# Evolution of opsin genes in closely-related species of butterflies specialized in different microhabitats

Joséphine Ledamoisel, Andrew Dang, Julien Devilliers, Tiphaine Marvillet, Sophie Lemoine, Manuela Lopez-Villavicencio, Adriana Briscoe, Vincent Debat, Violaine Llaurens

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Joséphine Ledamoisel, Andrew Dang, Julien Devilliers, Tiphaine Marvillet, Sophie Lemoine, et al. Evolution of opsin genes in closely-related species of butterflies specialized in different microhabitats. 2025. hal-05348341

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Evolution of opsin genes in closely-related species of
butterflies specialized in different microhabitats

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Joséphine Ledamoisel¹,², Andrew Dang³, Julien Devilliers², Tiphaine Marvillet⁴, Sophie Lemoine⁴,

Manuela Lopez-Villavicencio², Adriana Briscoe³, Vincent Debat², Violaine Llaurens¹

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1. Centre Interdisciplinaire de Recherche en Biologie (UMR 7241, Collège de France/CNRS/INSERM), Paris, France.

2. Institut de Systématique, Evolution et Biodiversité (ISYEB UMR 7205), Muséum National d'Histoire Naturelle, Paris, France.

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3. Department of Ecology and Evolutionary Biology, UC Irvine, Irvine, CA, USA.

4. GenomiqueENS, Institut de Biologie de l'ENS (IBENS), Département de biologie, École

normale supérieure, CNRS, INSERM, Université PSL, 75005 Paris, France

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*Corresponding author

Email: josephine.ledamoisel@outlook.fr (JL)

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**Abstract**

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Multiple selective pressures can shape the evolution of color vision in animals, by acting on the co-evolution of the opsin genes. How do adaptive processes shape the duplications of opsins, the evolution of their amino acids and the modification of their patterns of expression? At large phylogenetic scales, natural selection due to the contrasted light environments has been found to have a profound impact on the evolution of the opsin gene family. However, in closely-related species, species interactions due to sexual selection or competition may also influence opsin evolution. Here, we investigate the diversification of opsin sequences and their expression in closely-related blue *Morpho* butterfly species, living in different microhabitats, to shed light on the effect of biotic and abiotic selective pressures shaping the evolution of their opsin gene family. First, we combined genomics, transcriptomics and immunochemistry to precisely characterize the expression and the spatial distribution of the opsin proteins found in the eyes of *Morpho helenor*. We found unique ommatidial types compared to other butterfly species. We then investigated the evolution of opsin genes among 18 *Morpho* species, found signature of positive selection on two opsin genes, and identified key co-evolving amino-acids shaping the diversification of the *Morpho* visual system. We showed that such opsin evolution was correlated to both light environment and wing coloration, highlighting the joint effect of several selective pressures in the evolution of those proteins. Overall, our study underlines the peculiar evolution of visual systems in closely-related species specialized in divergent microhabitats.

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48 **Introduction**

49 The sensitivity to different wavelengths of light has been extensively studied in multiple species, and
50 these studies have revealed striking diversification of color discrimination capacities throughout
51 animals. Yet, the selective pressures generated by the light environment and the ecological interactions
52 within and among species shaping the diversification of the opsin gene family are largely unknown in
53 most animal species.

54 Opsin proteins have been shown to be much more diversified in the eyes of diurnal species compared to
55 nocturnal species in both vertebrates (Musilova et al. 2021) and invertebrates (Sondhi et al. 2021),
56 suggesting a prominent impact of selective pressures brought from the light environment on their
57 evolution. Evidence of concerted evolution, due to gene duplication, mutations and gene conversion has
58 been reported in the opsin gene family (Brandon et al. 2017). Variations in the amino-acid sequences of
59 opsins can also directly influence the sensitivity of the opsin-chromophore complex known as a visual
60 pigment to different light wavelengths, and the combination of opsins expressed within and among the
61 different photoreceptor cells within the eye strongly impacts color discrimination (Hofmann and Lamb
62 2023). In insects, the chromophore bound to the opsin protein is 11-cis-hydroxy retinal (Smith and
63 Goldsmith 1990) and the evolution of the light-absorbing properties of each visual pigment is strongly
64 linked to the nature of amino acids located around this chromophore (Hofmann and Lamb 2023).

65 In Lepidoptera, phylogenetic reconstructions inferred an ancestral visual system with three main opsin
66 proteins (Briscoe 2008): ultraviolet-sensitive opsins (UVRh), blue-sensitive opsins (BRh), and long
67 wavelength-sensitive opsins (LWRh). However, in many butterfly families, multiple events of opsin
68 gene duplications and mutations affecting the chromophore-interacting amino-acids have been
69 documented (Arikawa 2003; Chen et al. 2016). The duplication of opsin genes, accompanied by shifts
70 in their spectral sensitivity, has been shown to result in the absorbance of a wider wavelength range, as
71 for instance in Nymphalidae (Frentiu et al. 2007) or Lycaenidae (Sison-Mangus et al. 2008). Variation
72 in the combination of opsins with different amino-acid compositions in the binding pocket surrounding
the chromophore, as well as their distribution in different photoreceptive cells, shape the evolution of color discrimination capacities in butterflies (Stavenga and Arikawa 2006).

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The compound eyes of butterflies are composed of ommatidia, formed by a cluster of nine photoreceptive cells (named R1 to R9), which may contain different sets of opsin proteins. Variations in the spatial expression of the different opsins within the different cell types, as well as in the spatial distribution of ommatidial types throughout the compound eyes also strongly influence the evolution of sensitivity to light and color discrimination (Arikawa 2003). The co-expression of opsins in cells has been documented in *Heliconius* butterflies, with *LWRh* and *BRh* co-expression participating in spectral-tuning (McCulloch et al. 2022). However, the discrimination of a wider range of colors is also influenced by the evolution of filtering pigments occurring within the ommatidia (Wakakuwa et al. 2004; Zaccardi et al. 2006). The evolution of color discrimination might therefore stem from various molecular and cellular processes: reconstructing the evolutionary steps leading to divergent visual systems thus requires investigating not only the evolution of opsins sequences, but also their levels of expression as well as their spatial distribution within and among ommatidia.

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Identifying the respective contribution of the different selective pressures acting on the evolution of opsin properties and spatial distribution within the eyes is challenging, because vision is involved in multiple fitness-related traits in many animals, such as foraging (Zhang 2012), mate recognition (Smith et al. 2002; Gomez et al. 2010) and/or orientation (Franzke et al. 2020). Contrasted evolution of opsins has been shown among species living in different light environments (Horth 2007): for example, in aquatic environments, opsins were found to be differentially expressed between cichlids (Ricci et al. 2023) and cardinalfishes (Luehrmann et al. 2020) species living in different photic micro-habitats (shallow vs. deep waters). Ecological interactions can also shape the evolution of color discrimination capacities in animals. Visual cues are often used in mate choice and species recognition (in invertebrates; Detto 2007), and in vertebrates; (Dollion et al. 2020), potentially promoting the selection of different
99 visual sensitivities in different taxa. As the display of bright colorations is often associated with traits
100 contributing to fitness (e.g. boldness; Godin and Dugatkin 1996), low parasite load; (Maan et al. 2006),
101 body condition; (Pérez i de Lanuza et al. 2014)), sexual selection could favour the evolution of specific
102 color sensitivity. The evolution of color discrimination could also be promoted between closely-related
103 species sharing the same habitat, as poor species recognition could lead to substantial deleterious
104 reproductive interference (Gröning and Hochkirch 2008), although the literature still lacks empirical
105 evidence.

106 The rise of genomics has permitted the study of the evolution of opsin sequences at very large
107 phylogenetic scales (Musilova et al. 2021; Murphy and Westerman 2022; Schott et al. 2024),
108 highlighting an important effect of the light environment on the macro-evolution of visual systems.
109 However, sexual selection is more likely to influence opsin evolution at a smaller phylogenetic scale, as
110 revealed in fishes (Sandkam et al. 2015) and butterflies (Chakraborty et al. 2023). Investigating the
111 diversification of opsin gene sequences and patterns of expression in closely-related species with
112 contrasted ecologies can thus shed light on the respective effects of biotic and abiotic selective pressures
113 shaping the evolution of the opsin gene family.

