Pigment concentrations
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. We used a sequential carotenoid and pteridine pigment
extraction procedure which was previously developed for lizard skin
(McLean et al. 2017, full details in Supplementary Information).
In brief, samples were weighed and homogenised in methanol:ethylacetate
using a TissueLyser II system (with two 3mm tungsten-carbide beads;
Qiagen, Hilden, Germany), and the resulting carotenoid extract was
collected following centrifugation. Pteridines were then extracted from
the tissue pellet using 2% ammonium hydroxide. 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). 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.
Trans-β-apo-8’-carotenal and
biopterin-d3 were used as internal standards for carotenoids and
pteridines, respectively.
Carotenoids and pteridines were
quantified in separate LC-MS analyses on an Agilent 6490 triple
quadrupole MS system with a Jet Stream electrospray ionisation source
coupled to an Agilent 1290 series LC system (Agilent Technologies Inc,
Santa Clara, CA). Chromatographic separation of carotenoids was achieved
on an Agilent Zorbax Bonus RP column (2.1 x 50 mm, 1.8 µm.).
Chromatographic separation of pteridines was achieved on a Waters
Acquity UPLC BEH C8 column (2.1 x 100 mm, 1.7 µm).
Data were analysed using Agilent MassHunter Workstation Software
(version B.07.00). All peak assignments were matched against commercial
or purified (drosopterin) standards, confirmed with a qualifier ion and
quantified against the linear range from six-point calibration curves
(all R2 > 0.95). Lutein and zeaxanthin
cannot be baseline separated by this method, thus concentrations of the
isomer pair Lutein/Zeaxanthin were estimated from a zeaxanthin
calibration curve. 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 pigments except drosopterin, which we extracted and purified
from fruit flies, Drosophila melanogaster (per Wilson & Jacobson
1977). Consequently, we use the relative response for drosopterin, which
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 (β-carotene,
lutein/zeaxanthin and their common metabolite 3’-dehydrolutein), red
ketocarotenoids (astaxanthin, canthaxanthin), yellow pteridines
(xanthopterin), red pteridines (drosopterin) and other pteridines
(pterin, 6-biopterin, isoxanthopterin, pterine-6-carboxylic acid).
Although 3’-dehydrolutein is not a
dietary carotenoid, we included it is this category because it is a
common metabolite of the dietary xanthophylls lutein and zeaxanthin and
indicative of dietary carotenoid intake (Albert et al. 2008;
Nagao et al. 2015).