Comparative and Evolutionary Physiology Group, Department of Ecology and Evolutionary Biology, University of California, Irvine
Correspondence: E-mail: abriscoe{at}uci.edu.
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Abstract |
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Key Words: photoreceptor visual pigment color vision rhodopsin
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Introduction |
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Phylogenetic studies of the protein sequences of insect visual pigments have revealed that opsins fall into four major subfamilies, three of which match the physiologically identified short wavelengthsensitive, medium wavelengthsensitive, and long wavelengthsensitive visual pigment groups (for review, see Briscoe and Chittka [2001]). The blue-green opsins constitute the fourth group, which is most closely related to the long wavelength pigments, and includes such members as the Drosophila melanogaster majority pigment, Rh1 and the ocellar-specific, Rh2. The largest expansion of insect opsin genes reported so far has been in the LW opsin group of lepidopterans and dipterans. In the butterfly Papilio glaucus, for instance, four LW opsin genes were found but only a single blue and a single UV opsin gene were found (Briscoe 1998, 2000). One of these LW opsins (PglRh4) is now known to be expressed exclusively outside of the retina (Briscoe and Nagy 1999), whereas all the others have been localized to the eye (Kitamoto, Ozaki, and Arikawa 2000; Kitamoto et al. 1998). A search for G proteincoupled receptors in the recently published Anopheles gambiae genome identified 12 different opsin genes, of which seven belong to the LW Rh subfamily (Hill et al. 2002). The spatial distribution of these opsins has yet to be described.
In contrast to lepidopterans and dipterans, only one opsin in each spectral group has been reported in hymenopterans (Bellingham et al. 1997; Chang et al. 1996; Townson et al. 1998). All three of these transcripts were isolated from eye-specific mRNAs. One of these genes, the LW Rh gene, is extensively used to reconstruct phylogenetic relationships among hymenopteran tribes, families, and species (Ascher, Danforth, and Ji 2001; Cook et al. 2002; Kawakita et al. 2003; Mardulyn and Cameron 1999) as well as to compare patterns of nucleotide substitution between nuclear and mitochondrial genes (Lin and Danforth 2004). Physiological data, however, suggest that some hymenopterans should have more that one long wavelengthsensitive visual pigment in the retina. Peitsch et al. (1992), for instance, utilized intracellular recordings to determine the spectral properties of the visual receptors of 43 bee and wasp species and found several species in the families Andrenidae, Xiphydriidae, and Tenthredinidae with a fourth receptor type sensitive in the yellow to red part of the light spectrum. They proposed that these fourth spectral receptors were based on visual pigments with max between 570 and 596 nm (Peitsch et al. 1992). Although the observed distribution of these additional receptors favors their recent origin, they may also be the result of a single ancient gene duplication followed by loss in multiple lineages.
We were interested in determining whether the phylogenetic relationships of the recently reported Anopheles opsins relative to other insect opsin gene family members would provide evidence of additional hymenopteran opsins. (The Anopheles sequences were originally analyzed only with respect to other dipterans; see Hill et al. [2002] Supplementary Material). We, therefore, conducted phylogenetic analyses of all available insect LW opsin sequences and found that the branching pattern of the long wavelength opsin group suggested at least one early duplication of this gene in insects. We tested this hypothesis by conducting a PCR search for new and undetected LW opsin genes in bees, the most extensively investigated group among the hymenopterans. We extracted genomic DNA from Bombus, Diadasia, and Osmia bee species and used various combinations of degenerate primers to target possible novel genes. We found a new gene, which was present in all five species species that we studied (Bombus impatiens, B. terrestris, Diadasia afflicta, D. rinconis, and Osmia rufa). Phylogenetic analysis revealed that the new gene is paralogous to the already known LW Rh gene in hymenopterans and arose from an early gene duplication event within the insect LW-sensitive opsin gene family. We used Gu's (1999, 2001) method for identifying sites in the two bee genes that may be under altered selective constraints after duplication and have identified a number of sites that may be responsible for functional differences between the two genes. We discuss these sites in relationship to three-dimensional structural information inferred from a homology model of one of the hymenopteran opsins, as well as the possible role of the new gene in extraretinal photoreception.