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115 Here, we focus on the evolution of visual proteins within the neotropical butterfly genus *Morpho*, where
116 sympatric species are distributed across different vertical forest strata (Chazot et al. 2016; Chazot et al.
117 2021), as well as different temporal niches (Le Roy et al. 2021). Such specialisation into different spatio-
118 temporal microhabitats is associated with a strong heterogeneity in the light environment (Nilsson et al.
119 2022). The divergent light environment encountered by different *Morpho* species could thus influence
120 the evolution of their color discrimination capacities. Furthermore, these different species strongly differ
121 in the structural and pigmentary colors of their wings. Behavioral experiments carried out in blue
122 *Morpho* species showed that iridescent blue coloration can be a cue used by males in intra or interspecific
123 interactions, like male-male competition and female recognition (Le Roy et al. 2021; Ledamoisel et al.
124 2025). Consequently, as shades of iridescent blue vary between species, different sensitivities to blue
light could have evolved within this genus. In a lycaenid species displaying a blue wing color pattern, a
blue opsin duplication was indeed detected (Sison-Mangus et al. 2006). Similarly, some *Heliconius*
species displaying UV-colored pigments on their wings have accurate UV discrimination due to
duplicated *UVRh* (Briscoe et al. 2010). Visual modelling combined with behavioral experiments
revealed that this additional violet receptor could facilitate the discrimination of conspecifics during
mate choice (McCulloch et al. 2017; Finkbeiner and Briscoe 2021), highlighting the effect of sexual
interactions as selective agent acting on opsin evolution in butterflies.

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The recent sequencing of the genomes of three blue *Morpho* species has revealed duplications in the
*LWRh* opsins with three gene copies, in sharp contrast with other *Satyrinae* butterflies (Bastide et al.
2022), raising the question of the evolutionary origin of such duplications and of the selective regimes
acting on the five opsin genes throughout the *Morpho* genus. Recent studies also investigated the
different light sensitivities of the photoreceptor cells in the blue species *Morpho helenor* (Belušič et al.
2021; Pirih et al. 2022), also calling for an investigation of the spatial distribution of opsins in different
photoreceptors.

We thus investigate the evolution of opsin gene sequences and expression in the genus *Morpho*, where
divergent selection stemming from the light environment and from the wing coloration in closely-related
species might have shaped the evolution of opsins properties. We conducted a comprehensive study of
the visual system of *M. helenor* by characterizing all the sequences of opsin genes found in its genome,
quantifying opsin mRNA expression in the eyes of males and females, and localizing the corresponding
opsin proteins within the receptive cells in different ommatidia composing their retina. We then
investigated the evolution of opsin genes among 18 *Morpho* species, by (1) testing for signature of
selection against the null hypothesis of neutral evolution and (2) assessing the role of amino-acid co-
evolution in shaping the diversification of their visual systems. In particular, we tested whether the
ecological environment or the co-evolution with their wing coloration could have influenced the
150 diversification of their photosensitive proteins. Our study thus aims at identifying the ecological and
151 molecular mechanisms driving the evolution of visual systems.

152 **Results**

153 *Opsin diversity, expression and localization in the eye of M. helenor.*

154 The complete coding sequences of the butterfly opsins were retrieved from the previously published *M. helenor* genome (Bastide et al. 2022) allowing recovery of the three *LWRh* genes (further referred as *LW1Rh*, *LW2Rh* and *LW3Rh*), as well as the *UVRh* and *BRh* genes. We established contig-to-chromosome correspondence between the *M. helenor* genome assembly and the *Maniola jurtina* reference chromosomes and found that *LW1Rh*, *LW2Rh* and *LW3Rh* genes were located collinearly on the contig corresponding to chromosome 4 in *M. jurtina*. In contrast, *UVRh* and *BRh* were found on the contig corresponding to chromosome 9, separated by a distance of approximately 4 to 6 Mb (see Table S1). An Illumina analysis of the eye RNA expression confirmed that all five opsin genes (*UVRh*, *BRh*, *LW1Rh*, *LW2Rh* and *LW3Rh*) are expressed in the eye tissues of both males and females. A differential gene expression analysis did not reveal any differential expression of those opsin genes between sexes (Figure S1). However, individual ANOVAs performed on the expression levels of each opsin gene separately show that females express more *LW1Rh* (Kruskal-Wallis test; adjusted *p*-value = 0.045) and *LW2Rh* (Kruskal-Wallis test; adjusted *p*-value = 0.045) opsin genes than males (Figure S2).

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168 UVRh, BRh, and *LW1Rh* opsin proteins are expressed in the R1 and R2 photoreceptor cells of
169 Morpho helenor *eyes*.

170 Using custom polyclonal antibodies, we then specifically characterized the localization of the *UVRh*,
171 *BRh* and *LW1Rh* opsin proteins within *M. helenor* eyes using an immunochemistry approach. We found
172 expression of the *UVRh*, *BRh*, and *LW1Rh* opsin proteins in the R1 and R2 photoreceptors of both female
173 and male specimens of *M. helenor* (Figure 1 A & B). There was no cross reaction between these three
174 antibodies and we saw no evidence of co-localization of *BRh*, *UVRh*, or *LW1Rh*.
175 The proportion of cells expressing the different opsin proteins was strongly uneven: while more than
176 half of the photoreceptor cells expressed the *BRh* opsins in both males and females, the proportion of
177 photoreceptor expressing *UVRh* and *LW1Rh* was around 20% each, in both sexes (Figure S3). Although
178 the retina of both males and females expressed a majority of *BRh* photoreceptors, males expressed
179 significantly more *BRh* photoreceptors than females (Chi-square: *p-value* < 0.001).

180 Based on the proteins detected in the R1 and R2 photoreceptors, we then inferred that there are at least
181 six ommatidial types within the eye of *M. helenor* (Fig. 1 C-E), and we characterized the distribution of
182 these ommatidial types in males and females. Chi-square testing shows a significant difference between
183 male and female ommatidium distribution (Chi-square: *p-value* < 0.001). In males, the most abundant
184 ommatidial type is *BRh-BRh*, representing approximately 35% of receptors compared to approximately
185 25% of ommatidia in females (Figure S3). Overall, our results thus show similar ommatidial types in
186 males and females of *M. helenor*, and sexual dimorphism in the proportion of these different types of
187 ommatidia.

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<figure><img src="image_2.png" /><figcaption>[Figure 1. Characterization of opsin protein expression in the different ommatidia of males and females *M. helenor*. Anti-BRh (blue), anti-LW1Rh (green), and anti-UVRh (magenta) antibody staining of (A) female and (B) male *M. helenor* retina. Scale bars represent 20 microns. Individual ommatidium types identified in (C) female and (D) male retina based on BRh, LW1Rh, and UVRh opsin expression. (J) Cartoon of the at least six ommatidia types based on opsin expression in the R1 and R2 photoreceptor cells of *M. helenor*.]</figcaption></figure>
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198 *Opsin diversification at the genus scale in Morpho butterflies.*

199 In order to study the evolution of opsin genes throughout the *Morpho* genus, we used *Morpho* genome assemblies from (Bastide et al. 2022) and (López Villavicencio et al. 2024) to retrieve the opsin sequences from additional *Morpho* species. The *UVRh*, *BRh* and *LWRh* genes detected in the genome of *M. helenor* were also found in the assembled genomes of 14 other *Morpho* species (*M. marcus*, *M. eugenia*, *M. granadensis*, *M. amathonte*, *M. menelaus*, *M. deidamia*, *M. achilles*, *M. godartii*, *M. telemachus*, *M. hecuba*, *M. theseus*, *M. niepelti*, *M. cypris* and *M. rhetenor*). All 15 species contain the complete coding sequences of the five opsins, covering all 8 exons. Because the *LWRh* gene was found in three distinctive copies in all 15 assembled species, the two duplication events are not limited to *M. helenor*, but rather likely occurred before the diversification of the *Morpho* genus. The analysis of the transcriptome of the eyes of *M. achilles*, *M. deidamia*, *M. hecuba*, *M. rhetenor* and *M. marcus* males showed that all five opsin genes are expressed in the eyes of those species (Table S2). These results suggest that the five *UVRh*, *BRh*, *LW1Rh*, *LW2Rh* and *LW3Rh* genes could be fully functional and involved in color vision in understory species (*M. achilles*, *M. deidamia*), canopy species (*M. hecuba*, *M. rhetenor*) and phylogenetically basal species (*M. marcus*).

