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Warning signal properties covary with toxicity but not testosterone or aggregate carotenoids in a poison frog


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Aposematic (warning) coloration is a highly conspicuous trait that is found throughout the animal kingdom. In several aposematic species, warning signals have been co-opted for use in conspecific communication systems; for example, in the toxic and bright orange Solarte population of the strawberry poison frog (Oophaga [Dendrobates] pumilio), the brightness of male warning coloration serves as a sexual signal by both attracting females and repelling rivals. Here, we investigate correlations between bright male warning coloration and several physiological characteristics (e.g., circulating testosterone and carotenoids and noxious alkaloids in the skin), to gain insights into the mechanisms underlying the signal variation in this population and to inform hypotheses regarding the evolutionary stability of this trait. We find that although measures of male brightness (viewer-dependent or viewer-independent) do not correlate with two classic correlates of sexually selected traits (circulating testosterone and aggregate carotenoids in the skin), male reflectance does show a positive correlation with concentrations of two xanthophyll carotenoids. Total reflectance (a viewer-independent measure of male brightness) also shows a negative relationship with aggregate pumiliotoxin in the skin, which is considered one of the major classes of defensive alkaloids in this species. Because the alkaloids used in this species’ chemical defense are acquired from dietary sources, the magnitude of the reflectance intensity of a male’s warning signal can potentially provide viewers with reliable information regarding territory quality, health, and/or current condition.
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Evolutionary Ecology
ISSN 0269-7653
Evol Ecol
DOI 10.1007/s10682-016-9830-y
Warning signal properties covary with
toxicity but not testosterone or aggregate
carotenoids in a poison frog
Laura Crothers, Ralph A.Saporito,
Justin Yeager, Kathleen Lynch, Caitlin
Friesen, Corinne L.Richards-Zawacki,
Kevin McGraw, et al.
1 23
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Warning signal properties covary with toxicity
but not testosterone or aggregate carotenoids in a poison
Laura Crothers
Ralph A. Saporito
Justin Yeager
Kathleen Lynch
Caitlin Friesen
Corinne L. Richards-Zawacki
Kevin McGraw
Molly Cummings
Received: 16 November 2015 / Accepted: 25 March 2016
ÓSpringer International Publishing Switzerland 2016
Abstract Aposematic (warning) coloration is a highly conspicuous trait that is found
throughout the animal kingdom. In several aposematic species, warning signals have been
co-opted for use in conspecific communication systems; for example, in the toxic and
bright orange Solarte population of the strawberry poison frog (Oophaga [Dendrobates]
pumilio), the brightness of male warning coloration serves as a sexual signal by both
attracting females and repelling rivals. Here, we investigate correlations between bright
male warning coloration and several physiological characteristics (e.g., circulating
testosterone and carotenoids and noxious alkaloids in the skin), to gain insights into the
mechanisms underlying the signal variation in this population and to inform hypotheses
regarding the evolutionary stability of this trait. We find that although measures of male
brightness (viewer-dependent or viewer-independent) do not correlate with two classic
Electronic supplementary material The online version of this article (doi:10.1007/s10682-016-9830-y)
contains supplementary material, which is available to authorized users.
&Laura Crothers
University of California, Davis, Davis, CA 95616, USA
Department of Biology, John Carroll University, University Heights, OH 44118, USA
Department of Ecology and Evolutionary Biology, Tulane University, 400 Lindy Boggs Bldg.,
New Orleans, LA 70118, USA
Department of Biology, Hofstra University, Gittleson 324, Hempstead, NY 11549, USA
Department of Integrative Biology, University of Texas at Austin, 1 University Station C0990,
Austin, TX 78712, USA
Department of Biological Sciences, University of Pittsburgh, 4249 Fifth Avenue, Pittsburg,
PA 15260, USA
Smithsonian Tropical Research Institute, Apartado Postal 0843-03092, Panama
Republic of Panama
School of Life Sciences, Arizona State University, Life Sciences C-wing Room 522, Tempe,
AZ 85287, USA
Evol Ecol
DOI 10.1007/s10682-016-9830-y
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correlates of sexually selected traits (circulating testosterone and aggregate carotenoids in
the skin), male reflectance does show a positive correlation with concentrations of two
xanthophyll carotenoids. Total reflectance (a viewer-independent measure of male
brightness) also shows a negative relationship with aggregate pumiliotoxin in the skin,
which is considered one of the major classes of defensive alkaloids in this species. Because
the alkaloids used in this species’ chemical defense are acquired from dietary sources, the
magnitude of the reflectance intensity of a male’s warning signal can potentially provide
viewers with reliable information regarding territory quality, health, and/or current
Keywords Aposematism Chemical defense Conspicuousness Oophaga
[Dendrobates]pumilio Sexual selection Signal reliability
Colorful sexual ornaments can function as reliable signals of the health, body condition,
and territorial status of an animal (Andersson 1994; Maynard Smith and Harper 2003;
Whiting et al. 2003). Signal conspicuousness, for example, has been shown to correlate
with an individual’s health, condition, and/or nutritional state across a broad range of
taxonomic groups (e.g., Hamilton and Zuk 1982; Folstad and Karter 1992; Ryan and
Keddy-Hector 1992; Andersson 1994). The reliability of such ‘‘indicator’’ signals is often
governed by physiological costs, such as diet-limited pigment molecules (e.g., carotenoids;
Olson and Owens 1998). However, while many studies have focused on the physiological
correlates of sexual signals, they have largely ignored another class of conspicuous animal
signals. Aposematic ‘‘warning’’ signals, instead of evolving through sexual selection,
evolve through natural selection to advertise to predators that a chemically defended prey
item is unprofitable to attack (Wallace 1867).
Several lines of evidence indicate that physiological costs may also be associated with
aposematic signals. The sequestration, modification, and storage of noxious compounds by
chemically defended organisms are thought to be oxidatively stressful processes (Ahmad
1992; Blount et al. 2009; Santos and Cannatella 2011). Furthermore, many aposematic
signals comprise red, orange, and yellow coloring, which in non-aposematic organisms is
often controlled by pigments acquired from the diet that are involved in homeostatic redox
reactions (Hill and Johnson 2012; Kodric-Brown 1989; McGraw 2005; Olson and Owens
1998). Interestingly, whereas predators were historically thought to be the agents shaping
the evolution of aposematic signals (Mu
¨ller 1879), recent research suggests that apose-
matic signals can function in the context of conspecific communication and that sexual
selection may influence the direction of aposematic trait evolution (Jiggins et al. 2001;
Maan and Cummings 2009; Nokelainen et al. 2011). Because aposematic species are
highly conspicuous and can simultaneously signal to both predators and conspecifics with
the same trait (Jiggins et al. 2001; Maan and Cummings 2008; Nokelainen et al. 2011;
Saporito et al. 2007a,b), investigating the physiological correlates common to these traits
can help clarify the underlying constraints governing the evolution of signals.
A staggering intraspecific and interspecific diversity in signal expression is observed in
aposematic signals, causing some to speculate that they function as ‘‘magic traits’’ (sensu
Gavrilets 2004), so-called ‘‘speciation’’ traits that simultaneously drive non-random mating
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and are under divergent natural selection. The coloration of the phenotypically variable
strawberry poison frog (Oophaga [Dendrobates]pumilio), whose populations feature
15–30 unique color patterns throughout western Panama, is regarded as a magic trait
(Servedio et al. 2011). Both viewer-independent and viewer-dependent measures of signal
brightness function as honest indicators of toxicity among O. pumilio populations (Maan
and Cummings 2012). Females of this species show evidence of positive assortative mating
by male color pattern (Summers et al. 1999) and a widespread preference for brighter
males (Maan and Cummings 2009). However, populations do vary in these preferences;
females of some populations do not prefer the local male visual signal (Dreher and Pro
2014; Maan and Cummings 2008) and female preferences for acoustic signals override
visual signal preference in some populations (Dreher and Pro
¨hl 2014). Bright warning
coloration also appears to have been co-opted as an agonistic indicator signal within the
bright orange and highly toxic Solarte population (Crothers et al. 2011; Crothers and
Cummings 2015). Furthermore, males of this population also exhibit substantial variation
in brightness (Crothers and Cummings 2013) and brighter males appear to be more
aggressive (Crothers and Cummings 2015).
There are several biomolecules that commonly correlate with conspicuous sexual sig-
nals and are thus candidates for investigating as correlates of warning signals. First, the
development or expression of sexually selected bright signals is often testosterone
dependent (Folstad and Karter 1992; Johnstone and Norris 1993; Sinervo et al. 2000;
reviewed in Whiting et al. 2003). Second, carotenoid pigments predominate in orange/red
animal colors (Kodric-Brown 1989; McGraw 2005), and such pigment-based traits often
serve as honest signals because the carotenoids used in coloration can be limited dietarily
and have important health roles (Andersson 1994; Bezzerides et al. 2007; Blount et al.
2009; Hill and Johnson 2012; McGraw and Ardia 2003; Olson and Owens 1998). Finally,
color or brightness expression may correlate with skin toxins within a population (as
predicted by theoretical studies, e.g., Blount et al. 2009; Holen and Svennungsen 2012; Lee
et al. 2011), because they have been shown to correlate across populations of this and other
aposematic species (Bezzerides et al. 2007; Maan and Cummings 2012; Saporito et al.
