A re-evaluation of aff. Megaloolithidae eggshell fragments from the uppermost Cretaceous of the Pyrenees and implications for crocodylomorph eggshell structure

The Upper Cretaceous outcrops of the Pyrenees yield one of the most extensive and continuous records of paleoological remains anywhere in the world. Most of eggs and eggshells have been referred to the oofamily Megaloolithidae. In this study, we present a revision of eggshell fragments from the Blasi 2 locality, lattermost Maastrichtian in age, previously assigned to aff. Megaloolithidae. The presence of a blocky extinction pattern and basal knobs supports a crocodilian affinity of these materials. We classify them as Krokolithidae indet. Three structural layers can be recognised in the Blasi 2 eggshells, a feature that is shared with other recent eggshells (e.g. Crocodylus porosus and Crocodylus niloticus) and fossil crocodylomorph eggshells (Krokolitheswilsoni), which were previously described as single layered. The new proposed affinity of the Blasi 2 eggshells reduces the Megaloolithidae oodiversity of the last few million years of the Cretaceous in the Pyrenees to only two valid ootaxa, Megaloolithusmamillare and Megaloolithusbaghensis. The lack of more complete material precludes the erection of new ootaxa based on the Blasi 2 material.     

Two new theropod egg sites from the Late Jurassic Lourinhã Formation,Portugal

Two new Late Jurassic (uppermost Late Kimmeridgian) dinosaur eggshell sites are described, Casal da Rola and Porto das Barcas, both near Lourinhã, central-west Portugal. Casal da Rola yields eggshells with an obliquiprismatic morphotype comparable to those from a nest with the associated fossil embryos from Paimogo, tentatively assigned to the theropod Lourinhanosaurus antunesi. The Porto das Barcas eggshells have a dendrospherulitic morphotype with a prolatocanaliculate pore system. This morphotype was also recognised in eggshells from a clutch with associated Torvosaurus embryos at the Porto das Barcas locality. A preliminary cladistic analysis of eggshell morphology suggests theropod affinities for the Casal da Rola eggs, but is unable to resolve the phylogenetic position of the Porto das Barcas eggs. The eggshells at both sites are preserved in distal flood plain mudstones and siltstones. Carbonate concretions within the deposits indicate paleosol development.     

Parataxonomy and palaeobiogeographic significance of dinosaur eggshell fragments from the Upper Cretaceous strata of the Cauvery Basin,South India

Abstract

In the past, fossilised dinosaur eggshells have been extensively documented from the Upper Cretaceous Lameta Formation of Central India and as many as nine oospecies are known at present from this formation. Compared to this, only one dinosaur oospecies has been described from the Cretaceous succession of the Cauvery Basin. However, the first fossil egg from India, identified as a chelonian egg, was documented from the Aptian – Albian Karai Formation of the Cauvery Basin in 1957. Following this, a solitary titanosaurid dinosaur egg was described from the Upper Cretaceous (Lower Maastrichtian) Kallankuruchhi Formation, Cauvery Basin in 1996. More recently, we have recovered isolated eggshell fragments from the marine part of the Upper Cretaceous (Late Maastrichtian) Kallamedu Formation. Based on eggshell morphology, microstructure and ultrastructure, these eggshell fragments are assigned to the oospecies Fusioolithus baghensis. The new find from the Cauvery Basin is important from palaeobiogeographic point of view as the oofamily Fusioolithidae is found in the Upper Cretaceous strata of India, France, Argentina and Morocco. Based on the common occurrence of similar oospecies in South America, Africa, Europe and India, a Late Cretaceous palaeobiogeographic connection between India and South America as well as Europe via Africa is suggested.     

First report of fossil turtle eggshell west of the Colorado Plateau

The first fossil turtle eggshell from North America west of the Colorado Plateau (UCMP 218504) is described, adding to the growing global record of chelonian eggshell. Ten blocks of mudstone were collected from the Upper Miocene (Clarendonian) Orinda Formation, Contra Costa County, California, during the excavation of the Caldecott Tunnel Fourth Bore. They contain rigid eggshell averaging 500 μm in thickness. Closely packed shell units with a height/width ratio of 2:1 are composed of radiating, acicular crystals originating from a basal core, a set of characters that support the identification of the eggshell as chelonian, and further as testudinoid. Egg diameter is estimated at between 34.2 and 42.4 mm based on shell curvature. Adult carapace length is estimated to be 70.7 cm, a size similar to fossils of Hesperotestudo known from California and present in the Orinda Formation. UCMP 218504 greatly extends the geographic record of turtle fossil eggshell and adds to the limited number of mid-Neogene eggs. It also provides evidence for a warm climate in Miocene California and demonstrates the south-eastern contraction of conditions appropriate for tortoise reproduction during the last 10 million years.     

