Daniel Hanley

The cuticle modulates ultraviolet reflectance of avian eggshells

ABSTRACT Avian eggshells are variedly coloured, yet only two pigments, biliverdin and protoporphyrin IX, are known to contribute to the dramatic diversity of their colours. By contrast, the contributions of structural or other chemical components of the eggshell are poorly understood. For example, unpigmented eggshells, which appear white to the human eye, vary in their ultraviolet (UV) reflectance, which may be detectable by birds. We investigated the proximate mechanisms for the variation in UV-reflectance of unpigmented bird eggshells using spectrophotometry, electron microscopy, chemical analyses, and experimental manipulations. We specifically tested how UV-reflectance is affected by the eggshell cuticle, the outermost layer of most avian eggshells. The chemical dissolution of the outer eggshell layers, including the cuticle, increased UV-reflectance for only eggshells that contained a cuticle. Our findings demonstrate that the outer eggshell layers, including the cuticle, absorb UV-light, probably because they contain higher levels of organic components and other chemicals, such as calcium phosphates, compared to the predominantly calcite-based eggshell matrix. These data highlight the need to examine factors other than the known pigments in studies of avian eggshell colour.

Parents, predators, parasites, and the evolution of eggshell colour in open nesting birds

The colourful surface of birds’ eggshells varies dramatically between species, but the selective pressures driving this variation remain poorly understood. We used a large comparative dataset to test several hypotheses proposed to explain the evolution of eggshell colouration. We tested the hypothesis that predation pressure might select for cryptic eggshells by examining the relationship between predation rate and egg colouration. We found that predation rates were positively related to eggshell brightness. The blackmail hypothesis suggests that females lay colourful eggshells to coerce males into providing additional care during incubation to keep colourful eggs covered. According to this hypothesis, conspicuous eggs should be found in situations with high risk of visual detection from predators or brood parasites. In support of this hypothesis, proportional blue-green chroma was positively related to parasitism risk, and eggs with higher proportional blue-green chroma or higher ultraviolet chroma received higher combined parental nest attendance during the incubation period. The sexual signalling hypothesis states that blue-green colour indicates female quality; however, we did not find that blue-green eggshell colour was greater in species where males participate in any form of parental care, and relative male provisioning was unrelated to blue-green eggshell chroma. We found some support for the hypothesis that brood parasitism may select for high inter-clutch variation in eggshell colour to facilitate egg recognition. In our dataset, parasitism risk was negatively related to inter-clutch repeatability of blue-green chroma. Our study highlights the diversity of selection pressures acting on the evolution of eggshell colour in birds and provides suggestions for novel areas of future key research direction.

Conspicuous eggs and colourful hypotheses: testing the role of multiple influences on avian eggshell appearance

The diversity of eggshell colours and patterning across different birds is a fascinating example of the extended avian phenotype. The avian eggs background colours range from light green/yellow to brilliant blue and intense maroon, and with the scrawling patterns comparable to the assortment of eggs found in an Easter Bunnys basket. Here we briefly introduce the diversity of eggshell appearance, its perplexingly narrow chemical basis, and clarify how the a priori assessment of assumptions and potential hypotheses can shape new eggshell research. In addition, we highlight some of the recent hypotheses developed outside the visual paradigm that has dictated the most influential hypotheses of eggshell appearance to date. Finally, we indicate priority areas for future comprehensive research on eggshells based on adaptations to ongoing changes in environmental conditions.

Eggshell conspicuousness in ground nesting birds: do conspicuous eggshells signal nest location to conspecifics?

Predators can adversely impact nesting success and therefore the presence of seemingly conspicuous eggshells would appear maladaptive. The ratites, especially the tinamous, exhibit a diverse array of eggshell colours that appear to differ strikingly from their nesting substrate, while most ground-nesting species that do not build a nest lay camouflaged eggs. Surprisingly, there is little research on how these nest contents appear to ecologically-relevant viewers, including conspecifics and predators. Here we use visual modelling to compare eggshell conspicuousness in two distinct avian lineages differing in eggshell colour and breeding biology: ratites and galliformes. Ratites generally lay vibrant, unspeckled eggs directly on the ground, while galliformes tend to lay subtler, speckled eggs on nests built on the ground. We test the hypothesis that eggshell colours in ratites are more conspicuous, from the perspective of an avian conspecific, than those of galliformes. We found that the uniform colour covering the surface of the eggshell colour (hereafter, eggshell background colour) differs noticeably from the nesting substrate in both ratites and galliformes. However, the speckling pattern of galliform eggs often masks their conspicuous eggshell background colour, which contributes to a less conspicuous appearance overall. We tested the hypothesis that eggshell conspicuousness in ratites serves an intraspecific signalling function to advertise nest location to females in communally nesting species. We found no support for this hypothesis, suggesting that selection pressure for communal laying did not result in the diversity of conspicuousness found in avian eggs. Overall, we argue that future investigations of egg coloration should consider egg appearance (eggshell background colour and speckling) in the context of the natural nest substrate, all from the perspective of the relevant visual receiver.

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.

