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

The colourful appearance of bird eggshells has long fascinated biologists and considerable research effort has focused on the structure and biochemistry of the avian eggshell matrix. The presence of tetrapyrrole pigments was identified nearly a century ago. Surprisingly, how the concentrations of avian eggshell pigments vary among related species, and whether this variability is associated with either eggshell appearance and/or species life‐history traits, remains poorly understood. We quantified the concentrations of the two key eggshell pigments, protoporphyrin IX and biliverdin, from a diverse sample of eggshells stored at the Natural History Museum, Tring, UK. We explicitly tested how these two pigments are associated with physical measures of eggshell coloration and whether the pigment concentrations and colour diversity co‐vary with phylogenetic affiliations among species. We also tested a series of comparative hypotheses regarding the association between the concentrations of the two pigments and specific life‐history and breeding ecology traits. Across species, the average concentrations of protoporphyrin and biliverdin were positively correlated, and both strongly co‐varied with phylogenetic relatedness. Controlling for phylogeny, protoporphyrin concentration was associated with a higher likelihood of cavity nesting and ground nesting, whereas biliverdin concentration was associated with a higher likelihood of non‐cavity nesting habit and bi‐parental provisioning. Although unlikely to be explained by a single function, the breeding ecology and life history‐dependence of eggshell pigment concentrations in these comparative analyses implies that related species share pigment strategies, and that those strategies relate to broad adaptive roles in the evolution of variation in avian eggshell coloration and its underlying mechanisms. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106 , 657–672.     

Not so colourful after all: eggshell pigments constrain avian eggshell colour space

Birds'' eggshells are renowned for their striking colours and varied patterns. Although often considered exceptionally diverse, we report that avian eggshell coloration, sampled here across the full phylogenetic diversity of birds, occupies only 0.08–0.10% of the avian perceivable colour space. The concentrations of the two known tetrapyrrole eggshell pigments (protoporphyrin and biliverdin) are generally poor predictors of colour, both intra- and interspecifically. Here, we show that the constrained diversity of eggshell coloration can be accurately predicted by colour mixing models based on the relative contribution of both pigments and we demonstrate that the models'' predictions can be improved by accounting for the reflectance of the eggshell''s calcium carbonate matrix. The establishment of these proximate links between pigmentation and colour will enable future tests of hypotheses on the functions of perceived avian eggshell colours that depend on eggshell chemistry. More generally, colour mixing models are not limited to avian eggshell colours but apply to any natural colour. Our approach illustrates how modelling can aid the understanding of constraints on phenotypic diversity.     

Avian eggshell pigments are not consistently correlated with colour measurements or egg constituents in two Turdus thrushes

The colourful appearance of avian eggshells is a prominent aspect of maternal reproductive effort in birds. Some differences in eggshell coloration have been reported to co‐vary with various measures of maternal condition and these patterns support the hypothesis that, in some bird species, several aspects of eggshell colour (i.e. primary chroma and brightness) function as a signal of maternal and offspring quality to induce greater paternal investment. We directly quantified eggshell pigment concentrations of blackbird Turdus merula and song thrush T. philomelos eggshells and tested how the two key pigments (protoporphyrin IX and biliverdin) co‐varied with other eggshell traits and egg constituents as measures of maternal reproductive investment, including total yolk carotenoid concentration, total lipid concentration, yolk mass, and shell thickness. Contrary to predictions, we detected few statistical patterns overall. We found that protoporphyrin IX concentration was negatively associated with blue‐green chroma in blackbirds but not in song thrush. The concentration of protoporphyrin IX was significantly greater in blackbirds and also showed different patterns of association with total yolk lipids and yolk carotenoid concentrations between these two species (significant species interaction terms). Our results reveal that it is not appropriate to simply assume in these two avian species that reflectance‐based eggshell colour measures are a suitable proxy for eggshell pigment concentrations or can be used as consistent predictors of maternal reproductive investment. These results highlight the need to assess and validate the strength and direction of the statistical relationships between eggshell colour measures, pigment concentrations, and maternal resource deposition in the egg for other species of birds.     

