Carotenoid Access, Nutritional Stress, and the Dewlap Color of Male

Brown Anoles

John E. Steffen1, Geoffrey E. Hill2, and C. Guyer2

 Copeia 2010, No. 2, 239–246

Carotenoid Access, Nutritional Stress, and the Dewlap Color of Male

Brown Anoles

John E. Steffen1, Geoffrey E. Hill2, and C. Guyer2

Carotenoids are integumentary colorants that must be ingested by vertebrates to be used as colorants. In many studies
of fish and birds, carotenoid color serves as an indicator of foraging success, nutritional state, or parasite load. The
dewlap of male Brown Anoles (Norops sagrei) contains the carotenoid pigments lutein and zeaxanthin. We performed a
two-factor experiment to determine the effects of nutritional stress and xanthophyll supplementation on male dewlap
color in N. sagrei. We tested the hypotheses that dewlap color is dependent on access to carotenoids and good nutrition.
Contrary to our predictions we found no significant differences among groups of anoles that were either supplemented
or not supplemented with carotenoids. Similarly, post-experimental dewlap spectral variation did not differ
significantly among groups that were provided a standard or reduced diet. These findings demonstrate that dewlap
color of adult male Brown Anoles does not change in response to food access or carotenoid supplementation.

A
NIMALS use a variety of pigments to color their
integument (Bagnara and Obika, 1965; Bagnara and
Hadley, 1973; McGraw, 2006a, 2006b). Carotenoids
have been of interest to biologists because these molecules
must ultimately be obtained from the diet (Goodwin, 1984).
In fish and birds, carotenoid pigmentation acts as an honest
signal of an individual’s nutritional state (Frischknecht,
1993; Hill, 2000; Hill et al., 2002), parasite resistance (Zuk et
al., 1990; Houde and Torio, 1992; Thompson et al., 1997;
Brawner et al., 2000), or foraging success (Endler, 1980). The
function and proximate control of carotenoid coloration is
not nearly so well studied in other vertebrates including
lizards.
A central tenet in honest signaling theory is that cheating
must be minimized for a signal to be reliable (Zahavi, 1975,
1977; Andersson, 1982; Kodric-Brown and Brown, 1984;
Hill, 1994). It has been hypothesized that cheating is
minimized in those signals that incur a cost (Folstad and
Carter, 1992). Carotenoids may invoke a cost because they
must be obtained through foraging, and then once they are
ingested they must be extracted from food, absorbed from
the gut, modified (if necessary), transported, and deposited
(Hill, 2002). If these costs of acquisition and utilization are
necessary for elaboration of the coloration, then only strong
and healthy males will be highly ornamented (Frischknecht,
1993).
Two factors that have been shown to affect carotenoid
coloration are access to sufficient quantities of appropriate
pigments in food and overall nutrition. In both birds and
fish, if animals are maintained on diets that do not provide
abundant carotenoids, carotenoid coloration declines (End-
ler, 1983; Hill, 2006). Similarly, nutritional stress (in the
form of reduced food access) has been shown to adversely
affect red feather color in House Finches (Hill, 2000) and
yellow feather color in American Goldfinches (McGraw et
al., 2005). Evidence suggests that nutritional stress reduces
the caloric intake and energy available for enzyme-driven
biochemical steps involved in carotenoid utilization, and
reduces an individual’s ability to fully pigment its integu-
ment (Hill, 2002; McGraw et al., 2005).
All of the experimental tests to date on the effects of
carotenoid access and nutrition on expression of carotenoid
1
Department of Entomology and Plant Pathology, 301 Funchess Hall, Auburn University, Auburn, Alabama 36849; E-mail: steffje@
auburn.edu. Send reprint requests to this address.
2
Department of Biological Sciences, 331 Funchess Hall, Auburn University, Auburn, Alabama 36849.
Submitted: 10 April 2009. Accepted: 23 November 2009. Associate Editor: E. Schultz.