We predicted that conspicuous warning signals would show similar physiological
underpinnings to those of sexual signals. Here, we investigate several potential physio-
logical correlates of warning coloration in the phenotypically variable and sexually
dimorphic Solarte O. pumilio population, to begin to understand the underlying mecha-
nistic basis for inter-male color variation in this poison frog population. We predicted that
O. pumilio males with greater total reflectance or viewer-dependent (avian or O. pumilio)
measures of conspicuousness would have (1) higher circulating testosterone levels, (2)
greater quantities of skin carotenoids, and (3) greater quantities of noxious skin alkaloids.
Body measurements
Calling territorial adult males were located in the field during early daytime hours in
August to September of 2011 and July of 2012 on Isla Solarte, in Bocas del Toro, Panama
(N 09°20.0140,W82°13.1970). Males were captured and kept individually in plastic
475 mL deli containers moistened with ultraviolet (UV) purified water until body
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measurements were taken. All males (N =43) were measured for spectral reflectance and
were photographed on a standard background that included a ruler, using a Canon Rebel
10.1 XS DSLR camera, as done previously (Crothers et al. 2011; Crothers and Cummings
2015). In 2011, twenty-three males were measured beneath a tent in the field within several
hours of capture, and then transported to the Smithsonian Tropical Research Institute
(STRI) in Bocas del Toro, Panama, for blood sampling (see below). In 2012, twenty males
were first transported to the Smithsonian Tropical Research Institute (STRI) in Bocas del
Toro, Panama, and then were measured there within 24 h of capture, followed by skin
carotenoid and alkaloid measurements (see below). For a subset of the males caught in
2012 (N =10) we recorded perch height in the male’s territory prior to capture in order to
characterize any physiological correlates with microhabitat use.
Spectral reflectance measurements and analysis of brightness
Spectral reflectance measurements for each individual were taken at the head and dorsum
(two measurements per region in 2011; four measurements per region in 2012) using an
EPP2000 UV–VIS portable spectrometer and R600-8 UV–VIS-SR reflectance probe
(StellarNet Inc., Tampa, FL) and a PX2 Xenon flash lamp outfitted with a custom-made
50 Hz trigger input (Ocean Optics, Dunedin, FL). Spectralon white standard measurements
were taken frequently to account for lamp drift. Males captured in 2011 were briefly
housed individually at STRI until they were returned to their territories in the field.
We averaged measurements of the head and dorsum to calculate both the total reflec-
tance P
700 nm
300 nm
, a perceptually unbiased estimate of male brightness, and long-wave
chroma. Long-wave chroma (*redness) assesses the proportion of the total reflectance in
the long-wave band: P700nm
i¼600nm RkðÞ
i¼300nm RkðÞ
Because the conspicuousness of a frog depends both on the unique characteristics of a
viewer’s visual system and on the spectral properties of the signaling background, we also
evaluated viewer-specific measures of male brightness or luminance contrast (DL). In
addition, we evaluated viewer-specific measures of male color contrast (DS) and overall
conspicuousness (OC), which represents a bivariate measure of luminance and color
contrast (as in Maan and Cummings 2012). We estimated these parameters for a frog’s
dorsum when viewed against two common signaling backgrounds in the Solarte population
(palm leaf and Heliconia sp. leaf) and using an average irradiance measurement from that
population (see Crothers and Cummings 2013 for details). We used a Sturnus vulgaris-
specific visual model and an O. pumilio-specific visual model (as in Crothers and Cum-
mings 2013) to estimate conspicuousness for what is considered the species’ most common
class of predator (birds: Maan and Cummings 2012; Dreher et al. 2015) and for a con-
specific viewer, respectively. Overall conspicuousness (OC) for these two visual systems
was calculated as the Euclidean distance of two detection parameters (DL and DS) in the
viewer-specific color space.
Circulating testosterone
We housed the 23 calling males that we collected in 2011 in individual terraria at STRI
(*37 cm 922 cm 924 cm), containing water, leaf litter, and arthropods, for approxi-
mately 24 h prior to blood collection. Blood was collected from the orbital sinus using a
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heparinized capillary tube with a tapered end produced with a micropipette puller, as done
by Lynch et al. (2006). One frog died following blood collection, but the others were
returned to their points of capture in the field after sampling. In July of 2013, blood was
also collected from six additional adult males in a captive colony maintained in the
Richards-Zawacki laboratory at Tulane University; these samples were pooled and were
used to perform a serial dilution validation for the hormone analysis. These six frogs were
first sacrificed by double pithing and then blood was immediately collected from the orbital
sinus and from an incision in the right hind leg with a heparinized micropipette tip. All
blood samples were immediately centrifuged at *10,000 rpm for 6 min and the plasma
layer collected and frozen for several days on dry ice while in transport to the University of
Texas, where they were maintained at -20 °C until hormone analyses were performed in
February of 2014.
We measured circulating levels of total testosterone using enzyme-linked immunosor-
bent assay (Enzo Life Sciences, cat. # ADI-900-065). O. pumilio plasma samples were
analyzed for parallelism with the kit’s standard curve using a series of six dilutions from
the pooled plasma stock (1:10, 1:20, 1:40, 1:80, 1:160, and 1:320). The dilutions ran
parallel to the standard curve (homogeneity of slopes ANCOVA: F
P=0.895), validating the kit’s suitability for use with this species. Individual male plasma
samples were diluted at 1:64 (16 samples), 1:86 (5 samples), 1:142.5 (1 sample), and
1:213.5 (1 sample) in assay buffer and the kit protocol was strictly followed. Each sample
was run in duplicate. The use of lower dilution concentrations for some samples was due to
the exceptionally small available plasma quantities (\2lL) for those samples. The plates
were read with a conventional plate reader at 405 nm (SpectraMax M3, Molecular
Devices). To compute circulating testosterone levels, the percent bound for each of the
standards was calculated and a logarithmic curve was generated. This curve was then used
to compute the circulating testosterone levels for the males (in ng/mL). Circulating
testosterone levels ranged from 0.67 to 2.68 ng/mL (mean =1.34, SD =0.65). Intra-
assay variation was 17.64 %, and inter-assay variation was 7.14 %. Cross-reactivity in the
testosterone kit was \0.001 % for dihydrotestosterone.
Skin carotenoids
Immediately following body measurements at STRI (size and spectral measurements), all
20 males captured in 2012 were sacrificed by double pithing. A small sample of dorsal skin
was collected for skin alkaloid analysis from each male, as described below. The remaining
dorsal skin was dissected and frozen in liquid nitrogen for transport to the United States,
after which samples were stored at -80 °C until their carotenoid content was analyzed in
October 2013. Carotenoid levels in the dorsal skin tissue were quantified (in lg of car-
otenoid per g of tissue; tissue was weighed to the nearest 0.00001 g with a digital balance)
using high performance liquid chromatography (HPLC), following a modified version of
previously established protocols (McGraw et al. 2006).
In brief, carotenoids were extracted using a micronizer in the presence of solvent
(1.4 mL hexane:tert butyl methyl ether, 1:1, v/v), using 0.1 g of skin. Tissue and solvent
were centrifuged, and the supernatant was recovered and dried down for carotenoid
analysis. HPLC analyses follow those in McGraw et al. (2006), using a Waters 2695
instrument (Waters, Milford, MA). Because of the presence of ketocarotenoids in the
samples, the analytical method was slightly modified. First, the HPLC column (Waters
YMC Carotenoid column, 5 mm, 4.6 mm #250 mm) was pretreated with 1 %
orthophosphoric acid in methanol for 30 min at 1 mL/min. Secondly, solvent composition
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and flow rate were altered to optimize separation of different ketocarotenoids. At a con-
stant flow rate of 1.2 mL/min, an isocratic elution with 42:42:16 (v/v/v) methanol:ace-
tonitrile:dichloromethane was first used for 11 min followed by a linear gradient up to
42:23:35 (v/v/v) methanol:acetonitrile:dichloromethane through 21 min, holding those
conditions until minute 25, and finishing with a return to the original isocratic conditions
from 25 to 29.5 min. Carotenoid types were identified by comparison to authentic stan-
dards from CaroteNature (Ostermundigen, Switzerland). External standard curves were
used to quantify concentrations of each carotenoid type. One sample was lost during HPLC
analysis, resulting in a final sample size of 19 males for this dataset. Total carotenoid
quantities ranged from 86.04 to 1421.51 lg/g. We identified 17 unique carotenoids within
the analyzed samples (presented in Table 1).
