Visual pigments and oil droplets in diurnal lizards : a comparative study of Caribbean anoles
1
Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853,
USA
2
Department of Biology, Union College, Schenectady, NY 12308,
USA
3
Department of Biology, University of Virginia, Charlottesville, VA 22903,
USA
Present address: Department of Integrative and Molecular Neuroscience,
Division of Neuroscience and Psychological Medicine, Imperial College School
of Medicine, Charing Cross Hospital, Fulham Palace Road, London W6 8RF,
UK
Present address: Department of Anatomy, Physiology, and Genetics, Uniformed
Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda,
MD 20814, USA
* e-mail: ERL1{at}cornell.edu
Accepted 21 January 2002
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Summary |
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Key words: vision, microspectrophotometry, anoline, lizard, Anolis carolinensis, Polychrus marmoratus, visual pigment, oil droplet, photoreceptor
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Introduction |
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Animal visual systems exhibit tremendous diversity. A tenet of the
discipline of visual ecology is that this diversity is due, at least in part,
to differences among species in the environment in which their visual systems
function and in the visual tasks the systems are designed to carry out (see
Lythgoe, 1979). A common
approach has been to examine the visual and photic environment in which
different animals live and to compare it with the design features of their
visual systems, looking for correlations that might help explain the
functional significance of different elements in the system. As regards
spectral sensitivity (overall and of individual sets of photoreceptors), this
approach has been used most successfully in studies of fish and other aquatic
organisms in relation to the spectral quality of the waters they inhabit,
where it has been shown that these features have apparently evolved in
response to the color, water clarity and depth at which the animals are found
(Loew and Lythgoe, 1978
;
Bowmaker et al., 1994
;
Lythgoe et al., 1994
;
Levine and MacNichol, 1979
;
Shand, 1993
;
McDonald and Hawryshyn, 1995
;
Cronin et al., 1996
;
Partridge and Cummings,
1999
).
Attempts to apply the same process to terrestrial systems have generally
not yielded such clear relationships (e.g.
Partridge, 1989). For example,
Fleishman et al. (1997
)
measured overall spectral sensitivity and habitat light spectra for six
closely related species of Puerto Rican anoline lizard. Although the species
occupied microhabitats with distinctly different spectral irradiances, there
was little difference among the species in spectral sensitivity. Two possible
explanations were offered for the lack of apparent correlation between photic
environment and spectral sensitivity: (i) since the species evolved relatively
recently from a single common ancestor, they may all share the ancestral
condition and, for whatever reason (e.g. lack of time or lack of appropriate
genetic variation), they have not yet diverged in response to habitat
conditions; (ii) the visual systems have evolved in response to some specific
photic aspect (e.g. background radiance, which in all cases was dominated by
green vegetation) that is much more similar among habitats than is habitat
irradiance.
To deduce the influences of both ancestral state and environmental conditions on the evolution of specific traits, one would ideally like to compare species with distinctly different ecological conditions that share a common ancestry and, conversely, species under common ecological conditions with distinct ancestry.
The genus Anolis has experienced a massive radiation and contains
over 300 species. The best-studied species are those that occupy the islands
of the Greater and Lesser Antilles. Many of these islands appear to have been
colonized once or twice, and from these original colonists a number of new
species evolved on each island as the ancestral population diverged into
distinctly different ecological niches (see
Roughgarden, 1995). Detailed
studies of the evolutionary history and ecology of these species have shown
that many are found on different islands and occupy very similar ecological
niches (referred to as ecomorphs). However, the species on the different
islands occupying similar ecomorphs are generally not closely related. In
contrast, species from the same island occupying distinctly different
ecomorphs are typically closely related, reflecting the pattern of
colonization and radiation described above
(Jackman et al., 1999
;
Losos et al., 1998
).
The different ecological niches occupied are largely determined by
preferred substratum (e.g. tree trunks, grass, twigs, etc.) and range of
preferred temperatures (Losos et al.,
1998; Rand, 1964
;
Hertz et al., 1994
). An
indirect consequence of these shade and substratum preferences is that each
species occupies a habitat with a distinct and characteristic photic
environment. Fleishman et al.
