Department of Biology, Emory University, Atlanta, Georgia
Correspondence: E-mail: syokoya{at}emory.edu.
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Abstract |
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Key Words: Squirrelfishes rhodopsin adaptive evolution
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Introduction |
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Visual pigments usually consist of an opsin and the photosensitive molecule 11-cis-retinal chromophore and have been classified into five paralogous groups: (1) RH1 (consisting of mostly rhodopsins), (2) RH2 (RH1-like), (3) SWS1 (short wavelengthsensitive type 1), (4) SWS2 (SWS type 2), and (5) M/LWS (middle wavelengthsensitive and long wavelengthsensitive) pigments (Yokoyama and Yokoyama 1996; Yokoyama 2000a; Ebrey and Koutalos 2001). Among these, the molecular basis of specific wavelength sensitivity, or spectral tuning, of visual pigments has been reasonably well understood for the M/LWS pigments, where the observed max values of 510 to 560 nm can be explained fully by amino acid differences at five sites (Yokoyama and Radlwimmer 2001). The
max values of the SWS1 pigments are based on at least a total of 10 amino acid sites (Yokoyama, Radlwimmer, and Blow 2000; Wilkie et al. 2000; Shi, Radlwimmer, and Yokoyama 2001; Shi and Yokoyama 2003; Fasick, Applebury, and Oprian 2002). Because of strong synergistic interactions among these pigments, however, the molecular basis of spectral tuning of the SWS1 pigments is far from clear (Shi, Radlwimmer, and Yokoyama 2001; Shi and Yokoyama 2003). Additional eight amino acid sites are also known to be involved in the
max shift of RH1, RH2, and SWS2 pigments, but the mechanisms of spectral tunings of these pigments are not well understood (Yokoyama 2000a; Ebrey and Koutalos 2001; Takahashi and Ebrey 2003).
Toller (1996) cloned and sequenced the RH1 opsin cDNAs of a total nine squirrelfish species and two soldierfish species (Holocentridae: Beryciformes) and found that the max values of 481 to 502 nm of the corresponding pigments are closely associated with the depths of the holocentrid habitats (see also Munz and McFarland [1973]). These data provide a unique opportunity to explore the molecular basis not only of spectral tuning of RH1 pigments but also of possible adaptive evolution of these pigments to different water environments. Phylogenetic and mutagenesis analyses show that most of these
max values were generated by amino acid replacements at sites 122, 261, and 292. The results also suggest that the RH1 pigments in the common ancestor of the holocentrids had a
max value of approximately 493 nm, from which both red shifts and blue shifts in the
max value have been achieved by adaptive evolution.
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Materials and Methods |
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The amino acid sequences of ancestral pigments were inferred using a likelihood-based Bayesian method (Yang 1997). In the inference, we considered the phylogenetic relationship of (river lamprey (P500), (((goldfish (P492), zebrafish (P501)), cavefish (P503)), ((((N. sammara (P502), N. argent (P502)), S. punc (P494), (S. microst (P494), S. diadema (P491), S. xanther (P486))), (N. aurolin, (S. spinif (P490), S. tiere (P490)))), (M. viola (P499), M. berndti (P493))))) (see Yokoyama [2000a] and Results and Discussion).
In vitro Assays of the Bovine RH1 Pigment
Point mutations were generated by using QuickChange site-directed mutagenesis kit (Stratagene). To rule out spurious mutations, the mutated opsins were sequenced by using the Sequitherm Excel II long-read kits (Epicentre Technologies, Madison, Wis.) with dye-labeled M13 forward and reverse primers. Sequencing reactions were run on a LI-COR 4200LD automated DNA sequencer (LI-COR, Lincoln, Neb.). The bovine (P500) and its mutant cDNAs in an expression vector, pMT5, were expressed in COS1 cells by transient transfection. The visual pigments were regenerated by incubating these opsins with 11-cis-retinal (Storm Eye Institute, Medical University of South Carolina, Charleston) in the dark. The resulting visual pigments were then purified by immunoaffinity chromatography by using monoclonal antibody 1D4 Sepharose 4B (The Cell Culture Center, Minneapolis, Minn.) (Yokoyama 2000b). The absorption spectra of visual pigments were recorded at 20°C using a Hitachi (Tokyo) U-3000 dual beam spectrophotometer. Recorded spectra were analyzed by using SIGMAPLOT software (Jandel, San Rafael, Calif.).
