Perichordal Vertebral Column Formation in Rana kobai

The vertebral column of anurans exhibits morphological diversity that is often used in phylogenetic studies. The family Ranidae is one of the ecologically most successful groups of anurans, with the genus Rana being distributed broadly in Eurasia. However, there are relatively sparse detailed studies on the development of the vertebral column in Rana species, and images of the entire axial skeleton have seldom been illustrated till date. Here, we provide an illustrated description on the development of the entire vertebral column in Rana kobai, a Japanese small frog from the Amami Islands. Our observation of double-stained skeletal specimens revealed that in R. kobai, the original atlas and the first dorsal are fused into one vertebra, and the ninth neural arch is fused with the tenth arch in half of the examined larvae. Anuran vertebral column development is classified into two modes, perichordal and epichordal. Rana species undergo the typical perichordal mode of centrum formation. Kemp and Hoyt (1969) described that centrum formation in R. pipiens starts from a saddle-shaped bone on the dorsal half of the notochord. Nevertheless, our detailed observations revealed that centrum ossification initially emerges at the base of the paired neural arches and then forms the saddle-shaped bone. In Xenopus, a species with epichordal centra, centrum formation starts from a pair of ovoid bone elements at the base of the neural arches. Overall, our results imply that centrum ossification starts from the base of neural arches in anurans, irrespective of whether it is perichordal or epichordal. Our observations also revealed the presence of the crescent-shaped cartilage domain in the intervertebral region in R. kobai. The location of the crescent-shaped domain in R. kobai is consistent with that of the intercentrum in Ichthyostega and several temnospondyls. Based on our observations, we propose a hypothesis on the difference between perichordal and epichordal modes in light of evolution.