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From The Cover
Evolution
Molecular analysis of the evolutionary significance of ultraviolet vision in vertebrates
Department of Biology, Syracuse University, 130 College Place, Syracuse, NY 13244
Communicated by Masatoshi Nei, Pennsylvania State University, University Park, PA, April 28, 2003 (received for review January 9, 2003)
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max) at 
360
nm. Reconstructed SWS1 pigments of most vertebrate ancestors have
max values of 360 nm, whereas the ancestral avian
pigment has a max value of 393 nm. In the nonavian lineage,
UV vision in many modern species is inherited directly from the vertebrate
ancestor, whereas violet vision in others has evolved by different amino acid
replacements at 10 specific sites. In the avian lineage, the origin of the
violet pigment and the subsequent restoration of UV pigments in some species
are caused by amino acid replacements F49V/F86S/L116V/S118A and S90C,
respectively. The use of UV vision is associated strongly with UV-dependent
behaviors of organisms. When UV light is not available or is unimportant to
organisms, the SWS1 gene can become nonfunctional, as exemplified by
coelacanth and dolphin.
UV vision is mediated by visual pigments that absorb light maximally
(max)at 360 nm. These UV pigments and violet (or blue)
pigments with max values of 390–440 nm belong to a
short wavelength-sensitive type 1 (SWS1) pigment group
(11). Visual pigments consist
of a transmembrane (TM) protein, opsin, and the chromophore, usually an
11-cis-retinal. The variable max values are
generated by the interactions between the chromophore and various types of
opsins, which is referred to as the spectral tuning of visual pigments
(12). Experimental analyses of
the mechanisms of the spectral tuning of the SWS1 pigments started only
recently (13). So far, based
on a limited number of species, eight amino acid sites that determine the
max values of the SWS1 pigments have been identified
(14–19).
Comparative sequence analyses of SWS1 pigments suggest that most contemporary pigments have evolved from the UV pigment in the vertebrate ancestor (16, 17). This hypothesis has not been tested and, consequently, the evolutionary processes of the SWS1 pigments are not well understood. To explore these issues, we reconstructed ancestral pigments considering 10 representative SWS1 pigments from a wide range of vertebrates. The results show that most vertebrate ancestors indeed used UV pigments, whereas the avian ancestor made the UV pigment violet-sensitive, but some of its descendants have restored UV-sensitivity. Here, we also present a new amino acid site that is involved in the spectral tuning of the SWS1 pigments.
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Materials and Methods |
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So far, both amino acid sequence and max are
characterized for 21 SWS1 pigments. They are cloned from goldfish
(Carassius auratus), zebrafish (Danio rerio), Malawi fish
(Metriaclima zebra; GenBank accession no. AF191220
[GenBank]
), coelacanth
(Latemeria chalamnae), frog (Xenopus laevis), salamander
(Ambystoma trigrinum; AF038948
[GenBank]
), chicken (Gallus gallus),
pigeon (Columba livia), budgerigar (Melopsittacus
undulatus), zebra finch (Taeniopygia guttata), canary
(Serinus canaria), chameleon (Anolis carolinensis), gecko
(Gekko gekko; AY024356
[GenBank]
), human (Homo sapiens), macaque
(Macaca fascicularis), squirrel monkey (Sciurus
carolinensis), marmoset (Callithrix jacchus), bovine (Bos
taurus; U92557
[GenBank]
), dolphin (Tursiops truncatus), mouse (Mus
musculus), and rat (Rattus norvegicus) (for the date source, see
ref. 11). Among these, the
SWS1 genes in the coelacanth
(22) and dolphin
(23) are pseudogenes. The
evolutionary tree based on molecular and paleontological data
(20,
21) is shown in
Fig. 1, where the phylogenetic
positions of the avian and mammalian pigments are not specified.