213 Complementarily, we used PCR amplification to retrieve the opsin sequences of the *Morpho* species for which assembled genomes were not available. The PCR amplification allowed to retrieve some partial genomic *LW2Rh* and *LW3Rh* opsin sequences (6 exons) in 3 additional *Morpho* species (*M. aurora*, *M. sulkowskyi* and *M. cisseis*). We thus obtained opsin sequences for 18 *Morpho* species out of 30, including blue and non-blue species, inhabiting both the canopy and the understory (Figure 2).

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<figure><img src="image_3.png" /></figure>

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[Figure 2: Opsin duplications and ecological and morphological characteristics of the 18 species of the genus *Morpho* used in this study. The phylogenetic relationships among the studied species was retrieved from the phylogeny published in (Chazot et al. 2021)). The colored tiles show the retrieved *UVRh*, *BRh*, *LW1Rh*, *LW2Rh* and *LW3Rh* opsin genes for each *Morpho* species. The opsin sequences retrieved from the species marked with black stars were amplified using PCR and are thus only partial sequences. Note that the absence of some opsins in species where whole-genome sequence was not available is more likely due to a lack of amplification because of genetic divergence between species, rather than an actual absence of the genes. The male dorsal wing pattern (Wing color: filled blue symbols = blue-iridescent wings vs. open symbols = non blue-iridescent wings), and the habitat (Habitat: light-green symbols = understory species vs. dark-green symbols = canopy species) is provided for each studied species.]
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232 The phylogenetic reconstruction of the partial *LWRh* genes of *Morpho* butterflies (Figure 3) showed that

233 the *LW3Rh* gene is the most divergent gene compared to the other two *LW1Rh* and *LW2Rh* genes.

234 The phylogeny of the *LW3Rh* gene follows the phylogenetic species tree of the *Morpho* genus. However,

235 the extensive length of the branch of the *LW3Rh* opsin of *M. eugenia* and *M. marcus* might stem from a

236 particular selective regime acting on the visual sensitivities in these two species. The history of the

237 *LW1Rh* and *LW2Rh* duplications reveals a slightly more complex pattern. For instance, the *LW1Rh* and

238 *LW2Rh* genes of *M. eugenia* and *M. marcus* have a contrasted history as compared to the other species

239 and do not branch with their respective *LW1Rh* and *LW2Rh* clade formed by the rest of the *Morpho*

240 species. This pattern suggests that the 6 exons used to reconstruct this tree may undergo a different

241 regime of selection in *M. marcus* and *M. eugenia* than on the other branches. Note that when

242 reconstructing the phylogenetic tree of the *LWRh* opsins using only complete sequences (with less

243 individuals but covering 8 exons), *LW1Rh* and *LW2Rh* genes from *M. marcus* and *M. eugenia* both

244 branch at the base of their respective monophyletic clade (Figure S4), further suggesting that different

245 regime of selection might only affect some specific exons.

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<figure><img src="image_4.png" /></figure>

[Figure 3: Maximum likelihood gene tree for the partial 6 exon sequences of *LWRh Morpho opsins* and amino acid substitutions associated with putative spectral sensitivity shifts. The gene tree was inferred using IQtree under a TIM2e+I+G4 evolutionary model estimated with the ModelFinder option using nucleotide *LWRh* sequences. Bootstrap values for each node are represented in the tree. The long wavelength opsins of *Bicyclus anynana* (GenBank number: AY918895.2, (Frentiu et al. 2007)) and *Danaus plexippus* (GenBank number: AY605545.1, (Sauman et al. 2005)) were used as outgroups. *LW3Rh* genes are shown in dark-red, *LW2Rh* genes in orange and *LW1Rh* genes in yellow. We used colored dots to designate amino acid substitutions found on the *LWRh* sequences of *Morpho* associated with a spectral shift in other butterfly species (respective AA from (Frentiu et al. 2007) are numbered on *Limenitis archippus archippus* and AA from (Saito et al. 2019) are numbered on bovine rhodopsin. The homologous *Morpho* numbering can be found in Table S3).]
259 *Variations at spectral tuning sites: evidence of putative spectral sensitivity shifts in BRh and LWRh*
260 *opsin genes in the Morpho genus.*

261 Because the amino acid sequence of a photoreceptive protein determines its capacity to absorb specific
262 wavelengths, we checked whether variations of amino acids associated with tuning sites known in other
263 butterflies could be observed in *Morpho* species. Amino acid numberings are taken from the original
264 sources (Frentiu et al. 2007; Wakakuwa et al. 2010; Frentiu et al. 2015; Saito et al. 2019; Liénard et al.
265 2021) but the equivalent *Morpho* numbering is given in Table S3.
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267 *BRh opsin tuning sites*

268 Compared to previous observations made on other butterflies, *Morpho BRh* opsin sequences are
269 composed of amino acids associated with visual blue sensitivity shifts such as S116, F177 (Wakakuwa
270 et al. 2010; Frentiu et al. 2015; Liénard et al. 2021) and R216 (Frentiu et al. 2015). A F177Y substitution
271 is observed in two blue *Morpho* living in the canopy (*M. rhetenor* and *M. cypris*) and two blue *Morpho*
272 living in the understory (*M. amathonte* and *M. godartii*) only, suggesting that the absorption of the *BRh*
273 protein of those species might be slightly red-shifted compared to the other *Morpho* species. However,
274 the amino acid sequences of *M. rhetenor* and *M. cypris* are also composed of A301 (Frentiu et al. 2015),
275 an amino acid associated with subtle blue sensitivity shifts, suggesting that some amino acid shifts are
276 unique to blue canopy species (see Figure S5 and Figure S6). Functional validation is still needed to
277 understand the combined effect of substitutions associated with putatively opposite effects.
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279 *LWRh opsins tuning sites*

280 The amino acid changes involved in spectral shifts detected among *Morpho LWRh* opsins are shown in
281 Figure 3. The amino acid changes N70S and S137A are both correlated with a blue spectral shift of the
282 L-opsin absorption in nymphalid butterflies (Frentiu et al. 2007). In *Morpho*, the S137A amino acid
283 change occurred in the *LW1Rh* and *LW2Rh* lineages but not in the *LW3Rh* opsin, suggesting the *LW1Rh*
284 and *LW2Rh* opsin proteins might be sensitive to shorter wavelengths than the *LW3Rh* protein.
285 Interestingly, a A137S reversion was found in the *LW1Rh* opsin of the non-blue canopy species *M. hecuba*, which could thus have a *LW1Rh* protein with a closer sensitivity to the *LW3Rh* protein compared
286 to the other *Morpho* species.
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288 Amongst the amino acid changes linked with a spectral shift of the protein, some can also cause red
289 absorption shifts. For instance, the A116G amino acid change shifts the maximum of absorbance of
290 opsins toward red wavelengths (Saito et al. 2019). In *Morpho*, the corresponding amino acid change
291 occurs in the *LW3Rh* opsin protein for five species only, including 2 blue canopy species (*M. rhetenor*
292 and *M. cypris*) and 3 blue understory species (*M. marcus*, *M. eugenia* and *M. sulkowskyi*, see Figure 3,
293 Figure S7).
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295 The blue-shifting amino acid mutations in the *LW1Rh* and *LW2Rh* protein sequences and red-shifting
296 amino acid mutations in the *LW3Rh* opsin sequences strongly suggest that the duplicated *Morpho* *LWRh*
297 opsins might be sensitive to different long wavelengths, consistent with the evolution of divergent visual
298 systems in the *Morpho* genus.

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300 Evidence of positive selection on the BRh and LW3Rh Morpho opsin genes

301 We then studied the ratio between non-synonymous and synonymous substitutions in the coding
302 sequence of the different *Morpho* opsins ($dN/dS=\omega$) to characterize the selection regime acting on the
303 evolution of the five opsin sequences. No signal of pervasive positive selection was found on the 5
304 *Morpho* opsin genes using random site models implemented in PAML (Table S4). However, we found
305 significant evidence of episodic positive selection on the *BRh* opsin gene ($\omega_{BRh}= 4.890$, *p-value* = 0.002)
306 and the *LW3Rh* opsin gene ($\omega_{BRh} = 2.89$, *p-value* < 0.0001) using BUSTED at a gene-wide scale (Table
307 S5). A FUBAR analysis was run on those two genes to identify specific positively selected amino acid
308 sites (Table S6). The sites 113, 189, 224, 232, 246, 310 and 363 (site numbering relative to *Morpho*
309 opsins) were found to be under positive selection in the *BRh* opsin gene, and the sites 88, 133, 136, 179,
183, 205, 224, 229, 236, 305 and 318 showed signatures of positive selection in the *LW3Rh* gene. While none of these sites corresponds to known tuning sites in other butterfly species, they are either located near those sites or in the vicinity of the chromophore (see Figure 4).

