MBE Advance Access originally published online on July 3, 2007
Molecular Biology and Evolution 2007 24(9):2016-2028; doi:10.1093/molbev/msm132
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Research Articles |
Gene Duplication Is an Evolutionary Mechanism for Expanding Spectral Diversity in the Long-Wavelength Photopigments of Butterflies
* Department of Ecology and Evolutionary Biology, University of California, Irvine
Department of Electrical Engineering, University of Washington
Department of Biology, Middle Tennessee State University
E-mail: abriscoe{at}uci.edu.
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Abstract |
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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 (
max value). Here we present a comprehensive survey of L photopigment variation by measuring max in 12 nymphalid and 1 riodinid species using epi-microspectrophotometry. Together with previous data, we find that L photopigment max 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 max 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 max = 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 max values.
Key Words: visual pigment • gene duplication • Nymphalidae • Riodinidae • opsin • color vision
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsSupplementary MaterialAcknowledgementsReferences |
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Butterfly color vision, like that of other insects, is based on 3 major classes of photoreceptors, with peak sensitivity (
max) in the ultraviolet (UV, 300–400 nm), blue (B, 400–500), and long-wavelength (L, 500–600) regions of the light spectrum (reviewed in Briscoe and Chittka 2001
; Stavenga and Arikawa 2006). At the molecular level, these photopigments are comprised of a retinal-based chromophore (e.g., 11-cis-3-hydroxyretinal) surrounded by an opsin protein. The spectral tuning of the photopigment max value is achieved through the interaction of the chromophore with critical amino acid residues within the opsin, a mechanism that has been most extensively studied in vertebrate photopigments (Asenjo et al. 1994; Fasick and Robinson 1998; Yokoyama and Radlwimmer 1998; Wilkie et al. 2000). Changes in the polarity of amino acids in the chromophore-binding pocket of opsins, for instance, affect the distribution of electrons in the 
-electron system of the chromophore, producing a diversity of max values (Honig et al. 1976). In the case of the insect photopigments, the 3 major spectral classes are encoded by ancient duplications, which produced distinct UV, B, and L opsin genes.
Diversification of butterfly photopigments has also occurred through more recent lineage-specific gene duplications. For example, duplicate blue opsins have diversified into blue- and violet-absorbing photopigments in the Pieridae (Arikawa et al. 2005) and duplicate blue opsins have diversified into blue- and blue-green–absorbing photopigments in the Lycaenidae (Sison-Mangus et al. 2006). The extent to which spectral diversification has occurred via natural selection on a single opsin locus, however, is less clear.
To investigate the role of opsin gene duplication versus selection on a single opsin locus in producing spectral diversity, we focused on the L photopigments of butterflies. Earlier photochemical studies suggested some butterfly eyes contain 2 L photopigments, whereas others contain only 1 (Bernard 1979). In the adult compound eye, the papilionid Papilio xuthus has 3 L photopigments (max = 515 nm, 530 nm, and 575 nm; Arikawa et al. 2003), whereas the pierid Pieris rapae, the lycaenid Lycaena rubidus, and the nymphalid Vanessa cardui each have only one L photopigment (max = 563 nm, 568 nm, and 530 nm, respectively; Bernard 1983a; Bernard and Remington 1991; Wakakuwa et al. 2004). Of these families, we selected nymphalids for study because photochemical, molecular, and anatomical work has indicated that the typical nymphalid eye contains only one L photopigment (along with UV and blue photopigments mentioned above) (Briscoe et al. 2003; Sauman et al. 2005; Zaccardi et al. 2006) (see below) and physiological diversity of this photopigment in nymphalids ranges from 510–545 nm (Stavenga 1975; Bernard 1983b; Briscoe and Bernard 2005; Vanhoutte and Stavenga 2005; Frentiu et al. 2007).
Recently, we reported that the 31-nm range of L photopigment variation found within the nymphalid genus Limenitis is due to positive selection-driven adaptation at a single L opsin gene (Frentiu et al. 2007). In that analysis, we identified substitutions at 4 amino acid sites (Ile17Met, Ala64Ser, Asn70Ser, and Ser137Ala), which are strongly correlated with blueshifts in L photopigment max. We therefore sought to investigate the extent to which substitutions at these sites are correlated with blue spectral shifts in a taxonomically expanded data set of L opsin photopigments.