F 2010 by the American Society of Ichthyologists and Herpetologists
coloration in vertebrates have come from studies of birds
and fish. Many species of reptiles also use carotenoid
pigments to color their integument, and although several
studies addressing the evolution and signal function of
lizard coloration have been conducted, no experimental
study has yet addressed the environmental factors that
might shape coloration in lizards. We conducted an
experimental test of the effects of pigment access and
nutrition on the dewlap color of male Brown Anoles (Norops
sagrei, following the clade name in Nicholson [2002]).
Male Brown Anoles possess a large and extendable throat
fan, known as a dewlap, that derives its color from both
pterin and carotenoid pigments. The dewlap has a bright red
center with a yellow edge. The yellow along the edge is
colored primarily by two xanthophyll pigments (lutein and
zeaxanthin), while the red center is colored by pterins (e.g.,
especially drosopterin, isoxanthopterin, sepiapterin, pterin,
and biopterin to a lesser extent; Macedonia et al., 2000;
Steffen and McGraw, 2007, 2009). As in most anole species,
male Brown Anoles extend their colorful dewlaps to other
males in territorial contests (Jenssen, 1977; McMann, 2000;
Tokarz, 2002; Tokarz et al., 2003; Paterson and McMann,
2004), to females in courtship interactions (Jenssen, 1970;
Tokarz, 2002; Tokarz et al., 2003), and predators during an
attack (Leal and Rodriguez-Robles, 1997a, 1997b; Leal,
1999).
A common assumption is that the colors within an
individual’s dewlap are stable and constant throughout
adulthood (Losos, 2009). Here we investigate the impact of
xanthophyll supplementation and nutritional stress on the
stability of dewlap coloration of Brown Anoles. Specifically,
we assess the effects of xanthophyll supplementation and
nutritional stress on dewlap color to test the hypothesis that
adult male Brown Anole dewlap coloration is influenced by
access to xanthophyll and nutritional stress.
MATERIALS AND METHODS
Lizard care.—Adult male lizards were collected on 3 May
2008, from a parking lot 4.8 kilometers SW of Salt Springs,
Florida, near the Ocala National Forest. Upon return to the
lab two days later, lizards were assigned a unique toe-clip
identity (no more than two toes clipped per individual), and
DOI: 10.1643/CP-09-067
240
were placed into 37.9-liter terraria, each of which was
separated into four compartments so that lizards could be
separated. Compartment walls were constructed from solid
particle board which prevented lizards from seeing or
physically contacting other individuals. Each compartment
contained a perch, water dish, and sandy substrate. Lighting
strips containing full spectrum fluorescent tubes (Vitalite
T8, 32 watt) were suspended 30 cm above each terrarium
top. Terraria, and the lizards in them, were exposed to
natural light via sunlight that entered the holding room
through a large window. Water was sprayed into each
terrarium daily, and water bowls were refilled three times
per week. The room was maintained on a 14:10 hr light:dark
photoperiod, at an air temperature of 32.2uC, with relative
humidity ranging between 60 and 80%.
Experimental protocol, treatments, and controls.—Lizards were
housed for five weeks before the experiment began, during
which they were fed meal worms ad libitum and a minimum
of three crickets per day (dusted with Repta-vite), 3–4 times
per week. This feeding regiment served as the standard food
provisioning rate for treatment groups assigned to the
‘standard food access’ treatment groups.
The reduced food access groups received three crickets per
feeding, once per week. Crickets for all treatments were
three weeks old (Golden Crickets, Alabama; raised on a
protein-rich diet that lacked carotenoids) and were main-
tained on a diet of dog food (consisted of lamb, rice, oat, and
corn-based meals). Corn-based meal in the dog food may
contain xanthophylls and therefore be a source of carot-
enoids that crickets ingested, but crickets fed on this diet
were fed to all lizards. Moreover, if this potential source of
xanthophyll had an effect on dewlap color, then treatment
groups that were assigned to the reduced food provision rate
should show a decrease in xanthophyll-based color.
