A step-by-step method to quantify coloration with digital photography

Coloration is often used in biological studies, for example when studying social signaling or antipredator defense. Yet, few detailed and standardized methods are available to measure coloration using digital photography. Here we provide a step-by-step guide to help researchers quantify coloration from digital images. We first identify the do's and don'ts of taking pictures for coloration analysis. We then describe how to i) extract reflectance values with the software ImageJ; ii) fit and apply linearization equations to reflectance values; iii) scale and select the areas of interest in ImageJ; iv) standardize pictures; and v) binarize and measure the proportion of different colors in an area of interest. We apply our methodological protocol to digital pictures of painted turtles (Chrysemys picta), but the approach could be easily adapted to any species. More specifically, we wished to calculate the proportion of red and yellow on the neck and head of turtles. With this protocol, our main aims are to make coloration analyses with digital photography:• more accessible to researchers without a background in photography;• more consistent between studies.