Abstract
Spotted salamanders, Ambystoma opacum, contain bright yellow spots on their dorsal side.
Analysis of these spots compared to demographic information provided data that suggests
individuals that have spots with greater hue, saturation and lightness values arrival at the
breeding pond earlier in the season than do individuals with less-colorful markings.
Introduction
Colored spots on the backs of spotted salamanders, Ambystoma maculatum, vary greatly in
size and shape, and sometimes even color. Previous studies have examined the phenomena of
sexual dichromatism in marbled salamanders, Ambystoma opacum (Todd and Davis, 2007), as
well as red-spotted newts, Notophthalmus v.viridescens (Davis and Grayson, 2008). Both of
these aforementioned studies determined that a significant difference existed between the
coloration of spots from female animals compared to the coloration of spots from male animals.
For this study, the coloration of salamander spots was examined to determine if a sexual
dimorphism exists. We also looked at the relationship between individual body condition, spot
coloration, and date of arrival to breeding pond. In other species of animal, coloration has been
shown to aid in camouflage, function as a form of mimicry, as well as aid in courtship and mate
attraction (Davis and Grayson, 2008). In the class Amphibia, most relevant research has been
performed on the order Anura, frogs and toads. In some cases it has been determined that sexual
dichromatism has been attributed to sexual selection, aiding in mate choice (Summers et al,
1999).
On account of sexual dimorphism, we hypothesize that males and females will differ with
respect to the amount of total area covered by spots, and to brightness and other chromatic
characteristics such as hue and saturation. Taking this into account, we believe that males will
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have spots with a greater yellow hue (what people actually perceive as a color), a higher
saturation (the intensity of a hue, ranging from dark tones to full, vivid color), and a greater
brightness than spots of females (Davis and Grayson, 2008). In the relationship of overall body
condition, coloration, and breeding arrival, we hypothesize that animals with brighter and more
conspicuous spots will arrive earlier in season to the breeding pond due to better body condition
(indicated by animal robustness given by the residuals of mass regressed on snout-vent length).
Materials and Methods
Initial capture and processing
Spotted salamanders used in this study were intercepted at a permanent drift fence at
Windhover Pond in Bath Township, Ohio, the site of a long-term salamander demography
project. For the current study, we used captures from the 2008-2010 breeding seasons which
occur in early spring of each year.
Upon capture each salamander was identified according to a unique identification number
consisting of a series of clipped toes; animals not captured previously were given a new number.
Subsequent processing involved the determination of sex, and the recording of tail length, snoutvent length, and body weight. Finally, a digital image of each animal was obtained by of placing
the individual on a black stage where they were photographed from above at a fixed distance
with an Olympus e-500 controlled by Olympus Studio ver. 2.3 software. Image settings,
including camera control and lighting were kept constant for all images (see image 1). At the
conclusion of processing, all animals were released back into their natural habitat, usually within
12-24 hours.
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Image 1: Standard salamander image
Image processing
Salamander images from 2008 to 2010 were imported into the Mac application,
Macnification, an image processing program. Standard processing algorithms allowed us to
identify the number of spots on the dorsal surface of each salamander, the area of each of the
spots, relative spot circularity, and RGB values of each spot. The standard protocol that was used
for each image analysis is as follows:
1) each image was calibrated by inputting the
measurement of a color bar which was present in the photograph. 2) Spots of the salamander
were selected by adjusting a luminance bar (see image 2) until they had been highlighted yet the
majority of the salamander body remained unselected (see image 3). 3) During the selection
process, some of the lighter areas of the salamander body would become selected along with the
spots. These “fake spots” had to be removed from the spot list manually (see image 4). 4) The
resulting spot list was then exported into a Microsoft Excel spreadsheet and organized by animal
(see image 5). 5) This process was repeated for an image of each salamander.
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Image 2: Luminance bar.
Image 4: Example of a “fake spot.”
Image 3: Spots of salamander selected.
Image 5: Spot list.
Data analysis
For each animal of each year (2008, 2009, 2010), the number of spots, the total spot area
(cm2), and specific color properties for red, green and blue values served as the primary digital
data analysis. I used JMP, (SAS Institute) to test hypotheses about the relationship between spot
properties and various morphological and behavioral traits recorded for each salamander as part
of the demographic study. Comparisons for sexual dimorphism between sexes, and relationships
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between body condition, spot coloration and individual breeding arrival date were carried out
with a combination of correlation analyses and ANOVA/ANCOVA.
Results
Sexual dimorphism
It was observed that general variation in animal total spot area and all said color properties
was independent of the animal sex. No significant dimorphism exists in color parameters or the
total area of spots when compared to animal sex. Models serving to test this relationship
accounted for just 2-3% of total variance in each instance.
Body condition, coloration, and breeding arrival
For both female and male spotted salamanders, there was no significant negative correlation
between individual body condition (mass residuals of mass vs. SVL) and date of arrival to the
breeding grounds (Figure 1). That is, there was no evidence that salamanders in better condition
(as reflected as length specific mass) arrive at the pond earlier than other salamanders.
sex
arrival
spot property
correlation
n
P<
M
julian
Residuals Mass By Sex (M)
-0.0047298
192 0.948086
F
julian
Residuals Mass By Sex (F)
-0.0040616
372 0.937769
Figure 1: Male and female comparison of julian date and body condition.
.
Results comparing female and male date of arrival to the breeding pond (Julian day) and spot
characteristics both showed a significant negative correlation as presented in Figure 2. Even
though the results of comparison for male individuals were weaker, they were still significant.
Unique to males was that spot area was also found to be negatively related to date of arrival.