(1997
) identified four
distinct photic habitats for anoles on Puerto Rico. These included: (i) full
shade, closed forest canopy where habitat irradiance is dominated by the green
chlorophyll spectrum and total light intensity is quite low; (ii) partial
shade, dry, semi-open forest or forest edge where light intensity is
intermediate and the irradiance spectrum includes a large short-wavelength
component coming from blue sky; (iii) full sun, open habitats such as grassy
fields where light intensity is high and the irradiance spectrum is broadband
and essentially that of sunlight; and (iv) canopy, species that live near the
forest canopy, where the light environment is similar to that of partial
shade, except that the background tends to include a mixture of green
(vegetation) and blue (short wavelengths from the sky). The anoline species
from other islands can, in most cases, also be assigned to one of these
habitat types (Fleishman,
2000
) (see also Endler,
1992
,
1993
). However, as described
above, the occupants of similar light environments on different islands are
not usually closely related. For example, the Puerto Rican grass anole
Anolis pulchellus is more closely related to the fullshade Puerto
Rican species A. gundlachi than it is to the grass anoles of
Hispaniola or Cuba (Jackman et al.,
1999
).
The retina of anoles is considered to be pure cone with three classes
identified, double cones and large and small single cones. All cells apart
from the accessory member of the double cone contain an oil droplet (Walls,
1934,
1967
). Visual pigment and
opsin sequence data are available for one species, Anolis
carolinensis. Provencio et al.
(1992
) reported the presence
of three visual pigments: long-wavelength-sensitive (LWS) with maximum
absorbance (
max) at 625 nm, medium-wavelength-sensitive
(MWS) with a
max of 503 nm and short-wavelength-sensitive
(SWS) with a
max of 462 nm, all using vitamin A2
as the chromophore. An ultraviolet-sensitive (UVS) opsin was later identified
and expressed (see Yokoyama and Yokoyama,
1996
; Kawamura and Yokoyama,
1997
) together with a second pigment similar in
max to the MWS pigment but more closely related to
rhodopsin. However, nothing is known about the visual pigments of other
anoles, nor have there been any reports concerning the spectral
characteristics of the oil droplets.
In this paper, we present a study of the visual pigments and oil droplets
of 17 species of Anolis lizard from the Caribbean. In addition, we
have included data for Polychrus marmoratus, a South American lizard
that is widely considered to be one of the closest relatives to
Anolis (Jackman et al.,
1999). The species included are summarized in
Fig. 1, which also shows their
evolutionary relationships and typical photic habitat. Jackman et al.
(1999
) identified 17 distinct
monophyletic lineages in the genus. Of these, five are represented in our
sample. The majority of the species come from two monophyletic clusters of
ecologically diverse species, one from Jamaica and one from Puerto Rico, which
provide the opportunity to examine the relationship between the light
environment and retinal design in closely related species. The other species
in our sample provide the opportunity to examine the visual system in the
context of the overall evolutionary history of the group. Finally, we have
examined the outgroup species Polychrus marmoratus in an attempt to
identify the ancestral condition (i.e. those features common to all the anoles
and Polychrus marmoratus).
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Materials and methods |
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Chromophore analyses
Because vitamin A2 was found to be the chromophore for the
visual pigments in A. carolinensis
(Provencio et al., 1992), it
was deemed important to analyze as many other anoles as possible to determine
how widespread this usage is. Procedures identical to those described by
Provencio et al. (1992
) were
used. Briefly, dark-adapted animals were decapitated and enucleated, and the
the whole isolated eyes were frozen in liquid nitrogen and stored at -70°C
until analyzed. The 11-cis retinoids from whole eyes were converted
to their corresponding oxime isomers using hydroxylamine hydrochloride and
extracted using the method of Groenendijk et al.
(1980
). These retinoids were
separated from the crude extract by high-performance liquid chromatography
(HPLC) through a LichroSorb Si60 analytical column (Chrompack, Raritan, NJ,
USA) with a hexane:dioxane mobile phase. Eluent from the first 3 min of the
run was collected and evaporated under nitrogen, after which it was
redissolved in 200 µl of a 95:5 hexane:dioxane solvent mixture. Injection
volumes of 100 µl were run through an identical analytical column using a
95:5 hexane:dioxane mobile phase. A Waters HPLC system including a model 991
photodiode array detector was used for separation and analysis.