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Results and Discussion |
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Among the squirrelfish pigments, the max value of approximately 500 nm of N. sammara (P502), N. argent (P502), and M viola (P499) is suited to detect the light at the shallow water of type I habitat and that of 481 nm of N. aurolin (P481) is suited to detect the light at the much deeper type III habitat (see Materials and Methods). Most holocentrids with other pigments with
max values of 486 to 494 nm live anywhere at the depth between 0 and 70 m (fig. 1). Thus, there is a close association between the
max value of pigments and the wavelengths of light available to holocentrids possessing these pigments. S. punctatissimum is an exception and is supposed to live at the surface, but its RH1 pigment has a
max value of 494 nm. It should be noted that not only is there some possibility of misclassification of holocentrid habitats but also some
max values may be erroneous (see below). The phylogenetic tree in figure 1 also shows that the visual pigments sampled from type I and II habitats and those sampled from type II and III habitats are more closely related with each other, suggesting that the
max shifts of these visual pigments have occurred in a stepwise manner.
Spectral Tuning and Evolution of Holocentrid Pigments
The max values of most holocentrid pigments reported by Toller (1996) agree with those in Munz and McFarland (1973) (table 2). In both Toller (1996) and Munz and McFarland (1973), the
max value is determined by the difference between the prebleached and the fully bleached spectra. It should be cautioned, however, that this "extraction spectrophotometry (ESP)" method may not always provide the correct
max value of the unbleached visual pigments. For example, the
max value of the prebleached pigment in cavefish (P503) is 503 nm, but that estimated from the difference between the prebleached and fully bleached pigments is 508 nm (Yokoyama, Knox, and Yokoyama 1995). The absorption spectrum obtained by the ESP method also has a rather flat plateau with a large standard deviation and, therefore, the accuracy of the
max value is not always clear-cut. Indeed, the
max value of S. xanther (P486) is approximately 485 nm in Toller (1996) and Munz and McFarland (1973), but the corresponding value is 490 nm when the microspectrophotometry (MSP) method is used (Ellis R. Loew, personal communication; table 2). Thus, the
max values of the holocentrid pigments must be interpreted with caution. In the future, it would be helpful to reevaluate these
max values using in vitro assay (see Materials and Methods), where the prebleached (or dark) spectrum and the prebleached and bleached (or dark-light) difference spectrum can both be evaluated. Despite this uncertainty, table 2 suggests that the
max values of most holocentrid pigments are repeatable.
When the RH1 pigments from a wide range of vertebrates are surveyed, most pigments have a max value of approximately 500 nm (Yokoyama 2000a; Ebrey and Koutalos 2001). Therefore, the
max values of 480 to 494 nm of the squirrelfish and soldierfish RH1 pigments seem to have arisen in the past 120 to 130 Myr. Among the 23 functionally important amino acid sites (see Materials and Methods), only five sites (positions 97, 116, 164, 261, and 292) are polymorphic among the 11 pigments (fig. 2). In addition, amino acids M122 (methionine at site 122) are monomorphic among the holocentrid pigments, but those in the orthologous pigments in most other vertebrates are E122 (fig. 2; Yokoyama 2000a), showing that E122M (amino acid change from glutamic acid to methionine at site 122) occurred in the holocentrid ancestor (fig. 1). In fact, the maximum-likelihoodbased Bayesian method (Yang 1997) suggests that E122M occurred in the common ancestor of the holocentrids, followed by T97S, F116S, A164G, F261Y, and A292S during holocentrid evolution.
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Figure 1 shows that many contemporary holocentrids have inherited their max values of 493 nm directly from their common ancestor and live in the type II habitat (0 to 70 m), suggesting that the holocentrid ancestor was an "ecological generalist" (Toller 1996). From this ancestor, three major changes seem to have occurred during holocentrid evolution. First, although they are similar to that of the vertebrate ancestor, who lived some 400 MYA, the
max values of 502 nm of N. sammara (P502) and N. argent (P502) are of much more recent origin. The red shift in the
max value was caused by F261Y, and both species (N. sammara and N. argenteus) now live mostly near the surface of the ocean. Second, the common ancestor of three species (N. aurolineatus, S. spiniferum, and S. tiere) seem to have modified the
max value of their RH1 pigments from 493 nm to 483 nm using A292S. Today, the direct descendant (N. aurolineatus) lives mostly at the depth below 60 m, suggesting that the common ancestor may have lived in the deeper habitat as well. Interestingly, a much more distantly related coelacanth (Latimeria chalumnae) lives at the depth of 200 m and uses RH1 pigments with a
max value of 485 nm, and this blue shift was caused by E122Q and A292S (Yokoyama et al. 1999). Thus, both squirrelfish and coelacanth ancestors used the identical A292S mutation for shifting the
max values of their RH1 pigments toward blue. Third, the common ancestor of S. spiniferum and S. tiere reverted the
max value of its RH1 pigments from 483 nm to 493 nm using F261Y. These two species now live at the depth of 0 to 70 m. These evolutionary analyses reveal that the
max values of the holocentrid pigments have been modified possibly two times, depending on the species, suggesting frequent changes in the habitat choice of squirrelfishes in the last 120 to 130 Myr.
