Quantifying colour difference in animals with variable patterning

Colour pattern influences behaviour and modifies survival of organisms through perception of light reflectance. Spectrophotometric methods used to study colour navigate between precision and accuracy of reflectance across wavelengths, while photographic methods are generally used to assess the complexity of colour patterns. Here, we compare how colours characterised using point measurements (simulating spectrophotometry) on the skin of a sand lizard ( Lacerta agilis ) differ from colours estimated by clustering pixels in a photograph of the lizard’s body. We found that point measurements adequately represented the dominant colour of the lizard; however, where colour patterning influenced measurement geometry, image analysis outper-formed point measurement as regards stability between technical replicates on the same animal. The greater colour variation established from point measurements increased further under controlled laboratory illumination. Both methods revealed lateral colour asymmetry in sand lizards. 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.