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Construction of the Chimeric and Ancestral Pigments. To infer the amino acid sequences of ancestral pigments at nodes a–g in Fig. 1, we have selected 10 representative pigments: goldfish (P359), frog (P425), salamander (P359), chicken (P415), pigeon (P393), zebra finch (P358), chameleon (P358), human (P414), bovine (P436), and mouse (P359). In the inference, we considered essentially the same phylogenetic relationships of the 10 pigments given in Fig. 1. One exception, however, is the phylogeny of the three avian pigments: (chicken (P414), (pigeon (P393), zebra finch (P358))). This tree topology is based not only on the amino acid sequences of SWS1 pigments (14) but also on DNA–DNA hybridization data (24). When this tree topology was used, the seven ancestral amino acid sequences were inferred by using a likelihood-based Bayesian method (25, 26).
Point mutations were generated by using QuikChange site-directed mutagenesis kit (Stratagene). Various chimeric pigments were also constructed by recombining different SWS1 cDNAs using restriction sites SphI, NdeI, and MfeI (Fig. 2). NdeI and MfeI sites were introduced and removed after recombination by site-directed mutagenesis. To rule out spurious mutations, all WT and mutated opsins were sequenced by using the Sequitherm Excel II Long-read kits (Epicentre Technologies, Madison, WI) with dye-labeled M13 forward and reverse primers. Sequencing reactions were run on a LI-COR 4200LD automated DNA sequencer (LI-COR, Lincoln, NE).
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In Vitro Assays of Visual Pigments. The SWS1 cDNAs in an expression vector, pMT5, were expressed in COS1 cells by transient transfection (27). 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 using monoclonal antibody 1D4 Sepharose 4B (Cell Culture Center, Minneapolis) in buffer consisting of 50 mM N-(2-hydroxyethyl) piperazine-N'-2-ethanesulfonic acid (pH 6.6), 140 mM NaCl, 3 mM MgCl2, 20% (wt/vol) glycerol, and 0.1% dodecyl maltoside. The absorption spectra of visual pigments were recorded at 20°C by using a Hitachi (Tokyo) U-3000 dual beam spectrophotometer. Recorded spectra were analyzed by using SIGMAPLOT software (Jandel, San Rafael, CA).
In addition, visual pigments were not only exposed to a 366 nm UV light
illuminator and a 60-W room lamp with 440-nm cutoff filter, but also denatured
by sulfuric acid (H2SO4) at pH 1.8 in the dark. The
max values of all ancestral and mutant pigments under light
exposure and in sulfuric acid moved to new peaks at 380 and 440 nm,
respectively, showing that the observed max values are
caused by opsins covalently linked to 11-cis-retinal via a Schiff
base bond (27).
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Results and Discussion |
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max. All
amino acids that are involved in the spectral tuning of various visual
pigments have been localized in TM I–VII
(11). To evaluate the effects
of amino acid differences in the N and C termini on the max
shift of SWS1 pigments, we replaced the two segments of the mouse pigment with
those of the goldfish, chameleon, and human pigments separately. Here the N
and C termini are amino acids between sites 1 and 30 and between sites 313 and
348, respectively, where the amino acid site numbers are those of the bovine
rhodopsin (GenBank accession no. M21606
[GenBank]
). These two termini are separated from
the "internal segment" by NdeI and MfeI sites
(Fig. 2). The three chimeric
pigments have max values of 359–360 ± 1 nm and
are identical to the max of the mouse pigment
(13). These analyses
demonstrate that different amino acids at the two termini are not involved in
the spectral tuning of SWS1 pigments.
Ancestral Pigments. In inferring ancestral pigments, we consider the
tree topology of (goldfish (P359), ((frog (P425), salamander (P359)),
(((chicken (P415), (pigeon (P393), zebra finch (P358))), chameleon (P358)),
(human (P414), bovine (P436), mouse (P359))))), which is based on molecular as
well as paleontological data (see Materials and Methods). The amino
acid sequences of the pigments at nodes a–g (pigments
a–g) were inferred by using PAML
(26) with the JTT model of
amino acid replacements (28)
(Fig. 2). Most of these
inferred amino acids have posterior probabilities 
0.9, but those of many
amino acids in the N and C termini are <0.9
(Fig. 2). We have also inferred
the ancestral amino acid sequences by using the Dayhoff model of amino acid
replacements (29). The two
sets of inferred amino acid sequences of pigments a–g
differ at 11, 2, 3, 2, 3, 5, and 1 sites, respectively. Amino acids at most of
these sites have a posterior probability of <0.9, where amino acids with
the two highest probabilities in one inference are often reversed in another.