Structure of fossil dinosaur eggshell from the Aix Basin,France

At least four types of dinosaur eggshell are distinguished among samples of fossil eggshell from Late Cretaceous deposits of southern France. Recognition is based on shell microstructure, porosity, and shell thickness. Estimated values of water vapour conductance for these dinosaur eggshells are much greather than predicted for avian eggs, suggesting that the eggs were incubated under conditions of high humidity, such as would occur underground.     

Great spotted cuckoo eggshell microstructure characteristics can make eggs stronger

Obligate avian brood parasites lay stronger eggs than their hosts or non‐parasitic relatives because they are rounder and have a thicker eggshell. Additionally, some other characteristics of the brood parasitic eggshells related to their microstructure such as size and orientation of calcite crystal units could also contribute to generating even stronger shells. An eggshell microstructure formed by small randomly oriented calcite crystal units can increase the robustness of the eggshells of birds. Here, the eggshell microstructure of avian brood parasites as well as their hosts have been characterized in detail, using X‐ray diffraction analyses to estimate the size and degree of orientation of calcite crystal units making the eggshell. Specifically, the brood parasitic great spotted cuckoo Clamator glandarius and two hosts (jackdaws Corvus monedula and magpie Pica pica) and one non‐host species (the pigeon, Columba livia domestica) were considered. Calcite crystal of the eggshell of the brood parasitic species was smaller and more randomly oriented than those of the eggshells of non‐parasitic species, which suggest that eggshell microstructure would contribute to explain why parasitic eggs are more resistant to breakage than those of their hosts.     

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.     

A new peculiar dinosaur egg,Sankofa pyrenaica oogen. nov. oosp. nov. from the Upper Cretaceous coastal deposits of the Aren Formation,south‐central Pyrenees,Lleida, Catalonia,Spain

Abstract: A new type of small, ovoid dinosaur egg, Sankofa pyrenaica oogen. nov. oosp. nov., with a prismatic type eggshell is described from upper Cretaceous (upper Campanian–Maastrichtian) deposits of the Montsec area, South Pyrenean Central Unit, Lleida, Catalonia, Spain. This egg type was sub‐vertically laid in only two rich monospecific sites of a single stratigraphic layer from coastal deposits of the Aren Formation, interpreted as an emerged beach ridge of a barrier island – lagoon depositional system. The size and shape of these eggs with their asymmetric poles are roughly similar to modern hen eggs, which is unusual in the Cretaceous fossil egg record. Its phylogenetic position clusters with bird and Troodontid eggs. A morphospace analysis of egg shapes shows the similarity of the new egg to a Campanian fossil bird egg from Argentina, both being intermediate between modern‐bird eggs and extinct nonavian theropod eggs. However, the eggshell microstructure of Sankofa pyrenaica differs from that of bird eggs in its incipient squamatic texture. It has a peculiar pattern of interlocking small crystals in the middle of the palisade layer, instead of the thick squamatic structure commonly present in modern avian eggshells. This new egg type is attributed to a small theropod, probably with a single oviduct like birds and whose mosaic distribution of features is a combination between that of birds and nonavian theropods. This enhances the arguments supporting the close phylogenetic relationships between both groups.     

Fossile Reptil-Eischalen (Chelonia,Crocodilia, Dinosauria) aus dem unteren Barremium von Galve (Provinz Teruel,SE-Spanien)

The Lower Cretaceous vertebrate bearing sediments of Galve (province of Teruel, SE Spain) have yielded some hundred fossil eggshell fragments, all slightly diagenetically altered. Concerning their microstructure, an assignment of the material to different taxa of reptiles was possible. The eggshell assemblage of Galve consists of a-c) three types of chelonian eggshells, one of them probably representing the subfamily Batagurinae, which would extend this taxon back into the Lower Cretaceous, d) the oldest known crocodilian eggshells, with lacunae systems on the outer surface, comparable to Recent taxa, and e-f) two different types of dinosaurian eggshells. In correspondence to the suggested fluviatile paleoenvironment of Galve, the fossil eggshells described herein are fragmented and represent mostly turtles and crocodiles.     