Nature’s Palette: Characterization of Shared Pigments in Colorful Avian and Mollusk Shells

Pigment-based coloration is a common trait found in a variety of organisms across the tree of life. For example, calcareous avian eggs are natural structures that vary greatly in color, yet just a handful of tetrapyrrole pigment compounds are responsible for generating this myriad of colors. To fully understand the diversity and constraints shaping nature’s palette, it is imperative to characterize the similarities and differences in the types of compounds involved in color production across diverse lineages. Pigment composition was investigated in eggshells of eleven paleognath bird taxa, covering several extinct and extant lineages, and shells of four extant species of mollusks. Birds and mollusks are two distantly related, calcareous shell-building groups, thus characterization of pigments in their calcareous structures would provide insights to whether similar compounds are found in different phyla (Chordata and Mollusca). An ethylenediaminetetraacetic acid (EDTA) extraction protocol was used to analyze the presence and concentration of biliverdin and protoporphyrin, two known and ubiquitous tetrapyrrole avian eggshell pigments, in all avian and molluscan samples. Biliverdin was solely detected in birds, including the colorful eggshells of four tinamou species. In contrast, protoporphyrin was detected in both the eggshells of several avian species and in the shells of all mollusks. These findings support previous hypotheses about the ubiquitous deposition of tetrapyrroles in the eggshells of various bird lineages and provide evidence for its presence also across distantly related animal taxa.     

Effects of food limitation on the intensity of blue‐green and brown eggshell coloration: an experimental study with the canary

Variation in blue‐green and brown coloration of avian eggshells could be affected by several factors, including environmental nutritional constraints. Better availability of nutrients could influence the synthesis and deposition of pigments into the eggshell, so we may expect a link between the food availability during egg formation, the body condition of the female and intensity of eggshell coloration. This hypothesis has received mixed support so far: in some bird species a positive correlation between female body condition and eggshell blue‐green coloration could be demonstrated, but other studies failed to find a significant link. In this study, we experimentally limited the food availability for domestic canaries Serinus canaria prior to and during egg laying, and examined its effects on the biliverdin‐ and protoporphyrin‐based eggshell coloration. Treatment had no significant effect on eggshell blue‐green chroma and biliverdin concentration. However, we found a significant positive relationship between female body condition and eggshell background blue‐green chroma in the control group, but not in the food restricted group. Females possibly experiencing a decline in antioxidant capacity due to food limitation may not be able to produce a blue‐green eggshell colour intensity reliably indicating their body condition. Furthermore, food‐restricted canary females laid eggs with significantly higher eggshell brown chroma, spot intensity, and protoporphyrin concentration. Therefore, our results suggest that limitation in actual nutrient availability increases deposition of protoporphyrin into the eggshell, and it may also modify the association between female body condition and intensity of blue‐green eggshell coloration.     

Eggshell coloration and its importance in postmating sexual selection

Avian eggshell color seems to fulfill multiple functions, some of them being structural and others signaling. In this study, we tested whether or not eggshell coloration may play a role in sexual selection of Tree Sparrows (Passer montanus). According to the “Sexually selected eggshell coloration” hypothesis, eggshell coloration signals female, egg or chick quality and males adjust parental investment according to this signal. Eggs of this species are covered with brown spots and patches, and variation between clutches is high. We found that eggshell coloration correlates with both protoporphyrin and biliverdin, but protoporphyrin concentrations are ten times higher. Eggshell coloration reflects egg and offspring quality, but not female quality. Thus, eggshell coloration may signal female postmating investment in offspring rather than female quality. Furthermore, differential allocation in terms of maternal investment is supported by the fact that females lay more pigmented clutches when mated to males with bigger melanin‐based ornaments relative to their own. Moreover, males invested proportionally more to chicks that hatched from more pigmented clutches. Our correlative results thus seem to support a role of sexual selection in the evolution of eggshell coloration in birds laying brown eggs, pigmented mainly by protoporphyrin.     

Eggshells as hosts of bacterial communities: An experimental test of the antimicrobial egg coloration hypothesis