Skin alkaloids
A sample of mid-dorsal skin tissue, referred to herein as a ‘‘disc,’’ was removed from the
20 recently sacrificed males captured in 2012 (described above) using a 4 mm-diameter
circular biopsy punch. Biopsies were consistently performed on the same side and
approximate location on the dorsal skin surface. Skin samples were stored individually at
room temperature in methanol-filled glass vials for subsequent alkaloid analysis. Individual
alkaloid fractions were prepared in April 2013 from methanol extracts of each skin and
characterized using gas chromatography in combination with mass spectrometry (GC–MS)
Table 1 Skin carotenoids found in Solarte males
Carotenoid Median
Range (lg/g) SD (lg/g) % Indiv. Correlation
Apocarotenoid 5.74 5.67 0–13.75 4.88 68
Canary Xanthophyll 17.05 21.29 1.06–64.07 17.26 100 0.26
Canthaxanthin 6.05 11.93 0–73.74 16.73 79 0.22
Xanthophyll 3.77 6.47 0–24.78 6.73 84 0.48
cis-Ketocarotenoid 6.34 8.30 0–40.62 9.99 74 0.32
Echinenone 0 4.76 0–51.99 14.43 11
3-Hydroxy-echinenone 34.41 47.31 0–179.22 43.29 95 0.27
Lutein Ester (1) 23.79 34.76 2.2–83.87 27.05 100 0.03
cis-Xanthophyll 18.84 22.26 0–71.22 20.03 84 0.12
Canary Xanthophyll Ester (1) 47.94 56.55 4.01–214.11 55.92 100 0.03
B-Carotene 152.63 195.08 37.96–482.83 134.50 100 0.11
Canary Xanthophyll Ester (2) 60.04 76.67 5.98–226.29 63.44 100 0.23
Ketocarotenoid Ester (2) 0 2.63 0–35.21 8.45 16
Canary Xanthophyll Ester (3) 21.81 30.86 0–90.07 26.58 95 0.47
Canthaxanthin Ester 46.99 50.64 1.66–135.09 32.31 100 0.16
Ketocarotenoid Ester 18.82 24.77 0–95.38 22.81 84 0.25
Xanthophyll Ester 8.67 10.37 0–29.14 8.96 79 0.128
Total Carotenoids 459.73 610.32 86.04–1421.51 407.45 0.20
The penultimate column presents the percentage of individuals in the dataset (N =19 males) that had the
carotenoid present in their skin. The final column contains correlation coefficients for that carotenoid and
male brightness (total reflectance) for those carotenoids found in 70 % or more of males. Coefficients are
italicized for positive linear regressions in which P\0.05 (not corrected for multiple hypothesis testing)
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using the methods detailed in Saporito et al. (2010), with final dilution volumes modified to
accommodate the smaller tissue sizes. GC–MS was performed on a Varian 3900 GC with a
30 m 90.25 mm i.d. Varian Factor Four VF-5 ms fused silica column coupled to a Varian
Saturn 2100T ion trap MS instrument. Alkaloids were separated using a GC temperature
program from 100 to 280 °C at a rate of 10 °C per minute with helium as the carrier gas
(1 mL/min). Each alkaloid fraction was analyzed using electron impact MS and chemical
ionization MS with methanol as the ionizing reagent.
Comparisons of mass spectrometry properties and GC retention times with those
described in previous studies allowed us to identify individual alkaloids for each skin
sample (Daly et al. 2005; Saporito et al. 2007a,b,2010). All alkaloids within a fraction
were quantified (in lg/disc) by comparison of the alkaloid’s peak area to the peak area of a
nicotine internal standard using a Varian MS Workstation v.6.9 SPI. There was a broad
range of both alkaloid quantity (1.59–153.41 lg, median =5.64, SD =33.60) and alka-
loid diversity (8–48 unique alkaloids, median =13.5, SD =9.38) within the twenty
4 mm-diameter samples of dorsal skin (Table 2).
Statistical analysis
All statistical tests were performed in R 2.15.1 (R Development Core Team 2012). We first
assessed the relationship between circulating testosterone (in ng/mL) and male warning
coloration by fitting linear regressions (LM) with the viewer-dependent (DL, DS, OC) and
viewer-independent (total reflectance and long-wave chroma) measures of conspicuous-
ness as predictor variables. Two samples were off the standard curve and were not included
in these analyses, resulting in a final sample size of 21 males for the hormone analyses.
We then tested the relationship between total skin carotenoids (lg/g) and male col-
oration by fitting linear regressions with the conspicuousness measures described in the
paragraph above as predictor variables. For prevalent carotenoids (found in [70 % of
individuals), we performed individual linear regressions of those carotenoids on male total
reflectance (Table 1).
Because the alkaloid dataset contained two exceptionally toxic males ([6x that of the
median alkaloid quantity for the dataset), we converted alkaloid quantity in the skin (in
lg), and total alkaloid diversity (number of unique alkaloids) to rank data. Small sample
size (N =20) precluded the use of ordinal logistic regression, thus we modeled the
relationship between male color measurements (brightness, long-wave chroma, DL, DS,
OC) and these alkaloid measures using Kendall’s rank correlations (Kendall 1955). We
also modeled the relationship between viewer-independent estimates of conspicuousness
and alkaloids/carotenoids with Wilcoxon rank sum tests using a dichotomous total
reflectance and long-wave chroma measure (‘‘brighter’’ or redder than the median versus
‘duller’’ or less red than the median total reflectance or long-wave chroma for the dataset,
respectively; as in Crothers and Cummings 2013). We performed two additional analyses,
using Kendall’s rank correlations, to assess the relationships between male warning signals
and common alkaloids. First, we assessed whether conspicuousness measures co-varied
with aggregate quantity of pumiliotoxins, considered a major class of toxic alkaloids found
in the skin of poison frogs of the Dendrobates/Oophaga genera (Daly and Myers 1967;
Daly et al. 1999). Second, we assessed the relationship between male total reflectance and
the five specific alkaloids found in [70 % of male skins (results presented in Table 2).
Alkaloid sequestration, modification, and storage are believed to be oxidatively stressful
for chemically defended organisms (Ahmad 1992; Blount et al. 2009; Santos and Can-
natella 2011). We thus used two methods to assess whether the most abundant carotenoids
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Table 2 Skin alkaloids in Solarte males
Alkaloid Median
% Indiv. Correlation
3,5-I 195B 0.00 0.087 0–0.877 0.216 20
3,5-I 251K 0.00 0.010 0–0.202 0.045 5
3,5-P 209K 0.00 0.002 0–0.049 0.011 5
3,5-P 223H 0.00 0.479 0–8.509 1.896 25
3,5-P 223H (iso) 0.00 0.009 0–0.166 0.037 10
5,6,8-I 277E 0.00 0.009 0–0.172 0.038 5
5,6,8-I 277E (iso) 0.00 0.001 0–0.022 0.005 5
5,6,8-I 221U 0.00 0.001 0–0.019 0.004 5
5,6,8-I 223A 0.00 0.036 0–0.439 0.104 20
5,6,8-I 223X 0.00 0.001 0–0.015 0.003 5
5,6,8-I 231B 0.00 0.004 0–0.024 0.007 25
5,6,8-I 237C 0.00 0.109 0–1.424 0.320 40
5,6,8-I 249C 0.00 0.002 0–0.034 0.008 5
5,6,8-I 253H 0.00 0.021 0–0.235 0.056 25
5,6,8-I 263A 0.00 0.007 0–0.122 0.027 10
5,8-I (unidentified) 0.00 0.004 0–0.071 0.016 5
5,8-I 195A 0.00 0.046 0–0.920 0.206 5
5,8-I 195I 0.00 0.011 0–0.220 0.049 5
5,8-I 205A 0.00 0.018 0–0.183 0.048 15
5,8-I 207A 0.00 0.065 0–0.755 0.171 30
5,8-I 221A 0.00 0.001 0–0.030 0.007 5
5,8-I 223D 0.00 0.032 0–0.235 0.076 20
5,8-I 233D 0.00 0.352 0–5.880 1.327 10
5,8-I 233D (iso) 0.00 0.001 0–0.016 0.004 5
5,8-I 233D (iso) 0.00 0.007 0–0.075 0.018 30
5,8-I 233D (iso) 0.04 0.251 0–2.337 0.543 65
5,8-I 233D (iso) 0.00 0.023 0–0.336 0.078 10
5,8-I 235B 0.07 3.757 0–59.84 13.285 70 -0.03
5,8-I 243B 0.00 0.014 0–0.192 0.046 15
5,8-I 245B 0.00 0.014 0–0.289 0.065 5
5,8-I 251B 0.00 0.059 0–0.879 0.196 30
5,8-I 251B (iso) 0.00 0.002 0–0.036 0.008 5
5,8-I 253B 0.00 0.003 0–0.052 0.012 5
5,8-I 259B 0.00 0.003 0–0.067 0.015 5
aPTX 305A 0.00 0.052 0–0.798 0.178 20
aPTX 305A (iso) 0.00 0.001 0–0.027 0.006 5
aPTX 323B 0.00 0.014 0–0.273 0.061 5
aPTX 325A 0.00 0.291 0–4.892 1.095 25
aPTX 325B 0.00 0.017 0–0.346 0.077 5
d-5,8-I 221V 0.00 0.003 0–0.054 0.012 5
d-5,8-I 201A 0.00 0.003 0–0.039 0.010 10
DHQ 195A 0.10 2.852 0–37.37 8.314 75 -0.12
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Table 2 continued
Alkaloid Median
% Indiv. Correlation
DHQ 195A (iso) 0.00 0.005 0–0.106 0.024 5
DHQ 195A (iso) 0.00 0.011 0–0.205 0.046 15
DHQ 211A 0.00 0.123 0–1.352 0.317 25
Izidine 209D 0.00 0.002 0–0.037 0.008 5
Izidine 211C 0.00 0.002 0–0.015 0.005 20
Lehm 275A 0.00 0.003 0–0.058 0.013 5
Pip 211J 0.00 0.004 0–0.076 0.017 5
PTX 251D 0.00 0.026 0–0.513 0.115 5
PTX 277B 0.00 0.018 0–0.238 0.058 10
PTX 307A 0.66 2.356 0–21.02 5.107 75 -0.34
PTX 307B 0.00 0.090 0–1.176 0.265 35
PTX 307F00.00 0.007 0–0.109 0.024 15
PTX 307F00 0.00 0.022 0–0.222 0.067 10
PTX 307G 0.00 0.013 0–0.250 0.056 5
PTX 309A 0.00 0.027 0–0.196 0.060 20
PTX 321A 0.00 0.236 0–4.420 0.986 15
PTX 323A 0.04 1.495 0–10.60 3.196 50
PTX 323A (iso) 0.00 0.001 0–0.015 0.003 5
PTX 323A (iso) 0.00 0.046 0–0.455 0.112 20
PTX 323A (iso) 0.00 0.801 0–4.549 1.239 45
PTX 323A (iso) 0.00 0.006 0–0.082 0.020 10
PTX 325B 0.00 0.027 0–0.330 0.084 10
Spiro 236 0.00 0.011 0–0.219 0.049 5
Spiro 252A 0.00 0.019 0–0.258 0.057 30
Tri (unidentified) 0.00 0.003 0–0.061 0.014 5
Tri 191B 0.57 0.555 0–1.214 0.297 90 0.04
Tri 191B (iso) 0.00 0.005 0–0.029 0.010 25
Tri 203B 0.01 0.092 0–0.367 0.140 55
Tri 203B (iso) 0.00 0.039 0–0.517 0.126 10
Tri 205B 0.25 0.256 0.166–0.53 0.076 100 0.02
Tri 205B (iso) 0.03 0.200 0–0.952 0.313 55
Tri 205H 0.00 0.001 0–0.019 0.004 5
Unclassified 279I 0.00 0.072 0–0.812 0.197 20
Unidentified 0.00 0.011 0–0.127 0.034 10
Unidentified 0.00 0.004 0–0.084 0.019 5
Isomers are indicated by the designation (iso). The penultimate column contains the percentage of indi-
viduals in the dataset (N =20 males) that had the alkaloid present in their skin. The final column contains
the Kendall’s tau rank correlation coefficient of that alkaloid and male brightness (total reflectance), for the
few alkaloids found in 70 % or more of males
(3,5-I: 3,5-disubstituted indolizidine; 3,5-P: 3,5-disubstituted pyrrolizidine; 5,6,8-I: 5,6,8-trisubstituted
indolizidine; 5,8-I: 5,8-disubstituted indolizidine; aPTX: allopumiliotoxin; d-5,8-I: dehydro-5,8-disubsti-
tuted indolizidine; DHQ: 2,5-disubstituted decahydroquinoline; Lehm: lehmizidine; Pip: piperidine; PTX:
pumiliotoxin; Spiro: spiropyrrolizidine; Tri: tricyclic)
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co-varied with the most abundant alkaloids within samples; rank correlations were used to
assess the relationships between beta-carotene/xanthophyll (the most abundant car-
otenoids) and aggregate quantity of tricyclics/pumiliotoxins (the most abundant alkaloid
classes), and linear regression was used to determine whether the proportions of these
alkaloids co-varied with the proportions of these carotenoids.