To address this question, we determined the peak absorbance of L photopigments from 12 species of nymphalids and compared them with our previous studies of the L photopigments of 9 other nymphalids performed on the same experimental apparatus. We then examined L opsin transcripts from 8 of the 12 taxa as well as an expanded set of 11 nymphalid species for which no physiological data are available. We included the riodinid Apodemia mormo in our screen for L opsin transcripts because its eye contains an unusually redshifted L photopigment (max = 600 nm) as well as a second L photopigment (max = 505 nm) whose absorbance spectra we report below (Bernard 1979; Bernard et al. 1988). We discovered 3 novel L opsin gene duplications and further evidence that substitutions at the afore mentioned 4 amino acid sites are indeed involved in the evolution of L photopigment spectral shifts. Taken together, our data suggest that gene duplication is an evolutionary mechanism for expanding the spectral diversity of L photopigments in butterflies.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsSupplementary MaterialAcknowledgementsReferences |
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Photochemical and Physiological Measurements
Absorption spectra of L photopigment can be measured directly from eyes of completely intact butterflies, thanks to 2 unusual properties. The first is that photoisomerization of the dark-adapted L photopigment (rhodopsin) creates a blueshifted photoproduct (i.e., metarhodopsin) that is unstable (the metarhodopsin photopigment decays more rapidly than the rhodopsin photopigment regenerates). The second unusual property, in many species of butterfly, is that each ommatidium contains a multilayered tracheolar tapetum that creates eyeshine.
Each retinular cell has a rhabdomere, the microvillar membrane of which is packed with photopigment molecules. The 9 rhabdomeres of an ommatidium are fused into a single cylindrical rhabdom that functions as a fiber-optic waveguide. Eyeshine is created by light entering the cornea, traveling down the rhabdom waveguide, reaching the end of the rhabdom, reflecting from the tapetum, and then traveling back out of the eye where it is observable as eyeshine. Because the volume of the rhabdom is packed with photopigment, the reflectance spectrum of eyeshine is influenced by absorption spectra of all photopigments and their photoproducts contained within the volume of the rhabdom.
As described in the supplementary materials and methods (Supplementary Material online), we use an epi-microspectrophotometer to create partial bleaches of L photopigment, measure eyeshine reflectance spectra and difference spectra, and analyze those spectra to estimate the absorption spectrum of L photopigment and its wavelength for maximal absorbance, max. We used this method to survey the diversity of L photopigment absorbance spectra in 22 species from 7 nymphalid subfamilies (Asterocampa leilia, Archaeoprepona demophon, Danaus plexippus, Heliconius charithonia, Heliconius erato, Heliconius hecale, Agraulis vanillae, Anartia jatrophae, Euphydryas chalcedona, Hermeuptychia hermes, Limenitis archippus archippus, Limenitis archippus floridensis, Limenitis arthemis astyanax, Limenitis lorquini, Limenitis weidemeyerii, Inachis io, Junonia coenia, Nymphalis antiopa, Neominois ridingsii, Oeneis chryxus, Siproeta stelenes, and V. cardui; table 1).
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We also present the absorbance spectra of the R505 and R600 photopigments of the riodinid A. mormo including details not found in earlier reports (Bernard 1979
; Bernard et al. 1988). Estimates of absorbance spectra for H. erato R555 and A. mormo R505 were based on optophysiological measurements of spectral sensitivity. Details are provided in the supplementary materials and methods (Supplementary Material online).
Butterfly Tissue Sampling
We then sampled the L opsin gene from 9 of the 13 taxa for which we were able to measure L photopigment max values and combined these with previously published L opsin sequences for H. erato and Heliconius sara (Hsu et al. 2001; Zaccardi et al. 2006), and D. plexippus (Sauman et al. 2005). The L opsin genes of the riodinid butterfly, A. mormo, are of special interest taxonomically because this represents an exemplar of the only other butterfly family (out of 5) not yet examined for opsin genes, and this family together with the lycaenids are sister taxon to the nymphalids (Campbell et al. 2000). In addition, we sampled 11 other nymphalid taxa for which no physiological data are available. Butterflies were either caught by the authors as adults in the field (table 1) or kindly provided as pupae or adults by Carol Boggs (H. charithonia), John Emmel (A. mormo), Matthew Garhart (N. ridingsii), Antonia Monteiro (Bicyclus anynana), and Lincoln Brower (D. plexippus). Tissue was preserved for either RNA or genomic DNA extraction.
