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).