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Fossil Eggshell Cuticle Elucidates Dinosaur Nesting Ecology

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Fossil Eggshell Cuticle Elucidates Dinosaur Nesting Ecology

Tzu-Ruei Yang et al. PeerJ.

Abstract

The cuticle layer consisting mainly of lipids and hydroxyapatite (HAp) atop the mineralized avian eggshell is a protective structure that prevents the egg from dehydration and microbial invasions. Previous ornithological studies have revealed that the cuticle layer is also involved in modulating the reflectance of eggshells in addition to pigments (protoporphyrin and biliverdin). Thus, the cuticle layer represents a crucial trait that delivers ecological signals. While present in most modern birds, direct evidence for cuticle preservation in stem birds and non-avian dinosaurs is yet missing. Here we present the first direct and chemical evidence for the preservation of the cuticle layer on dinosaur eggshells. We analyze several theropod eggshells from various localities, including oviraptorid Macroolithus yaotunensis eggshells from the Late Cretaceous deposits of Henan, Jiangxi, and Guangdong in China and alvarezsaurid Triprismatoolithus eggshell from the Two Medicine Formation of Montana, United States, with the scanning electron microscope (SEM), electron probe micro-analysis (EPMA), and Raman spectroscopy (RS). The elemental analysis with EPMA shows high concentration of phosphorus at the boundary between the eggshell and sediment, representing the hydroxyapatitic cuticle layer (HAp). Depletion of phosphorus in sediment excludes the allochthonous origin of the phosphorus in these eggshells. The chemometric analysis of Raman spectra collected from fossil and extant eggs provides further supportive evidence for the cuticle preservation in oviraptorid and probable alvarezsaurid eggshells. In accordance with our previous discovery of pigments preserved in Cretaceous oviraptorid dinosaur eggshells, we validate the cuticle preservation on dinosaur eggshells through deep time and offer a yet unexplored resource for chemical studies targeting the evolution of dinosaur nesting ecology. Our study also suggests that the cuticle structure can be traced far back to maniraptoran dinosaurs and enhance their reproductive success in a warm and mesic habitat such as Montana and southern China during the Late Cretaceous.

Keywords: Chemometrics; Dinosaur reproduction; Elemental analysis; Raman spectroscopy; Soft tissue preservation.

Conflict of interest statement

The authors declare there are no competing interests.