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To investigate the potential change in evolutionary rates among the three *LWRh* opsin duplications, we performed branch-site models to test for positive selection on different *LWRh* tree branches (see Methods). All branch-site models testing for positive selection on the *LWRh* tree came out non-significant. We also tested for positive selection in the branch leading to the *M. rhetenor/M. cypris* pair (the only two blue iridescent *Morpho* species living in the canopy) vs. the rest of the tree in the *BRh* gene and found no signal of positive selection on these branches (Table S7).

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<figure><img src="image_5.png" /><figcaption>[Figure 4: Positive selection on the *BRh* (A) and *LW3Rh* (B) opsin proteins in *Morpho*. The amino acids detected by the FUBAR analysis are represented in stick figures, and colored depending on their probability of being under positive selection (posterior probabilities): from 65 to 80% (green), from 80 to 95% (orange) and from 95% to 100% (red). The approximated chromophore location is pictured in purple.]</figcaption></figure>
331

332 *Testing for ecological factors influencing the evolution of Morpho opsins.*

333 We looked for shifts in selective regimes acting on the sequences of the five *Morpho* opsin genes
334 depending on the species ecologies, using the CmC PAML model. We specifically annotated the *BRh*,
335 *UVRh*, and *LWRh* opsin trees depending on their wing phenotype and habitat (Figure 5, statistical
336 analyses in Table S8).

337

338 First, we found a significant effect of wing coloration on the evolution of the *LW2Rh* and *LW3Rh* genes,
339 with the species exhibiting blue wings having a higher ω than the non-blue species (*LW2Rh*:
340 ω<sub>LW3Rh</sub> = 0.61 vs. ω<sub>LW3Rh_Non-blue</sub> = 0.00 and *LW3Rh*: ω<sub>LW3Rh</sub> = 0.91 vs. ω<sub>LW3Rh_Non-blue</sub> = 0.03).

341 Second, the CmC analysis based on the habitat distribution of *Morpho* suggests that *UVRh* and *LW3Rh* opsins show different evolutionary rates between understory and canopy species, with canopy species having a higher ω *UVRh* than understory species (*ω*<sub>*UVRh*</sub> = 0.55 vs. *ω*<sub>*UVRh_understory*</sub> = 0.31), while the trend is reversed for the *LW3Rh* gene (*ω*<sub>*LW3Rh_canopy*</sub> = 0.22 vs. *ω*<sub>*LW3Rh_understory*</sub> = 0.77). Finally, when performing a clade model on the global phylogeny of the *LWRh* genes, comprising the data for *LW1Rh*, *LW2Rh* and *LW3Rh*, both the wing color partition and the habitat partition were significant, with blue species having a higher ω than the non-blue species (*ω*<sub>*LWRh*</sub> = 0.23 vs. *ω*<sub>*LWRh_Non-blue*</sub> = 0.14), and understory species having a higher ω than the canopy species (*ω*<sub>*LWRh_canopy*</sub> = 0.09 vs. *ω*<sub>*LWRh_understory*</sub> = 0.24).

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<figure><img src="image_6.png" /></figure>

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356

[Figure 5: Variation of Morpho opsin *dN/dS* depending on wing coloration (A), habitat (B) and Light environment (C). The *dN/dS* of blue species are marked in blue, in white for non-blue species, in dark-green in canopy species, in light-green in understory (Fg = foreground, Bg = background). The number of analyzed genes are shown below each opsin type.]

357

358

*Evidence of correlated amino acid evolution between LWRh opsin genes.*

359

360

361

362

363

Finally, we used the Evo-Scope pipeline (Godfroid et al. 2024) to determine whether the evolution of some amino acids in *Morpho* opsins could influence the evolution of other amino acids within the same protein or impact the evolution of the sequence of other opsin proteins. The Evoscope analysis showed signals of correlated evolution between amino acids both (1) within the same opsin protein and (2) among different *LWRh* proteins (Figure 6, statistical analysis in Table S9).

364

365

366

367

368

369

Within the *LW3Rh* opsin protein, several amino acids exhibiting a significant signal of correlated evolution were located in areas characterized by a high density of tuning sites and sites under positive selection (between sites 131 and 145), suggesting a hot-spot of adaptive evolution in this region of the protein. In particular, site 134, located near two sites under positive selection (133 and 136) and two tuning sites (137 and 140), has been identified as being linked to the evolution of site 138. The Evoscope results also highlighted that the evolution of the amino acids at site 134 can also induce the evolution of
370 amino acids at site 224, a site located near the chromophore of the protein, and showing signs of positive selection.
371

372 Interestingly, signals of correlated evolution were also found between amino acids belonging to different LWRh proteins: the evolution of two sites located in the LW1Rh protein and 3 sites in the LW2Rh protein was linked to the evolution of sites sitting in the LW3Rh protein. Among these sites of the LW3Rh proteins potentially influenced by the evolution of the other two LWRh opsins, two sites (site 135 and site 140) were located in the previously identified hotspot zone (between sites 131 and 145).
373
374
375
376

377

378 We separately analyzed the correlated evolution of the amino acids found within the BRh opsin protein (Figure S8) and the UVRh opsin protein (Figure S9) respectively. We found only two pairs of amino acids evolving under correlated evolution in the UVRh protein. In the BRh protein, some pairs of amino acids are located next to known tuning sites for this protein, but we found no hotspot zone similar to the one found in the LW3Rh protein.
379
380
381
382

383
<figure><img src="image_7.png" /><figcaption>[Figure 6: Correlated amino-acid evolution within and between the three *LWRh* opsins observed throughout the genus *Morpho*. The sequence of the *LW1Rh* protein is colored in yellow, the sequence of the *LW2Rh* is colored in orange and the *LW3Rh* protein is colored in dark-red. The tuning sites and the positively selected sites are located on each protein using blue and red arrows respectively. The amino acid correlations detected by Evo-scope are represented by arcs coloured depending on the p-value for each test, using arrows to represent the direction of the induction and displaying the amino acid numbering involved in the correlated evolution. The hotspot zone identified in the *LW3Rh* protein is highlighted in the box.]</figcaption></figure>

393

394

395

396
397 **Discussion**

398 *Duplications and patterns of expression of opsins form a peculiar visual system in M. helenor.*

399 The RNA expression analyses performed on *M. helenor* eye tissue confirmed that all 5 opsin genes,
400 including the three different *LWRh* copies, are expressed in the eyes of *M. helenor* butterflies from both
401 sexes. Our immunohistochemical assays then showed that *UVRh* and *BRh* opsins are expressed in R1
402 and R2 photoreceptive cells, consistent with previous observations in other butterfly visual systems (e.g.
403 *Vanessa cardui*, (Briscoe et al. 2003); *Heliconius*, (McCulloch et al. 2017); *Danaus plexippus*, (Sauman
404 et al. 2005); *Pieris rapae*, (Arikawa et al. 2005)). Surprisingly, *LW1Rh* expression in *M. helenor* was
405 restricted to the R1-R2 receptive cells, and we did not detect any co-expression of *BRh*, *UVRh* or *LW1Rh*
406 in any cell type. This *LWRh* pattern of expression is uncommon among various butterfly families as
407 long-wavelength sensitive opsin genes are usually expressed in R3 to R8 cells (Briscoe 2008). However,
408 these results are consistent with previous polarization sensitivity measurements performed on the eyes
409 of *M. helenor* (Pirih et al. 2022), showing that R1 and R2 cells are strongly sensitive to UV and Blue
410 light, and also sensitive to Green or Green/Yellow light, which could be absorbed by *LW1Rh* opsins.
411 Although we were not able to characterize the pattern of expression of the *LW2Rh* and *LW3Rh* proteins
412 in the eyes of *Morpho*, we assume these are likely to be expressed in cells R3 to R8 because these cells
413 are also sensitive to the Green and Yellow light according to the polarisation sensitivity reported in (Pirih
414 et al. 2022). We thus described 6 different ommatidial types in *M. helenor* (Figure 1), but unknown
415 *LW2Rh* or *LW3Rh* expression patterns may increase the number of possible ommatidia types in *M.
416 helenor*.

