Precise colocalization of interacting structural and pigmentary elements generates extensive color pattern variation in Phelsuma lizards

Abstract

Background: Color traits in animals play crucial roles in thermoregulation, photoprotection, camouflage, and visual communication, and are amenable to objective quantification and modeling. However, the extensive variation in non-melanic pigments and structural colors in squamate reptiles has been largely disregarded. Here, we used an integrated approach to investigate the morphological basis and physical mechanisms generating variation in color traits in tropical day geckos of the genus Phelsuma.

Results: Combining histology, optics, mass spectrometry, and UV and Raman spectroscopy, we found that the extensive variation in color patterns within and among Phelsuma species is generated by complex interactions between, on the one hand, chromatophores containing yellow/red pteridine pigments and, on the other hand, iridophores producing structural color by constructive interference of light with guanine nanocrystals. More specifically, we show that 1) the hue of the vivid dorsolateral skin is modulated both by variation in geometry of structural, highly ordered narrowband reflectors, and by the presence of yellow pigments, and 2) that the reflectivity of the white belly and of dorsolateral pigmentary red marks, is increased by underlying structural disorganized broadband reflectors. Most importantly, these interactions require precise colocalization of yellow and red chromatophores with different types of iridophores, characterized by ordered and disordered nanocrystals, respectively. We validated these results through numerical simulations combining pigmentary components with a multilayer interferential optical model. Finally, we show that melanophores form dark lateral patterns but do not significantly contribute to variation in blue/green or red coloration, and that changes in the pH or redox state of pigments provide yet another source of color variation in squamates.

Conclusions: Precisely colocalized interacting pigmentary and structural elements generate extensive variation in lizard color patterns. Our results indicate the need to identify the developmental mechanisms responsible for the control of the size, shape, and orientation of nanocrystals, and the superposition of specific chromatophore types. This study opens up new perspectives on Phelsuma lizards as models in evolutionary developmental biology.

Figure 1

Color and pattern variation in the genus Phelsuma. (a) Currently recognized species [19,20] with corresponding ranges of background coloration (indicated by filled squares), varying from gray/brown to yellow/green to turquoise-blue, and dorsal and lateral patterns (indicated for illustration on Phelsuma lineata with an arrow and an arrowhead, respectively). Empty shapes indicate absence of the corresponding color or pattern. Dorsal patterns vary in both color (typically different shades of red) and shape (spots, stripes, and transverse bars). Lateral patterns appear as spots (sometimes surrounded by a blue ring) or broad stripes. Species indicated in bold were used in this study. (b) Skin samples represent variation found across the genus. The skin on the belly (shown here for Phelsuma grandis, lowest panel) is off-white in the majority of species.

Figure 2

Pigmentary and structural colorations in Phelsuma geckos. (a) Semi-thin cross-sections of skins of different colors. Two types of melanophores are indicated (arrows and arrowheads, respectively), and iridophores are outlined with dashed lines. Ep, epidermis; X, xanthophores; Er, erythrophores. Bar = 10 μm. (b) Pteridin pigments were removed with NH₄OH (here in Phelsuma grandis, individual number 3), revealing the remaining structural color produced by the iridophores. Bar = 0.2 mm. (c) Red pigments in dorsal markings of Phelsuma quadriocellata and Phelsuma lineata can change color when the pH of the Ringer solution is lowered or when an oxidant (NaNO₂) is added, respectively. Bar = 0.2 mm. (d) Representative electron micrographs of iridophores in skin of different colors. Bar = 1 μm. Note the highly disordered guanine crystals in the white and red skin. (e) Mechanical pressure and dehydration (here applied to the green skin of P. grandis, individual number 2 after removal of the yellow pigment) lead to a blue shift of structural green (for supplementary movies, see Additional file 3; see Additional file 4).

Figure 3

Experimental and modeled reflectivities of Phelsuma skin. (a) Measured skin reflectivities (solid lines) after removal of pigments (skin colors varied from deep-blue to yellowish-green) compared with modeled reflectivities (dashed lines) based on crystal size and spacing (Table 1). For Phelsuma grandis number 4, the crystal geometry parameters were taken from the P. grandis number 1 neck sample that exhibited a similar structural color. Note that the UV peaks in the measured reflectivities are probably caused by scattering on melanosomes, on iridophore crystals, or on dermal collagen fibers. (b) Normalized reflectivity of green skin before and after yellow pigment removal (green and blue solid lines, respectively). Modeled multilayer responses for a crystal size of 70 nm and a spacing of 30 nm (assuming a standard deviation of 13 nm) are also shown with (dashed green line) and without (dashed blue line) a 3 μm thick pigment layer on top. The direct product of structural blue reflectivity with normalized yellow pigment transmittance (orange dotted line) generates the plain red line, confirming the mechanism of structural color filtering by the top pigment layer. (c) Reflectivity measured on P. grandis white skin (black line) and on red skin before (red solid line) and after (red dashed line) red pigment removal. Reflectivity intensities of ordered and disordered iridophores are comparable.

Figure 4

Colors simulated with the multilayer model. (a) Red colors simulated with varying thickness (0.1 to 4 μm) of a red pigment layer on top of a white reflector and comparison to red markings of different animals. (b)Phelsuma grandis individual number 1. (c) Simulated colors produced by a 7 μm yellow pigment layer on top of a multilayer interference reflector with varying spacing (40 to 105 nm) between layers of crystals 80 nm thick. Double arrows indicate the spacing measured between crystal layers on the dorsal and neck skin of the individual. (d) Simulated colors produced with varying thicknesses (0 to 7 μm) of a yellow pigment layer on top of a blue reflector.