Retinoid standards were prepared by dissolving 25 mg of all-trans
retinaldehyde (A1; Sigma, no. R-2500) or all-trans
3,4-didehydroretinaldehyde (A2; a gift from Hoffman-LaRoche, Inc.)
in 6 ml of ethanol. A 1 ml sample was illuminated with bright light (420-540
nm) for 3 h, after which 5 ml of a 1000-fold molar excess solution of
hydroxylamine hydrochloride (1.92 moll-1, pH 6.5) was added to
convert the retinoids to their respective oximes while retaining their
isomeric configuration. The oximes were extracted by the method of Groenendijk
et al. (1980) and stored at
-80°C.
Microspectrophotometry
Microspectrophotometric measurements were performed using methods identical
to those described by Loew
(1994) and Provencio et al.
(1992
). All procedures were
carried out under infrared illumination using appropriate image converters and
video cameras. Animals were dark-adapted for a minimum of 2 h, after which
they were cooled and decapitated. Enucleated eyes were hemisected, and the
posterior segments were incubated for 2 h in
Ca2+/Mg2+-free Puck's medium (Gibco) at 5°C. The
retinas were carefully teased from the retinal pigment epithelium and
macerated using razor blade fragments and tungsten needles. In some cases, the
posterior segment was immersed in a simple Sorensen's phosphate buffer (pH
7.2) with 6% sucrose or dextran added, and the retina was isolated and
prepared as above. No consistent differences in results were noted using these
two techniques. As regards cell dispersion, reptile retinas tend to be very
`dirty', with lots of free and adherent melanin granules and a paucity of
free, intact photoreceptors. As a general rule, the more gentle the cutting
and teasing, the better the preparation. In some cases, the best results were
obtained by folding a small piece of retina over on itself, receptor side out,
and working along the exposed edge. A drop of the dispersed retina or the
folded piece was sandwiched between two coverslips and transferred to the
stage of the microspectrophotometer, which has been described in detail
elsewhere (Loew, 1994
).
The criteria used for selecting data for inclusion into the analysis pool
were the same as those used by Loew
(1994). Dichroism was used for
differentiating the UVS pigment from the photoproduct. Each acceptable
spectrum was normalized by estimating a spectral maximum by eye and fitting a
Gaussian function to the data points 20 nm either side of this wavelength. The
peak of the Gaussian function was used to normalize the spectrum. Because the
number of usable recordings was small, it was decided to sum the individual,
normalized spectra at 1 nm intervals, calculate the mean at each nanometer and
use this generated spectrum for template-fitting. Smoothing was performed
using a digital filter routine (`smooft')
(Press et al., 1987
). The
smoothed spectrum was overlaid on the unsmoothed one and checked by eye to
make sure that over-filtering or spurious data points had not shifted the
apparent maximum. In cases where there was obvious distortion due to outlier
effects, the deviant point(s) was replaced with the mean of the 10 data points
surrounding the outlier.
max was obtained using the method
of Mansfield as presented by MacNichol
(1986
). The templates used
were those from Lipitz and Cronin
(1988
). Wavelength error of
the MSP measurements is ± 1.0 nm, so whole integer values of
max are reported here.
Wherever possible, oil droplet or inner segment pigment absorption was
measured together with visual pigment absorption in the same cell. Because of
the high concentration of pigment in the droplets and, sometimes, the
ellipsoids and the resulting high refractive index, it is not possible to
obtain true optical density values for most oil droplets. The result is
saturation of the MSP signal (the MSP method has been shown to give valid
density measurements up to an optical density of 2.0 based on calibrated
filters). In cases where this happens, the spectrum is normalized to the
highest absorbance, and the wavelength at 50 % of the maximum is reported (see
Lipitz, 1984). For
non-saturating spectra, the normalization step was omitted (e.g. accessory
member ellipsoid).