Finally, for the ten males on which we collected perch data, we used Kendall’s rank
correlation to assess whether measures of male conspicuousness may correlate with dif-
ferences in microhabitat by correlating male conspicuousness measures with a male’s
perch height (in m) in his territory.
Circulating testosterone
There was no relationship between circulating testosterone concentration and male total
reflectance (Fig. 1a; LM: N =21, t =0.545, P=0.592, r =0.12), long-wave chroma
(LM: t =-1.011, P=0.325, r =-0.23), luminance contrast (DL, all P[0.49; all
r\0.16), color contrast (DS, all P[0.27; all r \0.25), or overall conspicuousness (all
P[0.75; all r \0.07) for the bird or frog visual models (see table S1).
Skin Carotenoids
There was no relationship between male total reflectance or long-wave chroma and total
quantity of dorsal skin carotenoids, both when assessed as a linear relationship (see Fig. 1b
for total reflectance plot; LM: N =19, both t \0.94, both P[0.360, both r \=0.2) and
when these color characteristics were coded as dichotomous variables (Wilcoxon rank sum
test: total reflectance:W=39, P=0.661; long-wave chroma:W=46, P=0.968).
Analyses using the viewer-specific estimates of DL, DS, and overall conspicuousness (OC)
were also non-significant (LM: all P[0.55, all r \0.15; see table S1). However, the
quantity of two specific carotenoids exhibited a positive relationship with total reflectance
(xanthophyll: P=0.04, and a canary xanthophyll ester: P=0.04; Table 1; Fig. 1c, d),
though these relationships were no longer significant after correction for multiple
hypothesis testing (not shown). Xanthophyll and canary xanthophyll did not correlate with
viewer-dependent conspicuousness measures (Table S2).
Skin alkaloids
Alkaloid quantity and alkaloid diversity were positively correlated (Kendall’s rank cor-
relation: N =20, z =3.375, P=0.0007, tau coefficient =0.56). There was no correla-
tion between total reflectance and alkaloid quantity (Fig. 2a; Kendall’s rank correlation:
N=20, z =-1.3627, P=0.173, tau coefficient =0.22) or alkaloid diversity (Fig. 2a;
z=-1.212, P=0.225, tau coefficient =0.20), or between long-wave chroma and these
measures (Kendall’s rank correlations: both P[0.299, both tau coefficients \0.17);
results were similarly non-significant when assessing alkaloid quantity and diversity and
DL, DS, and overall conspicuousness (OC) for the bird and conspecific visual models (all
P[0.14; all tau coefficients\0.24; see table S1). However, when males were categorized
into dichotomous total reflectance categories, ‘‘brighter’’-than-average males had
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significantly less alkaloid diversity than ‘‘duller’’-than-average males (Fig. 2a; Wilcoxon
rank sum test: W =23, P=0.043), but did not differ significantly from duller males in
terms of alkaloid quantity (Fig. 2a; Wilcoxon rank sum test: W =25, P=0.063). There
was a significant negative relationship between male total reflectance and aggregate
quantity of pumiliotoxins (Fig. 2b; Kendall’s rank correlation: N =20, z =-2.160,
P=0.031, tau coefficient =-0.36), but no relationship between long-wave chroma and
aggregate pumiliotoxin (Kendall’s rank correlation: z =0.589, P=0.556, tau coeffi-
cient =0.10) or between aggregate pumiliotoxin and DL, DS, and overall conspicuousness
(OC) to the bird or frog visual models (all P[0.15, all tau coefficients \0.24; see
table S1). Finally, there was a significant negative relationship between the quantity of one
of the pumiliotoxins (PTX 307A; P=0.04) and total reflectance, but not the other
common alkaloids (Table 2). PTX 307A did not correlate with viewer-dependent measures
of conspicuousness (Table S2).
1.0 1.5 2.0 2.5
6000 7000 8000 9000 10000 11000
Testosterone (ng/mL)
Total Reflectance
200 600 1000 1400
5000 6000 7000 8000
Total Carotenoids (µg/g)
0 5 10 15 20 25
5000 6000 7000 8000
Xanthophyll (µg/g)
0 20406080
5000 6000 7000 8000
Canary Xanthophyll ester (µg/g)
Total Reflectance
Total Reflectance
Total Reflectance
Fig. 1 Plots of male total reflectance and acirculating testosterone, btotal carotenoid concentration,
cxanthophyll concentration, and dcanary xanthophyll ester concentration in the dorsal skin. For cand d, the
significant positive relationship detected between these carotenoids and total reflectance, and presented in
Table 1, has not been corrected for multiple hypothesis testing
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Relationships between alkaloids and carotenoids
There was no correlation between total carotenoid quantity and total alkaloid quantity
(Kendall’s rank correlation: N =19, z =0.105, P=0.916, tau coefficient =0.02).
Examining the proportional relationships between skin alkaloids and skin carotenoids, we
found that the proportion of tricyclic alkaloid quantity was positively correlated with the
proportion of beta-carotene (Fig. 3a; LM: N =19, t =2.426, P=0.027, r =0.51) and
negatively correlated with the proportion of xanthophyll (Fig. 3b; LM: N =19,
t=-2.548, P=0.021, r =-0.53). The proportion of pumiliotoxin alkaloids was neg-
atively correlated with the proportion of beta-carotene (Fig. 3c; LM: N =19, t =-2.609,
P=0.018, r =-0.53), but not correlated with the proportion of xanthophyll (Fig. 3d;
LM: N =19, t =1.639, P=0.119, r =0.37). Rank correlations of these measures were
all non-significant (all P[0.22, all tau coefficients \0.21).
Lastly, for the males with perch data, we found a significant positive relationship
between male dorsal total reflectance and perch height (Fig. 4a; N =10, Kendall’s rank
correlation: T =36, P=0.017, tau coefficient =0.6). However, a significant relationship
for the viewer-dependent conspicuousness measures was only found for overall conspic-
uousness (OC) to an avian viewer (but not O. pumilio viewer) and perch height against a
palm background (P=0.02, tau coefficient =0.6; Fig. 4b; see table S1 for other results).
We sought to address the relationships between measures of male warning signal bright-
ness and several common condition- or quality-related correlates of conspicuous sexual
and aposematic signals. We found that, though O. pumilio male dorsal total reflectance (an
510 15 20
Alkaloid Rank
Alkaloid Diversity
Alkaloid Quantity
Total Reflectance Rank
Total Reflectance Rank
Pumiliotoxin Rank
Fig. 2 Relationships between brightness (total reflectance) and skin alkaloids. aTotal reflectance (top as a
dichotomous measure, bottom as a rank measure) and rankings of alkaloid diversity (left the number of
unique alkaloids present in the skin) and alkaloid quantity (right total lg of alkaloids present in the standard-
sized skin samples) in the dataset of 20 males. Higher rankings indicate larger values. bRelationship
between ranked total reflectance and ranked quantity of aggregate pumiliotoxins (Kendall’s rank correlation:
N=20, z =-2.160, P=0.031)
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inherent and viewer-independent measure of brightness) does not correlate with total skin
carotenoids or circulating testosterone levels, it was positively linked to concentrations of
two specific xanthophyll carotenoids and negatively linked to quantities of a key class of
alkaloid toxins. Viewer-dependent estimates of conspicuousness did not correlate with
these measures.