Thus, the uncertain amino acid inference based on a certain model of amino
acid replacements and different amino acids inferred by the two models are
closely interrelated.
To reconstruct these ancestral pigments, we first cloned the cDNAs of the
chameleon, pigeon and mouse pigments into pMT5 separately. Before
reconstructing the ancestral pigments, however, the N and C terminal segments
of the pigeon and mouse pigments were replaced by those of the chameleon
pigment. Although the amino acid differences at these segments should not
affect the max values of the SWS1 pigments, this operation
will eliminate any possible differential effects of the interactions between
the TM and terminal segments on the max shift. The seven
ancestral pigments were then engineered by introducing a total of 70 aa
changes into the "internal segment" of the modified mouse and
pigeon pigments and chameleon pigment (Fig.
1). In vitro assays show that the engineered pigments
a–e and g are UV-sensitive, whereas pigment
f is violet-sensitive (Fig.
1). Standard errors associated with the max
values in Fig. 1 are all within
1 nm. It should be cautioned that these results are based on the amino acids
with the highest posterior probabilities, some of which are still <0.9.
Furthermore, they also depend on the phylogenetic relationship of (chicken
(P414), (pigeon (P393), zebra finch (P358))). Before we draw any conclusions,
therefore, these points must be checked.
To evaluate the effects of the ambiguous amino acids on the
max, we first replaced the amino acids 152–348 of
pigment a by those of the chicken and bovine pigments separately.
Compared with pigment a, each pigment has 31 different amino acids at
this region. These two chimeric pigments have max values of
362 ± 1 nm (data not shown), showing that different amino acids at
sites 152–348 do not cause any max shift in the SWS1
pigments (see ref. 17). At
amino acid sites 31–151, ancestral pigments a–g
contain 20, 5, 6, 7, 7, 8, and 3 amino acids with posterior probabilities of
<0.9, respectively. For pigment a, we replaced the 20 amino acids
with those with the second highest probabilities in six sets: 1, 1, 9, 1, 6,
and 2 changes in TM I, segment C–I, TM II, segment E–I, TM III,
and segment C-II, respectively, where C and E denote cytoplasmic and
extracellular loops (30). None
of the max values of these mutant pigments differs from
that of pigment a (Table
1). For pigment f, when we introduced eight mutations in
four sets, most of which have no effect on the max shift,
except that A118S (amino acid changes from A to S at site 118) in TM III
increases the max by 5 nm
(Table 1). In fact, with the
exceptions of an amino acid site 116 in pigment a, and 49 and 118 in
pigment f, none of the ambiguous amino acids in the seven ancestral
pigments are located at functionally important sites (see below). Thus, the
effect of the ambiguous amino acids on the max is not an
important factor.
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To test the effect of different tree topologies on the
max, let us now assume that the three avian pigments are
equally distantly related (Fig.
1). Then, for pigment a, of 20 amino acid changes, the
orders of amino acids with the two highest posterior probabilities are
reversed at three sites. For pigment f, the highest posterior
probability of each inferred amino acid increases significantly. For example,
the probability for A118 increases from 0.71
(Table 1) to 0.97. Because the
inference of A118 is now highly reliable, a slight increase in the
max caused by A118S may be disregarded. Importantly, the
new tree topology of the three avian pigments does not introduce any new amino
acids. Because amino acids with the two highest probabilities at ambiguous
sites do not change the max
(Table 1), the difference in
the effects of the two avian tree topologies on the max is
also negligible.
The order of ambiguous amino acids with the two highest posterior
probabilities in one inference is often reversed in the other and, therefore,
our mutagenesis results also imply that different amino acids inferred by the
different models of amino acid replacements do not change the
max significantly. All of these results show that most
ancestral pigments were UV-sensitive, whereas the ancestral avian pigment was
violet-sensitive (Fig. 1).