A calcium-binding sialoglycoprotein associated with an apparent eggshell membrane of Globodera rostochiensis

Energy dispersive X-ray microanalysis has been used to study the binding of two inhibitors of hatching in Globodera rostochiensis, ruthenium red and lanthanum, to a calcium binding site on the eggshell. Eggs were treated with either buffered ruthenium red or buffer only, and the levels of ruthenium and calcium that could be detected from the surface of eggshells were measured before and after repeated etching in a plasma oven. Counts for ruthenium increased during etching to a maximum after 8–12 min, just before the eggshell was eroded from the juvenile. The calcium levels on eggshells binding ruthenium red were suppressed relative to those from buffer. Similar experiments with lanthanum showed a significant increase in this inhibitor after 10 min etching and an accompanying suppression of calcium levels. The nature of the binding site was probed by measuring ruthenium red binding after pretreatment with selected enzymes. A non-specific protease, pronase, reduced the subsequent binding of this inhibitor but the site was resistant to trypsin in the presence of calcium. Neuraminidase also had an effect suggesting that the protein is a sialoglycoprotein. Phospholipase A2 but not Phospholipase C influenced the subsequent binding of ruthenium red. Phospholipase A₂ caused a loss of hatching ability and the degradation of a phospholipid from the eggshell. Ruthenium red binding was also reduced by treatment of eggshells with detergents or sonication. Apparently a calcium-binding sialoglycoprotein forms part of the integral proteins of an eggshell membrane located on the inner surface of the eggshell.     

Differential label‐free quantitative proteomic analysis of avian eggshell matrix and uterine fluid proteins associated with eggshell mechanical property

Eggshell strength is a crucial economic trait for table egg production. During the process of eggshell formation, uncalcified eggs are bathed in uterine fluid that plays regulatory roles in eggshell calcification. In this study, a label‐free MS‐based protein quantification technology was used to detect differences in protein abundance between eggshell matrix from strong and weak eggs (shell matrix protein from strong eggshells and shell matrix protein from weak eggshells) and between the corresponding uterine fluids bathing strong and weak eggs (uterine fluid bathing strong eggs and uterine fluid bathing weak eggs) in a chicken population. Here, we reported the first global proteomic analysis of uterine fluid. A total of 577 and 466 proteins were identified in uterine fluid and eggshell matrix, respectively. Of 447 identified proteins in uterine fluid bathing strong eggs, up to 357 (80%) proteins were in common with proteins in uterine fluid bathing weak eggs. Similarly, up to 83% (328/396) of the proteins in shell matrix protein from strong eggshells were in common with the proteins in shell matrix protein from weak eggshells. The large amount of common proteins indicated that the difference in protein abundance should play essential roles in influencing eggshell strength. Ultimately, 15 proteins mainly relating to eggshell matrix specific proteins, calcium binding and transportation, protein folding and sorting, bone development or diseases, and thyroid hormone activity were considered to have closer association with the formation of strong eggshell.     

Evidence for a calcium-binding site on the eggshell of Globodera rostochiensis with a role in hatching

Two inhibitors of hatching in Globodera rostochiensis, ruthenium red and lanthanum, have been shown to bind to the eggshell using the techniques of microdensitometry for ruthenium red and X-ray microanalysis for lanthanum. Neither inhibitor penetrated or adhered to unhatched or hatched viable juveniles. Scatchard analysis for binding of lanthanum and ruthenium red to eggshells gave dissociation constants (K) of K_La 32.5 ± 14.0 μM and K_Rured 33.5 ± 5.0 μM respectively. Both values are within the 95% fiducial limits of those shown to cause 50% inhibition of hatch in previous work. Pretreatment with sodium hypochlorite separated an outer part of the eggshell from an inner region which exclusively bound ruthenium red. It is the inner lipoprotein layer that is believed to include the membranes controlling the permeability of the tylenchid eggshell. The rate of binding of ruthenium red was similar for intact and isolated eggshells with 50% binding occurring after 6.11 ± 0.91 min and 4.95 ± 2.38 min but the latter gave a significantly higher maximum binding suggesting that rupture of the eggshells made available additional binding sites on their inner surface. The binding of ruthenium red to the eggshells was pH dependant over most of the range pH 2.8–8.5 with 50% binding, given with its standard deviation, occurring at pH 5.75 ± 0.85. Competitive binding of lanthanum influenced the binding of ruthenium red to the eggshells from which Scatchard analysis gave K_la of 176 ± 79 μM. Similarly, calcium influenced the binding but this caused a biphasic plot with high and low affinity binding sites of K“_ca of 0.423 ± 1.16 μm and K‘_ca of 1078 ± 462 μM. The existence of a high affinity site for calcium that also binds ruthenium red, suggests that the eggshell membrane includes a calcium binding glycoprotein as found in some other receptor mechanisms.     

