Histological evidence for muscle insertion in extant amniote femora: implications for muscle reconstruction in fossils

Since the 19th century, identification of muscle attachment sites on bones has been important for muscle reconstructions, especially in fossil tetrapods, and therefore has been the subject of numerous biological and paleontological studies. At the microscopic level, in histological thin sections, the only features that can be used reliably for identifying tendonbone or muscletendon-bone interactions are Sharpey's fibers. Muscles, however, do not only attach to the bone indirectly with tendons, but also directly. Previous studies failed to provide new indicators for muscle attachment, or to address the question of whether muscles with direct attachment can be identified histologically. However, histological identification of direct muscle attachments is important because these attachments do not leave visible marks (e.g. scars and rugosities) on the bone surface. We dissected the right hind limb and mapped the muscle attachment sites on the femur of one rabbit (Oryctolagus cuniculus), one Alligator mississippiensis, and one turkey (Meleagris cuniculus). We then extracted the femur and prepared four histological thin sections for the rabbit and the turkey and five histological thin sections for the alligator. Sharpey's fibers, vascular canal orientation, and a frayed periosteal margin can be indicators for indirect but also direct muscle attachment. Sharpey's fibers can be oriented to the cutting plane of the thin section at high angles, and two Sharpey's fibers orientations can occur in one area, possibly indicating a secondary force axis. However, only about 60% of mapped muscle attachment sites could be detected in thin sections, and frequently histological features suggestive of muscle attachment occurred outside mapped sites. While these insights should improve our ability to successfully identify and reconstruct muscles in extinct species, they also show the limitations of this approach.