Postcranial ossification sequences in 24 therian mammals and three outgroup taxa were obtained using clear staining and computed tomography to test the hypothesis that the marsupial forelimb is developmentally accelerated, and to assess patterns of therian postcranial ossification. Sequence rank variation of individual bones, phylogenetic analysis, and algorithm-based heterochrony optimization using event pairs were employed. Phylogenetic analysis only recovers Marsupialia, Australidelphia, and Eulipotyphla. Little heterochrony is found within marsupials and placentals. However, heterochrony was observed between marsupials and placentals, relating to late ossification in hind limb long bones and early ossification of the anterior axial skeleton. Also, ossification rank position of marsupial forelimb and shoulder girdle elements is more conservative than that of placentals; in placentals the hind limb area is more conservative. The differing ossification patterns in marsupials can be explained with a combination of muscular strain and energy allocation constraints, both resulting from the requirement of active movement of the altricial marsupial neonates toward the teat. Peramelemorphs, which are comparatively passive at birth and include species with relatively derived forelimbs, differ little from other marsupials in ossification sequence. This suggests that ossification heterochrony in marsupials is not directly related to diversity constraints on the marsupial forelimb and shoulder girdle.

Mammals show a very low level of variation in vertebral count, particularly in the neck. Phenotypes exhibited at various stages during the development of the axial skeleton may play a key role in testing mechanisms recently proposed to explain this conservatism. Here, we provide osteogenetic data that identify developmental criteria with which to recognize cervical vs. noncervical vertebrae in mammals. Except for sloths, all mammals show the late ossification of the caudal-most centra in the neck after other centra and neural arches. In sloths with 8–10 ribless neck vertebrae, the caudal-most neck centra ossify early, matching the pattern observed in cranial thoracic vertebrae of other mammals. Accordingly, we interpret the ribless neck vertebrae of three-toed sloths caudal to V7 as thoracic based on our developmental criterion. Applied to the unusual vertebral phenotype of long-necked sloths, these data support the interpretation that elements of the axial skeleton with origins from distinct mesodermal tissues have repatterned over the course of evolution.

We analyzed a comprehensive data set of ossification sequences including seven marsupial, 13 placental and seven sauropsid species. Data are provided for the first time for two major mammalian clades, Chiroptera and Soricidae, and for two rodent species; the published sequences of three species were improved with additional sampling. The relative timing of the onset of ossification in 17 cranial elements was recorded, resulting in 136 event pairs, which were treated as characters for each species. Half of these characters are constant across all taxa, 30% are variable but phylogenetically uninformative, and 19% potentially deliver diagnostic features for clades of two or more taxa. Using the conservative estimate of heterochronic changes provided by the program Parsimov, only a few heterochronies were found to diagnose mammals, marsupials, or placentals. A later onset of ossification of the pterygoid with respect to six other cranial bones characterizes therian mammals. This result may relate to the relatively small size of this bone in this clade. One change in relative onset of ossification is hypothesized as a potential human autapomorphy in the context of the sampling made: the earlier onset of the ossification of the periotic with respect to the lacrimal and to three basicranial bones. Using the standard error of scaled ranks across all species as a measure of each element's lability in developmental timing, we found that ossification of early, middle, and late events are similarly labile, with basicranial traits the most labile in timing of onset of ossification. Despite marsupials and placental mammals diverging at least 130 Ma, few heterochronic shifts in cranial ossification diagnose these clades.