Polymerase Chain Reaction, Cloning, and Sequencing
Total mRNA was extracted from 8 species (A. vanillae, Speyeria mormonia, E. chalcedona, B. anynana, Coenonympha tullia, O. chryxus, N. ridingsii, and A. mormo) using TRIzol (Invitrogen, Carlsbad, CA). Double-stranded complementary DNA (cDNA) was synthesized from total RNA using the Marathon cDNA Amplification Kit (BD Biosciences Clontech, Mountain View, CA). The 3'-RACE (rapid amplification of cDNA ends) products were amplified with a degenerate primer (5'-GAA CAR GCW AAR AAR ATG A-3') by polymerase chain reaction (PCR) (2 min at 94 °C; 35 cycles of 30 s at 94 °C, 30 s at 50 °C, and 1 min at 68 °C). Products were then ligated into the pGEM T-easy vector (Promega, Madison, WI), transformed into Escherichia coli (JM109 strain) and plasmids purified with the QIAprep Spin Miniprep Kit (Qiagen, Valencia, CA). Clones containing inserts were sequenced using the Big Dye Terminator 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). The 5'-RACE products were obtained by designing species-specific primers (available upon request) from 3'-RACE products. Products were amplified using the touchdown PCR protocol specified with the BD Advantage Polymerase Kit (BD Biosciences, San Jose, CA).
Genomic DNA was extracted from an additional 12 nymphalid species (Asterocampa celtis, A. demophon, H. charithonia, Marpesia chiron, Marpesia orsilochus, Philaethria dido, Libytheana carinenta, Adelpha bredowi, Morpho helenor, A. phidippus, A. jatrophae, and H. hermes) using a standard phenol–chloroform method. A region of approximately 300 bp of the opsin gene was amplified for each species using the same degenerate primer pair as above and a reverse primer (5'-CCR TAN ACR ATN GGR TTR TA-3') with the following PCR conditions: 94 °C for 1 min, then 35 cycles of 94 °C for 30 s, 50 °C for 1 min, and 68 °C for 10 min. PCR products were gel-purified, cloned, and sequenced as described above. Sequences served as templates for the design of species-specific reverse primers, which were then used with another degenerate primer (5'-CAY YTN ATH GAY CCN CAY TGG-3'). The fragment was then cloned and sequenced as above. Sequences were aligned, edited, and the introns manually removed using SeqMan in the Lasergene package (DNASTAR, Madison, WI).
Phylogenetic Inference
First, to test the utility of L opsin genes for assessing evolutionary relationships among butterfly families, phylogenetic hypotheses were inferred using full-length cDNAs for a set of 22 species (a total of 1152 bp). Second, in order to map spectral shifts in absorbance spectrum maximum across butterflies, phylogenetic relationships among L visual pigment lineages were inferred from nucleotide data for a set of 28 pigments for which both opsin sequence (795 bp of coding region) and max values were available (table 1 and supplementary fig. 1 [Supplementary Material online]). Lastly, we determined phylogenetic relationships among all available butterfly L opsins also using 795 bp of opsin-coding sequence (alignment shown in supplementary fig. 1, Supplementary Material online). All analyses were conducted on all codon positions, and Modeltest (Posada and Crandall 1998) as implemented in HYPHY (Kosakovsky-Pond et al. 2005) was used to determine the best-fit DNA substitution model for a maximum likelihood (ML) analysis using the Akaike information criterion. ML analysis was conducted in PHYML (Guindon and Gascuel 2003) with the GTR + I + G (invariant sites and gamma-distributed rates for sites) model, and the reliability of the tree was tested using 500 bootstrap replicates. Bayesian phylogenetic analysis was performed in MrBayes 3.1 with the GTR + I + G nucleotide substitution model, with 2 heated chains for 2 x 106 generations, and with a sampling frequency of 102 and a burn-in of 5 x 103 trees.
Character Mapping of Spectral Shifts and Ancestral State Reconstructions
The physiological diversity of butterfly L photopigment max values observed (table 1) suggested that adaptive evolution has played a significant role in their evolution. We were specifically interested in examining the hypothesis that amino acid substitutions at 4 sites (17, 64, 70, and 137) previously shown to be under positive selection in the Limenitis L opsins (Frentiu et al. 2007) may be correlated with the spectral diversification of L photopigments in this expanded data set. To test this hypothesis, we took 2 approaches. First, we mapped photopigment max values onto an L opsin phylogeny in order to correlate shifts in spectral phenotype with particular amino acid changes. Ancestral states at these 4 sites were also reconstructed for all available L opsins (N = 49) and mapped onto a phylogenetic tree to infer the frequency of their occurrence. Ancestral amino acids along each node in the phylogeny were inferred using maximum parsimony (MP) and ML methods in MacClade (Maddison DR and Maddison WP 2005) and CodonML in the PAML 3.15 package (Yang 1997), respectively.