Adaptive Evolution of Holocentrid RH1 Pigments
We have seen that the max values of most holocentrid pigments correspond to habitat depth. Thus, it is of considerable interest to evaluate whether this correlation arose because of positive selection. Such selective forces have often been inferred by demonstrating that the number of nonsynonymous changes per codon site is significantly larger than that of synonymous changes per codon site in a phylogenetic tree (e.g., Nei and Kumar 2000). Using the parsimony-based method (Suzuki and Gojobori 1999; Suzuki and Nei 2001) and likelihood-based Bayesian method (Yang 1997; Yang et al. 2000), we could not identify any positively selected codon sites. This is not surprising, because only a small number of amino acid changes are involved in the spectral tuning of visual pigments (Suzuki and Nei 2004).
To explore the possible adaptive changes in the holocentrid pigments, we shall evaluate the probability of observing E122M, F261Y, and A292S at certain branches under neutral evolution.
The probability of a specific amino acid change (AB) in a certain branch is calculated as the product of two quantities: (1) the probability (
) that the ancestral amino acid, A, is replaced by any other amino acid and (2) the probability (ß) that the amino acid change A
B occurs, given that A is replaced by another amino acid. To assess the
value, we first identify all amino acid sites (N) where the vertebrate ancestor had a specific amino acid, A, and count all amino acid replacements in the entire phylogenetic tree. Note that when we trace amino acid replacements at such critical sites in an opsin tree, not only is the number of changes small but also the changes are almost always unidirectional. Then, the average number of amino acid changes per site in the entire tree (K) can be evaluated by dividing the total numbers of amino acid replacements by N. Based on the amino acid sequences of ancestral organisms (see Materials and Methods), the K values for glutamic acid, phenylalanine, and alanine are 3/19, 9/29, and 11/25, respectively (table 3). Thus, without any selective force,
= 1exp (K x L/LT) for a specific branch of length of L and the total branch length of LT (= 0.344) for the entire tree. Table 3 shows that the
values for the three nonspecific amino acid replacements are less than 3%. The ß value is simply the proportion of the transition A
B among all amino acid changes from A to any other amino acids. Considering all amino acid changes from glutamic acid, phenylalanine, and alanine to others, the ß values of amino acid replacements E
M, A
S, and F
Y are given by 1/5, 8/19, and 6/18, respectively. Therefore, without any selective force, the probabilities (
x ß) of observing the specific amino acid change at branches a, b, c, and d in figure 1 are all less than 0.012 (table 3). Furthermore, the probability of observing the four independent amino acid replacements is 2.5 x 109.
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These analyses show that many holocentrids have RH1 pigments with blue-shifted max values. In fact, such species as marine eel, Conger eel, John Dory, coelacanth, chameleon, and dolphin are also known to have blue-shifted
max values (table 4; Yokoyama 2000a). With the exceptions of chameleon, these species live in the ocean and the blue-shifted
max values are most probably caused by their adaptation to the blue ocean environments. The chameleon pigments use the 11-cis-3, 4-dehydroretinal as the chromophore (Provencio, Loew, and Foster 1992). Visual pigments with the 11-cis-3, 4-dehydroretinal absorb more red-shifted wavelength than those with the 11-cis-retinal chromophore (Whitmore and Bowmaker 1989; Harosi 1994). Therefore, the RH1 pigments in this species must have a blue-shifted
max value to readjust the absorption spectrum toward the 500-nm wavelength. Again, the changes in the
max values of these RH1 pigments seem to have been affected by their photic environments. These functional changes seem to have been accomplished by a small number of amino acid replacements, such as D83N, E122Q, and A292S (table 4; Yokoyama 2000a). Thus, our results show that the molecular basis of spectral tuning in the currently characterized RH1 pigments at both amino acid and
max levels can be explained by a total of seven amino acid replacements at six sites: D83N, E122Q, E122M, H211C, F261Y, A292S, and A295S.
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Acknowledgements |
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Footnotes |
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