Association between ovocalyxin-32 gene haplotypes and eggshell quality traits in an F2 intercross between two chicken lines divergently selected for eggshell strength

Broken and cracked eggshells contribute significantly to economic losses in the egg production industry. We previously identified ovocalyxin-32 as a potential gene influencing eggshell traits, by analysing an intercross between two parent lines developed from the same founder population by a two-way selection for eggshell strength with non-destructive deformation (DEF) conducted over 14 generations. We determined the nucleotide sequences of six ovocalyxin-32 exons in the parent individuals and analysed the association between ovocalyxin-32 and eggshell traits in the F₂ individuals. We identified three haplotypes (W, M and S) of ovocalyxin-32 in the parent individuals. A mismatch amplification mutation assay was performed to distinguish six diplotype individuals (WW, MM, SS, WM, MS and WS) in the F₂ population. The egg weight (EW) of SS-diplotype individuals was significantly higher than that of WW-, WM- and WS-diplotypes. Short length of the egg (SLE) of SS-diplotype individuals was significantly higher than that of WW-, WM- and MS-diplotypes. Long length of the egg (LLE) of SS-diplotype individuals was significantly higher than that of WM- and WS-diplotypes. DEF of WW-diplotype individuals was significantly higher than that of SS-, WM, MS and WM-diplotypes. Haplotypic effect analyses showed significant differences between the W-haplotype and the S-haplotypes in the EW, SLE, LLE and DEF. The DEF of M-haplotype was significantly lower than that of W- and S-haplotypes. These results suggest that S- and M-haplotypes are critical for high quality of eggshells in the F₂ population. In conclusion, ovocalyxin-32 is a useful marker of eggshell traits and can be used to develop strategies for improving eggshell traits in commercial layer houses.     

Fine structure of the subitaneous eggshell of the sessile rotifer Stephanoceros millsii (Monogononta) with observations on vesicle trafficking in the integument during ontogeny

Rotifers that engage in cyclical parthenogenesis produce two types of eggs: subitaneous eggs that hatch as clonal females and meiotic eggs that hatch as haploid males, or if fertilized, as females after a period of diapause (resting eggs). The ultrastructure of resting eggshells is known for some motile species, but there are limited data on subitaneous eggshells, and no data on any eggshells of sessile rotifers. Here, we investigated the ultrastructure of the subitaneous eggshell of the sessile rotifer Stephanoceros millsii and its potential origins of secretion, the maternal vitellarium and embryonic integument. We also explored secretory activity in the larval and adult integuments to determine whether activity changes during ontogeny. The eggshell consists of a single layer with two sublayers: an external granular sublayer apparently derived from the maternal vitellarium, and an internal flocculent sublayer secreted by the embryonic integument that may form a hatching membrane or glycocalyx. Secretory activity remains high in both the larva and adult and appears to be the source of the thickening glycocalyx. Altogether, the subitaneous eggshell of S. millsii is the thinnest among monogonont rotifers. Thin eggshells may have evolved in response to the added protection provided by the mother’s extracorporeal tube.     

Influence of the evolution of viviparity on eggshell morphology in the lizard Lerista bougainvillii

In reptiles, the evolutionary transition from egg-laying to live-bearing is thought to involve a gradual increase in the duration of egg retention, with progressively more development occurring prior to oviposition, and culminating in the birth of fully developed offspring. However, prolonging the retention of fully-shelled eggs within the oviducts may pose serious gas-exchange problems for the embryos. Thus, evolutionary increases in the period of intrauterine retention may require correlated decreases in the thickness of eggshells and/or their degree of calcification to allow for adequate embryonic gas exchange. To test this evolutionary model, eggs of three distinct reproductive forms of the scincid lizard Lerista bougainvillii were examined to determine the evolutionary relationships between the thickness of the shell membrane, degree of eggshell calcification, and the duration of uterine egg retention. These comparisons revealed the predicted pattern of correlated shifts in eggshell morphology and embryonic stage at oviposition. Evolutionary increases in the duration of egg retention were accompanied by decreases in the thickness of the eggshell membrane and degree of eggshell calcification. This evolutionary model suggests that there may be a tradeoff between the advantages of extended egg retention and the disadvantages of a thinner eggshell. On the basis of this tradeoff, I propose that oviparous taxa with relatively thin eggshells may be preadapted to evolve viviparity. Comparative examination of the limited data available on eggshell thickness in lizards supports this possibility. © 1996 Wiley-Liss, Inc.     