Oviparous animals have evolved multiple defenses to prevent microbes from penetrating their eggs and causing embryo mortality. In birds, egg constituents such as lysozyme and antibodies defend against microbial infestation, but eggshell pigments might also impact survival of bacteria. If so, microbes could exert an important selective pressure on the evolution of eggshell coloration. In a previous lab experiment, eggshell protoporphyrin caused drastic mortality in cultures of Gram positive, but not Gram negative, bacteria when exposed to light. Here, we test this “photodynamic antimicrobial hypothesis” in a field experiment. In a paired experimental design, we placed sanitized brown, protoporphyrin‐rich chicken eggs alongside white eggs that lack protoporphyrin. We deployed eggs for 48 hr without incubation, as can occur between laying and incubation, when microbial infection risk is highest. Eggs were placed on the open ground exposed to sunlight and in dark underground storm‐petrel burrows. We predicted that the proportion of Gram‐positive bacteria on brown eggs should be lower when exposed to sunlight than when kept in the dark, but we expected no such difference for white eggs. Although our data revealed variation in bacterial community composition, the proportion of Gram‐positive bacteria on eggshells did not vary by egg color, and there was no interaction between egg color and location. Instead, Gram‐positive bacteria were proportionally more common on eggs on the ground than eggs in burrows. Overall, our experiment did not support the photodynamic antimicrobial hypothesis. The diverse range of avian egg colors is generated by just two pigments, but over 10 hypotheses have been proposed for the evolution of eggshell color. If our results are generalizable, eggshell protoporphyrin might not play a substantial role in defending eggs against microbes, which narrows the field of candidate hypotheses for the evolution of avian eggshell coloration.     

Eggshell coloration is an indicator of dietary calcium in Common Pheasants (Phasianus colchicus)

According to the structural-function hypothesis, the eggshell pigment protoporphyrin, deposited at weak spots, can strengthen the shell structure when calcium is lacking in avian species. However, this hypothesis has not been tested in species that produce pigmented eggs of uniform ground colour without spotting patterns. We tested the structural-function hypothesis using 435 eggs produced on seven calcium diets (0.2–4.5%) given to Common Pheasants Phasianus colchicus, a species that produces a large number of eggs on a low-calcium diet with unspotted eggshells composed of a uniform ground colour of mainly protoporphyrin. We found that pheasants on the lowest calcium diet (0.2%), which had thinner eggshells, produced eggs containing more protoporphyrin-based coloration than four of six other diets, suggesting this species employs pigmentation as ground colour to strengthen eggshells when available calcium is low. Our results provide the first, at least partial support for a structural function for eggshell pigments producing ground colour without spotting in a species that is often calcium-limited. This pattern may be more widespread in other ground-nesting taxa that also produce large numbers of eggs with protoporphyrin-based ground colour and are potentially limited by calcium during breeding.     

Eggshell Appearance Does Not Signal Maternal Corticosterone Exposure in Japanese Quail: An Experimental Study with Brown-Spotted Eggs

Reproduction is a critical period for birds as they have to cope with many stressful events. One consequence of an acute exposure to stress is the release of corticosterone, the avian stress hormone. Prolonged stress can have negative impacts on the immune system, resulting in, for example, increased oxidative stress. Through maternal effects, females are known to modulate their investment in eggs content according to their own physiological condition. Less is known about maternal investment in eggshells, especially in pigments. The two main eggshell pigments may possess opposite antioxidant properties: protoporphyrin (brown) is a pro-oxidant, whereas biliverdin (blue-green) is an antioxidant. In Japanese quail, we know that the deposition of both pigments is related to female body condition. Thus, a chronic stress response may be reflected in eggshell coloration. Using female Japanese quails that lay brown-spotted eggs, we explored whether physiological exposure to corticosterone induces a change in female basal stress and antioxidant factors, and eggshell pigment concentration, spectrophotometric reflectance, and maculation coverage. We supplemented adult females over a 2 week period with either peanut oil (control) or corticosterone (treatment). We collected pre- and post-supplementation eggs and analysed the effect of corticosterone treatment on female physiology and eggshell appearance parameters. Except for corticosterone-fed birds which laid eggs with brighter spots, supplementation had no significant effect on female physiology or eggshell pigment concentration, reflectance and maculation. The change in eggshell spot brightness was not detected by a photoreceptor noise-limited color opponent model of avian visual perception. Our data confirms that eggshell reflectance in spotted eggs varies over the laying sequence, and spot reflectance may be a key factor that is affected by females CORT exposure, even if the changes are not detected by an avian visual model.     

Variability in Avian Eggshell Colour: A Comparative Study of Museum Eggshells

Background

The exceptional diversity of coloration found in avian eggshells has long fascinated biologists and inspired a broad range of adaptive hypotheses to explain its evolution. Three main impediments to understanding the variability of eggshell appearance are: (1) the reliable quantification of the variation in eggshell colours; (2) its perception by birds themselves, and (3) its relation to avian phylogeny. Here we use an extensive museum collection to address these problems directly, and to test how diversity in eggshell coloration is distributed among different phylogenetic levels of the class Aves.

