Sources of variation in reflectance spectrophotometric data: a quantitative analysis using avian eggshell colours

Abstract

Summary1. The evolution of coloration has generated some of the most diverse and variable phenotypes, both within and between species, in animals and plants. The objective quantitative assessment of the physical, chemical and perceptual basis of coloration has been greatly improved by the application of portable full-spectrum spectrophotometers to measure reflectance data. Yet, the analysis and interpretation of physical measures of colour spectra must be conducted within the constraints of the uncertainty regarding the relative impacts of methodological vs. biological sources of variation. 2. Here, we characterise the components of variation in data on reflectance spectra, related to sample storage and measurement equipment, to characterise colourful pigmentation of eggshells (Class: Aves) of two Turdus species. We quantified longitudinal shifts in reflectance occurring over repeated measurements of the same sets of avian eggshells. Shells were sampled at the time of collection and again after 5 years of dark storage using the same equipment. These data were then compared with spectra obtained from the same eggs after the storage with a different model of spectrophotometer for three colour metrics [blue-green chroma (BGC), UV chroma and brightness]. 3. Blue-green chroma and brightness of the same eggs varied systematically between years at a similar magnitude to the biological variation among different eggs. This suggests the need for future research into the extent of chemical and physical deterioration of eggshell appearance even during relatively short-term storage. The variation introduced by using two different spectrophotometers also was significant but relatively small compared with biological levels of variation for UV and brightness. 4. Our results confirm quantitatively that museum eggshell specimens are suitable for interspecific comparative analyses, but also highlight the requirement to account explicitly for variation in storage duration and measurement equipment, when 'objectively' comparing biological variation in coloration across individuals in space and also in time.

Why are birds' eggs colourful? Eggshell pigments co-vary with life-history and nesting ecology among British breeding non-passerine birds

“…We have documented, previously, significant differences in a range of spectral reflectance‐based coloration‐metrics between eggshells stored in museums and freshly collected eggs (Cassey et al ., 2010a, 2011). These differences are probably due to temporal change in chemical structure of the pigments through storage exposure to light, and/or different preservation techniques.…”

First light for avian embryos: eggshell thickness and pigmentation mediate variation in development and UV exposure in wild bird eggs

“…The probe was fitted with a custom‐built light‐proof cap, which was fixed at a consistent measurement angle of 90° (Cassey et al . ). Spectra were expressed relative to a white Ocean Optics WS‐1 diffuse reflectance standard and measured in 0·4‐nm steps.…”

“…), and it is not straightforward to predict eggshell colour from the varying concentrations of the individual pigments (Cassey et al . ,b,c). Given that we are interested in the proposed influence of total pigment concentration hindering the transmission of light through the eggshell, we summed the total pigment concentrations (log 10 ‐transformed) for all subsequent analysis.…”

Condition-dependent strategies of eggshell pigmentation: an experimental study of Japanese quail (Coturnix coturnix japonica)

“…Yolk and albumen were separated and weighed (to the nearest 0.01g) on a digital balance to determine relative egg components by mass as a proxy for egg macronutrient content (Baumgartner et al, 2008). Eggshells were washed with distilled water and kept in a dark box to dry at room temperature and to avoid direct exposure to light that could cause pigment degradation (Cassey et al, 2011).…”

“…Egg collection began after 10days of mating, the period required to obtain fertile eggs (Adkins-Regan, 1995). Each cage was visited every morning and eggs were collected and placed in a dark box in a cold room (4°C) to avoid degradation of eggshell pigments by exposure to light (Cassey et al, 2011).…”