Quantifying colour difference in animals with variable patterning

Colour pattern influences behaviour and affects survival of organisms through perception of light reflectance. Spectrophotometric methods used to study colour optimise precision and accuracy of reflectance across wavelengths, while multiband photographs are generally used to assess the complexity of colour patterns. Using standardised photographs of sand lizards (Lacerta agilis), we compare how colours characterised using point measurements (using the photographs, but simulating spectrophotometry) on the skin differ from colours estimated by clustering pixels in the photograph of the lizard's body. By taking photographs in the laboratory and in the field, the experimental design included two 2-way comparisons. We compare point vs. colour clustering characterisation and influence of illumination in the laboratory and in the field. We found that point measurements adequately represented the dominant colour of the lizard. Where colour patterning influenced measurement geometry, image analysis outperformed point measurement with respect to stability between technical replicates on the same animal. The greater colour variation derived from point measurements increased further under controlled laboratory illumination. Both methods revealed lateral colour asymmetry in sand lizards, i.e. that colours subtly differed between left and right flank. We conclude that studies assessing the impact of colour on animal ecology and behaviour should utilise hyperspectral imaging, followed by image analysis that encompasses the whole colour pattern.