DISCUSSION
We tested whether pteridine pigments compensate for environmental
carotenoid availability among agamid lizards, using a large
interspecific dataset of pigment concentrations in coloured skin tissue.
We found that the total concentration of carotenoids was positively
associated with habitat productivity, and therefore presumably
environmental carotenoid availability. Individuals in more productive
environments not only had higher concentrations of total carotenoids,
but they also had a lower concentration of total pteridines and
consequently, a higher ratio of carotenoids to pteridines. Across all
species, the concentrations of carotenoid and pteridine pigments with
similar hue (red ketocarotenoids and drosopterin, yellow dietary
carotenoids and xanthopterin), were uncorrelated, although there was a
weak negative association between red ketocarotenoid and yellow
xanthopterin concentrations. This indicates that variation in carotenoid
availability is not compensated directly by replacing carotenoids with
pteridine pigments of the same hue (carotenoid mimicry). Instead,
compensation appears to be indirect. In environments where carotenoids
are scarce, pteridines are used in relatively higher concentrations but
different combinations of pteridine and carotenoid pigments are used to
produce different hues. Pigment concentrations correspond to variation
in yellow-red skin colours among species, and this was primarily driven
by pteridines: redder hues were associated with higher concentrations of
drosopterin, and more saturated colours were associated with higher
concentration of pteridines (xanthopterin, colourless and total). We
found no relationship between carotenoid or pteridine concentrations and
indices of sexual selection (sexual dichromatism and sexual size
dimorphism). This is consistent with the lack of association between
carotenoid concentration and skin colour and the hypothesised low
metabolic cost of pteridine synthesis (Bagnara & Matsumoto 2006). Taken
together, these results suggest that costs associated with the
production and allocation of pigments to skin colour are unlikely to
maintain colour signal honesty in agamid lizards.
In reptiles, yellow-red skin coloration can be produced through diverse
mechanisms and pigment compositions. Yellow, orange and red hues can all
be produced exclusively by carotenoids (Fitze et al. 2009) or
pteridines (snakes; Kikuchi & Pfennig 2012; Kikuchi et al.2014). Furthermore, red hues can be produced by a higher proportion of
red ketocarotenoids relative to both dietary yellow carotenoids and to
pteridines (McLean et al. 2019), or exclusively by drosopterin
(Merkling et al. 2018). In the majority of lizards, however, both
carotenoids and pteridines contribute to colour variation within and
between species, with yellow produced by relatively higher
concentrations of dietary carotenoids and orange-red produced by a high
relative proportion of red pteridines (usually drosopterin; Ortizet al. 1963; Ortiz & Maldonado 1966; Macedonia et al.2000; Steffen & McGraw 2009; Weiss et al. 2012; Haisten et
al. 2015; McLean et al. 2017; Andrade et al. 2019).
Although carotenoids contribute to skin coloration, carotenoid
concentrations are often uncorrelated with hue, saturation or luminance
(Steffen et al. 2010; Weiss et al. 2012). Instead, hue
frequently corresponds to the concentration of red pteridines,
particularly drosopterin (Steffen et al. 2010; Weiss et
al. 2012; Andrade et al. 2019). We found similar patterns among
the 28 taxa in our dataset: skin colour was associated with the
concentration of pteridines rather than carotenoids and there was no
correlation between the two. Thus, yellow-red ornamentation in agamid
lizards is not an indicator of carotenoid content.
Carotenoids vary in their dietary availability within and between
species; however, this does not necessarily mean that carotenoid
availability is limiting for integument coloration. Available
carotenoids may be sufficient to meet physiological and colour
signalling requirements (Koch & Hill 2018). Furthermore, environmental
availability can be compensated by more efficient carotenoid metabolism
(e.g. assimilation and transport; Craig & Foote 2001; Koch & Hill
2018). Indeed, the prevailing view is that carotenoid limitation, where
it exists, is due more to physiology (internal factors) than
environmental availability (McGraw et al. 2003; Hadfield & Owens
2006; Simons et al. 2014; Koch & Hill 2018). Accordingly, there
is limited and inconsistent evidence for an association between
carotenoid concentrations in the integument and diet, at least for birds
(Mahler et al. 2003; McGraw et al. 2003; Olson & Owens
2005). By contrast, we found that in agamid lizards, total carotenoid
concentrations in coloured skin tissue were associated with habitat
productivity. All species of agamid lizard in this study are
insectivorous, though some occasionally eat plant material including
yellow flowers (Cogger 2018; Melville 2019). Most species occupy
semi-arid to arid environments, often with little vegetation, suggesting
that environmental carotenoid availability may well be limiting in this
clade.
