Department of Molecular and Cellular Biology, University of Arizona
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Abstract |
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Introduction |
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The spectral properties of visual pigments can be measured using a variety of methods such as microspectrophotometry, single-cell recordings, and electroretinograms. The relative ease with which physiological data on the visual pigments can be collected, coupled with the availability of molecular sequence data, makes opsins a useful system for studying the relationship between a protein's function and structure. A comparative approach has been used to identify changes at amino acid sites that have spectral tuning effects in New World monkeys (Neitz, Neitz, and Jacobs 1991
; Shyue et al. 1998
), fish (Yokoyama and Yokoyama 1990
; Yokoyama et al. 1999
), and other organisms (Chang et al. 1995
). This approach makes use of a phylogeny upon which amino acids potentially involved in modulating the absorption spectrum of the visual pigment may be mapped. For instance, Chang et al. (1995)
identified an amino acid substitution correlated with a blue shift in vertebrate and invertebrate opsins that was subsequently tested and experimentally confirmed using resonance Raman spectroscopy (Lin et al. 1998
).
This study takes advantage of an available collection of lepidopteran long wavelengthsensitive opsin sequences (Chase, Bennett, and White 1997
; Kitamoto et al. 1998
; Briscoe 2000
), and contributes the sequences of several naturally occurring visual pigment spectral variants from a pair of butterfly and moth species.
max values of these pigments have been characterized by others (Goldman, Barnes, and Goldsmith 1975
; Langer, Haumann, and Meinecke 1979
; Bernard 1983
), whereas the sequences encoding the seven transmembrane domains are newly presented here. The data have allowed the examination of two sets of questions related to the description of the evolutionary history of lepidopteran opsins. First, what are the global patterns and processes governing long-wavelength (LW) opsin evolution following gene duplication and functional divergence? Second, can a comparative approach be taken for identifying amino acid sites under positive Darwininan selection correlated with changes in opsin spectral function?
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Materials and Methods |
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Phylogenetic Analyses
Opsins are a part of a large multi-gene family. To guard against the possibility that the cloned opsins belong to the UV or blue spectral classes, neighbor-joining (NJ) and maximum parsimony (MP) analyses of 43 sequences of spectrally diverse opsins were conducted. Their GenBank accession numbers are: Apis mellifera (Bee) (UV, AF004169; Blue, AF004168; LW, U26026), Cambarellus ludovicianus (AF003543), Camponotos abdominalis (UV, AF042788; LW, U32502), Cataglyphis bombycinus (UV, AF042787; LW, U32501), Drosophila melanogaster (Dmel) (Rh1, K02315; Rh2, M12896; Rh3, M17718; Rh4, M17730; Rh5, U67905; Rh6, Z86118), Drosophila pseudoobscura (Dpse) (Rh1, X56877; Rh2, X65878; Rh3, X65879; Rh4; X65880), G. mellonella (AF385330), Heliconius sara (OPS1, AF126753), Hemigrapsus sanguineus (Crab) (1, D50583; 2, D50584), Limulus polyphemus (lateral eye, L03781; ocelli, L03782), Manduca sexta (1, L78080; 2, L780801; 3, AD001674), Papilio glaucus (Papilio) (PglRh1, AF077189; PglRh2, AF077190; PglRh3, AF067080; PglRh4, AF077193; PglRh5, AF077191; PglRh6, AF077192), Papilio xuthus (Papilio) (PxRh1, AB007423; PxRh2, AB007424; PxRh3, AB007425), P. coenia (AF385332), Procambarus clarkii (Pclarkii) (S53494), Schistocerca gregaria (Locust) (1, X80071; 2, X80072), Sphodromantis spp. (Mantid) (X71665), S. exigua (AF385331), V. cardui (AF385333).
Amino acid sequences were aligned using Clustal W (Thompson, Higgins, and Gibson 1994
), and then nucleotide sequences were aligned accordingly. Calculation of p-distance using the NeiGojobori method (Nei and Gojobori 1986
) in MEGA (Kumar et al. 2000) suggested that the nucleotide sequences were too divergent to be reliably used in MP phylogenetic reconstruction (data not shown). Therefore, only amino acid sequences were used in phylogenetic analyses of this larger data set. A total of 282 amino acid sites were included in the MP and the NJ analyses. A stepmatrix of amino acid changes derived from a larger data set of G proteincoupled receptors (Rice 1994
) was employed as a weighting scheme to account for unequal probabilities of amino acid change in the MP analysis. For the NJ analysis, total character distance (uncorrected p-distance) was used along with minimum evolution as the objective function. The reliability of the trees was tested by bootstrap analysis in PAUP* (Swofford 1998
).