Although exaggerated color expression is due to high levels of testosterone in many
species of bird (Folstad and Karter 1992; Kimball and Ligon 1999; Whiting et al. 2006)
and lizard (reviewed in Cooper and Greenberg 1992), little is known of how testosterone
controls bright coloration in amphibians (Richards 1982). While brighter males of this
population are more aggressive (Crothers et al. 2011; Crothers and Cummings 2015), we
find here that males with higher total reflectance (and higher associated values of con-
spicuousness to bird and frog viewers) did not have higher testosterone levels. Several
mechanisms may explain the lack of relationship between variation in male signal qualities
and circulating testosterone. Though testosterone can correlate positively with amphibian
calling behavior (Emerson 2001; Marler and Ryan 1996) and induce changes in color
0.0 0.2 0.4 0.6
Proportion of Beta-carotene
Proportion Tricyclics (Tri)
0.0 0.2 0.4 0.6
Proportion of Beta-carotene
Proportion Pumiliotoxins (PTX)
0.0 0.2 0.4 0.6
Proportion Tricyclics (Tri)
Proportion of Xanthophylls
0.0 0.2 0.4 0.6
Proportion of Xanthophylls
Proportion Pumiliotoxins (PTX)
Fig. 3 Relationships of the most common alkaloids and carotenoids found in Solarte male skin samples.
Top Proportional tricyclic alkaloids (x-axis) and abeta-carotene and bxanthophylls (y-axis). Bottom
Proportional pumiliotoxins (x-axis) and cbeta-carotene and dxanthophylls. Lines and shaded areas flanking
the lines represent the predicted line and smoothed 95 % confidence intervals of the statistically significant
linear regressions, respectively
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pattern (Richards 1982), effects of testosterone on amphibian aggression have not been
well investigated (Wilczynski et al. 2005). It is possible that differences in circulating
testosterone levels among males may only be detected when the hormone is rapidly
modulated during short periods of social instability, as it is in other taxa (Goyman et al.
2007; Wingfield et al. 1990). However, we were unable to test this ‘‘challenge hypothesis’’
(Wingfield et al. 1990) with our dataset. Our experimental methodology may also have
precluded us from observing subtle differences in testosterone among males because of
stress-induced changes in their hormone profiles (e.g., Mosconi et al. 2007). Furthermore,
though the warning signal in this population has evidently been co-opted as a sexual signal,
the base color and pattern of various O. pumilio morphs are present prior to sexual mat-
uration in both sexes (L. R. Crothers, pers. obs.) and thus may not rely on gonadal steroid
Contrary to common perception, the maintenance of conspicuous signals is not always
testosterone-dependent (Owens and Short 1995) and other hormones have been implicated
in the control of both conspicuous male signals and male behavior. Corticosterone (Cote
et al. 2010; Moore and Jessop 2003) and melanocortins (Ducrest et al. 2008) both exhibit
complex relationships with aggression and male signal expression in other taxa, but their
impact on amphibian aggression and coloration are less understood (Wilczynski et al.
2005). Exogenous supplementation of arginine vasotocin has recently been shown to
increase aggressive calling in the Neotropical frog Eleutherodactylus coqui (Ten Eyck and
ul Haq 2012), suggesting that vasotocin may be an especially promising candidate hor-
mone for control of aggression in O. pumilio.
Although the skins of O. pumilio contain a strikingly complex mixture of carotenoids
(Table 1), skin color characteristics such as total reflectance, long-wave chroma, and avian
and frog DL, DC and OC did not correlate with total concentration of skin carotenoids.
Though these results were unexpected, a lack of correlation between plumage coloration
and aggregate carotenoid content in the integument has been observed in some birds (Saks
et al. 2003), and pigments other than carotenoids can often contribute to orange and red
2.5 5.0 7.5 10.0
Total Reflectance Rank
Perch Height Rank
2.5 5.0 7.5 10.0
Perch Height Rank
OC Rank (Avian Viewer)
Fig. 4 Ranks of amale total reflectance and boverall conspicuousness (OC) to an avian viewer against a
palm leaf background and males’ perch height in their territories in the field at the time of capture. Higher
rankings indicate larger values for those variables
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coloration in animals (e.g., pteridines; Bagnara 2003; McGraw 2005; Weiss et al. 2012).
However, we did find statistical support for a positive relationship between total reflectance
(but not other conspicuousness measures) and two carotenoids that are associated with
yellow coloration: xanthophyll and a canary xanthophyll ester (Saks et al. 2003; presented
in Table 1; Fig. 1c, d). While these two xanthophylls were not the most abundant car-
otenoids present in the skin samples, they may play an important function in regulating
total reflectance in this population. Little work has been conducted on pigmentary control
of amphibian coloration, though pteridines, carotenoids, and melanin can all contribute to
color (Bagnara 1976; Obika and Bagnara 1964; reviewed in Rudh and Qvarnstro
It should also be noted that although Solarte frogs are generally a uniform orange–red on
their dorsal and ventral surfaces, some individuals do also have small, pinprick-sized dark
spots on their dorsum. To our knowledge the impact of this spotting on conspicuousness
and the pigment molecules controlling this pattern have not yet been explored in this
population or in other populations of this species where patterning is more pronounced.
Ongoing investigations may elucidate whether other pigment molecules (e.g., pteridines, L.
Freeborn,pers. comm) or these two xanthophyll carotenoids control aspects of brightness
in this population of O. pumilio.
We found a significant negative relationship between one conspicuousness measure,
dorsal total reflectance, and the quantity of skin defensive chemicals (alkaloids) males of
this population. ‘‘Brighter’’-than-average males had a lower diversity of alkaloids and
exhibited a marginal trend for lower alkaloid quantities in their skin (Fig. 2a), but only
when total reflectance was treated as a dichotomous measure and not when analyzed as a
rank measure. Further testing is necessary to determine the magnitude of this relationship
and whether it is linear. Males with a higher total reflectance also had less aggregate
pumiliotoxin, which is considered one of the major classes of toxic alkaloids in the genus
(Daly et al. 2005). PTX 307A, a pumiliotoxin that we found negatively correlates with total
reflectance but not other measures of conspicuousness (Table 2), is one of the few noxious
alkaloids found in poison frogs that have been assessed for toxicity. Lethal dosage (LD50)
values for this alkaloid are 50 lg/mouse, indicating high toxicity (Daly et al. 2005).
Interestingly, we also detected correlations between the proportions of the most com-
mon alkaloids (pumiliotoxins and tricyclics) and the most common skin carotenoids (beta-
carotene and xanthophylls) found within these males. Correlations among chemical
defenses and pigments have also been observed in ladybirds, where elytral carotenoids
correlate negatively with some alkaloids and positively with others, sometimes in a sex-
dependent manner (Blount et al. 2012). There has been some speculation that the alkaloid
sequestration and chemical modification performed by chemically defended species is
oxidatively costly (Ahmad 1992; Blount et al. 2009). For example, there may be a
molecular tradeoff where carotenoids can be allocated to buffer the damaging effects of
alkaloid sequestration/modification or allocated to the integument as a visual signal. The
negative correlations we observe between particular carotenoids and alkaloids (Fig. 3b, c)
provide indirect support for this hypothesis, even though carotenoid quantity and alkaloid
quantity did not correlate.
There has been much disagreement over whether warning signals should be quantita-
tively honest, with a relatively tight correlation between conspicuousness and toxicity, or
qualitatively honest, where the presence of a signal, regardless of its magnitude, suffi-
ciently advertises secondary defense (Blount et al. 2009; Lee et al. 2011; Speed and Ruxton
2007; Speed et al. 2010). Theoretical investigations have predicted both negative and
positive relationships between toxicity and conspicuousness (Blount et al. 2009; Holen and
Svennungsen 2012; Speed and Ruxton 2007). The few empirical studies that have
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investigated the relationship between toxicity and conspicuousness have yielded seemingly
conflicting results, both interspecifically (e.g., Cortesi and Cheney 2010; Darst et al. 2006;
Summers and Clough 2001; reviewed in Summers et al. 2015) and intraspecifically
(Bezzerides et al. 2007; Daly and Myers 1967; Maan and Cummings 2012; reviewed in
Speed et al. 2012). Furthermore, it appears that the relationship between toxicity and color
can be relatively stochastic across time (as suggested by Daly et al. 2002; Saporito et al.