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Materials and Methods |
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DNA Extraction
All specimens were caught in the field except the B. impatiens specimen, which was obtained from a colony bought from a commercial breeder (Koppert, distributed by Plant Sciences, Inc., Watsonville, Calif.). The Diadasia rinconis and D. afflicta specimens were gifts of Jack Neff (table 1). All individuals were frozen in liquid nitrogen, except Osmia rufa, which was field collected directly into RNAlater (Ambion). One individual bee per species was used for DNA extraction. The heads were removed and homogenized in 400 µl of 50 mM Tris-Cl (pH 8.0), 20 mM EDTA, and 2% SDS and digested with 2 µl proteinase K. After incubating overnight at 37°C, 100 µl of 5 M NaCl was added and the tubes were placed on ice for 40 min. The tubes were then centrifuged for 15 min at 14,000 rpm and 4°C. Afterward the cellular debris was removed, and DNA was extracted by use of phenol/chloroform.
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On the basis of the sequences for B. terrestris and D. afflicta, which we cloned and sequenced first, we designed two gene-specific degenerate reverse primer (LWRH1 and LWRHR2) for the novel gene LW Rh2 to be able to target the gene by application of a direct sequencing strategy. The reverse primer was then paired with LWRHF. A single PCR product was obtained for both B. impatiens and D. rinconis (943 bp and 967 bp, respectively) with LWRH2 as the reverse, and both products were directly sequenced following the method described above. We found that the newly designed reverse primer LWRHR2, in combination with LWRHF, exclusively amplifies the new gene in Bombus and Diadasia, whereas amplification of the Osmia rufa LW Rh2 gene worked with LWRHF and LWRHR1.
Phylogenetic Analyses of the New Bee Sequences in Relation to Other Bees
LW opsin sequences from a large number of hymenopteran species have been published (Ascher, Danforth, and Ji 2001; Cameron and Mardulyn 2001; Cameron and Williams 2003; Cook et al. 2002; Danforth, Conway, and Ji 2003; Kawakita et al. 2003; Mardulyn and Cameron 1999). We were interested in determining whether any of these sequences are orthologous to LW Rh2, and, thus, whether the identification of this new gene has an impact on existing phylogenies. We, therefore, calculated synonymous substitution rates among 234 hymenopteran sequences downloaded from GenBank and the LW Rh1 and LW Rh2 sequence from one of our bees, B. terrestris. All pairwise comparisons between the B. terrestris LW Rh2 sequence and any of the published sequences resulted in a value larger than 0.75 and indicated saturation (data are not shown). However, no saturation of the rate of synonymous substitution was found within the data set when LW Rh2 was excluded. We, therefore, conducted a phylogenetic analysis with the LW Rh1 sequences from our five bee species and 231 published hymenopteran sequences to determine the relationship of the new LW Rh1 gene sequences and the already published LW-sensitive hymenopteran opsins. We used the NJ algorithm with p-distance and included all sites (for species names and GenBank accession numbers see Supplementary Material online at http://visiongene.bio.uci.edu/ABresearch.html). We also used the maximum likelihood algorithm with the general time reversible model + gamma as implemented in PhyML (Guindon and Gascuel 2003). The length of the sequences in the analysis was 471 nucleotides, and we excluded introns as well as four very short sequences (Ceratina sp., Exomalopsis completa, E. rufiventris, and Tetraloniella sp.).
Relative Rates Tests of the Duplicated Bee Gene Sequences
Possible site-specific rate changes that accompany gene duplication may indicate altered selective constraints (either enhanced or reduced) after diversification and provide information about potential amino acid residues that may count for functional divergence between the two genes (such changes are called type-I evolutionary functional divergence [Gu 1999]). (For a general review of functional divergence tests in protein evolution see Gaucher et al. [2002] and Yang and Bielawski [2000].) Hence, we attempted to detect possible site-specific rate changes between LW Rh1 and LW Rh2 by use of a nonhomogeneous gamma model that is implemented in the software DIVERGE version 1.04 (Gu 1999; Gu and Vander Velden 2002). For that, we calculated the coefficient of functional divergence () between the two gene clusters for each site and tested the null hypothesis
= 0. Conceptually,
is a measure of the degree of independence between the relative evolutionary rates at similar sites in the LW Rh1 and LW Rh2 clusters. The coefficient ranges from 0 to 1, where 0 indicates that the evolutionary rate is virtually the same in both gene clusters. Values of
significantly greater than 0 indicate rate shifts between homologous sites among the two gene clusters (Gu 1999, 2001).
