Marilou P. Sison-Mangus

Positive selection of a duplicated UV-sensitive visual pigment coincides with wing pigment evolution in Heliconius butterflies.

The butterfly Heliconius erato can see from the UV to the red part of the light spectrum with color vision proven from 440 to 640 nm. Its eye is known to contain three visual pigments, rhodopsins, produced by an 11-cis-3-hydroxyretinal chromophore together with long wavelength (LWRh), blue (BRh) and UV (UVRh1) opsins. We now find that H. erato has a second UV opsin mRNA (UVRh2)—a previously undescribed duplication of this gene among Lepidoptera. To investigate its evolutionary origin, we screened eye cDNAs from 14 butterfly species in the subfamily Heliconiinae and found both copies only among Heliconius. Phylogeny-based tests of selection indicate positive selection of UVRh2 following duplication, and some of the positively selected sites correspond to vertebrate visual pigment spectral tuning residues. Epi-microspectrophotometry reveals two UV-absorbing rhodopsins in the H. erato eye with λmax = 355 nm and 398 nm. Along with the additional UV opsin, Heliconius have also evolved 3-hydroxy-DL-kynurenine (3-OHK)-based yellow wing pigments not found in close relatives. Visual models of how butterflies perceive wing color variation indicate this has resulted in an expansion of the number of distinguishable yellow colors on Heliconius wings. Functional diversification of the UV-sensitive visual pigments may help explain why the yellow wing pigments of Heliconius are so colorful in the UV range compared to the yellow pigments of close relatives lacking the UV opsin duplicate.

Beauty in the eye of the beholder: the two blue opsins of lycaenid butterflies and the opsin gene-driven evolution of sexually dimorphic eyes

SUMMARY Although previous investigations have shown that wing coloration is an important component of social signaling in butterflies, the contribution of opsin evolution to sexual wing color dichromatism and interspecific divergence remains largely unexplored. Here we report that the butterfly Lycaena rubidus has evolved sexually dimorphic eyes due to changes in the regulation of opsin expression patterns to match the contrasting life histories of males and females. The L. rubidus eye contains four visual pigments with peak sensitivities in the ultraviolet (UV;λ max=360 nm), blue (B; λmax=437 nm and 500 nm, respectively) and long (LW; λmax=568 nm) wavelength range. By combining in situ hybridization of cloned opsinencoding cDNAs with epi-microspectrophotometry, we found that all four opsin mRNAs and visual pigments are expressed in the eyes in a sex-specific manner. The male dorsal eye, which contains only UV and B (λmax=437 nm) visual pigments, indeed expresses two short wavelength opsin mRNAs, UVRh and BRh1. The female dorsal eye, which also has the UV and B (λmax=437 nm) visual pigments, also contains the LW visual pigment, and likewise expresses UVRh, BRh1 and LWRh mRNAs. Unexpectedly, in the female dorsal eye, we also found BRh1 co-expressed with LWRh in the R3-8 photoreceptor cells. The ventral eye of both sexes, on the other hand, contains all four visual pigments and expresses all four opsin mRNAs in a non-overlapping fashion. Surprisingly, we found that the 500 nm visual pigment is encoded by a duplicate blue opsin gene, BRh2. Further, using molecular phylogenetic methods we trace this novel blue opsin gene to a duplication event at the base of the Polyommatine+Thecline+Lycaenine radiation. The blue opsin gene duplication may help explain the blueness of blue lycaenid butterflies.

Color discrimination in the red range with only one long-wavelength sensitive opsin

SUMMARY The basic precondition for color vision is the presence of at least two receptor types with different spectral sensitivities. The sensitivity of a receptor is mostly defined by the opsin-based visual pigment expressed in it. We show here, through behavioral experiments, that the nymphalid butterfly Heliconius erato, although it expresses short and medium wavelength opsins and only one long wavelength opsin, discriminates colors in the long-wavelength range (590 nm, 620 nm and 640 nm), whereas another nymphalid, Vanessa atalanta, despite having color vision, is unable to do so. In the eyes of H. erato we identified filtering pigments very close to the rhabdom which differ between ommatidia and produce the yellow and red ommatidial reflection seen under orthodromic illumination. The eyes of V. atalanta lack the filtering pigments, and reflect a homogeneous orange. We hypothesize that the filtering pigments found in the eyes of H. erato may shift the spectral sensitivity peak of the long wavelength receptors in some ommatidia towards longer wavelengths. The comparison of the signals between the two new receptor types makes color discrimination in the red range possible. To our knowledge, this is the first behavioral proof of color vision based on receptors expressing the same opsin.