Here we found that while there is a positive relationship between measures of bright-
ness/conspicuousness and toxicity across phenotypically distinct O. pumilio populations
(Maan and Cummings 2012), there is evidently a weak but negative relationship between
these traits within the Solarte population. Our results may be understood in the context of
Holen and Svennungen’s resource allocation tradeoff hypothesis (2012), which explains
that aposematic signals can function as handicaps, where a limited resource (e.g., pig-
ments) is used both to buffer the costs of chemical defense and as a component of the
visual display. A key prediction of this hypothesis is that there will not necessarily be a
perfect correspondence between physiological state and signal magnitude as long as there
is enough information for predators to respond appropriately. The across- and within-
population relationships between toxicity and coloration in O. pumilio fit with these pre-
dictions; across the archipelago, population-level conspicuousness positively correlates
with measures of toxicity, while within the exceptionally conspicuous and exceptionally
toxic Solarte population a tight correspondence between conspicuousness and chemical
defense may not be strategically beneficial. However, it is important to note a caveat when
interpreting our results: our inter- and intra-population assessments of ‘toxicity’ differ
significantly. For the inter-population study (Maan and Cummings 2012), the researchers
used a measure of noxiousness or irritability (assays using skin extracts injected into
sleeping mice), whereas in this intra-population study we assess toxicity based on alkaloid
identification and quantification and assume that having greater aggregate pumiliotoxin
imparts greater noxiousness/toxicity. Additionally, although pumiliotoxins are commonly
considered the important toxic class of alkaloids in this genus, it is not known which
particular alkaloids may be most important in chemical defense in O. pumilio, and whether
that differs depending upon the predator type.
There are several possible explanations for our finding that males with higher total
reflectance have less alkaloid diversity and aggregate pumiliotoxin in their skin. Brighter
males may be sampled more frequently by predators, and thus expel their toxins more
frequently, because they differ behaviorally or visually from duller males (Crothers et al.
2011; Crothers and Cummings 2013). Furthermore, there may be a metabolic tradeoff
between brightness and toxicity (e.g., Blount et al. 2009), and/or a correlation between
brightness, toxicity, and age. Finally, a strategic trade-off between conspicuousness and
toxicity, whereby individuals of a population can gain protection from predators through
investing in either toxicity or conspicuousness, is a pattern that has been observed among
poison frog species (Darst et al. 2006) but has not yet been identified within a species.
Because male warning signal reflectance exhibits a negative relationship with diet-de-
pendent skin alkaloids, it may function as a reliable indicator of territory quality or current
condition. Past findings that females prefer brighter males (Maan and Cummings 2009) are
thus somewhat surprising in this context, since females may prefer a more conspicuous but
physiologically compromised male phenotype within this population. However, further
investigations will need to be performed to see whether brighter males do indeed have
higher mating success in this population. It should also be noted that the patterns we find in
the highly conspicuous and toxic Solarte population may not be observed in other
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populations of this species. Sexual preferences and sexual signaling differ across the
populations of the archipelago (e.g., Dreher and Pro
¨hl 2014; Maan and Cummings 2008),
with some of these differences appearing to be explained by a population’s average level of
chemical defense and/or conspicuousness (Rudh et al. 2011,2013).
Cummings and Crothers (2013) argued that predators may impose a selective regime in
this species whereby, (1) populations above a toxicity–brightness threshold are at liberty to
diversify via sexual selection and below which populations are constrained to maintain a
stricter resemblance to a more cryptic population mean, and (2) synergistic/additive effects
of inter- and intrasexual selection could drive the evolution of brighter males within
populations above the toxicity threshold. Thus we would expect in populations of low
average toxicity that individuals deviating from the average aposematic signal of the
population would be more vulnerable to detection by predators. However, in populations
that are strongly defended, such as Solarte, predators may largely be tolerant to signal
variation due to predator generalization (Darst et al. 2006) or perceptual limitations
(Crothers and Cummings 2013). Predator permissiveness to signal variation in Solarte,
whether driven by predator generalization or the inability of predators to detect the vari-
ation, would result in the pattern we observe here—namely, the lack of relationship
between conspicuousness and chemical defense.
Intriguingly, brighter males were caught calling at higher locations within their terri-
tories (Fig. 4). Kime et al. (2000) found that frog call recordings played at higher locations
in the forest experience less degradation than calls played at ground level. The bright males
perched at higher locations may therefore be able to project their advertisement calls across
larger distances and communicate to a greater number of potential mates. Our within-
population findings also fit the across-population findings of Rudh et al. (2011) and Rudh
et al. (2013), where males of more conspicuous O. pumilio populations were found to
inhabit more exposed calling sites and to be more aggressive and exhibit more explorative
behavior. However, using higher perches likely comes with trade-offs. Higher males may
have less dietary access to alkaloid-containing arthropods found in the leaf litter or may be
sampled more frequently by avian predators—which are believed to be the major predator
of this species (Dreher et al. 2015; Hegna et al. 2011; Maan and Cummings 2012; Saporito
et al. 2007a,b)—and thus expel their accumulated toxins more frequently. A recent clay-
model predation study by Dreher et al. (2015) found that avian predation rates are espe-
cially high in this population compared to others of the archipelago. This could mean that
brighter males’ reduced toxicity may indeed be based on more frequent predator sampling
because they are more exposed to birds by virtue of their higher perch heights. Alterna-
tively, our past theoretical investigations with a model avian visual system revealed that
birds are unlikely to discriminate much of the brightness variation found within the Solarte
population, implying that there may be little fitness tradeoff for brighter, less toxic males in
terms of predation (Crothers and Cummings 2013).
A benefit of investigating signal evolution in O. pumilio is that it is evident that the
processes of selection are all impacting the same quantifiable trait: aposematic coloration.
In the highly conspicuous and toxic Solarte population, warning signal brightness has
seemingly been co-opted as an agonistic indicator trait (Crothers and Cummings 2015).
Many status-signaling models require that the signals be strategically costly in order to be
evolutionarily stable (Berglund et al. 1996; Maynard Smith and Harper 2003). Sexually
selected brightness in this population has previously been shown to correlate with body
temperature and a call characteristic that contributes to mating success (Crothers et al.
2011), and there also appears to be a tradeoff between long-wave chroma (*redness) and
measures of brightness in some orange/red taxa (e.g., Grether 2000), including this
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population (Crothers and Cummings 2013; Maan and Cummings 2009). Brightness,
however, does not appear to correlate with several classic measures of body condition,
such as length-mass residuals (Crothers et al. 2011), circulating testosterone or aggregate
amounts of pigments acquired from the diet. Instead, the costs of color expression may be
imparted through a physiological measure unique to chemically defended species: alka-
loids derived from the diet and sequestered in the skin. Future investigations may elucidate
the physiological or strategic trade-offs driving the relationships we describe here.
Acknowledgments This work complied with ANAM SE/A-112-08, SE/A-27-09, SEX/A-58-09, SE/A-30-
11 and SE/A-36-12 permits, and UT 07092101 and AUP-2010-00139, Tulane 0382R and STRI 2008-03-12-
05-2008 IACUC protocols. We thank Christina Buelow, Victoria Flores, Sara Mason and Anna Deasey for
their exceptional field help, and Clyde and Wilson Stephens for the generous use of their property over the
years. Special thanks to Hans Hofmann, Daniel Bolnick, Michael Ryan, Kyle Summers, William Wcislo,
John Christy, and David Cannatella for their advice on experimental protocols. Finally, we thank two
anonymous reviewers and the editors for helpful comments on previous versions of this manuscript. L.C.
was supported by a UT EEB grant, NSF DDIG #IOS 1110503, a Smithsonian Tropical Research Institute A.
Stanley Rand fellowship, an Animal Behavior Society Barlow Student Research Award, and an American
Association of University Women fellowship. L.C. and M.C. were supported by a National Geographic
Society Committee for Research and Exploration grant.
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... Another major question in aposematic evolution ---besides the contributions of natural and sexual selection ---regards violations of a key theoretical prediction. Aposematic organisms rely on detection by predators and subsequent predator education; otherwise, why possess such an energetically costly (i.e., requiring sequestration or production of color pigments [Crothers et al., 2016]) and visually obvious trait? Therefore, positive, frequency-dependent selection, whereby the fitness of a phenotype increases with its frequency, is expected in aposematic organisms. ...
... There is dissent over whether warning signals should be quantitatively honest, with a relatively tight correlation between conspicuousness and toxicity, or qualitatively honest, where the presence rather than magnitude of the aposematic signal advertises secondary defenses (Ruxton et al., 2018). Theoretical studies have predicted both negative and positive relationships between unpalatability and conspicuousness, and empirical studies are also conflicting, both intra-and interspecifically (Crothers et al., 2016;Ruxton et al., 2018). ...
... An explanation is higher signal efficacy and salience: evolution is expected to favor mate preferences for signals that stand out relative to the background (salient), because these signals take less energy to identify (efficacious). Perch calling as a more obvious and visible signal than calling from concealed location is salient and efficacious, and thus preferred (Rudh et al., 2011(Rudh et al., , 2013Crothers et al., 2016). ...
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This brief review applies classical theory in animal evolution and behavior to ask why the best-studied poison frog species Oophaga pumilio (Dendrobatidae) displays dramatic color variation among island populations of the Bocas del Toro archipelago, situated off the northwestern coast of Panama. The aims of the paper are to: (1) simplify a vast literature by narrowing down myriad explanations for color evolution in O. pumilio to an organized synthesis of evolutionary processes (neutral evolution, natural selection, and sexual selection) that integrates proximate (mechanistic) and ultimate (concerned with the broad fitness consequences) phenomena; and (2) to explore recent work on O. pumilio that may supply insight into several important questions about the evolution of aposematism. Aposematism is the use of conspicuous signals paired with some form of unprofitability or unpalatability to ward off predators. These questions concern the extent to which the three aforementioned evolutionary processes drive diversity in aposematic signals; how the selective regime expected to be at play in aposematic species may, contrary to expectation, allow for diversity in aposematic color; and whether aposematic signals are expected to be reliable indicators of unpalatability.
