Mammalian limb morphology: the role of the crawl in determining marsupial limb integration, development, and evolution

Marsupials and eutherians, though closely related sister taxa, differ greatly in the variety of morphologies found within their groups. This inequality has largely been argued to be due to the reproductive differences. The crawl most marsupial neonates complete after birth has constrained the evolution of the forelimb and the group as a whole. However, the hind limb, which does not participate in the crawl, may have “escaped” the crawl’s constraint. This strong selective pressure to develop the forelimb early has also led to a decrease in integration (i.e. covariance) between the serially homologous elements of the limb. While limb integration has been well studied in adults, there exists study quantifying developmental integration in any system, which is unfortunate as it is important to examine integration across contexts (genetic, developmental, functional) in order to understand the evolution of morphologies. The purpose of this study is to determine both if marsupials are more likely to adapt their hind limbs than their forelimbs as well as to determine developmental patterns of limb integration to better understand the role of the crawl in marsupial evolution. To determine whether or not marsupials are more likely to adapt their hind limbs, length and width measurements were taken from the skeletal elements of the girdles, stylopod, zeugopod, and autopod from 196 therian taxa (82 marsupials, 114 eutherians). This data were then incorporated into principal component analyses and correlation matrices compared with the species’ functional group. Marsupials were found to be less likely to specialize their forelimbs than eutherians and while also more likely to specialize their hind limbs than forelimbs, still less likely to specialize their hind limbs than eutherians. In order to determine developmental integration, embryos at different but comparable stages were cleared and stained to visualize bone and cartilage growth from three marsupials (Monodelphis domestica, Sminthopsis macroura, Trichosurus vulpecula) and one eutherian (M. musculus). A reflex scope was used to place four points on the stylopod, zeugopod, and autopod and these points were used to determine gross length and width for each element. A combination of correlation and covariance matrices, Mantel’s test, and Mann-Whitney U tests were used to determine the integration patterns, both within and between the limbs, for each embryonic stage. Integration patterns significantly differed between stages and for marsupials, between limb integration decreased over time. Overall this study found that the hind limb of marsupials has been constrained by the crawl and that developmental integration changes between stages, suggesting a post-birth period of limb flexibility in both marsupials and eutherians