Inheritance and variation in eggshell patterning in the great tit Parus major

The inheritance of patterns on avian eggshells is central to understanding the evolution of traits such as egg mimicry (e.g. in cuckoos). Yet little is known about the inheritance, or indeed function, of eggshell patterns. It has long been believed that the evolution of eggshell pattern mimicry required that patterns be determined by genes situated on the female-specific W chromosome. However, it has never been demonstrated for any bird that egg pattern traits (rather than ground colour) are female sex linked, or indeed that they are inherited. We studied the inheritance of three measures of egg-pigment patterns in a wild great tit population. Egg patterns were female specific but unrelated to female attributes such as age or condition and showed only weak environmental effects. Eggs of daughters resembled those of both their mothers and maternal grandmothers, but not of their paternal grandmothers. We conclude that this is the first demonstration of female sex-linked inheritance of avian eggshell patterning, so raising the probability that such a system operates in egg mimics and their hosts.     

Helicoidal architecture of fish eggshell

Previous publications show arced patterns in electron micrographs of either microfibrils or canals in sectioned fish eggshells, but these have been misinterpreted. We show here that such patterns in the inner layer of cod (Gadus morrhua), plaice (Pleuronectes platessa) and trout (Salmo gairdneri) eggs arise from a helicoidal structure. This consists of a laminate of protein microfibrils, with the direction of ply processing like the steps of a spiral staircase and with the same sense as a left-handed corkscrew. Mechanically, this is an ideal way to strengthen a spherical shell, to resist deforming forces equally from any direction. Radial canals which traverse this layer are forced into flattened and twisted ribbons. Both the helicoidal microfibrillar structure and the canal shape in fish eggshells show remarkable convergent evolution with similar structures in insect cuticles. Trout eggs were resistant to deforming forces as high as 380,000 N/m². Fish eggshells, like those of many other organisms, are mechanically well designed.     

Avian mitochondrial genomes retrieved from museum eggshell

Avian eggshell is a bio‐ceramic material with exceptional properties for preserving DNA within its crystalline structure, presenting an opportunity to retrieve genomic information from extinct or historical populations of birds. However, intracrystalline DNA has only been recovered from the large, thick eggshell of palaeognaths; members of their more‐diverse sister group (neognaths) lay smaller, thinner eggs that may not exhibit the same propensity for DNA preservation. Here, we use three 40–60‐year‐old museum eggshell specimens of Australian neognath birds to determine the minimum mass of eggshell from which intracrystalline DNA can be retrieved, and to characterize the yield and quality of such DNA. In doing so, we describe the first protocol for successful extraction of intracrystalline DNA from neognath eggshells, with the view to unlocking the potential of vast museum egg collections for genetic research. We were able to retrieve DNA fragments over 200 bp in length from 10 mg of eggshell powder from all three specimens, and demonstrate that expanding the existing blow‐hole can allow sufficient material to be collected for DNA extraction while minimizing damage to the appearance and structural integrity of the egg. Furthermore, we were able to reconstruct near‐complete mitochondrial genomes at a coverage of 40‐83X through shotgun sequencing of these extracts on three NextSeq lanes. Given the current extinction and extirpation rates of many avian species world‐wide, genetic data from eggshell could provide a rapid and cost‐effective approach to examining temporal changes in avian diversity, which is not only becoming crucial for conservation management, but also serve to deepen our understanding of genome‐wide evolutionary processes.     

The proteome of the insoluble Schistosoma mansoni eggshell skeleton

In schistosomiasis, the majority of symptoms of the disease is caused by the eggs that are trapped in the liver. These eggs elicit an immune reaction that leads to the formation of granulomas. The eggshell, which is a rigid insoluble structure built from cross-linked proteins, is the site of direct interaction between the egg and the immune system. However, the exact protein composition of the insoluble eggshell was previously unknown. To identify the proteins of the eggshell of Schistosoma mansoni we performed LC-MS/MS analysis, immunostaining and amino acid analysis on eggshell fragments. For this, eggshell protein skeleton was prepared by thoroughly cleaning eggshells in a four-step stripping procedure of increasing strength including urea and SDS to remove all material that is not covalently linked to the eggshell itself, but is part of the inside of the egg, such as Reynold’s layer, von Lichtenberg’s envelope and the miracidium. We identified 45 proteins of which the majority are non-structural proteins and non-specific for eggs, but are house-keeping proteins that are present in large quantities in worms and miracidia. Some of these proteins are known to be immunogenic, such as HSP70, GST and enolase. In addition, a number of schistosome-specific proteins with unknown function and no homology to any known annotated protein were found to be incorporated in the eggshell. Schistosome-specific glycoconjugates were also shown to be present on the eggshell protein skeleton. This study also confirmed that the putative eggshell protein p14 contributes largely to the eggshell. Together, these results give new insights into eggshell composition as well as eggshell formation. Those proteins that are present at the site and time of eggshell formation are incorporated in the cross-linked eggshell and this cross-linking does no longer occur when the miracidium starts secreting proteins.     

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.