... The first includes those studies that directly measure the concentration, quantity or diversity of specific compounds which invoke aversive responses in predators, such as alkaloids (e.g. [21,38,39]). The second general approach is to use bioassays in which proxy organisms are exposed to the relevant compounds or tissues containing them, such as skin extracts. ...
... Our final analysis included 127 effect sizes from 24 studies[21][22][23][26][27][28][29][30][31][32][38][39][40][46][47][48][49][50][51][52][53][54][55][56]. Most studies reported multiple effects, with a mean of 6 and range of 1-27. ...
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The combined use of noxious chemical defences and conspicuous warning colours is a ubiquitous anti-predator strategy. That such signals advertise the presence of defences is inherent to their function, but their predicted potential for quantitative honesty-the positive scaling of signal salience with the strength of protection-is the subject of enduring debate. Here, we systematically synthesized the available evidence to test this prediction using meta-analysis. We found evidence for a positive correlation between warning colour expression and the extent of chemical defences across taxa. Notably, this relationship held at all scales; among individuals, populations and species, though substantial between-study heterogeneity remains unexplained. Consideration of the design of signals revealed that all visual features, from colour to contrast, were equally informative of the extent of prey defence. Our results affirm a central prediction of honesty-based models of signal function and narrow the scope of possible mechanisms shaping the evolution of aposematism. They suggest diverse pathways to the encoding and exchange of information, while highlighting the need for deeper knowledge of the ecology of chemical defences to enrich our understanding of this widespread anti-predator adaptation.
... been putatively annotated (only to MS 1 level) recently on Phyllobates vitattus (Protti-Sánchez et al. 2019). The chemical analysis of non-alkaloid compounds, such as proteins (Caty et al. 2019;O'Connell et al. 2021;Alvarez-Buylla et al. 2022, 2023, peptides, volatile organic compounds (VOCs) , lipids, pigments (Crothers et al. 2016;Twomey et al. 2020b, a), among others, has been almost neglected in this superfamily. This was likely influenced by the initial focus on studying chemical defences in dendrobatoids, where alkaloids were expected to be the primary contributors. ...
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Amphibians are one of the most remarkable sources of unique natural products. Biogenic amines, peptides, bufodienolides, alkaloids, and volatile organic compounds have been characterized in different species. The superfamily Dendrobatoidea represents one of the most enigmatic cases of study in chemical ecology because their skin secretome is composed by a complex mixture (i.e. cocktail) of highly lethal and noxious unique alkaloid structures. While chemical defences from dendrobatoids (families Dendrobatidae and Aromobatidae) have been investigated employing ecological, behavioral, phylogenetic and evolutionary perspectives, studies about the analytical techniques needed to perform the chemical characterization have been neglected for many years. Therefore, our aim is to summarize the current methods applied for the characterization of chemical profiles in dendrobatoids and to illustrate innovative Eco-metabolomics strategies that could be translated to this study model. This approach could be extended to natural products other than alkaloids and implemented for the chemical analysis of different species of dendrobatoids employing both low- and high-resolution mass spectrometers. Here, we overview important biological features to be considered, procedures that could be applied to perform the chemical characterization, steps and tools to perform an Eco-metabolomic analysis, and a final discussion about future perspectives.
... Four phenotypically distinct populations were chosen encompassing a continuum of conspicuousness (to human viewers) from dull (Aguacate Peninsula, dark blue), intermediate (Isla Colon, yellow/green with black spots and light blue legs) and more highly contrasting (Solarte, orange; Almirante, red with blue legs) aposematic phenotypes. Alkaloids were extracted following Crothers et al. (2016), for full specifics see Yeager (2015). Our work utilizes a reduced version of that published dataset, composed of the 17 most abundant alkaloids reported in that study which represents approximately 73% of the total quantity of chemical defenses across populations (Fig. 1). ...
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Studies of the evolution of anti-predatory phenotypes such as aposematic signals have proven informative to demonstrate the role of selection on phenotypic divergence. Oophaga pumilio show high variance in both elements of their aposematic signals; visual cues consisting of color patterns, as well as their alkaloid chemical defenses where an individual frog can possess dozens of alkaloid types. Disentangling the function of individual alkaloids is therefore complicated due to various modes of chemical defenses, making simple comparisons between levels of relative ‘toxicity’ between populations difficult until we can elucidate the defensive capabilities of alkaloids. In this study we model binding affinity of the most abundant alkaloids found in divergent populations of O. pumilio which we discovered had a high affinity for binding with Muscarinic acetylcholine receptors in various potential predator classes. Molecular interaction and docking experiments indicate that interactions between alkaloid and muscarinic receptors are highly conserved, and muscarinic receptors themselves show evidence of strong purifying selection. Therefore, we predict functional redundancy is plausible among the most common alkaloids against common targets, and these alkaloids likely function similarly across diverse suites of predators. This affords a predictable baseline of defenses for this combination of alkaloids and receptors between divergent populations which vary in aposematic signals.
... Second, it can take > 9 months for newts to regenerate TTX in laboratory experiments (Cardall et al., 2004). If precursors to TTX are foraged from the environment, then variation in these resources might decouple the signal (Kodric-Brown, 1989;Mochida et al., 2013;Crothers et al., 2016;Casas-Cardonas et al., 2018). However, previous experiments have established that newts are able to maintain and regenerate TTX in laboratory conditions (Hanifin et al., 2002;Gall et al., 2022), suggesting that TTX is not a resource obtained from the diet. ...
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Colourful displays are used by diverse taxa to warn predators of dangerous defences (aposematism). Aposematic coloration is especially widespread among amphibians, which are often protected by harmful toxins. Pacific newts (Taricha) are considered a model of aposematism because when threatened, they arch the head and tail upwards to expose a vivid orange ventrum against a dark dorsum. Given that newts are defended by tetrodotoxin (TTX), a lethal neurotoxin, this signal is assumed to warn predators that an attack would be risky. However, colours have not been quantified in Taricha, and it remains unknown whether coloration provides qualitatively honest (signalling toxic defence) or quantitatively honest (signalling toxin level) warnings. We used two colour quantification methods (spectrometry and hyperspectral imaging) to measure chromatic (hue) and achromatic (brightness) qualities of ventral and dorsal coloration in two newt species (Taricha granulosa and Taricha sierrae). We assessed qualitative honesty using visual models of potential predators (snakes, birds and mammals). Next, we evaluated quantitative honesty by measuring TTX in newts and examining the potential correlation between defence level (amount of TTX) and colorimetrics. We found support for qualitative but not quantitative honesty. Selective pressures and evolutionary constraints might impede the evolution of honest quantitative signalling in this system.
... KEGG enrichment analysis showed significant enrichment in the cytochrome P450 family, CYP3A4, CYP3A5, and the cytochrome P450 pathway was also found to be enriched for differences in yellowness in skin RNA-seq results (Wu et al., 2021), and cytochrome P3A80 has been shown to be a strong candidate for carotenoid ketolase in amphibians (Twomey et al., 2020). CYP2J19 was found to convert yellow carotenoids to red carotenoids in birds (Kirschel et al., 2020), while birds may convert lutein to zeaxanthin (Crothers et al., 2016). Therefore, cytochrome P450related DEGs may be candidates for regulating skin yellowness in chickens. ...
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The yellow color of the skin is an important economic trait for yellow chickens. Low and non-uniform skin yellowness would reduce economic efficiency. However, the regulatory mechanism of chicken skin yellowness has not been fully elucidated. In this study, we evaluated the skin yellowness of 819 chickens by colorimeter and digital camera, which are from the same batch and the same age of 2 pure lines with significant differences in skin yellowness. A total of 982 candidate differential expressed genes (DEGs) were detected in duodenal tissue by RNA-seq analysis for high and low yellowness chickens. Among the DEGs, we chose fatty acid translocase (CD36) gene and identified a single nucleotide polymorphism (SNP) upstream of the CD36 gene that was significantly associated with skin yellowness at multiple parts of the chicken, and its different genotypes had significant effects on the promoter activity of the CD36 gene. These findings will help to further elucidate the molecular mechanism of chicken skin yellowness and is helpful for improving chicken skin yellowness.
... Intriguingly, Crothers et al. (2016) found no relationship between coloration and overall carotenoid abundance in the bright orange Solarte morph of O. pumilio, but did find a relationship between total dorsal reflectance and two carotenoids (xanthophyll and a canary xanthophyll ester). Thus, the evidence seems to indicate that overall carotenoid concentrations are not particularly important for red, orange or yellow colorations, and that instead these colours may be driven by a small subset of carotenoids or pteridines. ...
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A common goal in evolutionary biology is to discern the mechanisms that produce the astounding diversity of morphologies seen across the tree of life. Aposematic species, those with a conspicuous phenotype coupled with some form of defence, are excellent models to understand the link between vivid colour pattern variations, the natural selection shaping it, and the underlying genetic mechanisms underpinning this variation. Mimicry systems in which multiple species share the same conspicuous phenotype can provide an even better model for understanding the mechanisms of colour production in aposematic species, especially if comimics have divergent evolutionary histories. Here we investigate the genetic mechanisms by which vivid colour and pattern are produced in a Müllerian mimicry complex of poison frogs. We did this by first assembling a high-quality de novo genome assembly for the mimic poison frog Ranitomeya imitator. This assembled genome is 6.8 Gbp in size, with a contig N50 of 300 Kbp R. imitator and two colour morphs from both Ranitomeya fantastica and R. variabilis which R. imitator mimics. We identified a large number of pigmentation and patterning genes that are differentially expressed throughout development, many of them related to melanocyte development, melanin synthesis, iridophore development and guanine synthesis. Polytypic differences within species may be the result of differences in expression and/or timing of expression, whereas convergence for colour pattern between species does not appear to be due to the same changes in gene expression. In addition, we identify the pteridine synthesis pathway (including genes such as qdpr and xdh) as a key driver of the variation in colour between morphs of these species. Finally, we hypothesize that genes in the keratin family are important for producing different structural colours within these frogs.