Homology Modeling of Hymenopteran Opsins
Identifying polymorphic amino acid sites, correlating amino acid substitutions with shifts in spectral sensitivity, and mapping such substitutions onto a three-dimensional crystal structure of rhodopsin has been shown to be a useful approach for studying the relationship between opsin structure and spectral function (Briscoe 2002; Hunt et al. 2001). In such a way, the identified amino acid sites allow predictions about potential spectral-tuning effects in homologous opsin genes even if physiological data for these opsins are still missing. We, therefore, created a homology model of the complete Apis mellifera LW Rh1 opsin by the same procedure as described in Briscoe (2002) and mapped onto it variable sites that have previously been implicated in spectral tuning as well as the sites identified by method of Gu (1999, 2001).
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Results |
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Hymenopteran LW-Sensitive Opsin Phylogeny
To control for the possibility of PCR contamination and to test what impact the new gene has on existing phylogenies, the LW opsin nucleotide sequences from 231 species and 13 families were analyzed together with the LW Rh1 sequences from our five species. All new LW Rh1 sequences presented in this study cluster together with the other members of their genus (phylogeny available as Supplementary Material online at http://visiongene.bio.uci.edu/ABresearch.html). Diadasia afflicta forms a well-supported branch (86% NJ bootstrap support) with D. nigrifrons that is also recovered in the ML analysis. D. rinconis is the sister group to D. martialisD. diminuta in both the ML tree and the NJ tree. Osmia rufa clusters with other Megachilinae, Hoplitis, and Chelostoma spp. (61% bootstrap support). The two Bombus sequences cluster together with the previously published sequences from these species in both the NJ and ML trees and with all other published LW-sensitive opsin gene sequences from about 80 species of the same genus. In both species, our sequence data and the already published sequences showed variation at only six of the 711 nucleotide sites of common overlap. This analysis demonstrates that the LW Rh1 sequences we obtained from our species are most closely related to all previously reported LW Rh sequences in GenBank from similar species or genera. The identification of the new LW Rh2 gene in hymenopterans, therefore, does not impact the interpretation of previously published LW Rh1 phylogenies.
Rate Shifts Between LW Rh1 and LW Rh2
Pairwise comparisons of all 10 bee sequences by application of the Nei-Gojobori algorithm (Nei and Gojobori 1986) indicated saturation of synonymous substitutions (dS) between the LW Rh1 and LW Rh2 genes (fig. 4). (We also used the Li, Wu, and Luo [1985] method, which takes into account transition and transversion bias, and synonymous substitutions were incalculable between the paralogs). Mean dS was 0.44 and 0.47 within LW Rh1 and LW Rh2, respectively; dS between LW Rh1 and LW Rh2 was 0.81. The nonsynonymous substitutions per nonsynonymous site were 0.05 for LW Rh1 and 0.02 for LW Rh2 (we found no differences at the amino acid level between the B. terrestris and B. impatiens LW Rh2 sequences). Mean dN between LW Rh1 and LW Rh2 was 0.18. We used the Z-statistics implemented in MEGA 3.0 (Kumar, Tamura, and Nei 2003) to test the hypothesis that dS = dN; that is, both genes evolved neutrally and no selection is operating on them. For both genes, we could reject the hypothesis and we found that dN is significantly smaller than dS (LW Rh1: Z = 13.0, P < 0.001; LW Rh2: Z = 16.8, P < 0.001), which indicates a strong purifying selection pressure on both genes. We also tested whether synonymous and nonsynonymous substitution rates differ between the two genes (dS(LW Rh1) = dS(LW Rh2) and dN(LW Rh1) = dN(LW Rh2); same Z-statistics as above). Nonsynonymous substitution rate was found to be significantly lower in LW Rh2 compared with LW Rh1 (Z = 2.8, P < 0.01), and we could not reject H0 that dS(LW Rh1) = dS(LW Rh2) (Z = 0.84, P > 0.05). Our results indicate that the rate of synonymous nucleotide substitution does not differ between both genes (dS(LW Rh1) = dS(LW Rh2)), but the effect of purifying selection pressure seems to be stronger on LW Rh2 than on LW Rh1 (dN(LW Rh1) > dN(LW Rh2)). The higher rate of amino acid substitutions among the LW Rh1 sequences compared with LW Rh2 after speciation is apparent in the long branches at the tips of the LW Rh1 cluster compared with the equivalent LW Rh2 branches (fig. 1B). These results suggest that LW Rh2 may be a more useful gene for resolving higher-level taxonomic relationships than is LW Rh1.