To establish the evolution of opsin genes within the long-wavelength lepidopteran opsin group, a smaller data set (13 sequences) was used. Tree topologies were estimated from nucleotide data in which all three positions were pooled using maximum likelihood algorithms under a variety of models of evolution (Yang 2000
). The PAUP* computer program (Swofford 1998
) was used for conducting tree searches and making estimates of the model parameters. Significant differences in the fit of the models to the data were tested using the likelihood ratio test (Hasegawa, Kishino, and Saitou 1991
).
Statistical Analyses
The rate of molecular evolution has been observed sometimes to change following gene duplication. To explore this possibility, the two-cluster test of Takezaki, Rzhetsky, and Nei (1995)
as implemented in PhylTest 2.0 (Kumar 1996
) was used to test the constancy of the molecular clock between clusters consisting of the duplicated genes Papilio Rh1 and Rh3. As different parts of a protein may be under different levels of functional constraint, the data were divided into three categoriesall, transmembrane (TM), and non-TM domainsto examine the relative contribution of each domain to any rate difference, if detected.
Domains under higher levels of constraint are expected to be less variable than domains in which this assumption is relaxed. As an additional measure of differences in the level of variability between domains, the ratio of the total number of sites in the TM domain to the total number of sites in the non-TM domains was compared to the ratio of the number of variable sites in the TM to the number of variable sites in the non-TM domains using Fisher's exact test.
Reconstruction of phylogenies can be affected by changes in the pattern of substitution (heterogeneity) (Galtier and Gouy 1995
). To test the possibility that changes in the pattern of amino acid substitution have occurred following changes in the absorption spectra or following a duplication event, the Disparity Index (ID) (Kumar and Gadagkar 2001
) was calculated. The Disparity Index measures the extent of difference in evolutionary pattern between two sequences, and is a more powerful test than the
2 test (Kumar and Gadagkar 2001
). This method allows the identification of sequences that are evolving under patterns that violate assumptions used in phylogenetic reconstruction and which probably should be removed from phylogenetic analyses.
Codon-Based Tests of Selection
A maximum likelihood approach (Nielsen and Yang 1998
; Yang 2000
) was used to test first for the rate heterogeneity between sites and then for the presence of amino acid sites and branches that may have
> 1, a signature of positive Darwinian selection. Multiple models of site-specific evolution were compared using the likelihood ratio test. Additionally, the value of
was inspected to determine whether in the data a class of site under positive selection (
> 1) exists.
To test for the possibility of branch-specific > 1, a model in which all branches in the tree are assigned the same
(M0) was compared with a model in which all branches have a different
value (free-ratio) (Yang 1998
).
Ancestral State Reconstruction
The tree used for ancestral-state reconstructions was estimated using a maximum likelihood approach and a variety of models of evolution. The nucleotide sequences were then translated into amino acid sequences and imported into PAML (Yang 2000
) where the aaml program, empirical + frequencies model, with the Jones, Taylor, and Thornton (1992)
amino acid substitution rate matrix was used. Opsin amino acid ancestral states were also compared to reconstructions made in MacClade (Maddison and Maddison 1999
). Patterns of amino acid substitution correlated with wavelength shifts in opsin absorption spectrum were then examined in two ways. First, amino acid sites undergoing substitution in the lepidopteran LW opsin tree were examined for homology to amino acid sites with known spectral tuning effects in vertebrate opsins (Neitz, Neitz, and Jacobs 1991
; Asenjo, Rim, and Oprian 1994
; Sun, Macke, and Nathans 1997
). Secondly, the branches of the tree were examined for instances of convergent or parallel amino acid substitution. The CONVERGE program was used to test whether the observed number of parallel or convergent substitutions exceeded the expected (Zhang 1997
; Zhang and Kumar 1997
).
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Results |
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The null hypothesis of homogeneity of amino acid substitution patterns between opsins could not be rejected using the Disparity Index test. Four statistically significant (P < 0.05) comparisons were identified out of 80, a number that corresponds exactly to the expected Type-I error rate for this number of tests (5% of 80 tests = 4). Therefore, all lepidopteran LW opsin sequences were retained for further analysis.
Duplicated LW Opsins Are Evolving at Different Rates of Amino Acid Substitution
The hypothesis of equal rates of amino acid substitution between the duplicated gene clusters, PglRh1PxRh1 and PglRh3PxRh3, was rejected at the 5% level when all sites were included (Z statistic = 2.8225) in the two-cluster test. This elevated rate of substitution along the PglRh3PxRh3 branch seems to be primarily because of substitutions in the TM domains, as the Z statistic calculated with only the TM domain sites were significant at 5% level (Z = 2.7559), whereas the Z statistic calculated with the non-TM sites was not (Z = 0.93968).
