The evolution of red colour vision is linked to coordinated rhodopsin tuning in lycaenid butterflies

Abstract

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

Multiple mechanisms of photoreceptor spectral tuning following loss of UV color vision inHeliconiusbutterflies

“…Blue opsin alignment in the region around the spectral tuning site 116 (right). In distantly related butterflies ( Pieris, Lycaena ), a Ser116Ala substitution causes a blue-shift in blue opsin absorbance (Liénard et al, 2020; Sison-Mangus et al, 2006; Wakakuwa et al, 2010). Heliconius have a single blue opsin.…”

“…We therefore hypothesized that the blue receptor might be shifted toward shorter wavelengths in Heliconius species lacking the 390 nm receptor, to better discriminate colors in the blue/violet range. In Pieris rapae , a Ser116Ala mutation is solely responsible for a ~13 nm blue-shift in the peak absorbance of blue rhodopsin expressed in cell culture, from 450 nm to 437 nm (a Phe177Tyr substitution is responsible for a 4 nm blue-shift) (Frentiu et al, 2015; Liénard et al, 2020; Wakakuwa et al, 2010). The substitution at site 116 is present in one of the duplicated blue opsins in P. rapae and in the independently duplicated blue opsins of Lycaena rubidus, contributing to the difference between the two blue opsins’ peak absorbance within each species (Figure 3B) (Sison-Mangus et al, 2006; Wakakuwa et al, 2010).…”

True UV color vision in a butterfly with two UV opsins

“…As noted above, some clades (e.g., H. melpomene) do not express the UVRh2 protein at detectable levels in the adult compound eye despite expressing the UVRh2 mRNA, due to ongoing pseudogenization (McCulloch et al 2017). Opsin duplication events are not uncommon in butterflies (Sison-Mangus et al 2006, Lienard et al 2020). For example, the lycaenid butterfly Polyommatus icarus uses its duplicated blue opsin to see green, perhaps for discrimination of oviposition sites (Sison-Mangus et al 2008).…”

Molecular advances to study the function, evolution and spectral tuning of arthropod visual opsins

“…LW pigments were believed to be difficult to express in cultured cells on a large scale until optimized assays emerged to circumvent this problem [20,29]. To the best of our knowledge, two methods have been successful at consistently characterizing absorption spectra of arthropod LW opsins in vitro : heterologous action spectroscopy (HeAS), and parallel sensitive heterologous expression (PaSHE) [20,29].…”

“…Parallel sensitive heterologous expression . PaSHE is an in vitro assay that allows efficient purification of arthropod visual opsins for spectroscopic analysis [29]. PaSHE is essentially a heterologous expression system that builds upon previous protocols used to express opsins in HEK293T [26,27,30,87], but through a series of optimized steps is able to consistently recover active rhodopsin complexes for the different Gq opsin families, including the elusive LWS [29].…”