Insights from Contrast‐Enhanced MicroCT into the Formation of the Marsupial Hindlimb

Abstract

As the sister clade to placental mammals, marsupials offer insight into a completely separate experiment in mammalian evolution. As with many placental groups (e.g., ungulates, rodents, and lagomorphs), several marsupial groups like kangaroos and bandicoots evolved elongate limbs with reduced digit numbers for locomoting in open, arid habitats. However, unlike most placental lineages which specialized their limbs from a cursorial ancestor, marsupial lineages evolved their specialized hindlimbs from a grasp-climbing ancestor that was morphologically similar to extant didelphid marsupials. To examine the developmental changes involved in marsupial limb diversification we used contrast-enhanced microcomputed tomography (CE microCT) to characterise postnatal limb development in the grey short-tailed opossum and the tammar wallaby. We compared phosphotungstic acid (PTA) and Ruthenium Red (RR) as contrast agents for differentiating bone and cartilage in microCT scans. PTA staining was effective at contrasting soft tissues but it was difficult to differentiate bone and cartilage tissues without time-consuming manual segmentation. Preliminary results show that RR stains soft-tissues much more slowly than PTA and skin removal is required for stain penetration of postnatal specimens. Comparison of limb segment proportions between the opossum and tammar wallaby provides evidence that macropods undergo a pronounced late postnatal elongation of the distal hindlimb elements, especially the calcaneus, metatarsals and phalanges. Digital segmentation of the hindfoot of an early postnatal PTA-stained specimen of the tammar wallaby provides evidence that a cartilaginous condensation anterior to the reduced second and third toe is morphologically more similar to a prehallax than a first toe. Potential implications for the evolvability of tetrapod digit number and the ancestral constraints of arboreality on limb adaptation are considered.