Skeletal ontogeny of Monodelphis domestica (Mammalia: Didelphidae) : quantifying variation, variability, and technique bias in ossification sequence reconstruction

The field of evolutionary developmental biology (evo-devo) focuses on understanding the evolution of ontogeny and mechanisms of evolutionary change. Recently, taxonomic comparisons of the sequence of skeletal ossification have become prominent in evo-devo. However, most of these studies fail to consider two major issues: how the technique used to assay ossification and ontogenetic variation and variability may affect comparisons among taxa. This study focuses on the onset of ossification in the skeleton of Monodelphis domestica and quantifies the affects of variation, variability, and technique bias on reconstructions of ontogeny. Previous comparisons among mammalian taxa have used both computed tomography (CT) and clearing-and-staining (CS) to assess the presence or absence of skeletal elements (i.e., skeletal maturity). In this study, CT and CS were used on the same specimen to compare how these methods assess skeletal maturity. The comparisons of the same individual under reveal significant differences in how skeletal maturity is assessed by CT and CS techniques. Further, significant biases were recovered between techniques. CT is more likely to reveal cranial elements that CS does not, whereas CS is more likely to reveal appendicular elements that CT does not. To assess levels of variation and variability, Ontogenetic Sequence Analysis (OSA) was used to characterize the ontogeny of Monodelphis domestica. This revealed significant levels of variation with over 800 different ontogenetic pathways recovered for the onset of ossification of all skeletal elements studied. Additionally, high levels of variability were also reconstructed because the majority of specimens were found to exhibit non-modal ontogenetic sequences. This variability is more highly concentrated in the sequence of cranial ossification, suggesting potential modularity in ontogenetic variation and variability. Finally, OSA revealed that technique bias could importantly affect reconstructions of skeletal ossification sequences because no identical sequences were recovered by the CT and CS datasets. The results of this study demonstrate the importance of considering the primary nature of developmental studies, the specimen. Only by recognizing and quantifying the complexities of evo-devo research, especially natural variation and methodological biases, can more complete understandings of the evolution of ontogeny be had.