Methodology and Results

Spectrophotometric data on eggshell coloration were collected from a taxonomically representative sample of 251 bird species to determine the change in reflectance across different wavelengths and the taxonomic level where the variation resides. As many hypotheses for the evolution of eggshell coloration assume that egg colours provide a communication signal for an avian receiver, we also modelled reflectance spectra of shell coloration for the avian visual system. We found that a majority of species have eggs with similar background colour (long wavelengths) but that striking differences are just as likely to occur between congeners as between members of different families. The region of greatest variability in eggshell colour among closely related species coincided with the medium-wavelength sensitive region around 500 nm.

Conclusions

The majority of bird species share similar background eggshell colours, while the greatest variability among species aligns with differences along a red-brown to blue axis that most likely corresponds with variation in the presence and concentration of two tetrapyrrole pigments responsible for eggshell coloration. Additionally, our results confirm previous findings of temporal changes in museum collections, and this will be of particular concern for studies testing intraspecific hypotheses relating temporal patterns to adaptation of eggshell colour. We suggest that future studies investigating the phylogenetic association between the composition and concentration of eggshell pigments, and between the evolutionary drivers and functional impacts of eggshell colour variability will be most rewarding.     

Eggshell biliverdin concentration does not sufficiently predict eggshell coloration

Avian eggshell coloration may have arisen due to selection on the biological, chemical, or physical properties of the pigments embedded within the eggshell, or due to selection on the coloration that emerges due to pigment deposition. Within both hypothetical frameworks, pigment‐based eggshell coloration would be related to metrics of egg quality; however, no one has evaluated the relative strength of coloration and pigment concentration in predicting egg quality. Here, we examined 66 European starling Sturnus vulgaris eggs and quantified eggshell biliverdin concentration (an antioxidant that produces the eggshell's blue coloration) and used 28 different coloration metrics derived from both photographic and spectrophotometric data. We also measured egg size, eggshell thickness, concentration of carotenoids in the yolk, and concentration of lysozyme in the albumen to capture variation in egg quality. We found that throughout the laying sequence, biliverdin concentration increased while eggshell thickness, yolk carotenoid concentration, and lysozyme concentration in the albumen all decreased, but this variation was not captured by any eggshell coloration metric. Both eggshell coloration and biliverdin concentration were negatively associated with yolk carotenoid concentration, but only eggshell biliverdin concentration was negatively associated with yolk mass. Lastly, biliverdin concentration explained, at most, only 46% of the variation for all eggshell coloration metrics. Our results suggest biliverdin concentration is a better predictor of egg quality than egg coloration in European starlings, supporting the hypothesis that eggshell pigment concentration per se may be the target of selection.     

Experimental evidence that egg color indicates female condition at laying in a songbird

The signaling hypothesis of eggshell coloration in birds isbased on the assumption that females of species with blue-greeneggs signal their phenotypic quality to their mates throughdeposition of the antioxidant biliverdin as pigment. Egg pigmentationmay be an expression of the condition of females at laying orof genetic linkages between egg color and female performancevariables. We have supplemented 16 pied flycatcher, Ficedulahypoleuca, females with mealworms before and during laying andcompared the mass and color of their eggs as measured on theday of laying to those of 16 control females with the same nestconstruction and laying dates and clutch sizes. Supplementedfemales laid significantly heavier and more intensely blue-greeneggs than control females. Egg blue-green chroma was significantlyassociated with the amount of biliverdin in eggshells. Egg color,and thus biliverdin content, is an expression of female conditionat laying.     

A shared chemical basis of avian host-parasite egg colour mimicry

Avian brood parasites lay their eggs in other birds' nests and impose considerable fitness costs on their hosts. Historically and scientifically, the best studied example of circumventing host defences is the mimicry of host eggshell colour by the common cuckoo (Cuculus canorus). Yet the chemical basis of eggshell colour similarity, which impacts hosts' tolerance towards parasitic eggs, remains unknown. We tested the alternative scenarios that (i) cuckoos replicate host egg pigment chemistry, or (ii) cuckoos use alternative mechanisms to produce a similar perceptual effect to mimic host egg appearance. In parallel with patterns of similarity in avian-perceived colour mimicry, the concentrations of the two key eggshell pigments, biliverdin and protoporphyrin, were most similar between the cuckoo host-races and their respective hosts. Thus, the chemical basis of avian host-parasite egg colour mimicry is evolutionarily conserved, but also intraspecifically flexible. These analyses of pigment composition reveal a novel proximate dimension of coevolutionary interactions between avian brood parasites and hosts, and imply that alternative phenotypes may arise by the modifications of already existing biochemical and physiological mechanisms and pathways.     

