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.