Xanthophyll was supplemented in the form of a xantho-
phyll-nutrient powder designed by Mazuri diet (PMI Nutri-
tion, Richmond, IN). The powder was suspended in water by
mixing 512 grams of nutrient powder in 1.9 liters of warm
tap water. The powder consisted of 45% protein, 10% fat,
and 1% fiber to bind the xanthophylls in a fat-soluble
matrix to facilitate micellarization of the pigments and
subsequent absorption by the intestine (Kuksis, 1987;
Parker, 1996; Chavez et al., 1998). For lizards belonging to
the xanthophyll supplementation group, we fed individuals
3 ml of xanthophyll solution (0.028 mg of xanthophyll per
milliliter of water) with a blunt-tipped syringe. As a result,
each lizard in the xanthophyll treatment group received
0.084 mg of xanthophyll per supplementation. This is
roughly the same xanthophyll quantity used to influence
plumage and bill color of American Goldfinches (Navara
and Hill, 2003). The treatment groups that received no
xanthophyll solution received warm tap water via a separate
blunt-tipped syringe in a manner identical to the xantho-
phyll supplemented groups.
One day before the experiment’s initiation, we measured
lizard dewlap color with a spectrophotometer to serve as a
‘pre-experimental’ color measurement (see Spectrometry
details below). Lizards were then randomly assigned to one
of four groups: no xanthophyll supplementation and
standard food access (the control group), no xanthophyll
supplementation and reduced food access, xanthophyll
supplementation and standard food access, and xanthophyll
supplementation and reduced food access.
Copeia 2010, No. 2
The experiment was conducted from 5 June 2008 until 5
July 2008. We made a final series of spectrometric measure-
ments on 6 July 2008 as a post-experimental measure of
dewlap color. Lizards were weighed to the nearest 0.001 gram
at the beginning and end of the experiment using an
electronic balance. We weighed lizards three days after the
last meal to minimize the possibility that weights reflected
full stomachs instead of body mass. We performed a two-
way ANOVA on mass change, with food access and
xanthophyll supplement level as factors. There was a
significant main effect of food access on mass loss in lizards
over the experimental period (F3,36 5 8.56, n 5 40, P 5
0.007), indicating that the reduced food access treatment
used for this experiment was an effective form of nutritional
stress. Lizards that had reduced access to food lost an
average of 18% of body mass, while lizards that had standard
access to food maintained average body mass (i.e., they
gained 0.006% body mass).
Spectrometry.—Color of the center and edge dewlap regions
were measured with an Ocean Optics S2000 UV-visible
spectrometer equipped with OOIBase32 software. All reflec-
tance data were generated relative to a white reflectance
standard and were taken in an unlit room with tightly drawn
blinds covering the window. We placed a small black plastic
stopper on the tip of the reflectance probe, creating a 2 mm
gap between the probe tip and the dewlap, ensuring a
constant distance between probe and dewlap for all measure-
ments. To measure lizard dewlaps with the spec-
trometer, we placed each lizard ventral side up on a flat black
table and immobilized it with two pieces of athletic tape
placed across the belly and lower mandible. The dewlap was
kept maximally extended by attaching the edge to a small,
height-adjustable clamp that was attached to a horizontal
metal arm on a ring stand. We then placed the spectrometer
probe at a 90u angle, and flush with the exposed skin of the
dewlap. We measured spectral reflectance along two ‘regions’
of the dewlap: along the center and the edge. We took seven,
non-overlapping spectral measurements per dewlap region
for each lizard. These regions are commonly measured in
other dewlap color studies (Leal and Fleishman, 2002, 2004;
Fleishman et al., 2006; Nicholson et al., 2007).
