The sandwich structure of keratinous layers controls the form and growth orientation of chicken rhinotheca.

The upper beak bone of birds is known to be overlain by the rhinotheca, which is composed of the horny sheath of keratinous layers. However, the details of the structure and growth pattern of the rhinotheca are yet to be understood. In this study, the microstructure of the rhinotheca from chicken specimens of different growth stages (ranging from 1 to ~ 80 days old) was analyzed using a combination of thin section and scanning electron microscopy observations, and small-angle X-ray scattering analysis. We found that the rhinotheca comprises three different layers - outer, intermediate, and inner layers - throughout its growth. The outer layer arises from the proximal portion of the beak bone and covers the dorsal surface of the rhinotheca, whereas the intermediate and inner layers originate in the distal portion of the beak bone and underlie the outer layer. This tri-layered structure of the rhinotheca was also observed in wild bird specimens (grey wagtail, king quail, and brown dipper). On the median plane, micro-layers making up the outer and inner layers are bedded nearly parallel to the rostral bone at the base. However, more distally positioned micro-layers of the outer layer are more anteverted distally. The micro-layers of the intermediate layer are bedded nearly perpendicular to those of the outer and inner layers on the median plane. The growth of micro-layers in the intermediate layer adds thickness to the rhinotheca, which causes the difference in profile between the beak bone and the rhinotheca in the distal portion of the beak. Moreover, the entire intermediate layer grows distally as new proximal micro-layers form. The outer layer is dragged distally by the intermediate layer as a result of its distal growth, for the three layers are closely packed to each other at their boundaries. Furthermore, the occurrence of the intermediate and inner layers in the distal portion of the rostral bone may be because the distal end of the beak is frequently used and worn, and the rhinotheca therefore needs to be replaced more frequently at the distal end. The rhinotheca structure described here will be an important and useful factor in the reconstruction of the beaks of birds in extinct taxa.