Second, ancestral L photopigment max values were inferred using least-squares MP in the Mesquite package (Maddison WP and Maddison DR 2005). ML ancestral inferences, including the standard error (SE) estimates (equivalent to the standard deviation [SD] of the marginal distribution of the ML estimate; see Schluter et al. 1997) were performed in the program ANCML assuming a Brownian-motion model of evolution of continuously distributed characters. We note that both programs assume that proteins or phenotypic character states evolve in an additive manner, which is an assumption that is difficult to test directly. Our data must therefore be viewed in the context of this assumption.
Relative Rates Tests
We discovered 2 instances of lineage-specific gene duplication in the Nymphalidae (H. hermes, a member of the subfamily Satyrinae, and A. phidippus, a member of the subfamily Morphinae, table 1) and 1 instance in the Riodinidae (A. mormo). The 2 A. phidippus L opsin copies differed by 24 out of 300 bp (8%) of coding region (supplementary fig. 2, Supplementary Material online) and contained 2 introns of variable length. However, because we were unable to obtain more coding sequence from A. phidippus LWRh2, this gene was not used in further analyses. The hypothesis that duplicates may not be evolving at the same pace was investigated using phylogeny-based relative rates tests. Tests were conducted in the program RRTree (Robinson-Rechavi and Huchon 2000) on the H. hermes and A. mormo duplicates, with B. anynana and P. rapae as outgroups, respectively.
Homology Modeling
We employed homology modeling to investigate the structural proximity of specific amino acid residues to the chromophore because the mechanisms of spectral tuning of insect photopigments are poorly understood (Britt et al. 1993; Salcedo et al. 2003). Because the majority of L opsin sequences used in this study are from nymphalid butterflies, we used the L. a. astyanax L opsin homology model of Frentiu et al. (2007), which is based on the crystal structure of bovine rhodopsin (Palczewski et al. 2000; Okada et al. 2004) to map sites that appear to change frequently either in parallel or convergently among lepidopteran opsins correlated with spectral shifts in L photopigment absorbance (supplementary fig. 3, Supplementary Material online).
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Results and Discussion |
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Molecular studies of the nymphalid butterflies D. plexippus and V. cardui have shown that the UV-, B-, and L-sensitive photopigments are encoded by 1 UV, 1 B, and 1 L opsin gene likely present in the ancestor of all butterflies and moths. In the main retina of these species, the R1 and R2 photoreceptor cells express either UV or B opsin mRNAs and the R3-8 photoreceptors express the L opsin mRNA (Briscoe et al. 2003
; Sauman et al. 2005). (R9 has only been examined in V. cardui, and this cell expresses the same L opsin mRNA as the R3-8 photoreceptor cells [Briscoe et al. 2003].) This basic pattern of opsin expression within nymphalids, together with the presence of a tapetum, makes it possible to link the L photopigment absorbance spectra measured by microspectrophotometry to the L opsin gene sequences (see below).
Extensive Phenotypic Diversity of Butterfly L Visual Pigments
Our study represents the most taxonomically comprehensive investigation to date of the distribution of L photopigment spectra in butterflies measured microspectrophotometrically. Extensive spectral diversity at several taxonomic levels exists in these pigments, as the range of absorbance spectrum maxima (most estimates accurate to approximately ±1.5 nm) for 22 species indicates (fig. 1). For each of the 3 subfamilies, Apaturinae, Charaxinae, and Danainae, we obtained max measurements from 1 exemplar: A. leilia (530 nm), A. demophon (565 nm), and monarch D. plexippus (545 nm) (fig. 1A–C). We note that the 545 nm estimate for the L photopigment of monarch is in good agreement with the 540 nm estimate obtained from intracellular recordings (Stalleicken et al. 2006).
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Variation of
max within the subfamily Nymphalinae was as wide as the variation we observed across the entire Nymphalidae family and ranged from max = 510 nm in the buckeye J. coenia (Briscoe and Bernard 2005) to max = 565 nm in A. jatrophae (fig. 1F). Within nymphalids, we found photochemical evidence of only 1 L photopigment in all but 1 species, A. jatrophae, where in addition to the 565-nm pigment we found evidence of a 530-nm pigment. Even further blue and redshifted L visual pigments existed in the Riodinidae, with the metalmark butterfly A. mormo having 2 pigments with peak absorbance at 505 and 600 nm, respectively (fig. 1H). Indeed, a survey of insect L visual pigments that have been studied photochemically indicated that the A. mormo 600-nm value is the most redshifted max yet reported in any butterfly (reviewed in Briscoe and Chittka 2001).