In some cases, enough retina was available to allow the oil droplets from
pieces viewed in white or colored light to be categorized. A micrograph was
first obtained in white light using a color video camera and frame grabber to
show the true color of the droplets. The camera was switched to black and
white, and the background illumination was altered with glass cut-off filters
until a class of droplet was seen to turn black, indicating that the cut-off
for that droplet had been passed. A coincident image was obtained. The
background was again adjusted until the next class turned black. The process
could not be used to differentiate among the colorless droplet classes as this
would have required moving below 400 nm and there was neither enough light nor
enough camera sensitivity to work in this wavelength range. Fluorescence has
been used to differentiate the colorless droplet classes (see
Kolb and Jones, 1987);
however, a system suitable for this purpose was not available for this
study.
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Results |
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Cell types, visual pigments and oil droplets
Table 1 summarizes the MSP
results. All anoles possessed an LWS pigment with a max
between 560 and 570 nm (625 nm for A. carolinensis), an MWS pigment
with a
max between 487 and 503 nm and an SWS pigment with a
max between 446 and 467 nm. All but five of the species had
a UVS pigment with a
max between 364 and 367 nm. For these
five species, no cells meeting the inclusion criteria were measured. However,
the presence of type C2 droplets in these species strongly suggests that they,
too, have a UVS pigment (see below). Except for A. carolinensis, all
spectra were best fitted by a vitamin-A1-based pigment template.
Fig. 3 shows typical MSP
recordings for the four pigment classes; in this case, for A.
cristatellus. While the number of cells that met the selection criteria
for analysis was small, many spectral scans were made and recordings obtained
from both intact cones and isolated outer segments that could at least be
identified as to class even if the exact
max could not be
calculated.
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|
The three morphological cone classes known to exist in anoles were easily
identifiable in the MSP preparations, although only rarely were cones found
free with the outer segment attached. The rarest finds were free intact double
cones. As expected from the previous A. carolinensis study
(Provencio et al., 1992), the
long single cone and both members of the double cone contained the LWS
pigment. The finding of many free accessory members of the double cones,
distinguished by their granular ellipsoid, suggests that many of the isolated
long single cones measured may actually have been principal members of
separated doubles. The only way a cell could be identified as a true long
single cone was when measurements could be made along retinal edges. The short
single cones containing the SWS and UVS pigments were morphologically
identical. While these same cone morphotypes were observed in the MSP
preparations of Polychrus marmoratus, a previously unreported
photoreceptor class with relatively large, cylindrical, rod-like outer
segments was also observed in great numbers. However, unlike true rods, these
have a large oil droplet, as can be seen clearly in
Fig. 4. The outer segments
contain a visual pigment with a
max at 497 nm, a typical
terrestrial vertebrate rhodopsin position. However, bleaching of this pigment
with white light did not yield a long-lived photoproduct as usually seen for
`true' rods on the microspectrophotometer.
|
Three classes of oil droplet were easily distinguishable in retinal pieces from anoles viewed in white and colored light (see Fig. 4). These have been classified according to the spectral position of their cut-off wavelength and visual appearance as yellow (Y), green (G) and colorless (C). On the basis of MSP measurements, the G and C classes can be further divided into two sub-classes each, G1, G2 and C1, C2. Typical spectra for the five spectral types of oil droplet found in the anoles and Polychrus marmoratus are shown in Fig. 5AB together with the absorbance spectra recorded from the ellipsoid of the accessory member of the double cone. As seen in Table 1, all species had the G2 (502 nm cut-off), Y (467 nm cut-off), C1 (378 nm cut-off) and C2 (ultraviolet-transmissive) droplets. Only three species examined had the G1 (521 nm cut-off) type, but in these they were quite common. This suggests a real species difference in oil droplet complement, although failure to find the G1 class in other anoles could be due to sampling error and lack of adequate material for whole-retina flat preparation analysis using spectral imaging.
|
From Fig. 4, it would appear that the most numerous droplet class in anoles is class Y. This is found in the principal member of the double cones and a class of single cone. The G1 and G2 classes are found only in single cones. The C types are the least numerous and are found only in small single cones. In Polychrus marmoratus, C droplets are the most numerous because of their association with the rod-like receptors that are present in high density. This is followed by the G and Y classes.