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Homology Modeling of Apis mellifera LW Rh1
Specific amino acids in the chromophore-binding pocket are known to modulate the absorption spectrum maximum of the visual pigment to longer or shorter wavelengths of light (Salcedo et al. 2003). Therefore, we used the homology model of the Apis mellifera LW Rh1 opsin to map variable residues near the chromophore and identified five sites in our data set (residues 94, 97, 138, 185, and 186) that are homologous to sites that are involved in spectral shifts in human, New World monkey, crayfish, or insect pigments. We also mapped the five sites identified above as undergoing type I functional divergence and found that all of these sites face the exterior of the protein, away from the chromophore-binding pocket (data not shown). The significance of these sites for functional differentiation of the two opsins is discussed below.
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Discussion |
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Given the large number of papers in recent years that have utilized the LW Rh1 gene as a molecular marker (Hsu et al. 2001; Jiggins 2003; Ortiz-Rivas, Moya, and Martinez-Torres 2004), particularly for hymenopteran systematics (Mardulyn and Cameron 1999; Ascher, Danforth, and Ji 2001; Cook et al. 2002; Kawakita et al. 2003), the impact of a second hymenopteran LW opsin gene on all such previously published studies is worth briefly considering. First, we have shown that all available hymenopteran LW opsin sequences in GenBank belong to the LW Rh1 group. Therefore, our results do not challenge current opsin phylogenies. Second, we have found that LW Rh2 is evolving at a much slower rate than LW Rh1 (fig. 1B). This finding suggests that this new gene might usefully resolve higher-level phylogenetic relationships among hymenopterans. Third, we have developed gene-specific primers that permit the direct sequencing of this gene from genomic DNA (table 2). The availability of these primers should make direct testing of this hypothesis possible.
The impact of LW Rh2 on our original rationale for examining the hymenopteran genome for extra LW opsin copies is also worth considering. Does the discovery of LW Rh2 shed light on the origins of the extra LW photoreceptors found in the retina of some hymenopteran species by Peitsch et al. (1992) in so far as this new gene may have given rise to those receptors? We think that the answer is likely to be no because LW Rh1 is expressed in the retina, whereas LW Rh2 is likely extraretinal in origin. Therefore, it is more parsimonious to assume that the retinal receptors described by Peitsch et al. (1992) evolved from other retinal opsins, such as LW Rh1, rather than from a (more distantly related) extraretinal opsin. Duplicated LW opsins that have given rise to new retinal photoreceptor classes have been found in butterflies (e.g., PglRh1 and PglRh3) (Kitamoto et al. 1998), and as shown in figure 1, these genes are closely related to each other. Furthermore, these gene duplications evolved after the split between the common ancestor of nymphalid and papilionid butterflies (Briscoe 2001). Therefore, in the absence of screening pigments, the extra receptors found in the Andrenidae, Xiphydriidae, and Tenthredinidae are very likely the result of recent, independent duplications of the LW Rh1 gene (see discussion below).
Where then might the new LW Rh2 gene be expressed? From the original study reporting the cloning of the Apis mellifera LW Rh1 gene from eye-specific cDNA (Chang et al. 1996), it is clear that this gene is expressed in the photoreceptor cells in the retina. We have attempted RT-PCR using the gene-specific LW Rh2 primers on cDNA synthesized from Bombus impatiens head tissue mRNA. It is also clear from these experiments that the LW Rh2 transcript is not very abundant, because we were unable to amplify this gene from our cDNA. By contrast, we easily amplified, cloned, and sequenced the LW Rh1 transcript and localized it by in situ hybridization to the retina (Spaethe and Briscoe, unpublished data). We speculate that LW Rh2 may have a specialized function as an extraretinal opsin, expressed in a small number of light-sensitive neurons in the brains of bees. What evidence do we have to support this hypothesis? First, an extraretinal opsin, PglRh4, has been cloned from butterflies (Briscoe and Nagy 1999). This transcript is expressed at exceedingly low levels, such that it was undetectable on a Northern blot of mRNA from a single individual butterfly head (Briscoe 2000). (Low mRNA abundance seems to be the case also for Anopheles GPRop7, [C. A. Hill, personal communication]). Second, polyclonal antibodies specific to this protein have been developed and show that this opsin is localized to the optic lobes of butterflies and moths (Briscoe and Nagy 1999; Lampel, Briscoe and Wasserthal, unpublished data.). This opsin appears to have evolved before the radiation of lepidopterans (moths and butterflies) (fig. 1B). Therefore, it is possible that extraretinal opsins evolved early in invertebrate evolution, but gene conversion and gene loss in some lineages has obscured this development. (Gene conversion, for example, has played a role in the evolution of some dipteran LW opsins [Spaethe and Briscoe, unpublished observation]). This finding makes sense from a functional point of view because generalized light detection is a necessary component for the entrainment of photoperiodic and circadian rhythms. We note that the Drosophila melanogaster Rh6 opsin has been detected in the extraretinal eyelet structure (Yasuyama and Meinertzhagen 1999) and that this class of photoreceptor has been proposed to be part of the light sensitive input pathway for the photic entrainment of the circadian clock (Malpel, Klarsfeld, and Rouyer 2002; Shimizu, Yamakawa, and Iwasa 2001). We speculate that LW Rh2 together with Anopheles GPRop7 represent a novel and ancient class of extraretinal opsins.