417

418 This peculiar expression pattern of opsins is also associated with a sexually dimorphic expression pattern
419 of some opsins in *M. helenor*. In particular, higher RNA *LW1Rh* expression was found in the eyes of
420 females, supported by a higher *LW1Rh* photoreceptor count at the protein level. Furthermore, a high
421 number of *BRh* photoreceptors was found in males *M. helenor* eyes, associated with a high frequency
422 of *BRh-BRh* ommatidia types in males compared to females (see Figure S3). Such sexual dimorphism
423 in opsin protein expression has been reported in several taxa, and can take many forms: sexual
dimorphism can encompass variation in opsin co-expression in different eye photoreceptor cells (Sison-
Mangus et al. 2006), the lack of opsin expression in a photoreceptor cell of one sex compared to the
other (McCulloch et al. 2017), and variations in the number of photoreceptor cells (Hilbrant et al. 2014).
427 Sexual dimorphism could arise from different selective pressures affecting the evolution of the visual
systems of males and females. For instance, the behaviors of *Morpho* males and females strongly differ:
428 in the wild, males display a typical patrolling behavior and show a striking response to blue stimuli (Le
430 Roy et al. 2021). On the other hand, females tend to fly in dense forest areas and to spend time locating
431 and flying around host-plants. Those different ecological conditions encountered by males and females
432 might generate divergent selection on visual systems. Long-distance detection of blue color might be
433 advantageous in males searching for females and might promote increased sensitivity to blue
434 wavelengths. Although *M. helenor* males do not have duplicated *BRh* opsin genes as other blue sensitive
435 butterflies (Lycaenids, (Sison-Mangus et al. 2006); Pieridae, (Arikawa et al. 2005; Awata et al. 2009;
436 Wakakuwa et al. 2010)), their retina is composed of a higher proportion of *BRh-BRh* photoreceptors
437 than other blue butterflies (Sison-Mangus et al. 2006). In contrast, discrimination in the green
438 wavelength might be promoted in females, because of the significance of host-plant detection in this
439 sex, as suggested in other butterflies with sexually-dimorphic visual sensitivities (Finkbeiner and
440 Briscoe 2021).

441 The novelty of the *M. helenor* eye organization compared to other butterflies and the number of
442 expressed opsin genes in its visual system shows a divergent evolution of opsin gene expression in
443 *Morpho* butterflies as compared to other Nymphalidae, that may result in the evolution of color
444 discrimination capacities.

445

446 *Light environment and wing iridescence strongly influence the evolution of the genes involved in the*
447 *visual system of Morpho.*

448
The five opsin genes described in *M. helenor* were also found in the genome of 14 additional *Morpho* species. RNA expression data confirmed that those five genes are expressed in the eyes of all tested species, suggesting that this specific visual system organization might likely be ancestral to the diversification of *Morpho*. While neutral evolution contributes to the divergence of opsin genes among species, the divergent selection generated by the different biotic and abiotic conditions encountered in the different micro-habitats occupied by the different species could also impact the evolution of the opsin sequences across the genus.

Models testing for episodic positive selection on the 5 opsin genes revealed significant signals of positive selection mostly on the sequences of *BRh* and *LW3Rh* genes. Since several positively selected sites detected in our study are located close to previously-documented tuning sites and/or close to the chromophore, our results are consistent with an effect of selection on the diversification of visual sensitivities in those genes.

First, the analysis of the amino acid sequence of the *BRh* gene in *Morpho* showed that four species exhibit amino acid changes involving known tuning sites. In particular, *M. rhetenor* and *M. cypris*, the only two blue *Morpho* species living in the canopy in this dataset, display amino acid substitutions at two different tuning sites, that could putatively cause shifts in visual perception. This result suggests that *M. rhetenor* and *M. cypris* could have a different visual perception than the rest of the *Morpho* genus. However, no significant signal of selection specific to this lineage was found, suggesting that this difference might be due to phylogenetic divergence only. Overall, we found that the evolutionary rate of the *BRh* opsin was not significantly different between canopy and understory species, and surprisingly did not find a correlation between the evolution of the *BRh* opsin sequences and the presence or absence of blue iridescent coloration on the wings of *Morpho* butterflies. In the context of mate choice or male-male competition, we would expect that blue visual perception would be different between blue and non-blue species. Interestingly, while spectral sensitivity data suggest that the blue absorbing cells of *M. helenor* might be sensitive to approximately 450 nm (Pirih et al. 2022), iridescence quantification
475 showed that the peak of reflectance of *M. helenor* wings can vary from 350 nm to 500 nm (UV/purple
to green) depending on the angle of illumination (Ledamoisel et al. 2025), suggesting that different color
signals generated by the wings might be processed by other opsin genes during mate searching.
476
477

478 In line with this hypothesis, we found that *LWRh* genes are very diversified among *Morpho*. Our results
479 show that some amino acid substitutions found on those proteins could be associated with different
480 spectral shifts affecting the three *LWRh* gene copies: indeed, *LW1Rh* and *LW2Rh* tend to bear blue-
481 shifting amino acids as compared to *LW3Rh*. Although functional validation is needed, the *LW3Rh*
482 protein could thus be responsible for the absorption peak found in *Morpho* at 570 nm in (Pirih et al.
483 2022), while the *LW1Rh* and *LW2Rh* proteins could be associated with the absorbance peaks found at
484 505nm and 551nm (Pirih et al. 2022). Long-wavelength sensitive opsin diversification has been reported
485 in a number of taxa (fishes: (Watson et al. 2011); Hemiptera: (Xu et al. 2021); Coleoptera: (Sharkey et
486 al. 2021); Anurans: (Schott et al. 2022)) including butterflies (*Papilio xuthus*, (Briscoe 2008)), and is
487 often associated with change in light habitats and/or niche differentiation (Carleton et al. 2020). In
488 *Morpho*, we found that the *dN/dS* of the *LW2Rh* and *LW3Rh* opsins of the blue iridescent species is
489 significantly higher than the *dN/dS* of the *LW2Rh* and *LW3Rh* genes of the non-blue iridescent species,
490 consistent with higher rate of adaptive evolution of *LWRh* genes in blue iridescent species. Such
491 evolution might have been promoted by selection favouring detection and discrimination of the blue-
492 green iridescent signals displayed by the wings of blue *Morpho* species. Interestingly, we also found
493 that the evolutionary rate of the *LW3Rh* gene is significantly higher in the *Morpho* species living in the
494 understory compared to those living in the canopy. The understory habitat is dominated by vegetation:
495 as the discrimination of subtle long-wavelength color variation might be advantageous in a forest
496 environment in terms of foraging and oviposition (Kelber 1999), this habitat is likely an important driver
497 of green/red color discrimination in *Morpho*. The understory is also a darker environment than the
498 canopy: in particular, the amount of UV-light is significantly reduced in the understory in tropical forests
499 compared to the canopy (Brown et al. 1994; Nilsson et al. 2022). The higher evolutionary rate of the
500 *UVRh* opsin in the canopy as compared to understory species suggests that heterogeneous light
501 availability might have influenced the evolution of this specific opsin gene in those habitats.
502

503 *The co-occurrence of mutations between sites located on different opsin proteins suggests a joint*
504 evolution of LWRh genes.

505

506 Because wavelength discrimination by the visual systems depends on the shape of sensitivity peaks and
507 on the distance among them, controlled by the respective sensitivities of each opsin protein, its evolution
508 might involve integrated changes across the different opsin genes. To assess this coevolution, we
509 specifically tested for correlated shifts in amino acids both within and among opsins.