Spectral measurements were gathered as percent reflec-
tance at 1 nm wavelength increments from 300–700 nm
(which represents the lower range of photon absorption by
UV-sensitive photoreceptor cones published for anoles
[Fleishman et al., 1993]), and this output was smoothed
and processed using CLR v 1.0 (Montgomerie, 2008). For
both the pre- and post-experimental color measurements,
we generated a spectral reflectance curve for every measure-
ment, and we established a mean reflectance curve for each
dewlap region of every individual lizard by averaging all
seven reflectance curves per dewlap region. We then
established a pre- and post-experimental mean reflectance
curve for each dewlap region of each treatment group. Next,
we calculated two of the ‘tristimulus’ colorimetric variables
commonly referred to as chroma and brightness. We used
CLR v 1.0 to determine mean values of chroma, and we used
MS Excel, along with formulas described in Montgomerie
(2006) to determine brightness. These variables describe
different aspects of the spectral curve and have been used
extensively in the literature (Endler, 1990; Hill, 1998;
Montgomerie, 2006). Brightness was calculated as the
percent total reflectance in UV (300–400 nm) and yellow
Steffen et al.—Nutrition and dewlap color
(550–624 nm) and red wavebands (605–700 nm). UV,
yellow, and red chroma were calculated as proportion of
light in these wavebands relative to total reflectance across
the 300–700 nm spectrum. Hue was not used in the analyses
because correlative data on xanthophyll concentration and
its influence on hue are non-significant in the Brown Anole
(Steffen and McGraw, unpubl. data).
Statistical analysis.—We studied the effects of xanthophyll
supplementation and reduced food access on post-experi-
mental dewlap color when pre-experimental dewlap color
was treated as a covariate. By using this covariate approach,
we could determine if the independent variables (referred to
as IVs hereafter) had significant effects on the dependent
variables (referred to as DVs hereafter) after the covariate
influence was removed.
Before this can be done, however, data need to meet all
assumptions of ANOVA and ANCOVA, multivariate signifi-
cance needs to be investigated, and covariates need to be
assessed for reliability. If covariates are reliable, then one
usually wants to determine the ability of specific covariates
to adjust specific DVs. Only after these steps have been
performed can the researcher reliably evaluate the depen-
dent variables, and infer statistical significance of the effects
of factors on DVs (Tabachnik and Fidell, 2001).
We used JMP to evaluate normality of distributions of
spectral variation. Several variables did not meet assump-
tions of normality; when yellow, red, or UV chroma was
non-normally distributed, the values were arc-sin trans-
formed because the values are expressed as a ratio between 0
and 1. When yellow, red, or UV brightness was non-
normally distributed, the values were log-transformed. The
transformations were retained and used for the analyses
only if they improved the normality of the variable
(Shapiro-Wilks W test, available in JMP). Otherwise, the
original, untransformed variables were used. After appro-
priate transformations were retained or rejected, the data
met all assumptions of homogeneity of variance–covariance
matrices.
The following analyses were all performed in SPSS in
syntax mode (with syntax provided by Tabachnik and Fidell,
2001). We used SPSS’s MANOVA command to perform
MANOVA and determine multivariate significance. We also
used SPSS MANOVA to assess the reliability of covariates. In
this assessment, we used Wilks’ lambda to test for significant
relationships between the linear combinations of DVs
(representing post-experimental dewlap color) and the
covariates (i.e., pre-experimental dewlap color).
We used SPSS MANOVA to investigate the ability of
covariates to adjust specific wavebands of post-experimental
spectral variation (i.e., the DVs) by performing multiple
regressions for each DV in turn, with the covariates acting as
IVs in the model. Regression coefficients (beta coefficients)
and statistical tests of covariate significance were provided
as output in SPSS.
Once the contributions of covariates to adjust the DV
were evaluated, we wanted to see if the DVs were affected by
the treatments once the effect of covariates were removed.