Spectral Tuning of the SWS1 Pigments. One curious aspect of the evolution of SWS1 pigments is that the avian ancestor achieved violet vision, but some of its descendants changed it back to UV vision (Fig. 1). It is of considerable interest to find some biological significance associated with these changes (see below). At the same time, these evolutionary changes provide an excellent opportunity to test our current understanding of the spectral tuning of the SWS1 pigments: amino acid sites at 46, 49, 52, 86, 90, 93, 114, and 118 mediate the spectral tuning of the SWS1 pigments (14–19).
Of the eight critical sites, amino acids at 49, 86, and 118 differ between
pigments e and f. When F49V/F86S/S118A (amino acid changes
F49V, F86S, and S118A) are introduced into pigment e, the mutant
pigment has a max of 374 ± 1 nm
(Fig. 3A). Thus, it is
clear that the eight amino acid sites are not sufficient to explain the
transition from pigment e to pigment f. It turns out that
pigments e and f have six different amino acids in addition
to those at 49, 86, and 118 at the "internal segment." When the
amino acids at these six sites in pigment e with F49V/F86S/ S118A
were replaced by those of pigment f individually, all but one
quadruple mutant have max values of 374–375 ±
1 nm (Fig. 3A). The
pigment e with F49V/F86S/L116V/S118A (referred to as pigment
e') has a max of 393 ± 1 nm
(Fig. 3A), which is
identical to that of pigment f. When the four reverse mutations are
introduced into pigment f, the mutant pigment has a
max of 360 ± 1 nm
(Fig. 3A). The
acquisition of violet sensitivity by the ancestral avian pigment can now be
fully explained by F49V/ F86S/V116L/S118A. Thus, we have discovered a new
amino acid site that is involved in the spectral tuning of the SWS1
pigments.
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It has been claimed that the SWS1 pigments of zebra finch, canary, and
budgerigar achieved their UV sensitivities by S90C
(14,
15). Indeed, when S90C is
introduced into pigment e', the mutant pigment becomes
UV-sensitive (Fig.
3B). However, in addition to S90C, F46L/S86C/A114G
probably occurred in the common ancestor of the zebra finch and canary
pigments. When they are introduced singly into pigment e',
F46L, S86C, and A114G shift the max by 0, -27, and 5 nm,
respectively (Fig.
3B). When S86C/S90C and F46L/S86C/S90C/A114G are
introduced into pigment e', the mutant pigments have
max values of 360 ± 1 nm
(Fig. 3B), identical
to the effect of S90C alone
(14). Thus, both S86C and S90C
decrease the max by 30 nm either separately or jointly.
The budgerigar, zebra finch, and canary pigments, and ancestral pigment
f have amino acids Ala, Cys, Cys, and Ser at site 86, and Cys, Cys,
Cys, and Ser at site 90, respectively. Thus, it is most likely that S90C
occurred in the common ancestor of the three avian pigments, whereas S86C
occurred in that of the zebra finch and canary pigments, strongly suggesting
that S90C preceded S86C. Thus, despite its ability, S86C probably was not
involved in the max shift.
Having new information that the max values of SWS1
pigments are determined mainly by nine amino acid sites 46, 49, 52, 86, 90,
93, 114, 116, and 118, we can identify a total of 43 amino acid replacements
at these sites during vertebrate evolution. At these sites, with the exception
of the three amino acids, all amino acids in pigments
a–g have a posterior probability of >0.97. Amino
acid changes that are known to cause some max shifts in
some mammalian and avian SWS1 pigments are shown in
Fig. 1. Clearly, the
contemporary UV pigments in fish, salamander, chameleon, gecko, mouse, and rat
are mostly free of these amino acid replacements, showing that these pigments
have maintained their UV-sensitivities during vertebrate evolution.
F49L/T93P/L116V/S118T and L116V seem to have increased the
max values of the frog and Malawi fish pigments,
respectively (Fig. 1). On the
other hand, V49L in the pigeon pigment and G114A in the macaque pigment do not
seem to change the max. The actual effects of these and
other amino acid changes at the nine critical sites on the
max shift remain to be evaluated.