510 Within the *LW3Rh* protein, five pairs of amino acid sites displayed a significant signal of correlated
511 evolution, meaning that a change of amino acid at site A was found to coincide with a change of amino
512 acid at site B more often than expected by chance given the *Morpho* phylogeny. Because purifying
513 selection tends to promote mutations that would stabilize the structure or the function of a protein,
514 coupled amino acid evolution within a protein is expected: amino acid substitutions at a site could
515 compensate for deleterious substitutions at other sites of the protein (Pál et al. 2006). However, amino
516 acid changes could also be driven by adaptation. Considering the direct link between amino acid
517 sequence and wavelength sensitivity (Hofmann and Lamb 2023), this seems particularly plausible in
518 opsin proteins. Amino acid variations in the *LW3Rh* revealed correlated evolution between 2 pairs of
519 sites located very close (1 or 2 sites away) to either known tuning sites in butterflies, or positively
520 selected sites detected by our previous analysis. Among those, site 134 was found to induce amino acid
521 changes at the positively selected site 224, highlighting the intertwined evolution of key-sites in specific
522 areas of the protein.

523 Moreover, we also found co-occurring mutations between amino acid sites belonging to different *LWRh*
524 proteins, suggesting concerted evolution among those three genes. Specifically, we show that mutations
525 found on specific amino acid sites in *LW1Rh* co-evolved with mutations found on LW3Rh amino acid
526 sites close to tuning sites, in what looks like a hotspot of adaptive evolution. Correlated mutations
527 between proteins have been extensively observed between proteins part of a network and physically
528 interacting with one another (Pazos et al. 1997), but correlations in the evolution of genes sharing
529 functional relationships are much less documented. Yet, visual perception can be seen as the result of
530 the integration of visual signals absorbed by different opsin proteins, potentially creating an indirect link
531 between the evolution of those genes, whether in the form of shared regulatory mechanisms or similar
532 evolutionary forces. For example, following an opsin duplication, the gain of function of the new protein
533 could impact the selection regime of the other to maintain fine-tuned spectral sensitivity. It is the case
534 in Lycaenidae where coordinated shifts of amino acids have been found in the *BRh* and *LWRh* opsins,
535 enhancing their visual perception (Liénard et al. 2021). Although our approach is only correlative and
536 functional validation is needed, it underlines the importance of studying the molecular co-evolution of
537 opsins, because of the extensive evolutionary tinkering involved in the diversification of visual system
538

538

539 **Conclusions**

540 Altogether, our results evidence the peculiar evolution of visual systems in closely-related species
541 evolving in divergent microhabitats and illustrate how the evolution of the different opsins protein
542 properties and patterns of expression may be shaped by the specific selective pressures generated by
543 different micro-habitats or contrasted sexual selection. By jointly studying the correlated evolution of
544 different genes as well as their spatial pattern of expression, our study highlights the multiple pathways
545 enabling the evolution of visual discrimination capacities finely-tuned by selective processes.
546

546

547 **Materials and Methods**

548 **Characterisation of opsins expression and spatial localisation in *Morpho helenor***

549 *Testing RNA differential expression between males and females M. helenor*

550 We used the previously assembled genome of *M. helenor* (Bastide et al. 2022) to retrieve the sequence
551 of the 5 opsin genes (*BRh*, *UVRh*, *LW1Rh*, *LW2Rh* and *LW3Rh*) found in this *Morpho* species. In order
to test whether opsin genes were expressed in *Morpho helenor* tissues, the eye transcriptome of five
males and five females *M. helenor theodorus* were sequenced. The specimens were purchased from a
breeding farm located in Ecuador (Quinta De Goulaine, https://quintadegoulaine.com/es/papillons.php)
and raised in insectaries at STRI in Gamboa, Panama, between January and March 2023. The Qiagen
RNAeasy Mini Kit was used to extract the RNA of each sample following the manufacturer's
instructions. Library preparation and Illumina sequencing were performed at the Ecole normale
supérieure GenomiqueENS core facility (Paris, France). Messenger (polyA+) RNAs were purified from
250 ng of total RNA using oligo(dT). Libraries were performed using the strand specific RNA-Seq
library preparation *Stranded mRNA Prep, Ligation kit* (Illumina) and were multiplexed by 39 on a P3
flowcell (Illumina). A 118 bp single end read sequencing was performed on a NextSeq 2000 device
(Illumina). The analyses were performed using the Eoulsan pipeline (Jourdren et al. 2012), including
read filtering, mapping, and alignment filtering. Before mapping, poly N read tails were trimmed, reads
≤40 bases were removed, and reads with quality mean ≤30 were discarded. Reads were then aligned
against the *Morpho helenor* genome using STAR version 2.7.8a (Dobin et al. 2013). Alignments from
reads matching more than once on the reference genome were removed using Java version of samtools
(Li et al. 2009). To compute gene expression, the *Morpho helenor* genome annotation was used. All
overlapping regions between alignments and referenced exons were counted and aggregated by genes
using FeatureCounts v2.0.6 (Liao et al. 2014). The sample counts were normalized using DESeq2 1.42.1
(Love et al. 2014). Statistical treatments and differential analyses were also performed using DESeq2
1.42.1, using an adjusted p-value threshold set at 0.05.

The number of opsin reads was extracted and their relative expression was analyzed separately from the
rest of the transcriptome, and Kruskall-Wallis tests were used to test for the effect of sex in the number
of expressed opsin reads. To account for the multiple testing on the 5 opsin genes, we use a Bonferroni
test to compute adjusted p-values.

576

577 *Spatial distribution of opsin protein within Morpho helenor eyes revealed by immunohistochemistry*
We used an immunohistochemistry approach to locate the opsin proteins within the eyes of *M. helenor*.
First, we used the translated amino acid sequences of *M. helenor UVRh*, *BRh*, *LW1Rh*, *LW2Rh*, and *LW3Rh* opsins to identify antigenic, accessible, and unique peptides as candidate targets for polyclonal antibody production. An antibody against the peptide GIVKQVFAHEAALRE in the loop domain between helix 5 and 6 of *UVRh* was generated in guinea pigs, an antibody against the peptide GWNIPEEHQDLVHE in the N-terminus domain of *BRh* was generated in goat, an antibody against a peptide GSDTGPGISC in the N-terminus domain of *LW1Rh* was generated in rabbit (Biosynth International, Gardner, MA, USA).

Then, we purchased *M. helenor* pupae from Costa Rica Entomological Supply to test these specific antibodies on fresh eyes. Upon delivery, pupae were hung in greenhouse enclosures where they were housed post-eclosure and until processing. Butterflies freely flew in the enclosures and fed on moistened orange slices. Five male and five females were processed for immunohistochemistry.

Methods were adapted from previous studies (Hsiao et al. 2012; Perry et al. 2016; McCulloch et al. 2017; Chakraborty et al. 2023). Butterflies were euthanized by quickly crushing the thorax and beheading them. In 1x PBS, the heads were bisected and excess tissue was removed under a dissecting scope. The eyes were fixed in 4% paraformaldehyde for 30 minutes in a cold room (approximately 15 °C) with rotation. Fixed eyes then underwent step-wise sucrose baths (10, 20, then 30% sucrose in 1x PBS) for two hours for each step in a 15°C cold room with rotation. The corneal lenses of the eyes were carefully removed under a dissecting scope, and each eye was then embedded in gelatin-albumin blocks. Blocks were then fixed in 4% formalin (in 1x PBS) for 15 hours at 4 °C. Embedded tissue gelatin-albumin blocks were sliced with a Precisionary Compresstome VF-310-0Z to 60 micron slices. Slices were blocked in a blocking solution of 5% (v/v) normal donkey serum and 0.3% triton-X in 1x PBS (0.3% PBST) for one hour at room temperature. The slices were incubated overnight at 4 °C with primary antibodies (1:200 goat anti-Blue, 1:100 guinea pig anti-UVRh, and 1:100 rabbit anti-LW1 in blocking solution). Afterwards, the slides underwent five washes with 0.3% PBST for 15 minutes per wash at room temperature. The slides were then incubated overnight in darkness at 4 °C with secondary antibodies (1:250 donkey anti-goat AlexaFluor 488, 1:250 donkey anti-guinea pig AlexaFluor 647, and
605 1:250 donkey anti-rabbit Cy3 in blocking solution). Again, they were washed five times with 0.3%
606 PBST for 15 minutes per wash and mounted in 70% glycerol. Images were taken using a Zeiss LSM
607 900 Airyscan 2 confocal microscope under a 20x/0.8NA dry objective in the UC Irvine Optical Core
608 Facility and exported using ZenBlue 3.5.

