Biogenic origin of secondary eggshell units in dinosaur eggshells elucidates lost biomineralization process in maniraptoran dinosaurs

Although rarely seen in modern avian eggshells, secondary eggshell units are remarkable structures in non-avian dinosaur eggshells and provide paleobiological information. However, the origin of secondary eggshell units has rarely been studied, which has resulted in controversies in paleontology because abiogenic origin has also been suggested for the formation of secondary eggshell units. Here, we show that secondary eggshell units of non-avian dinosaur eggshells are biogenic structures using a combination of microscopic techniques, mainly electron backscatter diffraction (EBSD) and scanning electron microscope (SEM). The features of secondary eggshell units suggest that the formation mechanism of non-maniraptoran dinosaur eggshells is comparable to that of turtle and crocodile eggshells. Matrix fibers appear to be the initiators of secondary eggshell units. A weakening trend of secondary eggshell units toward non-avian maniraptoran dinosaurs supports that maniraptoran dinosaurs evolved a more organized physiological mechanism of eggshell formation.

Methods
The thin sections are prepared following the usual thin section preparation methods of geological studies and the images were obtained using a polarized light microscope.

EBSD analysis of fossil eggshells followed an established protocol (see Choi et al. 2023, Journal of Vertebrate Paleontology).