Blue‐green eggshell coloration reflects yolk antioxidant content in spotless starlings Sturnus unicolor

The hypothesis that eggshell colouration is a sexually selected trait of female birds is based on the fact that biliverdin, the pigment responsible for blue‐green colours of the eggshell, is a potent antioxidant and that only females with high antioxidant capacity can deposit higher concentrations of biliverdin as eggshell pigment. Antioxidants (e.g. carotenoids, vitamins) are also abundant in the egg yolk, which serve as nutrient reserves for the developing embryo, and eggshell colour intensity may reflect maternal investment in yolk antioxidants. Here, we test the relationship between blue‐green eggshell colour intensity and concentration and amount of carotenoids, vitamin A, and vitamin E in the egg yolk of spotless starling Sturnus unicolor, a species for which we have previously shown good evidence of sexual selection driving egg coloration. As could be extrapolated from the hypothesis of sexual selection driving the evolution of blue‐green eggshells, we found that eggshell colour intensity was positively related to the concentration and amount of carotenoids and vitamin E in the yolk. Thus, mothers may use egg colour intensity to signal to fathers the antioxidant status of their offspring. Moreover, we provide evidence suggesting that maternal yolk investment in more coloured eggs can also explain the detected association between feeding decisions of males and egg colour intensity that we have found previously in this species.     

Latitudinal variation in biophysical characteristics of avian eggshells to cope with differential effects of solar radiation

Solar radiation is an important driver of animal coloration, not only because of the effects of coloration on body temperature but also because coloration may protect from the deleterious effects of UV radiation. Indeed, dark coloration may protect from UV, but may increase the risk of overheating. In addition, the effect of coloration on thermoregulation should change with egg size, as smaller eggs have higher surface‐volume ratios and greater convective coefficients than larger eggs, so that small eggs can dissipate heat quickly. We tested whether the reflectance of eggshells, egg spottiness, and egg size of the ground‐nesting Kentish plover Charadrius alexandrinus is affected by maximum ambient temperature and solar radiation at breeding sites. We measured reflectance, both in the UV and human visible spectrum, spottiness, and egg size in photographs from a museum collection of plover eggshells. Eggshells of lower reflectance (darker) were found at higher latitudes. However, in southern localities where solar radiation is very high, eggshells are also of dark coloration. Eggshell coloration had no significant relationship with ambient temperature. Spotiness was site‐specific. Small eggs tended to be light‐colored. Thermal constraints may drive the observed spatial variation in eggshell coloration, which may be lighter in lower latitudes to diminish the risk of overheating as a result of higher levels of solar radiation. However, in southern localities with very high levels of UV radiation, eggshells are of dark coloration likely to protect embryos from more intense UV radiation. Egg size exhibited variation in relation to coloration, likely through the effect of surface area‐to‐volume ratios on overheating and cooling rates of eggs. Therefore, differential effects of solar radiation on functions of coloration and size of eggshells may shape latitudinal variations in egg appearance in the Kentish plover.     

Global diversity and adaptations of avian eggshell thickness indices

The amniote eggshell is a fundamental aspect of the embryo life-support system, protecting it from UV light, microbes and mechanical damage, while regulating gas exchange and providing calcium for growth. The thickness of eggshells is highly diverse across modern birds and influences multiple eggshell functions, yet the selective pressures driving eggshell thickness have not been clearly identified. Here, we use a global dataset of avian eggshell thickness indices for 4260 (> 41%) avian species to assess trends in eggshell thickness across the phylogeny, as these indices are strongly correlated with direct measures of eggshell thickness and are non-destructive to the sample. We analysed the dataset within a phylogenetic framework to assess the relative importance of climatic and ecological explanations for variation in eggshell thickness indices. The distribution of avian eggshell thickness indices across species was found to be primarily driven by phylogenetic relatedness, in addition to evolutionary processes that do not match a Brownian model of evolution. Across modern birds, thicker eggshells were more prevalent in species (1) with precocial young, (2) which exhibit a scavenger-based diet, (3) which primarily feed on vertebrates or plants (excluding nectivores, seed and fruit specialists) and (4) which breed in open habitats. Thicker eggshells found in species with precocial young probably enable higher rates of calcium removal for the more advanced embryo development. Excessive light transmission through the shell damages developing embryos, while too little light transmission can impede development. Eggs in shaded habitats experience low light exposure, and thus thinner shells are more prevalent in these environments potentially to increase light transmission through the shell. Overall, variation in eggshell thickness indices appears to be driven largely by phylogeny, with certain life-history traits linked to embryo growth rate, calcium content of their food, and the need to mitigate excessive light transmission through the shell.     

