The calcitic layers of the eggshells of archosaurs (including crocodilians and birds) and turtles are composed of distinctive crystalline structures known as eggshell units. Those growing from the shell membrane are called primary eggshell units (PEUs), while those forming within the calcitic layer are called secondary eggshell units (SEUs). Although rare in modern bird eggs, SEUs are common in dinosaur eggs. Due to the lack of in-depth research on this structure, however, scientists are uncertain whether they are biogenic or abiogenic in origin.

To tackle this issue, an international research team led by Dr. Zhang Shukang and postdoctoral researcher Dr. Choi Seung from the Institute of Vertebrate Paleontology and Paleoanthropology (IVPP) of the Chinese Academy of Sciences, has conducted a comprehensive study of SEUs in dinosaur eggshells using techniques including electron backscatter diffraction (EBSD), polarized light microscopy (PLM), and scanning electron microscopy (SEM). For comparison, they examined some eggshells of modern birds, turtles, and crocodiles.

The study revealed that the crystallographic characteristics of dinosaur egg SEUs were almost identical to those of PEUs. Notably, they also matched the crystallographic characteristics of the SEUs in modern turtle and crocodile eggshells.

Additionally, the SEUs in dinosaur eggs exhibited numerous grooves and vesicles, similar to those in modern bird eggshell units. These grooves and vesicles were interpreted as remnants of spaces left by the degradation of organic matrix fibers during fossilization. These findings, published in the journal Science Advances, strongly suggest that the SEUs in dinosaur eggs are biogenic structures.

Furthermore, in some dinosaur eggshells with well-developed pore canals, the researchers discovered that the SEUs either overlapped the PEUs or grew inside the pore canals. Although the growth of these SEUs was undisturbed, the c-axes of their calcite crystals still extended parallel to the growth direction of the eggshell.

This phenomenon challenges the “competition hypothesis” derived from modern bird eggshells, and it reveals that the c-axis orientation of eggshell units is likely regulated by organic matrix fibers rather than the result of the competitive growth between adjacent calcite crystals.

Notably, when it comes to evolutionary dynamics, the researchers discovered that SEUs were present in the eggshells of sauropods, hadrosaurs, and potential basal Tetanurae while being conspicuously rare in those of maniraptorans (including birds), suggesting that the formation mechanism may have changed during the evolution of eggshells from non-avian theropods to modern birds.

From a macroevolutionary perspective, SEUs appear in turtles, crocodilians, as well as ornithischian, sauropod, and theropod dinosaur lineages, indicating that these structures may have evolved independently within the calcitic layers of the eggshells. However, at the molecular level of biomineralization, the possibility of a deep homology of SEUs across all these lineages cannot be ruled out.

This study sheds light on the characterization of SEUs in dinosaur eggshells and offers new insights into the evolutionary plasticity of eggshell structures.