Eggshell spot scoring methods cannot be used as a reliable proxy to determine pigment quantity

Eggshell maculation of most passerines is due to the deposition of the pigment protoporphyrin which is produced during biosynthesis of blood haem. Its functional significance has only received empirical attention in recent years. This interest has generated a number of hypotheses of which some remain untested partly because the quantification of protoporphyrin is analytically challenging and can be prohibitively expensive. Many studies have therefore used the extent of eggshell spotting as a proxy for total eggshell protoporphyrin concentration, although this has not been formally tested. Pigment scoring involves recording visible eggshell pigment attributes, such as spot intensity, distribution and size. Since even immaculate eggs can contain some protoporphyrin, there remains doubt over the degree to which visible pigment correlates with total pigment content of the shell. In this study, we test whether visible pigment scoring can be used as a proxy for protoporphyrin concentration of an eggshell. We use pigmented eggshells of two common British passerine species to compare eggshell spot intensity, distribution and spot size (as used by the visual pigment scoring method) with direct measures of eggshell protoporphyrin concentration. In addition, we compared an alternative method of pigment scoring, the pixel pigment scoring method, using a computer programme to quantify the number of pixels exceeding a specified colour threshold. We demonstrate that although results from both scoring methods were positively correlated with eggshell protoporphyrin concentrations, the correlations were not sufficiently strong to be used as surrogates in studies where actual pigment concentrations are required.