Significant spectral diversity was observed within 2 other nymphalid subfamilies, the Satyrinae and the Limenitidinae (fig. 1G and E, respectively). For Satyrinae, max ranged from 515 nm in N. ridingsii to 530 nm in O. chryxus. Interestingly, we found photochemical evidence of only one L pigment in the eye of the satyrine H. hermes (max = 530 nm), even though we did clone 2 L opsin genes in this species (potential reasons for this discrepancy are discussed below). As mentioned previously, there was a 31-nm range of spectral diversity in the L photopigments of North American Limenitis species (fig. 1E; max = 514–545 nm; Frentiu et al. 2007).
In contrast to Limenitis, none of the L photopigments sampled within the genus Heliconius (i.e., H. erato, H. charithonia, and H. hecale) showed evidence of significant physiological diversification (fig. 1D; max = 550–560 nm). Both the North American Limenitis and the 2 principal Heliconius clades diverged 
4 MYA (Brower 1994; Mullen 2006), and both groups are known for their mimicry-driven diversification of wing patterns. However, evolution of L visual pigment max has proceeded at different paces in the 2 groups, suggesting factors other than mimicry-related wing change are mediating visual pigment absorbance spectra, at least in Heliconius.
L Opsin Gene Tree Recovers Familial-Level Relationships
Full-length coding L opsin cDNAs (encoding 380–384 amino acids) were obtained from 8 species. Using these data along with full-length coding sequences from GenBank, we investigated the utility of L opsins for reconstructing the broad phylogenetic relationships within Lepidoptera using ML and Bayesian inference. Despite the sparse sampling of most genera within families, most nodes were well supported (>50%; fig. 2) and the overall phylogeny was similar to other trees obtained from blue opsins (Sison-Mangus et al. 2006) and other nuclear and mitochondrial genes (Brower 2000; Wahlberg et al. 2003, 2005). Most butterfly families appeared as monophyletic clades, except for Lycaenidae and Riodinidae in which a duplicate L opsin (LWRh1) was evident in the riodinid A. mormo and was not found in the lycaenid L. rubidus. This unusual relationship may reflect the difficulty in resolving relationships between the 2 sister groups based on single exemplars (Wahlberg et al. 2005). Alternatively, the ancestral opsin may have duplicated prior to the split between the lycaenid and riodinid lineages and 1 copy (LWRh1) was lost in the lycaenids.
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Ancestral Nymphalid L Photopigment
max Estimates and Inferred Blue Spectral ShiftsFor the remaining nymphalid species in this study, we obtained between 1365–3012 bp of L opsin sequence from genomic DNA, including coding sequence for 266 amino acids spanning 6 transmembrane domains (supplementary fig. 1, Supplementary Material online). Using these data, we investigated the pattern of L photopigment spectral shifts among butterflies by mapping all
max values available onto an L opsin phylogeny (fig. 3). Both Bayesian and ML analyses returned the same topology, but most nodes received higher support in the Bayesian analysis. Separate MP and ML methods returned estimates of max values for L photopigments at ancestral nodes that were in good agreement with each other. The most parsimonious max estimates at nodes 1, 2, and 3 (fig. 3) were 542 nm, 540 nm, and 537 nm, respectively. ML estimates and their SE estimates (equivalent to the SD of the marginal distribution of the ML estimate; see Schluter et al. 1997), at the same nodes were 540 nm (±10 nm), 539 nm (±9 nm), and 541 nm (±13 nm). The estimated values at nodes 1 and 2 (fig. 3) suggest that the ancestral nymphalid L photopigment max was 540 nm (nodes 1 and 2, fig. 3). Interestingly, this ancestral state estimate is similar to the mean max = 534 nm (SD ± 10 nm) of L photoreceptors measured from 46 extant bee species (Peitsch et al. 1992).