An experimental test of the effect of food and antioxidants on blue‐green eggshell coloration in mountain bluebirds Sialia currucoides

Evolutionary biologists have long debated the functional relevance of the ornamental traits that occur in many animal taxa, and yet, female ornaments have received relatively little attention compared to those produced by males. A greater understanding of these traits, particularly those that are unique to females, may shed light on the potential for sexual selection to shape female phenotypes. Recently, blue‐green eggshell colour, derived from the antioxidant pigment biliverdin, has been proposed as a candidate trait that advertises female quality to males in species of birds with biparental care. However, studies have been equivocal in their support for blue‐green eggshell colour being an informative signal, and correlations between eggshell colour and other female characteristics have been inconsistent. We conducted a supplementation experiment to test if improving the access of female birds to food resources and micronutrients, thereby improving their condition prior to egg laying, would intensify the blue‐green colouration of the eggs they laid. We provided mountain bluebirds Sialia currucoides with food and carotenoids during nest building and egg laying in two breeding seasons, and assessed both within‐ and among‐clutch variation in colour. Supplementation did not affect patterns of within‐clutch variation, but did result in differences in colour among clutches. Specifically, we found that food, but not carotenoids, resulted in higher colour saturation, and decreased the brightness of blue‐green eggshells. Although this trend was observed in both years, the effect was statistically significant only in one year. Our results suggest that food supplementation influences eggshell colour, but that conditions, such as weather and natural food availability, which can vary annually, may also determine how female birds allocate pigment to eggshells.     

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.     

Quantification and variability of eggshell pigment content

A high-performance liquid chromatography method was used for the determination of the eggshell pigment constituents. Using this method, the content protoporphyrin IX in seven bird species was quantified. It was found that this compound is most abundant in the eggs of the blackbird (65.55 nmol per egg), but when the amount is related to the eggshell mass, then the greatest amount is in the egg of the yellowhammer (301.0 pmol/mg eggshell), while lowest values were found in the eggs of the swift (0.59 nmol); dunnock egg shells also contained biliverdin (10.25 nmol). Eggs of the red-backed shrike show a high variability of porphyrin content (9.40 ± 6.27 nmol; range 1.03–20.61 nmol per egg). The high intra-species variability of the porphyrin content is likely to reflect physiological influences, e.g. order of egg laying and nesting conditions rather than the effect of the environment.     

Effects of experimental calcium availability,egg parameters and laying order on Great Tit Parus major eggshell pigmentation patterns

Many bird species lay eggs speckled with protoporphyrin‐based spots, however, for most of them the function of eggshell spotting is unknown. A plausible hypothesis is that protoporphyrin might have a structural function in strengthening the eggshell and is therefore deposited when calcium is scarce. In this study, we experimentally provided Great Tit Parus major females with supplemental calcium to examine its effect on the protoporphyrin‐based maculation of their eggs. In addition, we studied variation in eggshell pigmentation patterns in relation to other egg parameters and laying order. Calcium‐supplemented females laid larger eggs but shell thickness was not significantly affected by the treatment. Calcium supplementation may reduce the time and energy females devote to searching for calcium‐rich material, so that they can collect more nutrients and so lay larger eggs. Furthermore, pigment darkness was associated with egg volume and shape, which suggests that female quality and environmental food availability may also influence the shell pigmentation pattern. Within clutches, later‐laid eggs had larger and darker spots that were distributed more unevenly on the shell surface. This within‐clutch pattern could be explained by the increase in egg volume and egg shape and a decline in shell thickness with egg‐laying order, which characteristics were all related to shell‐spotting pattern. Eggs with a coronal ring had thinner shells, but pigment intensity and spot size were not related to shell thickness. Thus, our results suggest that concentrated spotting distribution may have a mechanical function, supporting the structural‐function hypothesis.