Pattern of Amino Acid Substitution and Candidate Spectral-Tuning Sites
Finally, we consider whether the observed amino acid variation between the two hymenopteran proteins is likely to result in spectral tuning or other functional differences. By use of the Gu (1999, 2001) method, we identified five sites as undergoing type I functional divergence (amino acids 129, 143, 170, 177, 263). Each of these sites, when mapped onto the homology model of the Apis mellifera LW Rh1 opsin, faces the exterior of the protein, and the significance of these sites for rhodopsin function is unknown. Eighteen fixed (or nearly fixed) differences between LW Rh1 and LW Rh2 exist. A number of these sites (e.g., 117, 120, 228, and 229) are located in cytoplasmic loops II and III and are presumably involved in the recognition of the G-protein (Teller et al. 2001). This finding suggests that functional differences in G-protein binding or activation may exist between the two opsins. Five variable sites (residues 94, 97, 138, 185, and 186) were found to be homologous to sites that are involved in spectral shifts in human, New World monkey, crayfish, or insect pigments (fig. 3).
Amino acid sites 94 and 97 have undergone parallel/convergent changes that are correlated with spectral shifts in peak sensitivity in several butterfly opsins (Briscoe 2001) and have also undergone similar changes in crayfish (Crandall and Cronin 1997) and bees (Briscoe 2002). We observed a Phe to Cys substitution at amino acid 94 and a Gly to Ala substitution at amino acid 97 between LW Rh1 and LW Rh2 (fig. 3), which suggests that these sites are undergoing correlated evolution. Mapping residue 94 onto a homology model of the full-length Apis mellifera LW opsin showed that this amino acid is located in the third transmembrane domain of the protein and faces the chromophore-binding pocket, which makes it highly likely to have an effect on spectral tuning in the bee opsins (fig. 5B). (Indeed, site-directed mutagenesis of this site was shown to affect wavelength regulation [Zhukovsky, Robinson, and Oprian 1992]).
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The Phe to Tyr substitution at amino acid site 185 in transmembrane domain V is also found in butterflies and crayfish and is correlated with a red shift (Briscoe 2001; Crandall and Cronin 1997) (fig. 5C). In New World monkeys, an Ile to Phe substitution at residue 229 (which is equivalent to amino acid position 185 in our sequences) appears to be responsible for a 2-nm blue shift (Shyue et al. 1998). In addition, residue 186 (corresponding to human red cone pigment amino acid 230), which is highly variable in our four bee species and might be coevolving together with the adjacent residue 185, has been shown to cause a 1-nm blue shift in human cone pigments (Asenjo, Rim, and Oprian 1994; Merbs and Nathans 1993). Altogether we identified three residues (F94C, S138A, and F185Y) at which identical amino acid substitutions have been reported in other vertebrate and insect species and were shown to affect spectral tuning. Convergent evolution of vertebrate and invertebrate opsin spectral-tuning mechanisms has recently been experimentally verified by Salcedo et al. (2003), at least for UV vision. These results suggest that spectral diversification both within and between LW Rh1 and LW Rh2 may have played a role in the evolution of these opsins.
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Conclusion |
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Acknowledgements |
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Footnotes |
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