609

610 **Investigating the evolution of opsin sequences throughout the Morpho genus**

611 *Retrieving the opsin sequences from 15 Morpho reference genomes*

612 The visual opsin sequences of 8 *Morpho* species observed in the understory habitat (*M. helenor*, *M. marcus*, *M. eugenia*, *M. granadensis*, *M. amathonte*, *M. menelaus*, *M. deidamia*, *M. achilles*) and 3 *Morpho* species found in the canopy habitat (*M. telemachus*, *M. hecuba*, and *M. rhetenor*) were retrieved from the annotations of the *Morpho* genome assemblies published in (Bastide et al. 2022) and (López Villavicencio et al. 2024). Four additional genomes including 1 understory species (*M. godartii*) and 3 canopy species (*M. theseus*, *M. niepelti* and *M. cypris*) were specifically obtained for this study and their sequencing, assembly and annotation were performed following the same pipeline as described in (López Villavicencio et al. 2024). Among the 30 *Morpho* species described in the literature (Chazot et al. 2021), we thus had access to the genomic data of 15 *Morpho* species scattered across the phylogeny. Overall, one *UVRh*, one *BRh* and three *LWRh* (referred to as *LW1Rh*, *LW2Rh* and *LW3Rh* hereafter) opsin genes were identified within each of these 15 genomes. To compare opsin gene positions across all *Morpho* species with available chromosome level assemblies, we scaffolded *Morpho* contigs against the *Maniola jurtina* chromosome-level assembly (ilManJurt1.1) using RagTag (Alonge et al. 2022). All scaffolds were renamed to match the chromosome nomenclature of *M. jurtina*.

626

627 *Sequencing of eye transcriptome of six Morpho species*

628 To check for opsin RNA expression in different *Morpho* species scattered across the phylogeny of the
629 genus, the eyes of freshly caught males belonging to 5 sympatric species from French Guiana (*M*.
achilles, M. deidamia, M. hecuba, M. rhetenor and M. marcus), were dissected and stored in RNAlater at -80°C before extraction. RNA was extracted using the Qiagen RNeasy Mini Kit according to the manufacturer's instructions and stored at -80°C until use. The transcriptome of each sample was sequenced using Nanopore technology. Library preparation and Nanopore sequencing were performed at the Ecole normale supérieure GenomiqueENS core facility (Paris, France). 10 ng of total RNA were amplified and converted to cDNA using a modified version of the protocol described in (Guilcher et al. 2021). Briefly, the ligation and rRNA depletion steps were skipped and the oligo GCAGGGGAAATCATCAGCGTATAACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN was used for the first strand cDNA synthesis. The oligos TTTCTGTTGGTGCTGATATTGCAAGCAGTGGTATCAACGCAGAGTAC and ACTTGCCTGTCGCTCTATCTTCGCAGGGGAAATCATCAGCGTATAAC were used for the amplification of the full-length cDNAs. Afterwards an average of 15 fmol of amplified cDNA was used for the library preparation. After the PCR adapter ligation, a 0,6X Agencourt Ampure XP beads clean-up was optimised and 2 fmol of the purified product was taken into PCR for amplification and barcodes addition with a 17 minutes elongation at each 18 cycles (barcodes from SQK-PCB111, ONT). Samples were pooled in equimolar quantities to obtain 25 fmol of cDNA and the rapid adapter ligation step was performed. Libraries were multiplexed by 9 on one flowcell FLO-PRO002 according to the manufacturer's protocol. Sequencing was performed with the SQK-PCB111 72-hour sequencing protocol run on the PromethION P2 solo, using the MinKNOW software (versions 5.9.12). A mean of 11 ± 2,8 million passing ONT quality filter reads was obtained for each of the samples. Base-calling from read event data was performed by dorado-0.7.1 sup. Structural annotation was performed following a custom protocol published in protocols.io (DOI: dx.doi.org/10.17504/protocols.io.36wgqd5qyvk5/v1). The extracted reads were assembled on the respective Morpho reference genomes from (Bastide et al. 2022) and (López Villavicencio et al. 2024) and the RNA counts were extracted using FeatureCounts v2.0.6 (Liao et al. 2014). As we collected data for only one individual per species, this analysis was used to assess the presence of expressed opsin genes in the eyes of Morpho but not their absence, using TPM counts.
657

658 *Amplification of additional opsin sequences throughout the Morpho genus*

659 In order to retrieve the opsin sequences of the 15 *Morpho* species for which assembled genomes are not
660 available, the coding sequences of the *UVRh*, *BRh* and *LWRh* opsins from *M. helenor* and *M. telemachus*
661 were used to design specific PCR primers (see Table S10) targeting these three genes with Primer3
662 (Untergasser et al. 2012).

663 PCRs were performed on these 15 *Morpho* species, using DNA samples from collection specimens
664 stored in the National Museum of Natural History in Paris, France. The DNA of those *Morpho* was
665 extracted using the Qiagen extraction DNeasy Blood Tissue Kits. Each PCR reaction was run using
666 20µL of solution containing 11.25µL of water, 4µL of Buffer 5X for Taq LongAmp, 1.25µL of dNTPs
667 (6.6mM), 1µL of BSA (5mg/mL), 0.625µL of DMSO, 0.475µL of primers (10pM), 0.375µL of Taq
668 LongAmp (ThermiFischer) and 3µL DNA. The thermal cycling conditions for the amplification started
669 with a 30s denaturation at 94°C, followed by a gradual decrease of the hybridization temperature during
670 18 PCR cycles, from 61°C to 52°C, and a 10 min elongation step at 65°C (touch-down method). For the
671 following 50 cycles, the hybridization temperature was fixed to 52°C.

672 The PCR products were sequenced using the short-read Illumina technology. The Geneious Prime
673 program (2022.1.1 version) was then used to reconstruct the whole opsin sequences. Adding to the 15
674 *Morpho* species already studied from *Morpho* genomes, this method allowed to retrieve partial *LWRh*
675 opsin sequences for 2 other understory species (*M. aurora*, *M. sulkowskyi*) and 1 canopy species (*M.
676 cisseis*).

677

678 Sequence alignment and tree reconstruction

679 The coding opsin sequences (exons only) of each opsin, retrieved from 18 *Morpho* species, were aligned
680 using the MUSCLE algorithm (Edgar 2004) implemented in MEGA X (Tamura et al. 2021). As our
681 *UVRh* and *BRh* opsin sequence dataset is composed of complete sequences only, the phylogenetic tree
682 of the *UVRh* and *BRh* opsins were generated using the alignment of those complete genes (8 exons)
683 across 15 *Morpho* species. However, because the *LW1Rh*, *LW2Rh* and *LW3Rh* opsins sequence dataset
684 contains both complete (i.e. whole-genome based) and partial opsin (i.e. PCR based) sequences, we
685 generated the associated *LWRh* phylogenetic tree using an alignment of the 6 exons sequences in all
686 species studied (n = 18). Independent phylogenetic trees were also generated for the *LW1Rh*, *LW2Rh*
687 and *LW3Rh* genes respectively, allowing for separate analyses within each *LWRh* copy. IQtree (Nguyen
688 et al. 2015) was used to generate the phylogenies, and the ModelFinder option (Kalyaanamoorthy et al.
689 2017) was used to select the best respective evolutionary models for each tree. Nodes' robustness was
690 estimated using Ultrafast Bootstrap (Hoang et al. 2018) on 10000 iterations.

691

692 *Detection of amino-acids changes acting on spectral sensitivity*

693 To identify amino acid changes associated with changes in spectral sensitivity in other butterfly species,
694 we used data from the literature (Frentiu et al. 2007; Wakakuwa et al. 2010; Frentiu et al. 2015; Saito et
695 al. 2019; Liénard et al. 2021) to locate so-called tuning sites within the *BRh* and *LWRh* opsin sequences
696 of the *Morpho* butterflies studied here. We used the MUSCLE algorithm (Edgar 2004) implemented in
697 MEGA X (Tamura et al. 2021) to align the *Morpho* opsin sequences to the opsin sequences of other
698 butterfly species to find the tuning-sites in *Morpho*. This method allows identifying whether the amino
699 acids variations observed among *Morpho* species detected in our positive selection analyses are likely
700 to modify the sensitivity to different wavelengths.