Fig. 6 summarizes pictorially the correlation between cell type, visual pigment and oil droplet type in anoles. Both members of the double cone house the LWS pigment, with a Y droplet in the principal cone and dispersed yellow pigment in the accessory cone. Three classes of large single cone were always present. Two contained the LWS pigment with either the Y or G2 droplet and one contained the MWS pigment with the G2 droplet. As mentioned above, three species had an additional large single class with the MWS pigment and the G1 droplet. The MWS pigment was never associated with the Y or C1/C2 droplets. Small single cones contained either the SWS pigment with the C1 droplet or the UVS pigment with the C2 droplet. The strict association between the UVS pigment and the C2 droplet makes us confident that, even in those five species in which we failed to record spectra from any ultraviolet-sensitive cells, the finding of C2 droplets supports their presence.
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Discussion |
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Clearly, if there are no differences in max among the
species and the species come from a full range of photic habitats, there can
be no correlation between visual pigment
max and habitat.
This situation contrasts with that for fish, in which strong correlations
between habitat light and visual pigment
max have been
reported (for reviews, see Bowmaker,
1998
; Partridge and Cummings,
1999
). Endler
(1993
) demonstrated that there
are large differences in the spectral quality of downwelling irradiance in
terrestrial habitats with different degrees of shade. Our failure to find
differences in visual pigment
max suggests either that the
anoline visual system is, for some reason, highly conservative in an
evolutionary sense or that the irradiance differences are not as important to
vision in terrestrial species as one might have predicted. However, if one
examines background lighting (i.e. horizontal radiance) rather than
downwelling irradiance, the differences in spectral quality among these
different habitats are greatly reduced since all are dominated by green
vegetation (Fleishman et al.,
1997
). This is not the case in aquatic systems, in which
horizontal radiance can show a great deal of variation.
Of course, other factors besides visual pigment absorption, such as optical
filtering, waveguiding or scatter, will affect spectral sensitivity (see
Loew, 1995). However, even
using electrophysiological techniques that would be influenced by these other
factors, all anoles studied to date, except the A2-utilizing A.
carolinensis, have similar spectral sensitivities
(Fleishman et al., 1997
) (L.
J. Fleishman, unpublished data). HPLC failed to detect vitamin A2
in any of the Puerto Rican species tested, and the similarity in
max between this group and the other anoles measured with
microspectrophotometry makes the widespread use of a vitamin A2
chromophore doubtful. However, this does not exclude the possibility of
vitamin-A2-based visual pigments being present in species other
than A. carolinensis. A small or localized population of
vitamin-A2-based cones could have been missed because of the
sampling limitations of microspectrophotometry or could have been below the
detection level of the HPLC technique.
As mentioned above, five different opsins have been identified in A.
carolinensis and have been sequenced and expressed using the COS-1 cell
cDNA system (Kawamura and Yokoyama,
1998). They have been designated SWS1AC,
SWS2AC, RH1AC, RH2AC and LWSAC
(see Kawamura and Yokoyama,
1997
) and are orthologous to the chicken violet, blue, rhodopsin,
green and red opsins, respectively. Visual pigments reconstituted from these
products using 11-cis retinal were spectrally characterized and,
after calculation of the expected effect of substituting
A2-aldehyde using the formula of Harosi
(1994
), were found to have
expected values of
max at 377, 444, 511, 517 and 626 nm,
respectively. However, the MSP data of Provencio et al.
(1992
) and those presented
here identified only four classes of cone in A. carolinensis with
values of
max at 365, 462, 502-503 and 625 nm. Even given
the admittedly low signal-to-noise ratio of the microspectrophotometry
recordings from the small anole cones (Fig.
3) and the low values of N
(Table 1), both conspiring to
produce large standard deviations, the correlation between the expressed and
reconstituted pigments and those measured in situ, except for the LWS
pigment, is not very good.
The match is even worse if the conversion formula of Whitmore and Bowmaker
(1989) is used. Assuming that
Kawamura and Yokoyama (1998
)
are correct in saying that RH2AC corresponds to our reported MWS
pigment, none of the mean values for this class seen in
Table 1 comes very close to the
max of 511 nm calculated for RH2AC. We agree
with Kawamura and Yokoyama
(1998
), who suggested that the
discrepancy could be due to inappropriateness of the
A1-to-A2 conversion formulae with regard to anoles.