Dynamic egg color mimicry

Abstract Evolutionary hypotheses regarding the function of eggshell phenotypes, from solar protection through mimicry, have implicitly assumed that eggshell appearance remains static throughout the laying and incubation periods. However, recent research demonstrates that egg coloration changes over relatively short, biologically relevant timescales. Here, we provide the first evidence that such changes impact brood parasite–host eggshell color mimicry during the incubation stage. First, we use long‐term data to establish how rapidly the Acrocephalus arundinaceus Linnaeus (great reed warbler) responded to natural parasitic eggs laid by the Cuculus canorus Linnaeus (common cuckoo). Most hosts rejected parasitic eggs just prior to clutch completion, but the host response period extended well into incubation (~10 days after clutch completion). Using reflectance spectrometry and visual modeling, we demonstrate that eggshell coloration in the great reed warbler and its brood parasite, the common cuckoo, changes rapidly, and the extent of eggshell color mimicry shifts dynamically over the host response period. Specifically, 4 days after being laid, the host should notice achromatic color changes to both cuckoo and warbler eggs, while chromatic color changes would be noticeable after 8 days. Furthermore, we demonstrate that the perceived match between host and cuckoo eggshell color worsened over the incubation period. These findings have important implications for parasite–host coevolution dynamics, because host egg discrimination may be aided by disparate temporal color changes in host and parasite eggs.

Who moved my eggs? An experimental test of the egg arrangement hypothesis for the rejection of brood parasitic eggs

Avian brood parasitism is an exceptional reproductive strategy whereby parasites reduce their own costs associated with parental care and impose them on the host parents. Consequently, host species have evolved multiple defensive mechanisms to combat parasitism. The vast majority of research attention to date has examined host defenses to recognize and reject parasitic eggs. The recently proposed “egg arrangement hypothesis” suggests that hosts may not focus solely on individual eggs’ features, but instead the overall arrangement of the clutch may also provide a cue that parasitism has occurred. Correlative data revealed that host females maintaining a consistent egg arrangement across the incubation period were more likely to reject foreign egg models than females that did not keep a consistent egg arrangement. Here, we provide the first experimental test of this hypothesis in the European blackbird (Turdus merula). We experimentally parasitized nests such that the egg arrangement was either disrupted or not disrupted. We found no evidence that altered egg arrangement was used as a cue for egg rejection by host females. Therefore, we suggest that females that keep consistent egg arrangement are more likely to eject foreign eggs for other correlated reasons. Thus, egg arrangement does not serve as an independent cue to trigger egg rejection responses to parasitism in this host species.

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

Abstract 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.

Analysing avian eggshell pigments with Raman spectroscopy

ABSTRACT Avian eggshells are variable in appearance, including coloration. Here, we demonstrate that Raman spectroscopy can provide accurate diagnostic information about major eggshell constituents, including the pigments biliverdin and protoporphyrin IX. Eggshells pigmented with biliverdin showed a series of pigment-diagnostic Raman peaks under 785 nm excitation. Eggshells pigmented with protoporphyrin IX showed strong emission under 1064 nm and 785 nm excitation, whereas resonance Raman spectra (351 nm excitation) showed a set of protoporphyrin IX informative peaks characteristic of protoporphyrin IX. As representative examples, we identified biliverdin in the olive green eggshells of elegant crested tinamous (Eudromia elegans) and in the blue eggshells of extinct upland moa (Megalapteryx didinus). This study encourages the wider use of Raman spectroscopy in pigment and coloration research and highlights the value of this technique for non-destructive analyses of museum eggshell specimens. Summary: The major constituents of bird eggshells (mineral, matrix, pigment) can be analysed without sample destruction, which is ideal for ancient and other precious eggshell specimens.

Eggshell conspicuousness is related to paternal brood patch vascularisation in the American thrashers

Conspicuous blue-green egg colouration has been of interest to scientists and naturalists for more than a hundred years; however, the function of this trait has never been fully understood and has spurred extensive debate. The American thrashers exhibit great variation in egg colouration and patterning, and also show variation in male brood patch development. A recent hypothesis suggests that conspicuous egg colouration has evolved as the product of sexual conflict, where females may create conspicuous eggs to obtain greater amounts of male care, either in the form of male incubation or incubation assistance to prevent their eggs from being vulnerable to visually orienting predators and brood parasites. The American thrashers, a genus comprising of 10 species, exhibits striking variation in egg colouration and patterning, and also show variation in male brood patch development. By using avian visual modelling, I show that blue-green chroma of American thrasher eggs is positively related to their degree of conspicuousness against a nest background, while brown chroma is negatively related. Interestingly, the degree of male brood patch vascularisation is directly related to eggshell conspicuousness in American thrashers. I suggest that in the American thrashers, male brood patch vascularisation has evolved in response to conspicuous eggshell colouration to mitigate visual risks. Here I show that the degree of male brood patch vascularisation is directly related to relative paternal incubation effort in more than 300 North American breeding birds. This suggests that American thrasher species that possess both conspicuous eggs and brood patches, have most likely evolved to keep these conspicuous nest contents concealed, thereby reducing risk of visual detection. I suggest that these patterns are not unique to thrashers, but may extend to several avian families.