Concentrations of ketocarotenoids were generally low (particularly
astaxanthin) relative to other carotenoids. Although ketocarotenoids are
common in red integument tissue in vertebrates and may be obtained from
dietary sources, they are rare in the diets of most terrestrial
vertebrates. Despite being rare in the diet, in some species,
ketocarotenoids can accumulate when enzymes responsible for carotenoid
breakdown, such as the β-carotene oxygenase enzymes BCMO1 and BCO2, are
disrupted or deactivated (Twomey et al. 2020a). More commonly,
ketocarotenoids are metabolically converted from dietary yellow
xanthophylls through oxidation reactions catalysed by ketolation enzymes
(ketolases; Lopes et al. 2016; Mundy et al. 2016; Twymanet al. 2016). Metabolic conversion of dietary yellow xanthophylls
to red ketocarotenoids has not been demonstrated in lizards, and the
CYP2J19 gene that encodes the primary ketolase in birds and turtles is
absent in squamates, tuataras and crocodilians (Twyman et al.2016). A similar P450 enzyme (encoded by the gene CYP3A80) may act as a
ketolase in the dendrobatid poison frog Ranitomeya sirensis and
possibly other amphibians (Twomey et al. 2020a) but whether this
may be the case in reptiles is not currently known. In this species of
frog, the carotenoid cleavage enzyme BCO2 is also disrupted, possibly
facilitating accumulation of ketocarotenoids and their dietary
precursors (Twomey et al. 2020a). BCO2 is associated with yellow
coloration in the wall lizard, but not other polymorphic lacertids
(Andrade et al. 2019). Therefore, it is unclear whether agamid
lizards have evolved mechanisms to enhance assimilation or enable
conversion of dietary carotenoids to ketocarotenoids. The positive
association we identified between the concentration of dietary
carotenoids and ketocarotenoids could indicate increased ketocarotenoid
conversion when dietary carotenoid availability is high, or that
ketocarotenoids are similarly more available through diet. An absence of
a mechanisms for ketocarotenoid conversion may explain the prevalence of
drosopterin to produce orange and red hues in lizards and some other
groups of poikilothermic vertebrates.
We found that the ratio of carotenoids to pteridines was higher in more
productive environments and concentrations of total pteridines were
lower in habitats with higher productivity. Variation in pteridine
synthesis is likely to have a genetic basis. This is the case among
populations of guppies in which genetic differences in pteridine
synthesis among populations compensate for environmental carotenoid
availability (Grether et al. 2005). Furthermore, the positive
correlation of carotenoid and drosopterin concentrations in guppies is
driven by female preference for a specific orange hue (Deere et
al. 2012). We found that higher concentrations of red ketocarotenoids
were associated with lower concentrations of yellow xanthopterin in
agamid lizards, which further indicates compensation, though not
carotenoid mimicry. This may be due to selection for specific hues,
particularly in species in which ketocarotenoids contribute to
integument coloration.
Variation in the ratio of carotenoids to pteridines in association with
habitat productivity among agamid lizards may reflect variation in
sexual or natural selection in different environments. Sexual selection
can vary in relation to environmental gradients via, for example,
environmental effects on population density (Littleford-Colquhounet al. 2019). However, we found no relationship between pigment
concentrations and indices of sexual selection, apart from a weak trend
for increased total carotenoids with higher sexual dimorphism. By
contrast, we found that species with darker colours (lower luminance)
were more likely to be found in more productive or vegetated
environments. This pattern has been documented in birds and butterflies
and may enhance camouflage (Dalrymple et al. 2018). More
saturated and darker colours were associated with a higher concentration
of colourless pteridines. Thus, one possibility is that variation in
pteridine synthesis among species partly reflects variation in selection
to minimise predation risk in different environments, leading to lower
pteridine concentrations in productive habitats.
Our comparative analysis uncovered broad patterns in pigment
concentration; however, mechanisms underlying skin colour in reptiles
are complex and influenced by structural components. In ectothermic
vertebrates, colour is produced by the combination of chromatophore
cells containing different pigment types or crystalline structures and
structural components of the dermis (e.g. collagen and connective
tissue). Xanthophores containing yellow to red carotenoid and/or
pteridine pigments comprise the upper layer of chromatophores and may be
underlain by iridophores containing periodically arranged guanine
crystals, and melanophores containing melanin pigments (reviewed in
Grether et al. 2004; Bagnara & Matsumoto 2006; Olsson et
al. 2013; Ligon & McCartney 2016). The extraordinary diversity of
integument colours in reptiles and other animals is produced by the
interaction of pigments and structural components, or by structural
colour alone, but never by pigments alone (Kemp et al. 2012). For
example, within a mimicry complex of poison frogs (Dendrobatidae), model
species and different morphs of the mimic species use different
combinations of dietary and metabolically converted carotenoids (Twomeyet al. 2020b). In these species, drosopterin contributes to
orange coloration but variation in hue across the group is predominantly
associated with the thickness of platelets within iridophores (i.e.
structural; Twomey et al. 2020b). Furthermore, skin tissue
commonly contains high concentrations of colourless pteridines such as
isoxanthopterin, pterin and biopterin (Bagnara & Matsumoto 2006; McLeanet al. 2017; McLean et al. 2019; Twomey et al.2020b). We found an association between the concentration of colourless
pteridines and skin colour saturation and luminance. However, it is not
clear whether or how colourless pteridines contribute to skin coloration
(e.g. light scattering). For example, isoxanthopterin (a pteridine
analog of guanine that forms the crystalline platelets in iridophores)
forms crystalline structures that act as reflectors in the eyes of
crustaceans (Palmer et al. 2018), but whether it contributes to
structural coloration in vertebrates is unknown. The role and
contribution of such colourless pteridines to integument coloration is a
fascinating area for future research.
Overall, our results support a scenario where limited carotenoid
availability in low productivity environments is compensated by higher
concentrations of pteridines. This has important implications for honest
colour signalling. A dominant paradigm is that costs associated with
carotenoid signalling maintain the honesty of yellow, orange and
particularly red colour signals; however, this paradigm derives largely
from literature on birds. Our results suggest that expression of
yellow-red signalling colours in agamid lizards is unlikely to convey
information on individual quality related to carotenoid acquisition or
allocation. Instead, the honesty of these colour signals may be
maintained by other costs such as predation risk associated with
conspicuous coloration. Our study
suggests that the paradigm of honest carotenoid signalling may not apply
broadly to other major vertebrate groups, such as reptiles, that use a
combination of pteridine and carotenoid pigments to generate yellow-red
hues and have complex colour generation mechanisms.