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sex
arrival
spot property
correlation
n
lower 95 CL
upper 95 CL
P<
F
F
julian
julian
Sum(area)
Hue
0.09169236
-0.4100977
193
193
-0.0501979
-0.5211516
0.22995445
-0.2853879
0.20472
0.00000
F
F
julian
julian
Saturation
Lightness
-0.2871789
-0.1565104
193
193
-0.4117193
-0.291311
-0.1521085
-0.0156155
0.00005
0.02974
M
M
julian
julian
Sum(area)
Hue
-0.2054496
-0.2160095
372
372
-0.300844
-0.3108609
-0.1059845
-0.1168979
0.00007
0.00003
M
M
julian
julian
Saturation
Lightness
-0.1176429
-0.1006647
372
372
-0.2167294
-0.2002937
-0.0161571
0.0010248
0.02325
0.05239
Figure 2: Male and female comparison of julian day and spot properties
Lastly, results comparing the female and male julian day and spot characteristics (corrected
for by principle component analysis of mass and SVL to help give an estimate of individual
robustness) (Figure 3) showed very similar results as presented in Figure 2.
sex
F
size corrected spot property
Residuals Sum(area)
arrival correlation
n
julian 0.09316597 192
lower 95 CL
-0.04909
upper 95 CL
0.2317172
P<
0.19868
F
F
Residuals Hue
Residuals Saturation
julian
julian
-0.4117821
-0.2928553
192
192
-0.5228992
-0.4171635
-0.2869036
-0.1577907
0.00000
0.00004
F
M
Residuals Lightness
Residuals Sum(area)
julian
julian
-0.1561906
-0.1863585
192
372
-0.2913548
-0.2826811
-0.0149122
-0.086315
0.03051
0.00030
M
M
Residuals Hue
Residuals Saturation
julian
julian
-0.2227626
-0.1542475
372
372
-0.3172557
-0.2519746
-0.1238897
-0.053406
0.00001
0.00286
M
Residuals Lightness
julian
-0.0808985
372
-0.181088
0.02095286
0.11932
Figure 3: Male and female comparison of julian day and corrected spot properties.
Discussion
While we are not the first to test for a sexual dimorphism of spot properties of A. maculatum,
we went a step deeper and examined the relationship between body condition, coloration and
breeding arrival. There has been previous spot analysis performed on A. maculatum, where the
study used spot symmetry on the back of spotted salamanders to indicate overall individual body
condition (Davis and Maerz, 2007). Through analysis for a sexual dimorphism of spot color
parameters between male and female spotted salamanders, it was determined that a significant
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difference does not exist. Results suggested that neither male nor female salamanders showed
variation in either spot area or chromatic properties as a function of variation of overall
individual size. This finding is opposite of the conclusion drawn for both Ambystoma opacum
and Notophthalmus v.viridescens of the studies Todd and Davis of 2007 and Davis and Grayson
of 2008 respectively.
Although no evidence of sexual dimorphism was found examining color parameters (hue,
saturation, and lightness) against animal sex between spotted salamanders, we did find
significant results in, coloration and breeding arrival date. It was concluded that in both males
and females, early arrival to the breeding pond is significantly associated with higher hue,
saturation, and lightness values, as well as a greater total spot area in male animals. In order to
determine that these findings were not attributable to the body size of an individual, but rather
spot properties, a statistical test was performed that compared the date of arrival with spot
characteristics that had been corrected to provide an estimate of individual overall size. These
results, in Figure 3 of the Results section, are very similar to the data presented in Figure 2 in
both the degrees of correlation and p-values for each spot property. Since the values are
relatively unchanging, the overall size of an individual does not seem to be driving the
relationship have an inverse relationship with julian day. Providing further evidence that earlier
arrival to the breeding grounds appears to be related to salamander spot properties.
This phenomenon is not limited to the class Amphibia by any means. For example, a study
was conducted that examining epaulettes (shoulder colorations) of male red-shouldered
widowbirds. In specific, the study was looking if males with greater coloration of the shoulders
resulted in a higher competitive ability. Results concluded that males with larger and redder
epaulettes were able to out-compete males with smaller and less red coloration when establishing
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territory (Pryke and Andersson, 2003). These results are similar to the study we have completed
in the sense that greater coloration seems to play a role in greater individual success (be it
courtship or territoriality).
The arrival of such animals to the breeding pond at an earlier date may be related to an
increased mating probability. It is reasonable to assume that increased spot color values are
positively related to mate interest, such as is the male courtship dance of red-spotted newts
(Verrell, 1982). Animals with higher chromatic values may arrive early to the breeding site so
they are able to have the greatest selection of potential mates to court. This is important because
it has been seen in species such as A. opacum that male:female ratios are nearly always maleleaning during breeding season. Females to court would be in lesser numbers, making them the
limiting variable during breeding season (Scott, 2005). The exact meaning of this relationship
still remains unknown and aforementioned assumptions are just that. Future studies could be
conducted in order to determine if sexual selection is actually responsible for the determined
correlation between body condition, spot coloration and date of breeding arrival.
References
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Davis, A. K., and J. C. Maerz. "Spot Symmetry Predicts Body Condition in Spotted
Salamanders, Ambystoma maculatum." Applied Herpetology 4 (2007): 195-205. Print
Davis, A. K., and K. L. Grayson. "Spots of Adult Male Red-spotted Newts Are Redder and
Brighter than in Females: Evidence for a Role in Mate Selection?" Herpetological
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Pryke, S.R., and S. Andersson. "Carotenoid-based Epaulettes Reveal Male Competitive Ability:
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Todd, B. D., and A. K. Davis. "Sexual Dichromatism in the Marbled Salamander, Ambystoma
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Verrell, P. A. "The Sexual Behaviour of the Red-spotted Newt, Notophthalmus viridescens."
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