Because many of the dependent variables were significantly
correlated to each other (i.e., they showed a correlation
coefficient .0.30 [Tabachnick and Fidell, 2001]), we used
SPSS MANOVA to perform Roy-Bargmann step-down analy-
sis in which the DVs are assigned priority based on
theoretical or practical considerations, and the highest
241
priority DV is analyzed as a univariate ANOVA where the
covariates are entered as IVs. The second priority DV is then
entered into ANCOVA as a DV, while the highest priority
DV is entered as a covariate (along with covariates from
previous ANOVA). Following a first ANCOVA, a second
ANCOVA is performed, with the higher priority DV from the
previous ANCOVA being entered as a covariate along with
previous covariates. This succession of ANCOVAs is per-
formed until the lowest priority DV is entered as a DV, and
all previous DVs are entered into ANCOVA as covariates
(along with initial covariates, so that adjustment is made for
the previous DVs as well as the pre-experimental spectral
variation (i.e., the covariates). In the study presented here,
we assigned priority to these DVs in the following order:
yellow, red, and UV brightness, then yellow, red, and UV
chroma. This order was chosen because in most UV-sensitive
spectrometric studies of animal coloration (as well as the
present study), PC1 typically describes variation in overall
brightness (Cuthill et al., 1999). Since yellow brightness
correlated with xanthophylls in a correlative study investi-
gating how pigments generate color in Brown Anole
dewlaps (Steffen and McGraw, 2009), we assigned it the
highest priority. Red brightness was assigned second highest
priority because xanthophylls also likely influence portions
of the red spectral curve (i.e., yellow and red make orange).
By default, UV brightness was assigned third highest
priority. PC2 in the present study describes long wavelength
(red and yellow) chroma (which were assigned fourth and
fifth priority), and PC3 describes UV chroma (and was given
lowest priority) because it explained the least amount of
variance in the data. Thus, yellow chroma was assigned
fourth priority, red chroma fifth priority, and UV chroma
sixth priority.
After performing Roy-Bargmann stepdown analysis, we
used SPSS MANOVA to perform univariate ANCOVA on
each dependent variable, with xanthophyll supplementa-
tion, food access, and the xanthophyll supplementation and
food access interaction as IVs, and pre-experimental mea-
sures of yellow, red, and UV chroma and brightness as
covariates. To test for significant effects of food access and
xanthophyll supplementation on the linear combination of
DVs that represent post-experimental dewlap spectral varia-
tion, one needs to compare significant differences in
spectral variation between univariate ANCOVA and step-
down F tests. According to Tabachnik and Fiddell (2001),
dependent variables that show both significant univariate
and stepdown F statistics are statistically pure tests of
significance because they demonstrate that there is unique
variability shared by the treatments after adjustments for
differences created by the covariates. We combined F
statistics, and effect and error degrees of freedom from the
univariate and stepdown analyses, and present them side by
side in two tables to facilitate comparison and evaluate the
significance of each analysis type. When results show
significant univariate but not significant stepdown F
statistics, there is not unique treatment variability after
covariate adjustment. Thus, significance is not reliable. If
there is a significant stepdown F but not a significant
univariate F, significant DVs take on importance in the
presence of higher order DVs, and the results may be related
to the order in which DVs were entered into the stepdown
analysis. Thus, significance is not reliable in this case, either.
We controlled for increased likelihood of Type I inflation
error due to multiple comparisons by dividing a typical P
242 Copeia 2010, No. 2

Table 1. Multiple Regression Beta Coefficients (Regression Coefficients) to Assess Ability of Specific Covariates to Adjust DVs in the Dewlap Center
and Edge. YB, RB, UVB = yellow, red, and UV brightness, respectively. YC, RC, UVC = yellow, red, and UV chroma, respectively. * represents beta
coefficient values where P , 0.05, and covariates significantly adjust the DV.

DV
Region Covariate YB RB UVB YC RC UVC
Center YB 20.197 20.323 0.425 0.371 20.001 1.176
RB 0.864 1.922 0.925 23.678* 20.977 20.368
UVB 20.319 21.626 21.629 5.635* 1.818 21.081
YC 0.583* 0.267 0.325 0.372 20.404 20.087
RC 20.646 20.861 20.843 2.030* 1.087 20.116
UVC 0.367 0.939 1.253 23.117* 21.251 0.984
Edge YB 22.356* 22.180* 22.381* 1.317 0.990 21.461
RB 3.852* 3.550* 4.883* 22.455 21.811 3.951*
UVB 2.476 2.105 3.857 21.638 21.340 3.328
YC 23.737 23.346 27.729 4.590 2.852 27.480
RC 21.670 21.266 23.086 1.997 1.584 22.589
UVC 1.834 1.654 4.998 23.092 21.720 5.100

value indicating an experiment-wise error rate of 0.05 by 6
to accommodate the increased probability of making type I
errors among six separate tests (i.e., one separate test for
each DV).