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From these data, we propose that blueshifts in peak absorbance from the ancestral nymphalid L photopigment
max value of 540 ± 10 nm to max approximately 
530 nm have evolved in 4 lineages within the Nymphalidae (fig. 3). (We chose to categorize blueshifted pigments as having max approximately 530 nm due to the uncertainty in the estimate of the ancestral L photopigment max values.) To more rigorously define blue spectral shifts, we examined ancestral max values at all nodes in the tree. Blueshifts were inferred from the branch leading from node A (max = 536 ± 4 nm) to node B (max = 530 ± 2 nm) (error estimates as noted above are equivalent to the SD of the marginal distribution of the ML estimate); from node C (max = 534 ± 7 nm) to node D (max = 529 ± 7); from node E (max = 539 ± 7 nm) to node F (max = 533 ± 6 nm); and from node G (max = 542 ± 10 nm) to node H (max = 528 ± 5 nm), which was at the base of the clade comprising N. ridingsii and O. chryxus (fig. 3). A further blueshift was inferred from node D (max = 529 ± 7 nm) to the branch leading to J. coenia, max = 510 nm (fig. 3; see table 2 below). These data also suggest that the A. mormo photopigment with max = 505 nm (LWRh1) was likely blueshifted compared with its 540 ± 13-nm ancestor (results not shown).
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Amino Acid Substitutions Correlated with Blue Spectral Shifts
Along the same pigment lineages, parallel and convergent amino acid substitutions were inferred to have also occurred in the L opsins in tandem with spectral shifts (table 2). Some of the same amino acid sites previously found to be under positive selection in Limenitis L photopigments (sites 64, 70, and 137; Frentiu et al. 2007
) were found in this expanded analysis as well as several new sites. A total of 10 sites (positions 26, 64, 70, 131, 137, 145, 165, 170, 177, and 260; supplementary fig. 1, Supplementary Material online) displayed parallel amino acid substitutions along at least 2 lineages and 2 sites (61 and 177) displayed convergent evolution. Along the branches connecting nodes E to F and nodes G to H on the tree shown in figure 3, 3 sites had acquired parallel substitutions (Ala64Ser, Phe131Leu, and Phe165Leu), which were more sites than expected by chance (P < 0.000) using the method of Zhang and Kumar (1997) (table 2). Site 177 is in transmembrane domain V facing outward though in the same plane as the chromophore such that it might influence spectral tuning via steric effects, and site 165 is in extracellular loop domain II that folds deep into the chromophore-binding pocket of bovine rhodopsin (Palczewski et al. 2000). All other sites (26, 131, 145, 170, and 260), except for sites 64, 70, and 137 as previously reported (see below) (Frentiu et al. 2007), are further removed from the chromophore.
Most strikingly, the Ala to Ser substitution at site 64 was associated with at least 4 phylogenetically independent blueshifts (table 2). Using a homology model of a nymphalid opsin protein, we previously found that site 64 was located immediately adjacent (within 5 Å) to the chromophore, suggesting a principal spectral tuning role for this site (Frentiu et al. 2007). A Pro91Ser mutation in its bovine rhodopsin equivalent causes a shift of 10 nm toward the blue in vertebrate short wavelength–sensitive visual pigments (Takahashi and Ebrey 2003). Mutagenesis experiments using native butterfly pigments as a template are needed to confirm the hypothesized spectral tuning effects of the Ala to Ser substitution at site 64.
Mapping of Substitutions at Candidate Spectral Tuning Sites Indicates Frequent Shifts in max
Vertebrate middle- and long-wavelength (MWS and LWS, respectively) visual pigment spectra can be determined by inspecting amino acids present at 5 sites within the opsin (Merbs and Nathans 1992; Asenjo et al. 1994; Yokoyama and Radlwimmer 1998, 1999). Assuming effects are roughly additive, concordance between particular amino acid residues at butterfly opsin site 64 and blue spectral shifts suggests that it may be possible to use this site to predict spectral shifts in nymphalid L photopigments in which physiological information is lacking, as is currently possible for vertebrate MWS and LWS visual pigments (Yokoyama 2000). The pattern of change in absorbance spectra observed in the L visual pigment phylogeny (fig. 3) suggested that shifts in max occur frequently in butterflies. To evaluate this hypothesis, we reconstructed phylogenetic relationships across all 49 butterfly L opsin sequences available and then mapped amino acid substitutions at key candidate spectral tuning sites found in this (i.e., site 64) and previous analyses (sites 17, 64, 70, and 137; Frentiu et al. 2007) (Briscoe and Bernard 2005).
Phylogenetic relationships among the 49 taxa as indicated by L opsins were generally in agreement with previously published evolutionary relationships (Brower 2000; Wahlberg et al. 2003, 2005), with most nymphalid subfamilies forming well-supported monophyletic groups (fig. 4). The Satyrinae was found to be polyphyletic, with members of the subfamilies Morphinae and Charaxinae clustering within this group (fig. 4), reflecting the close relationship among these groups reported previously (Wahlberg et al. 2003; Peña et al. 2006). However, our phylogenetic tree suggested that the Danainae is not the basal nymphalid family, as implied by other genes (Wahlberg et al. 2003, 2005). This pattern may reflect the fact that our phylogeny is a single-gene tree because the position of the Danainae in other studies also changes depending on which gene is used to reconstruct evolutionary relationships (Wahlberg et al. 2005).
