Gene Duplication Is an Evolutionary Mechanism for Expanding Spectral Diversity in the Long-Wavelength Photopigments of Butterflies

Butterfly long-wavelength (L) photopigments are interesting for comparative studies of adaptive evolution because of the tremendous phenotypic variation that exists in their wavelength of peak absorbance (λmax value). Here we present a comprehensive survey of L photopigment variation by measuring λmax in 12 nymphalid and 1 riodinid species using epi-microspectrophotometry. Together with previous data, we find that L photopigment λmax varies from 510-565 nm in 22 nymphalids, with an even broader 505- to 600-nm range in riodinids. We then surveyed the L opsin genes for which λmax values are available as well as from related taxa and found 2 instances of L opsin gene duplication within nymphalids, in Hermeuptychia hermes and Amathusia phidippus, and 1 instance within riodinids, in the metalmark butterfly Apodemia mormo. Using maximum parsimony and maximum likelihood ancestral state reconstructions to map the evolution of spectral shifts within the L photopigments of nymphalids, we estimate the ancestral pigment had a λmax = 540 nm ± 10 nm standard error and that blueshifts in wavelength have occurred at least 4 times within the family. We used ancestral state reconstructions to investigate the importance of several amino acid substitutions (Ile17Met, Ala64Ser, Asn70Ser, and Ser137Ala) previously shown to have evolved under positive selection that are correlated with blue spectral shifts. These reconstructions suggest that the Ala64Ser substitution has indeed occurred along the newly identified blueshifted L photopigment lineages. Substitutions at the other 3 sites may also be involved in the functional diversification of L photopigments. Our data strongly suggest that there are limits to the evolution of L photopigment spectral shifts among species with only one L opsin gene and that opsin gene duplication broadens the potential range of λmax values.