Colour patch size and measurement error using reflectance spectrophotometry

Abstract

Summary Over the past 20 years, portable and relatively affordable spectrophotometers have greatly advanced the study of animal coloration. However, the small size of many colour patches poses methodological challenges that have not, to date, been assessed in the literature. Here, we tackle this issue for a reflectance spectrophotometry set‐up widely used in ecology and evolution (the beam method). We reviewed the literature on animal coloration reporting the use of reflectance spectrophotometry to explore how the minimum measurable size of a colour patch is determined. We then used coloured plastic sheets to create artificial colour patches, and quantify the relationship between colour patch size and distortions induced by resulting chimeric spectra (spectra contaminated by an adjacent colour patch). Finally, we assessed the generality of our findings using natural colour spots in the lizard Podarcis muralis, as a biologically realistic model. We found a lack of consensus in the literature, frequently resulting in the rejection of valid data or the potential inclusion of unreliable data. As expected, we show that decreasing colour patch size reduces the reliability of reflectance measurements, but also that spectral distortions resulting from chimeric spectra depend on patch/background colour combinations. We found similar results using natural colour spots in P. muralis. We propose a series of steps to avoid the pitfalls described above. First, we provide guidelines on how to identify chimeric spectra and estimate the minimum size of a measurable colour patch in order to avoid them. Second, we show that reducing the probe‐to‐surface distance allows for more accurate measurements and therefore improves the spectrophotometric assessment of small colour patches. Third, we suggest that, as a general rule of thumb, very small (< 2 mm) colour patches should be avoided when using traditional spectrophotometry methods.

Thermal melanism explains macroevolutionary variation of dorsal pigmentation in Eurasian vipers

“…Such a hypothesis would need to be formally tested in light of the phylogeny of Eurasian vipers to further reinforce the conclusions of this study, through the direct quantification of scale colour characteristics in living specimens (e.g. 30,31 ). Such a complementary approach would also allow a more profound assessment of the precise genetic and developmental mechanisms that control the macroevolutionary patterns and signals identified here.…”

Trait differences among discrete morphs of a color polymorphic lizard,Podarcis erhardii

“…Measurements were calibrated with a white WS-1-SL Labsphere Diffuse Reflectance Standard (Spectralon, Ocean Optics). Spectra were collected by placing the spectrometer illumination probe perpendicular to the surface of the throat 5 mm away from the skin ( Pérez i De Lanuza, Font & Carazo, 2013 ; Badiane et al, 2017 ). Measurements were a circular point sample three mm × three mm, and we took six measurements of lizard throat color patches at landmark throat scale locations to avoid measurement bias and capture potential variation across the entire signal ( Pérez i de Lanuza et al, 2019 ).…”

Through the eye of a lizard: hue discrimination in a lizard with ventral polymorphic coloration

“…Spectra were compiled from previously published studies (Pérez i de Lanuza et al, 2013Pérez i de Lanuza and Font, 2015; raw data are available from the corresponding author on request), resulting in a dataset of 643 adult lizards showing pure morphs (199 white males, 88 white females, 135 yellow males, 57 yellow females, 131 orange males and 33 orange females). Reflectance spectra were obtained with a USB-2000 portable spectrometer and a PX-2 xenon strobe light source (Ocean Optics Inc., Dunedin, FL, USA), calibrated with a Spectralon white diffuse reflectance standard (Labsphere) (for more details, see Font et al, 2009;Pérez i de Lanuza et al, 2013Badiane et al, 2017). Irradiance inside the experimental terraria was measured with a second USB-2000 spectrometer calibrated by means of a LS1-CAL calibration light source (Ocean Optics), using a cosine-corrected irradiance probe (Ocean Optics CC-3-UV).…”