An evaluation of different methods for assessing eggshell pigmentation and pigment concentration using great tit eggs

Abstract

The striking variation in colour and maculation of bird eggs has fascinated biologists since centuries, and many hypotheses based on mechanical, physiological or signalling functions have been proposed for its evolution by natural and sexual selection. Protoporphyrin is the main eggshell pigment found in brown maculated eggs, and is assumed to function as a mediator of these selection processes. It is a precursor of heme with pro‐oxidant properties, and hence a link between brown maculation and female condition has been proposed and tested in a number of studies, albeit with contrasting results. A variety of different visual methods have been used to quantify outer eggshell pigmentation, which has been assumed to correspond to overall quantity of protoporphyrin in the shell. Yet, this relationship has rarely been tested. The aim of this study was to apply four commonly used methods to assess pigmentation in great tit eggs with protoporphyrin as the predominant eggshell pigment, and to compare the results of these methods. Specifically, we 1) ranked eggshell pigmentation by human naked eye, 2) applied a granularity approach and 3) measured spectrophotometric reflectance of eggshell pigments. Second, we estimated the relationship between outer eggshell pigmentation (i.e. estimated by three different methods above) and true protoporphyrin concentration deposited in the entire shell measured by HPLC. Among‐method estimates were significantly correlated for the traits describing pigment ‘darkness’ only. While the model including scores based on human naked eyes explained 16% of the variance of pigment concentration in the entire shell, spectrometry explained 27%, and the granularity approach explained 40%. Thus, the estimation of true pigment concentration in the entire shell from the visible outer side of the shell is most reliable with the granularity approach. It is relevant for studies where the maintenance of the integrity of the eggs is essential.