Pigment concentrations
To identify the specific metabolites responsible for yellow-red coloration, we extracted pigments in tissue samples (approx. 3 x 3 mm) from 1 to 5 body regions of each lizard depending on the colour pattern of the species (186 tissue samples in total). This included 150 tissue samples from body regions that had a component of yellow-red (i.e. including shades of brown) and 36 samples from body regions that were black, grey, white or cream. The pigment extraction protocol was based on McLean et al. (2017). In brief, we sequentially extracted carotenoids then pteridines: Samples were weighed and then homogenized in methanol:ethylacetate (6:4 v/v + 0.01% butylated hydroxy toluene) using a TissueLyser II system (with two 3mm tungsten-carbide beads; Qiagen, Hilden, Germany). The resulting carotenoid extract was collected following centrifugation and the extraction repeated once on the remaining tissue pellet. Pteridines were subsequently extracted from the remaining tissue pellet using 2% ammonium hydroxide in two serial extractions. Respective fractions were combined. Internal standards were added directly to respective extraction solvents. We quantified concentrations of 5 carotenoids (lutein/zeaxanthin, 3’-dehydrolutein, β-carotene, astaxanthin, canthaxanthin) and 6 pteridines (drosopterin, xanthopterin, pterin, 6-biopterin, isoxanthopterin, pterine-6-carboxylic acid) in two separate liquid chromatography-mass spectrometry analyses on an Agilent 6490 triple quadrupole MS system with a Jet Stream electrospray ionization source coupled to an Agilent 1290 series LC system (Agilent Technologies Inc, Santa Clara, CA). The yellow carotenoid β-cryptoxanthin and very low levels of the yellow pteridine sepiapterin have also been identified in skin tissue of agamid lizards (McLean et al. 2017; McLean et al. 2019); however runs for these pigments were inconsistent, so they were not analyzed further.
Data were analysed using Agilent MassHunter Workstation Software (version B.07.00). All peak assignments were matched against commercial or purified standards, confirmed with a qualifier ion and quantified against the linear range from six-point calibration curves (all R2 > 0.95). Final concentrations were normalized against tissue weight: i.e. ‘pigment per gram of tissue’ referred to as “concentration” throughout for brevity. Commercial standards were used for all metabolites except drosopterin, which we extracted and purified from fruit flies, Drosophila melanogaster(per Wilson & Jacobson 1977). Peaks for lutein and zeaxanthin could not be reliably distinguished chromatographically so they were combined into a single category). For drosopterin we use the relative response because we lacked a commercial standard, thus, we refer to this as “level”. For subsequent analyses, we calculated the total concentration of carotenoids and pteridines, as well as the concentration of 5 subcategories: dietary yellow-orange carotenoids (lutein/zeaxanthin, 3’-dehydrolutein, β-carotene), red ketocarotenoids (astaxanthin, canthaxanthin), yellow pteridines (xanthopterin), red pteridines (drosopterin) and colourless pteridines (pterin, 6-biopterin, isoxanthopterin, pterine-6-carboxylic acid).