When only the duplicated Papilio opsins were considered (PglRh14, PxRh13), the ratio of variable sites within the TM domain to non-TM domain (56:26) was significantly different (P = 0.039) from the ratio of the total number of TM domain sites to non-TM domain sites (160:122) using Fisher's exact test. This suggests that following gene duplication there has been a change in the relative level of variability in different domains, where there appears to be a higher level of variability in the TM domains relative to the non-TM domains. As a control for this scenario, the nonduplicated non-Papilio moth and butterfly opsins were similarly examined and displayed no significant difference in the variability between TM and non-TM domains (P = 0.269).
Evolutionary Relationships Within the Lepidopteran Long-Wavelength Opsin Family
Eight models of molecular evolution were used in maximum likelihood estimates of the lepidopteran LW opsin gene tree. The relative goodness-of-fit of the models to the data was tested using the likelihood ratio test (Hasegawa, Kishino and Saitou 1991
; Yang 1995
). The goodness-of-fit of the model to the data increased with increasing complexity of the model. The largest improvement in likelihood score of the recovered tree topology occurred between the HKY85 and HKY85 +
models (data not shown). HKY85 assumes a single substitution rate at all sites, whereas HKY85 +
assumes a site-to-site rate variation that follows the gamma distribution. This significant improvement in score between the two models is consistent with differences in %GC content among the first (42%), second (43%), and third (54%) positions of the data set, as well as with differences in the numbers of parsimoniously informative sites at first (69/282), second (44/282), and third (245/282) positions. Models that fit the data best (HKY85 +
, HKY85 +
+ I, GTR +
, GTR +
+ I) recovered an identical tree topology (fig. 3
). Models that were a significantly worse fit to the data (JC69, F81, HKY85, and GTR) differed from this topology in their placement of Rh4. In these models, Rh4 clustered with other butterfly opsins as the most basal member of a monophyletic butterfly opsin clade. However, the difference between the two estimated topologies is not expected to have affected the handful of sites undergoing convergent evolution at branches near the tips of the tree (identified later), so the topology recovered using more realistic models of evolution (shown in fig. 3
) was used in subsequent ancestral-state reconstruction.
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Ancestral-State Reconstructions
Similar shifts in visual pigment absorption spectrum maximum have evolved in vertebrates through convergent or parallel amino acid substitutions at specific sites in the opsin protein (e.g., Yokoyama and Radlwimmer 1999
). To identify sites that may result in spectral shifts in the lepidopteran LW opsins, ancestral states were reconstructed using a maximum likelihood approach (data not shown). Most ancestral amino acid states were reconstructed with posterior probabilities between 90% and 100%: only 54 out of 1,128 sites had lower posterior probabilities. Comparison of ancestral states reconstructed in PAML using either a poisson correction or the empirical + base frequencies model, with those reconstructed in MacClade reveal a near-perfect degree of overlap. Only four sites (aa 41, 74, 143, 169) differed between the analyses. Maximum likelihood analyses have the added advantage of assigning differential probabilities to alternative ancestral states that are given equal weight in MP (Yang, Kumar, and Nei 1995
), therefore these reconstructions were preferentially used to identify parallel or convergent changes.
Of particular interest are branches leading to opsins that have undergone large (2045 nm) spectral shifts in absorption (max) relative to ancestral opsins. Table 3
presents the reconstructed amino acid sites along branches undergoing large shifts in absorption spectrum. In the branch leading to the blue-shifted Precis opsin, there is an S to A substitution at aa 138. This substitution in human cone pigments (aa 180) has been shown to cause a 5-nm blue shift in site-directed mutagenesis experiments (Asenjo, Rim, and Oprian 1994
). In a study of crayfish LW (520530 nm) opsins, Crandall and Cronin (1997)
found an F to C substitution at aa 102 (aa 94 in Lepidoptera) associated with a blue shift, a site that is also changing in parallel in the blue-shifted Precis lineage. Along the branch leading to the red-shifted Heliconius opsin sequence, an F to Y substitution has occurred at an amino acid site (aa 185) with known spectral tuning effects in New World monkeys (aa 229) (an F to I substitution at this site is associated with a 2-nm red shift) (Shyue et al. 1998
). Parallel substitutions at aa 185 have also been correlated with blue shifts in crayfish LW opsins (Crandall and Cronin 1997
).
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Discussion |
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Using maximum likelihoodbased tests of selection, an attempt was made to determine whether accelerated rates in the Papilio Rh3 gene were initially driven by positive Darwinian selection for functional divergence (Ohta 1993
) or by relaxation of selective constraints. In the latter case, referred to as the DykhuizenHartl effect (Zhang, Rosenberg, and Nei 1998
), random fixation of neutral changes eventually leads to a novel function for one or both copies. The results of the branch-based test of selection, where the
< 1 for the Papilio Rh3 lineage, so far suggest a role for the latter model.