701

702 *Detection of signature of selection acting on the evolution of opsin sequences*

703 To characterize the selection regime acting on the evolution of opsin sequences and assess departure
704 from neutral evolution, we studied the ratio between the non-synonymous (dN) and synonymous (dS)
705 mutations in the coding sequences of the different opsins ω=dN/dS. A gene with a higher non-
706 synonymous substitution rate compared to its synonymous substitution rate (ω>1) is said to be under
707 positive selection. The *codeml* program from the software package PAML 4.9j (Yang and others 1997)
708 was used to test for gene-wide pervasive selection acting on the evolution of visual opsins. To investigate
709 whether selection is acting on specific codons in every branch of the opsin gene trees, we performed
710 model comparisons using several optional models proposed by PAML (Site Models M0, M1a, M2a,
711 M7, M8). First, to ensure the selective pressure varies among the codons of opsin genes, the M0 model
712 was compared to the M1a model. Then, the M2a and M8 models were used to assess positive selection
713 on each opsin gene, by comparing their prediction to their respective null models M1a and M7. A Bayes
714 Empirical Bayes (BEB) analysis was used to identify the positively selected amino acids among the
715 genes under positive selection. As PAML's Site Model is not suited to detect gene-wide selection
716 occurring only in a few subsets of branches and sites, we also used the BUSTED program (Murrell et
717 al. 2015) from the software HYPHY v2.5.65 (Kosakovsky Pond et al. 2020) to test for episodic selection
718 on *Morpho* opsin genes. This method uses a mixed effects model allowing $\omega$ to vary across sites and
719 branches, and detects signature of selection happening on at least one branch. For genes showing
720 significant results, we applied the FUBAR program (Murrell et al. 2013) to identify specific amino acids
721 evolving under positive selection across lineages. The gene trees used for PAML's site model, BUSTED
722 and FUBAR are available in Figure S10.

723

724 Additionally, PAML's branch-site models were also computed to estimate the selective pressures acting
725 at specific codon sites within different lineages of the *LWRh* gene phylogeny. In order to test whether
726 the duplicated *LWRh* opsin genes follow distinct regimes of selection, the branches of the gene
727 phylogeny were partitioned into three clades comprising the branches leading to (1) the *LW1Rh* genes,
728 (2) the *LW2Rh* genes and (3) the *LW3Rh* genes, excluding *M. marcus* and *M. eugenia* branches. We
729 generated three distinct branch-site models to test whether the $\omega$ of those clades were different from the
730 $\omega$ of the branches of the rest of the phylogeny. As *M. eugenia* and *M. marcus*, the most basal species of
731 the phylogeny, seem to follow a peculiar evolution, the branches leading to (1) the *LW1Rh* genes, (2)
732 the *LW2Rh* genes and (3) the *LW3Rh* genes of those two species were also used as "foreground" branches
733 and to generate three additional branch-site models (see Figure S11 for branch annotations).
734 Furthermore, we also used PAML's branch-site model to test for positive selection in specific branches
735 of the *BRh* gene, especially to look for any specific pattern of selection in the pair *M. rhetenor*/*M. cypris*
736 vs. the rest of the phylogeny. To assess positive selection, the results of the branch-site models were
737 compared to their respective null models, which assume the same ω among all branches.

738

739 Finally, in order to test the effects of ecological factors on the evolution of opsin sequences, while
740 accounting for phylogenetic relationships between species, we used a clade model (PAML's Cmc
741 Model: M2a_rell (null) vs. Model C) to analyze putative differential evolution of the opsin genes
742 between species sharing a blue iridescent phenotype and others (Blue vs. Non-Blue), species living in
743 the canopy or the understory (Canopy vs. Understory).

744

745 Detection of correlated amino acid evolution

746 We used the Evo-Scope pipeline described in (Godfroid et al. 2024) to determine whether the evolution
747 of some amino acids in a given opsin protein could (i) influence the evolution of other amino acids
748 within the same protein or (ii) impact the evolution of the sequence of other opsin proteins. The Evo-
749 Scope pipeline was designed to study correlated evolution of biological discrete traits by accounting for
750 the phylogenetic structure of the data. After a step of ancestral character reconstruction, a first tool
751 (*epics*, (Behdenna et al. 2016) is used to identify pairs of co-occuring mutations on each branch of the
752 tree. Second, after selecting the pairs of traits showing a significant signal of correlated evolution, the
753 *epocs* method (Behdenna et al. 2022) determines which trait influences the evolution of the other under
754 different scenarios among each pair.

755 In order to apply this method to our data, we considered each amino acid of the 3 partial *LWRh* opsin
756 proteins (6 exons) as discrete traits to analyze the co-occurence of amino acid shifts found across the
757 phylogeny. We applied this method only for the species for which we had the opsin data for the 3 LWRh
758 genes (*n* = 15). As the gene trees of the 3 *LWRh* opsins are similar but not identical, we first performed
759 Shimodaira-Hasegawa tests implemented in IQ-tree (Nguyen et al. 2015) to evaluate whether the
760 likelihood difference between each gene's own topology and alternative trees was statistically different
761 (Table S11). As the *LW3Rh* gene tree showed the highest compatibility with the *LW1Rh*, *LW2Rh* and *LW3Rh* alignments, we used this phylogeny to study the correlated evolution of the amino acids found in the *LW1Rh*, *LW2Rh* and *LW3Rh* opsin genes. After identifying amino acid pairs showing signals of correlated evolution, we compared whether some of those amino acid sites were associated with known tuning sites or positively selected sites, to understand the global impact selection can have on the evolution of *LWRh* opsin proteins.

767 As *BRh* and *UVRh* genes are more divergent compared to the *LWRh* genes, we did not measure the correlated evolution of the amino acids of those three proteins. However, we computed the correlated evolution of the amino acids among *BRh* and *UVRh* opsins respectively.

771 *Structural representation of opsin proteins*

772 To locate the positively selected amino acid sites and the sites associated with a signal of correlated evolution within the protein and specifically their proximity to the chromophore, we predicted the 3D structure of the different opsins of *M. helenor* using the online platform CollabFold v 1.5.2 (Mirdita et al. 2022). The platform combines the *Alphafold2* algorithm (Jumper et al. 2021) for protein structure prediction, and the model *MMseq2* to generate the sequence alignments. The protein structures were edited in *PyMOL* (The PyMOL Molecular Graphic System, Version 2.6 Schrödinger, LLC) to highlight the amino acids previously detected as under positive selection. The putative chromophore location was inferred from the chromophore location determined in the jumping spider rhodopsin (Varma et al. 2019). Complementarily, we used *Protter* (Omasits et al. 2014) to generate a 2D representation of each gene and annotated it with the relevant amino acid sites detected in this study.

783 **Data availability:**

784 All sequences used for the analyses will be available on GenBank upon publication.

785
786 Acknowledgements:

787 The authors would like to thank Guillaume Achaz for the advice provided on the use of the EvoScope
788 pipeline. We are also grateful to Owen McMillan from the Smithsonian Tropical Research Institute
789 (Panama) for providing facilities to raise *M. helenor theodorus*. We thank Etienne Delannoy from the
790 POPS facility (IPS2) for conducting with GenomiqueENS the adaptation of the ONT RNA-seq protocol.

791 All the bioinformatic analyses were performed on the Plateforme de Calcul Intensif et Algorithmique
792 PCIA (Muséum national d'histoire naturelle, Centre national de la recherche scientifique), the MeSU
793 platform at Sorbonne-Université and the Genotoul bioinformatics platform Toulouse Occitanie (Bioinfo
794 Genotoul, https://doi.org/10.15454/1.5572369328961167E12). We exported the eyes of *M. helenor*
795 *theodorus* from Panama using exportation permit number PA-01-ARB-028-2023, and declared to the
796 French authorities the exportation of butterfly eyes from French Guiana to the French metropole. J.L
797 PhD was funded by an IBEES grant from Sorbonne Université. The GenomiqueENS core facility was
798 supported by the France Génomique national infrastructure, funded as part of the "Investissements
d'Avenir" program managed by the Agence Nationale de la Recherche (contract ANR-10-INBS-0009).

800 This study was funded by the European Union (ERC-2022-COG - OUTOFTHEBLUE - 101088089)
801 and by the Human Frontier Science Program grant (RGP005/2023,
802 https://doi.org/10.52044/HFSP.RGP0052023.pc.gr.168591). Views and opinions expressed are
803 however those of the authors only and do not necessarily reflect those of the European Union or the
804 European Research Council. Neither the European Union nor the granting authority can be held
805 responsible for them.

806

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