Figures

Figure 1
Figure 1. Cross-sectional view, SEM images, and Raman imaging and spectrum of a Gallus gallus domesticus egg and eggshell.
The Raman image and spectrum were collected using 532 nm excitation wavelength and a 50x objective with RI. (A) The generalized anatomy of an egg. (B) The chicken eggshell comprises three crystalline layers, including the mammillary layer, prismatic layer, and external layer. The cuticle layer overlying the calcareous eggshell is further divided to two layers, including a HAp inner layer and a proteinaceous outer layer. The shell membrane, namely membrane testacea, is also characterized by two layers. (C) SEM image of the cuticle on the surface of the Gallus eggshell, showing a patchy and cracked pattern. (D) SEM image of the radial section of the Gallus eggshell. The white arrow indicates the cuticle layer that lies on the calcitic eggshell. (E) Raman chemical image with peak targeting at 967 cm−1 which is attributed to HAp. The yellow area represents the patchy distribution of the inner HAp cuticle layer on the Gallus eggshell. The calcitic eggshell that is not covered by the inner HAp cuticle layer is shown in the brown area. The dotted circle corresponds to the spectrum shown in (F). (F) Spectrum collected in the dotted-circle area of the Raman chemical image shown in (E). Two significant peaks at 967 cm−1 and 1,087 cm−1 represent HAp and calcite, respectively. Credit of the SEM images and drawings: Tzu-Ruei Yang (Universität Bonn).
Figure 2
Figure 2. Raman spectra and chemometric analysis.
(A) Raman spectra derived from chicken, crocodilian, fossil dinosaur eggshells, and surrounding sediments. The peaks around 972–986 cm−1 and 1,063–1,097 cm−1 are marked by yellow bars, indicating the calcite and phosphate bonds. (B) The principle component chemometric analysis on the spectral area of 800–1,200 cm−1 shown in (A) demonstrates significant disparities between dinosaur/chicken eggshells, crocodilian eggshells, and sediments. Credit of the drawings: Tzu-Ruei Yang (Universität Bonn).
Figure 3
Figure 3. Microscopic images of Macroolithus yaotunensis eggshell from Henan, Jiangxi, and Guangdong and their line-scan spectra.
(A) The Macroolithus yaotunensis eggshell (NMNS CYN-2004-DINO-05-I) from Jiangxi under the polarized light microscope. The red arrows indicate the indistinct boundary between the sediment and an enigmatic layer. This enigmatic layer probably was formed by the interaction between organics of the egg and surrounding sediment. The location of the EPMA line-scan spectra in (D) is indicated by the white solid line. In this eggshell image, a smooth boundary between the prismatic and mammillary layers (PL and ML, seen as dark and light zones) is clearly observed. (B) SEM image of radial untreated fracture of the Macroolithus yaotunensis eggshell. The boundary between the sediment and prismatic layer is marked by yellow dashed line based on their distinct structural features. The boundary between ML and PL is marked by the red dashed line. The ML shows the distinct vertical mammillae structure. (C) Close-up image of the area in white box in (B). Possible preservation of cuticle is indicated by the white arrow, showing patchy and flaky structures similar to the cuticle on the modern chicken eggshell (Figs. 1C and 1D). (D, F, H) The microscopic image shows the radial cut section of studied Macroolithus yaotunensis eggshells seen in the SEM coupled with the EPMA and elemental line-scan track marked as black lines. (D) The spectra illustrate the distribution of Ca and P across the eggshell from the Hongcheng Basin in Jiangxi shown in (A), from innermost (left) to outermost, as indicated by the white line in the microscopic image. The Ca spectrum clearly marks the extent of the calcitic eggshell. A significant peak that is marked by a black arrow demonstrates a relatively high concentration of P, indicating the possible preservation of the cuticular HAp layer. (G) The spectra illustrate the distribution of Ca and P across the eggshell from the Liguangqiao Basin in Henan, from innermost (left) to outermost. The Ca spectrum clearly marks the extent of the calcitic eggshell. A slightly higher amount of P near the interface between the eggshell and sediments, as indicated by a black arrow, is observed. (I) The spectra illustrate the distribution of Ca and P across the eggshell from the Nanxiong Basin in Guangdong, from innermost (left) to outermost. A significant peak that is marked by a black arrow demonstrates a relatively high concentration of P, indicating the possible preservation of the cuticular HAp layer. ML, mammillary layer; PL, prismatic layer. Credit of the SEM images and drawings: Tzu-Ruei Yang (Universität Bonn).
Figure 4
Figure 4. SEM images of the Triprismatoolithus eggshell and EPMA line-scan spectra.
(A) The radial cut section of studied Triprismatoolithus eggshell in the SEM coupled with the EPMA. (B) The probable flake-like cuticle structure atop the eggshell, marked by the white arrow. The red dotted line marks the outermost boundary of the prismatic layer. (C) An enigmatic structure (white arrow) protruding from the mammillary layer, possibly a fiber of the membrana testacea extending into the mammillary layer. (D) Enlargement of the possible fiber. (E) Line scans across eggshell in (A). The scan shows a high concentration of P at the boundary between the eggshell and surrounding sediment. Two other peaks of P were observed on the boundary between the external and prismatic layer and in the sediment. The P concentration does not differ between the mammillary and prismatic layers; however, the external layer showed a significantly lower concentration of P than the mammillary and prismatic layers. EL, external layer; ML, mammillary layer; PL, prismatic layer. Credit of the SEM images and drawings: Tzu-Ruei Yang (Universität Bonn).

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Grant support

This research is supported by the scholarship for studying abroad from the Ministry of Education, Taiwan and research grant from the Jurassic Foundation to Tzu-Ruei Yang. Tzu-Ruei Yang and P. Martin Sander are both funded by the Deutsche Forschungsgemein-schaft (DFG, German Research Foundation)—project number 348043586. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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