtering pigments from red-shifted LW and blue short-wavelength opsins expressed in distinct photoreceptor cells of individual ommatidia. We use in situ hybridization to visualize six ommatidial classes in the compound eye of a lycaenid butterfly with a four-opsin visual system. We show experimentally that certain key tuning residues underlying green spectral shifts in blue opsin paralogs have evolved repeatedly among short-wavelength opsin lineages. Taken together, our results demonstrate the interplay between regulatory and adaptive evolution at multiple Gq opsin loci, as well as how coordinated spectral shifts in LW and blue opsins can act together to enhance insect spectral sensitivity at blue and red wavelengths for visual performance adaptation.

1Colour vision is largely mediated by changes in number, expression, and spectral 2 properties of rhodopsins, but the genetic mechanisms underlying adaptive shifts in 3 spectral sensitivity remain largely unexplored. Using in vivo photochemistry, 4 optophysiology, and in vitro functional assays, we link variation in eye spectral sensitivity 5 at long wavelengths to species-specific absorbance spectra for LW opsins in lycaenid 6butterflies. In addition to loci specifying an ancestral green-absorbing rhodopsin with 7 maximum spectral sensitivity (λ max ) at 520-530 nm in Callophrys sheridanii and 8Celastrina ladon, we find a novel form of red-shifted LW rhodopsin at λ max = 565-570 nm 9in Arhopala japonica and Eumaeus atala. Furthermore, we show that Ca. sheridanii and 10Ce. ladon exhibit a smaller bathochromic shift at BRh2 (480-489 nm), and with the 11 ancestral LW rhodopsin, cannot perceive visible red light beyond 600 nm. In contrast, 12 molecular variation at the LW opsin in A. japonica and E. atala is coordinated with tuning 13 of the blue opsin that also shifts sensitivity to longer wavelengths enabling colour 14 discrimination up to 617 nm. We then use E. atala as a model to examine the interplay 15 between red and blue spectral sensitivity. Owing to blue duplicate expression, the spatial 16 distribution of opsin mRNAs within an ommatidium defines an expanded retinal 17 stochastic mosaic of at least six opsin-based photoreceptor classes. Our mutagenesis in 18 vitro assays with BRh1 (λ max = 435 nm) chimeric blue rhodopsins reveal four main 19 residues contributing to the 65 nm bathochromic shift towards BRh2 (λ max = 500 nm). 20Adaptations in this four-opsin visual system are relevant for discrimination of conspecific 21 reflectance spectra in E. atala. Together, these findings illustrate how functional changes 22 at multiple rhodopsins contribute to the evolution of a broader spectral sensitivity and 23 adaptation in visual performance. 24 Keywords 25 molecular evolution, ecological adaptation, visual system/vision, rhodopsin, spectral sensitivity, in vitro (27, 60, 61, but see 62). Red receptors are intriguingly very common in butterflies 126 compared to other insect groups such as bees or beetles (30), raising the possibility that 127 perception of longer wavelengths plays an important role in the context of foraging (31, 128 46, 63), oviposition (64, 65) and mate recognition (25) for species equipped with them. 129Lycaenids comprise the second largest family of butterflies, representing almost 130 thirty percent of all species, and exhibiting considerable ecological and morphological 131 diversity (66, 67). Pioneering work showed that species of Lycaenidae in the genera 132Lycaena and Polyommatus have expanded spectral sensitivity at long wavelengths, and