RESULTS
Multivariate significance.—In the dewlap center, post-experi-
mental spectral variation was significantly different between
reduced and standard food access (approximate F6,22 5
2.829, P , 0.05), but not with xanthophyll supplementation
(approximate F6,22 5 0.834, P 5 0.556). In addition, the
interaction of xanthophyll and food was not significant
(approximate F6,22 5 0.784, P 5 0.592).
Along the dewlap edge, post-experimental experimental
spectral variation was not significantly different in response
to food access (approximate F6,22 5 1.840, P 5 0.137) or
xanthophyll supplementation (approximate F6,22 5 1.862, P
5 0.133), and the interaction of xanthophyll and food was
not significant (approximate F6,22 5 0.652, P 5 0.688).
Covariate reliability and assessment.—Although MANOVA
showed multivariate significance, pre-experimental dewlap
color was found to be a reliable covariate of post-experi-
mental dewlap color in both dewlap regions (dewlap center:
approximate F36,99 5 3.113, P , 0.001; dewlap edge:
approximate F36,99 5 2.235, P 5 0.001). Multiple regression
showed that several covariates significantly adjusted DVs. In
the dewlap center, post-experimental yellow chroma was
significantly adjusted by pre-experimental readings (i.e.,
covariates) of UV and red chroma, as well as UV and red
brightness (Table 1). Post-experimental yellow brightness
was significantly adjusted by pre-experimental yellow
chroma. The remaining post-experimental measures of
spectral variation (UV or red chroma, or UV and red
brightness) were not affected by any covariates.
Along the dewlap edge, post-experimental UV chroma was
significantly adjusted by pre-experimental readings of UV
brightness (Table 1). Post-experimental yellow, red, and UV
brightness was significantly adjusted by pre-experimental
readings of yellow brightness. Post-experimental yellow,
red, and UV brightness as well as red chroma was
significantly affected by pre-experimental readings red
brightness. Post-experimental yellow and UV chroma were
not significantly affected by any covariates.
Statistical inference.—Results of the univariate and step-down
analyses are combined and summarized by dewlap region in
Table 2. In the dewlap center, reduced food access showed a
significant effect on post-experimental red chroma in a
univariate F test. However, covariates (pre-experimental
dewlap color) had significant effects on post-experimental
measures of yellow and red chroma, as well as yellow
brightness. Indeed, post-experimental dewlap spectra did
not significantly differ with respect to xanthophyll supple-
mentation, reduced food access, or xanthophyll-by-food
interaction after adjusting for covariates (i.e., pre-experi-
mental dewlap spectral variation) via successive stepdown F
tests and correcting for inflated Type I error due to multiple
comparisons (i.e., Bonferroni corrections).
Along the dewlap edge, pre-experimental dewlap color
covaried significantly with measures of post-experimental
yellow and UV chroma (Table 2). Reduced food access had a
significant effect on post-experimental red brightness in a
univariate ANCOVA. Similar to the dewlap center, however,
post-experimental dewlap spectra did not significantly differ
with respect to xanthophyll supplementation, reduced food
access, or xanthophyll-by-food interaction after adjusting
for differences in pre-experimental spectral variation, and
via successive stepdown F tests and Bonferroni multiple
comparisons.
