Peer Review #1 of "Digital dissection of the pelvis and hindlimb of the red-legged running frog, Phlyctimantis maculatus, using Diffusible Iodine Contrast Enhanced computed microtomography (DICE μCT) (v0.1)"

Abstract

Background. The current study applies both traditional and Diffusible Iodine Contrast Enhanced computed microtomography (DICE μCT) techniques to reveal the musculoskeletal anatomy of Phlyctimantis maculatus. DICE μCT has emerged as a powerful tool to visualise intricate musculoskeletal anatomy. By generating 3D digital models, anatomical analyses can be conducted non-destructively, preserving the in situ 3D topography of the system, therefore eliminating some of the drawbacks associated with traditional methods. We aim to describe the musculature of the spine, pelvis, and hindlimb, compare the musculoskeletal anatomy and pelvic morphology of P. maculatus with functionally diverse frogs, and produce 3D digital anatomy reference data. Method. An adult frog was stained using an aqueous Lugol's solution and scanned in a SkyScan1176 in vivo µCT scanner. Scan images were reconstructed, resampled, and digitally segmented to produce a 3D model. A further adult female frog was dissected traditionally for visualisation of tendinous insertions. Results. Our work revealed three main findings: 1) P. maculatus has similar gross muscular anatomy to Rana catesbeiana (bullfrog) but is distinct from those species that exhibit ancestral traits (leopelmids) and those that are highly specialised (pipids), 2) P. maculatus's pelvic anatomy best fits the description of Emerson's walking/hopping pelvic morphotype IIA, and 3) a split in the semimembranosus and gracilis major muscles is consistent with the reported myology in other anuran species. Discussion. While DICE μCT methods were instrumental in characterising the 3D anatomy, traditional dissection was still required to visualise important structures such as the knee aponeurosis, tendinous insertions, and fasciae. Nonetheless, the anatomical data presented here marks the first detailed digital description of an arboreal and terrestrial frog. Further, our digital model presents P. maculatus as a good frog model system and as such has formed a crucial platform for further functional analysis within the anuran pelvis and hindlimb.