... It is important to also note that the perception of color contrast is not the only factor that may change in human modified environments. Poison frogs rely on dietary derived carotenoid pigments and chemical precursors to synthesize the colors and toxins underlying their aposematic defenses (Saporito et al. 2012;Crothers et al. 2016). In disturbed habitat, frog diets, color, and alkaloid composition may change along with the availability of their prey (McGugan et al. 2016;Moskowitz et al. 2020;Yeager et al. ...
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Animals use color both to conceal and signal their presence, with patterns that match the background, disrupt shape recognition, or highlight features important for communication. The forms that these color patterns take are responses to the visual systems that observe them and the environments within which they are viewed. Increasingly, however, these environments are being affected by human activity. We studied how pattern characteristics and habitat change may affect the detectability of three frog color patterns from the Bocas del Toro archipelago in Panama: Beige-Striped Brown Allobates talamancae and two spotted morphs of Oophaga pumilio, Black-Spotted Green and Black-Spotted Red. To assess detectability, we used visual modeling of conspecifics and potential predators, along with a computer-based detection experiment with human participants. Although we found no evidence for disruptive camouflage, we did find clear evidence that A. talamancae stripes are inherently more cryptic than O. pumilio spots regardless of color. We found no evidence that color pattern polytypism in O. pumilio is related to differences in the forest floor between natural sites. We did, however, find strong evidence that human disturbance affects the visual environment and modifies absolute and rank order frog detectability. Human-induced environmental change reduces the effectiveness of camouflage in A. talamancae, reduces detectability of Black-Spotted Green O. pumilio, and increases chromatic contrast, but not detectability, in Black-Spotted Red O. pumilio. Insofar as predators may learn about prey defenses and make foraging decisions based on relative prey availability and suitability, such changes may have wider implications for predator–prey dynamics.
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Many animals bear conspicuous warning signals that advertise toxin-mediated unpalatability to predators; this is known as aposematism. Frogs in particular have evolved aposematism repeatedly. These so-called “poison frogs” sequester a diverse array of alkaloid toxins into their skin from their diet of small arthropods. The frogs secrete the alkaloids when faced with the threat of predation. Poison frogs are therefore ideal for asking about how unpalatability predicts the evolution of aposematic signals. To evolve alkaloid defenses, poison frogs must withstand the adverse effects of the alkaloids. The mechanisms underlying this alkaloid resistance remain poorly understood. In this review, we synthesize literature to theorize about how alkaloid resistance and aposematism might have evolved. We note goals for research on possible toxin resistance modes in poison frogs (target-site insensitivity, compartmentalization, and detoxification). While recent work exploring the co-evolution of single alkaloids and proteins that support resistance is important, examining the myriad interactions between poison-frog alkaloids and resistance-conferring proteins is essential for a more complete understanding. We conclude by pointing out how answering proximate questions about the molecular basis of poison-frog alkaloid resistance will allow us to answer ultimate questions fundamental to ecology and evolutionary biology.
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Rapid adaptation to global change can counter vulnerability of species to population declines and extinction. Theoretically, under such circumstances both genetic variation and phenotypic plasticity can maintain population fitness, but empirical support for this is currently limited. Here, we aim to characterize the role of environmental and genetic diversity, and their prior evolutionary history (via haplogroup profiles) in shaping patterns of life history traits during biological invasion. Data were derived from both genetic and life history traits including a morphological analysis of 29 native and invasive populations of topmouth gudgeon Pseudorasbora parva coupled with climatic variables from each location. General additive models were constructed to explain distribution of somatic growth rate (SGR) data across native and invasive ranges, with model selection performed using Akaike's information criteria. Genetic and environmental drivers that structured the life history of populations in their native range were less influential in their invasive populations. For some vertebrates at least, fitness‐related trait shifts do not seem to be dependent on the level of genetic diversity or haplogroup makeup of the initial introduced propagule, nor of the availability of local environmental conditions being similar to those experienced in their native range. As long as local conditions are not beyond the species physiological threshold, its local establishment and invasive potential are likely to be determined by local drivers, such as density‐dependent effects linked to resource availability or to local biotic resistance.
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Sexual signals indicate species identity and mate quality, and their importance for mate attraction is largely recognized. Recently, research in animal communication has started to integrate multiple signal modalities and evaluate their interactions. However, mate choice experiments across animal taxa have been limited to laboratory conditions, and assessments of multiple sexual signals under field conditions are still lacking. We take advantage of the divergence in visual and acoustic signals among populations of the Neotropical poison frog Oophaga pumilio to evaluate the importance of male advertisement calls and color patterns in female mate selection. Previous mate choice experiments in this species suggested color-assortative female mate preferences across many populations. Nevertheless, acoustic signals are crucial for sexual selection in frogs, and males of O. pumilio use advertisement calls to attract females. We hypothesize that both advertisement calls and coloration affect female mate selection in O.pumilio. To test this hypothesis we tested 452 receptive females from six populations in Costa Rica and Panama in their natural home ranges for preferences regarding local vs. non-local advertisement calls and color patterns. Overall, the calls overrode the effect of coloration, whereby most females preferred local over non-local calls. We found a tendency to prefer brighter (but not necessarily local) males in two populations. Furthermore, the strength of preferences varied geographically, and thus might be involved in prezygotic isolation among populations. The stronger effect of calls on mate attraction is associated with acoustic divergence between genetic groups in the species, while color pattern diversity is mostly located within one genetic group, i.e., not linked to large-scale population structure. Finally our data highlights the importance to consider an array of signal modalities in multiple wild populations in studies of behavioral isolation.
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Natural selection is widely noted to drive divergence of phenotypic traits. Predation pressure can facilitate morphological divergence, for example the evolution of both cryptic and conspicuous coloration in animals. In this context Dendrobatid frogs have been used to study evolutionary forces inducing diversity in protective coloration. The polytypic strawberry poison frog (Oophaga pumilio) shows strong divergence in aposematic coloration among populations. To investigate whether predation pressure is important for color divergence among populations of O. pumilio we selected four mainland populations and two island populations from Costa Rica and Panama. Spectrometric measurements of body coloration were used to calculate color and brightness contrasts of frogs as an indicator of conspicuousness for the visual systems of several potential predators (avian, crab and snake) and a conspecific observer. Additionally, we conducted experiments using clay model frogs of different coloration to investigate whether the local coloration of frogs is better protected than non-local color morphs, and if predator communities vary among populations. Overall predation risk differed strongly among populations and interestingly was higher on the two island populations. Imprints on clay models indicated that birds are the main predators while attacks of other predators were rare. Furthermore, clay models of local coloration were equally likely to be attacked as those of non-local coloration. Overall conspicuousness (and brightness contrast) of local frogs was positively correlated with attack rates by birds across populations. Together with results from earlier studies we conclude that conspicuousness honestly indicates toxicity to avian predators. The different coloration patterns among populations of strawberry poison frogs in combination with behavior and toxicity might integrate into equally efficient anti-predator strategies depending on local predation and other ecological factors.
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We explore the relevance of honest signalling theory to the evolution of aposematism. We begin with a general consideration of models of signal stability, with a focus on the Zahavian costly-signalling framework. Next, we review early models of signalling in the context of aposematism (some that are consistent and some inconsistent with costly honest signalling). We focus on controversies surrounding the idea that aposematic signals are handicaps in a Zahavian framework. Then we discuss how the alignment of interests between signaller and predator influences the evolution of aposematism, highlight the distinction between qualitative and quantitative honesty, and review theory and research relevant to these categories. We also review recent theoretical treatments of the evolution of aposematism that have focused on honest signalling as well as empirical research on a variety of organisms, including invertebrates and frogs. Finally, we discuss future directions for empirical and theoretical research in this area. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
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Aposematic species use conspicuous “warning” signals to communicate unprofitability to potential predators. Although warning signals are classic examples of communication systems that evolved by natural selection, they can also function in the context of sexual communication and are therefore particularly useful for investigating conspicuous trait evolution under multifarious selection. To test whether aposematic signals also serve to mediate intrasexual disputes, we observed males from a highly territorial poison frog species (Oophaga pumilio) in their native territories and in experimental dyadic contests to assess the influences of body characteristics such as warning signal brightness and body size on the outcomes of territorial interactions. We report here that although neither male size (snout-vent length) nor mass significantly predicted male aggressiveness (latency to call) in dyadic contests, a male’s dorsal brightness was a significant predictor of willingness to initiate aggressive interactions, with brighter males exhibiting a shorter latency to call than duller males. Furthermore, brightness asymmetries between males predicted the outcomes of contests such that asymmetries were smaller in escalated aggression trials (where both males called), and brighter males were more likely to be the sole aggressor in trials with large asymmetries. These tests, combined with previous work, provide evidence that warning coloration has been co-opted as an agonistic indicator trait in this aposematic amphibian and reveal the potential evolutionary lability of conspicuous traits that arise through natural selection.