DISCUSSION
The data presented here demonstrate that dewlap color in
adult Brown Anoles does not change significantly over the
course of one month in response to carotenoid supplemen-
tation or food access. These results differ from most other
studies of birds and fish, which show that integumentary
carotenoids change in response to both carotenoid supple-
mentation and food access. For instance, if Trinidadian
Guppies (Poecilia reticulata; Endler, 1983) and Brown Trout
(Salmo trutta; Steven, 1947a, 1947b) are fed a carotenoid-free
(or carotenoid-poor) diet, then these colors fade over the
duration of a few weeks, and if carotenoids are provided
again, the colors become brighter and more intense. In
Steffen et al.—Nutrition and dewlap color 243

Table 2. Univariate and Stepdown F Tests for Assessment of Statistically Significant Differences in Post-experimental Dewlap Color (DVs) between
Factors (Xanthophyll Supplementation, or ‘Xanth’, Food Access, or ‘Food’, and Their Interaction, ‘Xanth by Food’). ‘Center’ refers to results of
Univariate and Stepdown F tests for the dewlap center. ‘Edge’ refers to the results of these F tests for the dewlap edge. Covariates are the pre-
experimental dewlap spectral wavebands (and are the same wavebands described for DVs below). YB, RB, UVB = yellow, red, and UV brightness,
respectively, and YC, RC, UVC = yellow, red, and UV chroma, respectively. a Significance level cannot be evaluated but would reach statistical
significance in univariate context. * P , a (Bonferroni adjusted significance level). Italics represent F values that were statistically significant at P ,
0.05, but which were no longer significant after Bonferroni multiple comparisons test.

Center Edge
Effect DV Univariate F Stepdown F Univariate F Stepdown F df a
Covariates YB 6.046a 6.046* 2.009 2.009 6/27 0.0083
RB 5.408a 5.525* 2.053 0.923 6/26 0.0083
UVB 5.585a 2.564 2.893 8.613** 6/25 0.0083
YC 3.222 3.105 4.775a 3.626 6/24 0.0083
RC 4.039a 1.784 2.342 0.542 6/23 0.0083
UVC 2.419 0.839 6.206a 0.567 6/22 0.0083
Xanth by Food YB 0.000 0.000 0.123 0.123 1/27 0.0083
RB 0.344 1.521 0.219 0.141 1/26 0.0083
UVB 0.002 0.675 0.146 1.403 1/25 0.0083
YC 0.211 0.000 0.059 0.535 1/24 0.0083
RC 0.152 0.230 0.026 1.508 1/23 0.0083
UVC 0.030 2.321 1.041 0.300 1/22 0.0083
Food YB 0.057 0.057 4.138 4.138 1/27 0.0083
RB 2.899 9.641* 7.700a 4.772 1/26 0.0083
UVB 1.011 0.105 0.889 0.106 1/25 0.0083
YC 3.604 5.189 0.592 1.401 1/24 0.0083
RC 8.312a 1.141 0.259 0.306 1/23 0.0083
UVC 1.720 0.136 0.344 0.477 1/22 0.0083
Xanth YB 1.340 1.340 0.415 0.415 1/27 0.0083
RB 3.455 2.903 1.644 3.235 1/26 0.0083
UVB 0.341 0.348 1.071 3.267 1/25 0.0083
YC 0.655 0.040 0.937 0.001 1/24 0.0083
RC 1.007 0.109 0.302 3.831 1/23 0.0083
UVC 3.297 0.683 1.449 0.027 1/22 0.0083

Zebra Finches (Taeniopygia guttata) and American Gold-
finches (Carduelis tristis) xanthophyll supplementation leads
to an increase in feather and bill saturation (i.e., chroma)
and is condition dependent (McGraw and Ardia, 2003;
Navara and Hill, 2003).
Without additional study, there is no way to know why
the color of dewlaps of Brown Anoles responded differently
to experimental manipulation of carotenoid and food access
compared to various species of fish and birds, but a few
speculations seem in order. First, it is possible that even the
poorest diet—low food and low carotenoids—provided
adequate carotenoid pigments for full ornament expression.
Crickets that formed the base diet for all animals ate corn-
based foods and may have sequestered sufficient carotenoids
for lizard color production. This idea could be tested by
feeding anoles a carotenoid-free diet during a one-month
period and assessing the effect on color.