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indicates Bayesian topology fails to resolve this node. Sequences contributed by this study shown in bold. Newly identified duplicate genes are underlined.Mapping of amino acid substitutions onto the expanded L opsin phylogeny indicated that there have been multiple occasions of parallel changes at key spectral tuning sites implicated in blue spectral shifts (fig. 4): Ile17Met occurred 5 times (open red circles), Asn70Ser occurred 3 times (purple), and Ser137Ala occurred 3 times (yellow dots). Most striking, however, was the frequency of the Ala64Ser substitution, which occurred 8 times across the whole tree (fig. 4, blue dots). Because site Ala64Ser is strongly associated with shifts in
max toward the blue, the pattern observed suggests that these types of shifts occur commonly in the Nymphalidae.
We also observed an intriguing pattern of reverse substitution at 2 key putative spectral tuning sites. The Ser64Ala change occurred 2 times (open blue circles) and was associated with an L pigment showing a redshift in max as in L. a. astyanax (fig. 4). The Met17Ile substitution also occurred twice (fig. 4, red dots), including a branch in which there was a red spectral shift because both A. jatrophae and E. chalcedona had max = 565 nm, but the ancestral pigment may have had max = 534 ± 7 nm (results not shown). These patterns indicate that the reverse mutation may result in the opposite spectral shift and have a symmetrical effect, at least qualitatively. For example, the substitution A269T in bovine rhodopsin shifts the max by 14 nm toward the red (Chan et al. 1992), but the reverse substitution at the corresponding site in human L pigments causes a 16-nm blueshift (Asenjo et al. 1994).
Given the strong associations between key sites and spectral shifts, it may be possible to use ancestral reconstructions and the presence of particular amino acid residues at these sites to predict shifts absorbance spectra in taxa whose spectra have not yet been measured. But these predictions can only be clarified once the hypothesized spectral tuning effects of these substitutions have been validated by site-directed mutagenesis and functional characterization. For example, an Ala64Ser substitution occurred along the branches leading to the Marpesia opsins and the A. phidippus LWRh1 opsin, implying that the visual pigments may be blueshifted in sensitivity (fig. 4). Similarly, the substitution Ser137Ala occurred along the branch leading to M. helenor (fig. 4), suggesting that photo pigment may also blueshifted. Although in vitro mutagenesis studies are needed to test the hypothesized spectral tuning effect of these sites, in vivo physiological data on the max values of L pigments in species like A. phidippus and M. helenor provides an additional, complementary test.
Parallel Evolution of Red-Sensitive Photoreceptors through Gene Duplication
Our sequence data indicate that L opsin gene duplication has occurred several times in butterflies. Independent duplicates were found in 3 new species: A. phidippus, A. mormo, and H. hermes, showing that L opsin gene duplication has occurred in 3 out of 5 butterfly families (Briscoe 1998; Kitamoto et al. 1998). Physiological data revealed the presence of 2 L visual pigments in the nymphaline species A. jatrophae (fig. 1F), but we were unable to isolate a second L opsin gene from genomic DNA. Either we missed a second L opsin gene in our screen or the 530-nm photopigment is encoded by a different opsin gene family member, such as by a duplicate blue opsin gene, for which we did not screen. Precedent for such a pigment has been found in the lycaenid L. rubidus, in which a 500-nm photopigment is encoded by a blue opsin (Sison-Mangus et al. 2006). A screen of additional samples, including eye-specific cDNA, may help resolve this issue.
Curiously, we discovered the presence of 2 L opsin genes in H. hermes but only identified physiologically 1 L visual pigment in the eye (fig. 1G). The amino acid sequences of the H. hermes duplicate genes suggest that both copies are functional opsins (supplementary fig. 1, Supplementary Material online). The absence of a second L visual pigment in the eye suggests that the second H. hermes opsin is expressed in another body tissue, such as the brain, as has been found in Papilio glaucus (Briscoe 2000) and moths (Shimizu et al. 2001; Lampel et al. 2005).
Duplicate copies of the H. hermes and A. mormo L opsins have evolved at significantly different rates (table 3), particularly at nonsynonymous codon sites. Interestingly, A. mormo LWRh2 appears to have evolved much faster than the A. mormo LWRh1 opsin at nonsynonymous (P < 0.002) rather than synonymous sites (P = 0.538). This pattern of evolution suggests strong diversifying selection acting on the LWRh2 copy and is consistent with it encoding the 600-nm photopigment.