However, it is also possible that the discrepancy arises from differences
between the biochemical/biophysical environment of the visual pigment in the
expression system and those in situ.
max aside,
there is also the problem of locating the photoreceptor class expressing
RH1AC. This is like classic `rod rhodopsin' and differs very little
from RH2AC, which is expressed in cones. Given a difference of only
6 nm in
max between the two RH pigments and the noisy
nature of the microspectrophotometry data, it is possible that both exist in
separate, but morphologically indistinguishable, cones both classified as MWS.
It is also possible that only one MWS class exists, but that it is expressing
both opsins. The data from Polychrus marmoratus are useful in this
context for here there is evidence for two very similar pigments differing in
max and clearly present in separate types of cell. We
therefore feel that the anoles studied here have two RH-containing
photoreceptor types, an MWS photoreceptor containing RH2AC and a
morphologically similar cell containing RH1AC. However, unlike
Polychrus marmoratus, the RH1AC-containing cell has a
colored oil droplet.
The outgroup Polychrus marmoratus was added to this study to try
to identify the ancestral condition. The fact that its cone pigments and oil
droplets are similar to those of the anoles and use vitamin A1 as
the visual pigment chromophore suggests that the A2 condition is a
derived characteristic. However, its retina is unlike that of any of the
anoles studied because it contains a class of single photoreceptor with large,
rod-like outer segments in addition to the four cone classes found in the
anoles (see Table 1 and
Fig. 4). Neither Walls
(1967) nor Underwood
(1968
) describe such a cell in
any of the numerous diurnal reptiles they studied. The outer segment is
attached to an inner segment containing a large, colorless oil droplet similar
to the C2 droplet of the UVS cone in that it shows no appreciable absorbance
below 400 nm. The cell's appearance is somewhat similar to the cells in
nocturnal geckos such as Aristelliger praesignis (see
Crescitelli, 1972
), but in
these retinas the outer segments in all photoreceptor classes are large, as
befits the nocturnal condition. Several interesting questions arise from these
observations. (i) Is this cell functionally a `rod' or a `cone'? (ii) Where
does this cell fit within the reptilian visual cell sequences created by Walls
(1967
) and Underwood
(1968
) and would they call
this a `transmuted' rod or a `transmuted' cone? (iii) What happened to this
class of photoreceptor during the evolutionary transition to the anoles? (iv)
How general is this pattern among other species of Polychrus? It
would be very interesting to apply the techniques of Kawamura and Yokoyama
(1998
) to
Polychrus.
Oil droplets
Except for the G1 and C2 classes, there was considerable variability among
the species within an oil droplet class
(Table 1). We believe this
variability is real and not due to the problems of measuring the
high-optical-density, highly refractive droplets. The differences were obvious
in those species in which there was enough material for small pieces of retina
to be examined microscopically in white light. No retinal whole-mounts were
examined, so it is not possible to say whether droplet color or density was
uniform over the entire retina. For two of the Jamaican anoles and
Polychrus marmoratus, in which it occurs, the G class of droplet
clustered into two non-overlapping groups, hence the creation of the G1 and G2
classes. However, as pointed out above concerning the presence of
vitamin-A2-based pigments, the failure to identify G1 droplets in
other species may be due to selection error. The C1 droplet was identifiable
by having a cut-off below 400 nm, so it appears colorless to the human eye.
The C2 class showed no appreciable absorbance down to 340 nm, where MSP
measurements stopped (Fig.
5A,B).
As mentioned above, the accessory members of double cones do not contain an
oil droplet, but instead a dispersed, yellow pigment
(Fig. 5A,B). However, even
here, there was considerable variation in the density (`yellowness') among the
species. The functional significance of this dispersed pigment is unknown, but
Underwood (1968) made the
intriguing suggestion that it could play a role in polarized light detection
as a result of scatter.