Second, anoles may have used their alternative pigment
system—pterins—to compensate for spectral changes that
result from low carotenoid intake or poor nutrition. Grether
et al. (2001) considered how drosopterin-to-carotenoid
ratios vary in color patches of male Trinidadian Guppies
(P. reticulata). In P. reticulata, drosopterin concentration
correlated positively with carotenoid (tunaxanthin) avail-
ability. Moreover, pterin-to-carotenoid ratio was responsible
for a particular orange hue that was maintained between
several streams, where genetic variation in drosopterin
compensated for environmental variation in carotenoids
(Grether et al., 2005). We did not have the opportunity to
test the pigment content of dewlaps in our experimental
animals, so this idea cannot be assessed with the data on
hand. However, Steffen and McGraw (2009) found that
drosopterin concentration did not correlate with xantho-
phyll concentration in another central Florida population
sampled two years earlier, and this lack of inter-pigmentary
correlation suggests an absence of such compensation in
Brown Anoles.
Third, the coloration of anoles may be fixed by the time
they are adult. If carotenoid coloration is fixed by adult-
hood, then the carotenoids responsible for dewlap color
must be obtained as juveniles or embryos. According to a
dietary analysis of Biminian Norops sagrei (Schoener, 1968),
phytophagous insects known to sequester carotenoids
(Mummery et al., 1975; Rothschild and Mummery, 1985;
Czeczuga, 1986, 1990; Rothschild et al., 1986), such as
lepidopteran larvae and adults comprise low volumetric
percentages of juvenile stomach volumes compared to
adults. For example, lepidopteran larvae and adults com-
bined comprised only 10.1% of juvenile lizard stomach
volumes, and plant matter was non-existent. In contrast,
lepidopteran larvae and adults comprised 40.7% and 32.2%
of adult male and female stomach volumes, respectively.
244
Plant matter comprised 16.3% of stomach volume in males
and 4.9% of stomach volume in females. The reduced
volumetric percentage of lepidopteran larvae and adults, as
well as the lack of whole plant matter in juvenile lizard
stomachs suggest that juveniles are not going out of their
way to obtain carotenoids.
One possibility is that carotenoids used for dewlap
spectral variation are obtained during vitellogenesis or
embryogenesis by the mother. Dewlap color obtained from
the mother’s diet might indicate something about the
mother’s genes or diet, and this maternal source of
environmentally obtained pigment may influence the son’s
ability to maximally contrast in a given light environment
(Leal and Fleishman, 2002, 2004).
Whatever the reason that our results differ from previous
studies of fish and birds, the lack of color change in anoles
in response to manipulation of pigment access and nutrition
has important implications. If they are not used as a signal
of recent xanthophyll foraging success or nutritional
condition in adult males, why use carotenoids as integu-
mentary colorants? In the fishes and birds described above,
the variation in carotenoid-based color serves as a visual
signal to communicate information to conspecifics about
foraging success (Endler, 1980), parasite resistance (Zuk et
al., 1990; Houde and Torio, 1992; Thompson et al., 1997;
Brawner et al., 2000), and nutritional state (Frischknecht,
1993; Hill, 2000; Hill et al., 2002). It is unknown whether
xanthophyll-based dewlap color communicates any fitness-
related information in Brown Anoles, but the present study
suggests that xanthophylls are not deposited into the
integument and do not communicate information about
immediate nutritional states or xanthophyll accumulation
in adult males.
Future research should investigate integumentary xantho-
phyll deposition as a maternal effect, and should attempt to
determine physiological benefits of xanthophyll acquisition
and utilization relative to pterin deposition. In union with
studies of pterin synthesis and deposition, findings of these
sorts would contribute a great deal to the study of dewlap
color evolution because they elucidate the underlying
physiological causes of dewlap color and help clarify
potential uses of dewlap color as a visual signal.
ACKNOWLEDGMENTS
JES is grateful to L. McBrayer, J. Sergi, and members of the
McBrayer lab for help collecting lizards. Thanks go to L.
Siefferman, W. Hood, and members of C. Guyer’s and G.
Hill’s labs for revision suggestions, B. Tabachnik for reading
the Roy-Bargmann step down analysis revision and con-
firming that it was done correctly, and K. Nicholson for
insightful discussions about dewlap color diversity. We
would also like to thank L. Koutsos of Mazuri diet (PMI
Nutrition, Richmond, IN) for designing the xanthophyll
diet. All care of lizards followed animal care and use
guidelines and IACUC approval (PRN 2006-0961).
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