Impact of duplicate gene copies on phylogenetic analysis and divergence time estimates in butterflies

BackgroundThe increase in availability of genomic sequences for a wide range of organisms has revealed gene duplication to be a relatively common event. Encounters with duplicate gene copies have consequently become almost inevitable in the context of collecting gene sequences for inferring species trees. Here we examine the effect of incorporating duplicate gene copies evolving at different rates on tree reconstruction and time estimation of recent and deep divergences in butterflies.ResultsSequences from ultraviolet-sensitive (UVRh), blue-sensitive (BRh), and long-wavelength sensitive (LWRh) opsins, EF-1α and COI were obtained from 27 taxa representing the five major butterfly families (5535 bp total). Both BRh and LWRh are present in multiple copies in some butterfly lineages and the different copies evolve at different rates. Regardless of the phylogenetic reconstruction method used, we found that analyses of combined data sets using either slower or faster evolving copies of duplicate genes resulted in a single topology in agreement with our current understanding of butterfly family relationships based on morphology and molecules. Interestingly, individual analyses of BRh and LWRh sequences also recovered these family-level relationships. Two different relaxed clock methods resulted in similar divergence time estimates at the shallower nodes in the tree, regardless of whether faster or slower evolving copies were used, with larger discrepancies observed at deeper nodes in the phylogeny. The time of divergence between the monarch butterfly Danaus plexippus and the queen D. gilippus (15.3–35.6 Mya) was found to be much older than the time of divergence between monarch co-mimic Limenitis archippus and red-spotted purple L. arthemis (4.7–13.6 Mya), and overlapping with the time of divergence of the co-mimetic passionflower butterflies Heliconius erato and H. melpomene (13.5–26.1 Mya). Our family-level results are congruent with recent estimates found in the literature and indicate an age of 84–113 million years for the divergence of all butterfly families.ConclusionThese results are consistent with diversification of the butterfly families following the radiation of angiosperms and suggest that some classes of opsin genes may be usefully employed for both phylogenetic reconstruction and divergence time estimation.

The lycaenid butterfly Polyommatus icarus uses a duplicated blue opsin to see green.

SUMMARY The functional significance of gene duplication is rarely addressed at the level of animal behavior. Butterflies are excellent models in this regard because they can be trained and the use of their opsin-based visual pigments in color vision can be assessed. In the present study, we demonstrate that the lycaenid Polyommatus icarus uses its duplicate blue (B2) opsin, BRh2, in conjunction with its long-wavelength (LW) opsin, LWRh, to see color in the green part of the light spectrum extending up to 560 nm. This is in contrast to butterflies in the genus Papilio, which use duplicate LW opsins to discriminate colors in the long-wavelength range. We also found that P. icarus has a heterogeneously expressed red filtering pigment and red-reflecting ommatidia in the ventral eye region. In behavioural tests, the butterflies could not discriminate colors in the red range (570–640 nm). This finding is significant because we have previously found that the nymphalid butterfly Heliconius erato has filter-pigment mediated color vision in the long wavelength range. Our results suggest that lateral filtering pigments may not always influence color vision in insects.

Reply to Nozawa et al.: Complementary statistical methods support positive selection of a duplicated UV opsin gene in Heliconius

Statistical methods used to test for positive selection have a long history and continue to evolve (1–4). In their letter, Nozawa et al. (5) question our use of the branch-site method in our recent paper in PNAS (6). As experimental biologists, we welcome all methods that facilitate the detection of interesting parts of the genome for functional exploration. In this case, a statistically significant result using the branch-site method combined with structural modeling and the identification of a few biochemically relevant substitutions provided us with an incentive for the in vivo physiological characterization of the UV-sensitive rhodopsins in Heliconius. Had we not had that first hint from sequence data alone, it is unlikely that we would have made that functional discovery, which makes the eyes of Heliconius unique compared with all other studied butterflies.