DISCUSSION
We tested whether pigment concentrations are associated with
environmental gradients indicative of carotenoid availability among
agamid lizards, using a large interspecific dataset of pigment
concentrations in coloured skin tissue. We found that species in more
arid environments with high summer temperatures and radiation and lower
vegetation productivity had lower concentrations of total carotenoids,
higher concentrations of total pteridines and consequently, a lower
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, indicating that carotenoids are not simply replaced with
pteridine pigments of a similar hue (carotenoid mimicry). Although the
concentration of dietary carotenoids was of similar magnitude to the
concentration of drosopterin or xanthopterin, only pteridine
concentrations predicted colour variation among species: redder hues
were associated with higher concentrations of drosopterin, and more
saturated colours were associated with higher concentration of
pteridines (xanthopterin, other and total). We found no relationship
between carotenoid or pteridine concentrations and indices of sexual
selection (sexual dichromatism and sexual size dimorphism), which is
consistent with the lack of association between carotenoid concentration
and skin colour. Taken together, these results suggest that
environmental carotenoid availability may alter the relative cost of
acquiring and sequestering carotenoids vs synthesising pteridines to
generate yellow-red skin colours.
In a series of pioneering studies, Grether and colleagues showed that
genetically determined pteridine synthesis can compensate for
environmental carotenoid availability among populations of guppies
(Grether et al. 1999; Grether et al. 2001; Gretheret al. 2005). In this species, carotenoid and pteridine
concentrations are positively correlated among populations to maintain a
consistent ratio of tunaxanthin (yellow carotenoid) to drosopterin (red
pteridine). This ratio produces the specific orange hue preferred by
females (Grether et al. 1999; Grether et al. 2001; Deereet al. 2012). Stabilising selection acting on hue within guppies
can explain the positive correlation between pigment types across
streams. Across lizard species, however, hue varies greatly, and
stabilising selection would not be expected as different species use
different signalling colours. Our results instead suggest that pteridine
synthesis balances geographic variation in carotenoid availability,
irrespective of hue. In carotenoid-scarce environments, it may be less
costly to synthesise pteridines than to acquire and metabolise
carotenoids and vice versa in carotenoid-rich environments.
However, the specific combination of pigments and skin colour pattern of
each species is likely to depend on local selective pressures.
The strongest drivers of the association between total carotenoid
concentrations in coloured skin and environmental PC1 were aridity and
summer radiation. Most species of agamid lizards occupy semi-arid to
arid environments, often with very little vegetation. All species of
agamid lizard in this study are insectivorous, though some occasionally
eat plant material including flowers (Cogger 2018; Melville 2019).
Insects sequester carotenoids in proportion to their dietary
availability (Heath et al. 2013); thus carotenoid availability
may well be limited for both insects and their predators in arid
environments. Limited dietary availability of carotenoids, however, 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). This view is primarily derived from the literature on birds, in
which there is limited and inconsistent evidence for an association
between feather carotenoid concentrations and diet (Mahler et al.2003; McGraw et al. 2003; Olson & Owens 2005). However,
selection on carotenoid metabolism may differ greatly for birds compared
to poikilothermic vertebrates (fish, amphibians, reptiles) because birds
have different colour producing mechanisms and do not use pteridines to
colour feathers. Thus, carotenoid availability may well be limiting for
skin coloration in lizard species occupying arid environments.
We found that in agamid lizards, concentrations of ketocarotenoids were
generally low (particularly astaxanthin) relative to other carotenoids.
Astaxanthin is produced by a number of bacteria, fungi and algae, and
can also be found in large quantities in some red flower petals (Ohmiya
2011). Agamid lizards are known to seek out and eat flower petals so
could potentially obtain astaxanthin from the diet; however astaxanthin
and other ketocarotenoids are generally rare in the diet of terrestrial
animals (Svensson & Wong 2011; Heath et al. 2013; Koch & Hill
2018). Nevertheless, in some
species, ketocarotenoids from dietary sources can accumulate when
enzymes responsible for carotenoid breakdown, such as the β-carotene
oxygenase enzymes BCMO1 and BCO2, are disrupted or deactivated (Twomeyet 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; Twyman et 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 (Twomeyet 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.
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. In most other lizards, yellow is produced
by high relative concentrations of dietary carotenoids and orange-red is
produced by a high relative proportion of red pteridines (usually
drosopterin; Ortiz et 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; Weisset al. 2012; Andrade et al. 2019). Our data is consistent
with these studies and suggests that yellow-red coloration is seldom a
reliable indicator of carotenoid content in lizards. This suggests in
turn that expression of yellow-red signalling colours in lizards is
unlikely to convey information on individual quality through mechanisms
of honest carotenoid signaling such as resource trade-offs or indicator
mechanisms (Koch et al. 2017; Koch & Hill 2018). Instead, the
honesty of these colour signals may be maintained by other costs such as
predation risk associated with conspicuous coloration (Stuart-Foxet al. 2003; Amdekar & Thaker 2019). More generally, honest
carotenoid signalling may not apply to the many species of
poikilothermic vertebrates that use a combination of pteridine and
carotenoid pigments to generate yellow-red hues and have complex colour
generation mechanisms.
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 (Kemp et al.2012). For example, within a mimicry complex of poison frogs
(Dendrobatidae), drosopterin contributes to orange coloration but
variation in hue across the group is predominantly associated with the
thickness of crystalline platelets within iridophores (i.e. structural;
Twomey et al. 2020b). Furthermore, skin tissue commonly contains
high concentrations of crystalline pteridines such as isoxanthopterin
and pterin (Bagnara & Matsumoto 2006; McLean et al. 2017; McLeanet al. 2019; Twomey et al. 2020b). We found an association
between the concentration of these pteridines and skin colour saturation
and luminance. This represents novel evidence that pteridines that are
often assumed to be colourless are associated with variation in
integument coloration and may contribute to skin colour due to their
crystallinity rather than spectral absorption (Oliphant & Hudon 1993;
Palmer et al. 2018).
Overall, our results support a scenario where environmental carotenoid
availability influences the relative concentrations of carotenoid and
pteridine pigments used to generate yellow to red skin colours in
lizards. Environmental gradients may shape the ecology and evolution of
animal coloration by altering the relative cost of environmentally
acquired and self-synthesised pigments.