These results do not rule out the possibility that positive Darwinian selection has occurred at a handful of amino acid sites responsible for opsin spectral tuning, however. The Papilio Rh3 gene has in fact evolved a novel function relative to its ancestral gene, Papilio Rh2: it has evolved to be red-sensitive (575 nm) relative to its green-sensitive (520 nm) ancestor. Evidence for positive Darwinian selection on this protein is derived from the identification of several convergent and parallel amino acid substitutions (aa 70, 94, 97) that are correlated with red shifts along two independently evolved branches of the tree: the Papilio Rh3 (575 nm) and Heliconius (550 nm) lineages. At least one class of change, F to Y at aa 94, is of the category of amino acid substitution known to have spectral tuning effects in vertebrate opsins (i.e., the substitution of a hydroxyl-bearing Y for a nonhydroxyl bearing F). Indeed, this substitution, when mapped onto the three-dimensional crystal structure of bovine rhodopsin (Palczewski et al. 2000
) is located in the third transmembrane domain of the protein (fig. 4
), facing the chromophore-binding pocket. It would be worth testing the spectral tuning effects of this substitution in vitro.
Processes shaping the evolution of the paralogous Papilio Rh1 lineage may be a good example of what Force et al. (1999)
refer to as the partitioning of ancestral [gene] functions rather than the evolution of new functions. Consider the spatial expression pattern of Rh1 relative to its ancestral gene Rh2. Rh2 is expressed in three classes of photoreceptor cells in the Papilio retina: the R34 class, the R58 class, and the R9 class (Kitamoto et al. 1998
). Rh1, the descendant gene, is expressed only in a subset of these photoreceptor cell classes, R34 and R9. Moving to a scale beyond individual photoreceptor cell classes within an ommatidium (the structural unit of the compound eye) to global opsin expression patterns across the eye, Rh2 is expressed both dorsally and ventrally in the R34 photoreceptor cells, whereas Rh1 expression is restricted ventrally (Kitamoto et al. 1998
). Additionally, Rh1 and Rh2 are coexpressed in the R34 cells in the ventral part of the retina, suggesting that they may share overlapping regulatory elements. In the R9 cells, these duplicated genes seem to have nonoverlapping expression patterns.
Spectrally, Rh1 and Rh2 are very similar in absorption spectrum (520 nm), though probably not identical (see earlier paragraphs of Discussion). Therefore, changes in the regulatory elements of Rh1 seem to have predominated following duplication, leading to the partitioning of ancestral Rh2 spatial expression patterns, rather than changes in the coding region of the gene which could lead to diversification of spectral function. In contrast, both partitioning of ancestral Rh2 spatial expression patterns and the evolution of novel function, i.e., red-sensitivity, appears to have occurred to the duplicated Rh3 gene. Rh3 is expressed in a subset of photoreceptor cells (R58) that also express Rh2 (Kitamoto et al. 1998
). In some ommatidia Rh3 is coexpressed with Rh2 in the R58 cells, in other ommatidia only one of the two gene transcripts are expressed. It would appear that both complementary loss of gene subfunctionalizations and the acquisition of novel functions are mechanisms for the preservation of the Papilio opsin gene duplicates (Force et al. 1999
).
Has the null hypothesis of neutral evolution been ruled out for any other part of the lepidopteran LW opsin clade? The observation of a parallel S to A change between vertebrate red and green cone pigments and the blue-shifted Precis (510 nm) opsin at an amino acid site in vertebrates (aa 180) with a known spectral tuning effect of 5 nm (Asenjo, Rim, and Oprian 1994
), suggests that this site (aa 138 in Precis) may be under positive Darwinian selection in the Precis lineage. Similarly, another candidate site under positive Darwinian selection is the F to Y change from node 18 to the red-shifted Heliconius opsin at a site (aa 185) that in New World monkey cone pigments is associated with a 2-nm red shift (Shyue et al. 1998
).
In conclusion, spectral diversification of the lepidopteran LW opsin clade (510575 nm) appears to be driven by a handful of amino acid sites, some of which are shared with known spectral tuning sites in primates and fish, and others of which have so far only been identified in other invertebrate opsins, i.e., crayfish (Crandall and Cronin 1997
). It will be interesting to see whether these same sites are involved in the spectral diversification of the UV and the blue insect opsins, but to do so will involve sampling a broader spectral range of pigments than are currently known.
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Acknowledgements |
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Footnotes |
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1 Present address: Department of Cellular and Structural Biology, University of Colorado Health Sciences Center.
Keywords: visual pigment
color vision
rhodopsin
photoreceptor
convergent evolution
Address for correspondence and reprints: Adriana D. Briscoe, Department of Cellular and Structural Biology, University of Colorado Health Sciences Center, B-111, 4200 East 9th Avenue SOM Rm 4547, Denver, Colorado 80262. adriana.briscoe{at}uchsc.edu
.
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