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The presence of P. xuthus and A. mormo visual pigments with
max = 575 nm and max = 600 nm, respectively, and their independent origin (fig. 3), indicates the parallel evolution in the Papilionidae and Riodinidae families of photoreceptors, which are considerably redshifted compared with their ancestral pigments, and compared with the most redshifted photopigment observed in nymphalids (565 nm). Interestingly, the far redshifted photopigments are both encoded by independently arising duplicate opsin gene copies (fig. 2). More strikingly, the evolution of these parallel spectral phenotypes has been accompanied by parallel changes at amino acids sites 10 (His to Tyr), 23 (Ile to Thr), 29 (Gly to Ala), and 82 (Ala to Phe) (supplementary table 1 and supplementary fig. 1, Supplementary Material online). The number of parallel amino acid changes between the opsin sequences was significantly greater than expected by chance (P < 0.001) using the test of Zhang and Kumar (1997), suggesting that they may have been under positive selection.
We used the homology model of L. a. astyanax L opsin from Frentiu et al. (2007) (alignment shown in supplementary fig. 3, Supplementary Material online) to investigate the position of sites inferred to change in parallel between the A. mormo LWRh2 sequence and P. xuthus PxRh3 (supplementary table 1, Supplementary Material online). Sites 23, 29, and 82 are located away from the retinal-binding pocket, whereas site 10 is located in the vicinity of the Schiff base. Recent work indicates that 10 amino acids modulate absorbance spectra in vertebrate violet/UV (SWS1) pigments, yet 7 of these are outside the binding pocket and act in a synergistic fashion (Wilkie et al. 2000; Yokoyama and Shi 2000; Parry et al. 2004; Yokoyama et al. 2006). It is therefore possible that substitutions at these 4 sites may be important for the evolution of these redshifted absorbance spectra.
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Conclusions |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsSupplementary MaterialAcknowledgementsReferences |
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Spectral shifts toward the blue in butterfly L photopigments have been shown to be the result of positive selection (Frentiu et al. 2007
), although the reasons for these changes remain an open issue. In other animals, spectral changes in L visual pigments may be due to sexual selection (e.g., guppies) (Hoffmann et al. 2007) or adaptation to different light environments (e.g., Lake Victoria cichlids) (Terai et al. 2006). It is harder to establish a direct link between light environment and visual pigment spectra in terrestrial animals, as has been possible for fish (Yokoyama et al. 1999; Carleton et al. 2005), because the light environments are more complex (Endler 1993). Nonetheless, as more comparative data on visual pigment spectra and the opsin genes that encode them become available, it may be possible to link shifts in max to particular ecological factors.
Our finding that the ancestral nymphalid, riodinid, and lycaenid photopigments appear to have had a similar max 540 nm allows us to speculate on the role of gene duplication in the evolution of spectral shifts. The presence of very redshifted photoreceptors in addition to green-sensitive ones in Papilionidae (575 nm and 515 nm) and Riodinidae (600 nm and 505 nm) suggests that gene duplication may permit the evolution of photoreceptors with absorbance spectrum maxima well outside the typical range of max values achieved with only one L photoreceptor. In support of this idea, we note that in the Nymphalidae duplicated L visual pigments have not been observed in the majority of taxa and the most extreme max that is achieved in the long-wavelength region is 565 nm. Consequently, there appears to be an upper limit to the evolution of max in the long-wavelength range for butterflies with only one L photoreceptor.
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Supplementary Material |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsSupplementary MaterialAcknowledgementsReferences |
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Supplementary Materials and Methods, figures 1–3, and table 1 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
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Acknowledgements |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsSupplementary MaterialAcknowledgementsReferences |
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We thank Lincoln Brower, Carol Boggs, Matthew Garhart, Antonia Monteiro, and John Emmel for providing specimens; Furong Yuan, Lawrence Lee, Emily N. Yee, Lisa Inouye, and Wei-Hsi Kao for technical assistance. We especially thank Steven Reppert for helpful discussions. This research was funded in part by research grants from the National Institutes of Health (EY01140 and EY00785) and National Science Foundation (NSF) (BNS-8719220) to G.D.B., NSF DEB 0640301 to A.V.Z.B., and NSF IOB-0346765 and IOS-0646060 to A.D.B.
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Footnotes |
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Billie Swalla, Associate Editor
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References |
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