The presence of oil droplets changes the spectral sensitivity of the cells
that contain them and is believed to improve color discrimination
(Govardovskii, 1983) (see also
Vorobyev et al., 1998
). By
multiplying the normalized visual pigment absorbance by the normalized oil
droplet transmission, an approximate spectral sensitivity can be calculated,
as seen for A. cristatellus in
Fig. 7. It is easy to
rationalize the pairing of Y and G2 droplets with the LWS pigment since the
overall effect is to remove the blue and ultraviolet sensitivity. The
reduction in ultraviolet sensitivity is obviously the reason for the SWS/C1
pairing. The association between MWS and the G1 and G2 droplets greatly
decreases the capture area for the MWS cone while moving the sensitivity peak
to longer wavelengths (Fig. 7).
We can only assume that the improvement in color discrimination provided by
this pairing outweighs the reduction in capture area.
|
Ecological considerations
The spectral location of the visual pigments reported here could be
rationalized using the same model and reasoning recently applied to birds by
Vorobyev et al. (1998) and to
bees by Vorobyev and Menzel
(1999
). Even spacing of
pigments across the spectrum would allow for reasonable color discrimination
with no particular spectral region being singled out for increased
discrimination. Thus, the spacing fits well with a `gray world' model.
However, models including green leaves among the targets and backgrounds to be
discriminated favour an LWS pigment further into the red than 570nm (Zhang,
1997
,
1999
), the apparent
long-wavelength cut-off for vitamin-A1-based visual pigments (see
Loew, 1995
). Sensitivity to
longer wavelengths would take advantage of the steep increase in reflectance
for chlorophyll-containing targets that exists between 700 and 800 nm (see
Gates, 1980
), which could
increase contrast for non-infrared-reflecting or - absorbing objects viewed
against the bright leaf background. In fact, the LWS pigments predicted by the
Zhang (1997
,
1999
) model for viewing
non-chlorophyll-containing targets against a leafy background would have a
max extending above 650nm. Only A. carolinensis
comes close to satisfying this prediction.
It was hoped that the finding of a broader use of
vitamin-A2-based visual pigments among the anoles might shed some
light on the possible utility of the dramatic red-shift in spectral
sensitivity noted in A. carolinensis. In particular, Provencio et al.
(1992) speculated that the use
of vitamin A2 might be related to the red color of the A.
carolinensis dewlap. However, A. sagrei and A.
pulchellus also have red dewlaps
(Fleishman, 2000
), yet do not
use A2 as their chromophore. Thus, having
vitamin-A2-based pigments is not a prerequisite for `seeing' red
dewlaps. The use of vitamin A2 also does not correlate with such
obvious variables as body color or habitat selection. Thus, we remain ignorant
as to what benefit, if any, A. carolinensis derives by having
extended red sensitivity.
It is clear from the Polychrus marmoratus results and the
cladogram (Fig. 1) that vitamin
A2 is not the ancestral condition among the anoles. Jackman et al.
(1999) identified 17 distinct
clades among the anoles. In our sample, A. carolinensis is the only
representative of its clade. It would be of obvious interest to study other
members of this (mostly Cuban) radiation to determine whether A2
utilization is shared by close relatives and to try to pinpoint where in the
anole radiation the use of vitamin A2 evolved.
The possibility that oil droplet color is the adaptational variable in anoles cannot be discounted. While there was no apparent correlation between cut-off position and photic habitat or dewlap color, there could be other visual tasks we have not considered driving oil droplet cut-off position.
These results, together with recent studies of birds (see
Bowmaker et al., 1997;
Vorobyev et al., 1998
), draw
into question the fundamental assumption made by visual ecologists that visual
pigment
max should correlate with the photic environment,
at least for terrestrial vertebrates. Rather than concentrating on irradiant
and visual pigments, one can look for relationships between visual signals and
tasks and overall spectral sensitivity. This seems to work for rationalizing
the presence of UVS cones in birds and anoles with
ultraviolet-reflecting/absorbing targets such as feathers and dewlaps
(Bennett et al., 1997
;
Fleishman et al., 1993
). As
anoles have radiated over evolutionary time, there have been only relatively
modest changes in the design of their visual system. In contrast, what is
known to be a highly significant visual signal, the colored dewlap, shows a
great deal of variation among the species. We find no support for the idea
that signal color variation evolved as a result of spectral sensitivity. This
further supports that idea that the anoles are `color generalists' with regard
to the positioning of their spectral sensitivities.
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