Ecological and Physiological Requirements for UV Vision. A wide range of species has maintained UV vision during vertebrate evolution and used it for such basic behavioral traits as foraging, mate choice, and communication. This finding suggests that organisms with UV vision have a selective advantage over those without it. However, we have also seen that many other species exchanged UV vision by violet vision. Therefore, the selective advantage of organisms having UV vision may occur under special circumstances. Because UV vision works under UV light, it is reasonable for organisms to switch UV vision to violet vision when UV light is not available to them. In the extreme cases, as we have seen in the coelacanth and dolphin (Fig. 1), the SWS1 gene can become nonfunctional when UV and violet light are not available or are unimportant to them.
Given an abundance of UV light in their environment, why have so many
organisms switched from UV vision to violet vision? Two major reasons can be
considered for this change. First, UV light, even at 360 nm, can damage
retinal tissues (31). Indeed,
the yellow pigments in the lenses or corneas in many species, including human,
are devised to obviate most UV light from reaching the retina
(2). This change in the eye
structure must be responsible for the transition from UV vision to violet
vision. Second, by achieving violet vision, organisms can improve visual
resolution and subtle contrast detection
(2). On the other hand, in the
avian lineage, its ancestor lost UV vision, but some of its descendants
restored it (Fig. 1). This
restoration of UV vision seems to have been caused by avian migration. For
migratory birds, the pineal gland senses changes in day length and releases
hormones that cause the restless behavior that precedes each period of
migration (32). Indeed, in a
"relatively closely related" American chameleon (Anolis
carolinensis), some opsin genes, including the UV gene, are expressed in
the pineal gland (33). In
addition, UV pigments in the migratory birds seem to be essential in the
orientation based on the "sun compass"
(34).
Rodents distinguish themselves from other mammals by using UV vision (2). It has been observed that voles (Microtus agrestis) mark their runaways with urine and feces, which can be detected much more easily under UV light than under visible light (7). Importantly, a high proportion of the light available to animals around dawn and dusk is of short wavelength (35). Consequently, nocturnal rodents that are also active at these times of the day can use UV pigments for a range of tasks requiring vision. Under similar dim light conditions, UV pigments are also important, but in another simple but highly structured photoreceptor organ, called the third (or parietal) eye (36). The UV pigments are the major visual pigments expressed in the third eye of the American chameleon (33), strongly suggesting that UV detection through this special organ is important to lizards in addition to UV vision.
It should also be noted that many fish have UV vision, but this does not necessarily mean that they use UV vision their entire lives. On the contrary, UV vision in many of them may decline during development. For example, young brown trout (Salmo trutta) has UV vision, but the adults do not use it (37). This change in gene expression is closely related to the change in their habitats: young fish live in shallow water and feed on plankton, where UV light is essential, whereas adults live in deeper water and do not receive much UV light.
Taken together, the use of UV pigments by organisms is strongly associated with their light environments and behaviors. Compared with organisms with violet vision, those with UV vision have an advantage of recognizing certain UV-reflecting objects much more quickly, but they lack precision in viewing their surroundings and are also subjected to a higher chance of developing retinal damages caused by UV light. Whether organisms use UV vision or violet vision must depend mostly on a relative importance of these and other conflicting characteristics associated with UV vision to them. Expression of UV opsins in the pineal gland and third eye in lizards strongly suggests that the importance of UV pigments is not limited to vision. To appreciate the evolution of UV pigments in nature, it is necessary to study the roles of UV and violet pigments of many species in different photic environments. The functional differentiation of these SWS1 pigments must be related not only to ecological and behavioral changes of organisms, but to physiological changes of the eye and other photosensitive organs during evolution.
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Footnotes |
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See commentary on page 8045.
* To whom correspondence should be sent at present address: Department of Biology, Emory University, 1510 Clifton Road, Atlanta, GA 30322. E-mail: syokoya{at}emory.edu.
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