Background Mammalian skeletons are largely formed before birth. Heterochronic changes in skeletal formation can be investigated by comparing the order of ossification for different elements of the skeleton. Due to the challenge of collecting prenatal specimens in viviparous taxa, opportunistically collected museum specimens provide the best material for studying prenatal skeletal development across many mammalian species. Previous studies have investigated ossification sequence in a range of mammalian species, but little is known about the pattern of bone formation in Carnivora. Carnivorans have diverse ecologies, diets, and biomechanical specializations and are well-suited for investigating questions in evolutionary biology. Currently, developmental data on carnivorans is largely limited to domesticated species. To expand available data on carnivoran skeletal development, we used micro-computed tomography (micro-CT) to non-invasively evaluate the degree of ossification in all prenatal carnivoran specimens housed in the Harvard Museum of Comparative Zoology. By coding the presence or absence of bones in each specimen, we constructed ossification sequences for each species. Parsimov-based genetic inference (PGi) was then used to identify heterochronic shifts between carnivoran lineages and reconstruct the ancestral ossification sequence of Carnivora.Results We used micro-CT to study prenatal ossification sequence in six carnivora species: Eumetopias jubatus (Steller sea lion, n = 6), Herpestes javanicus (small Indian mongoose, n = 1), Panthera leo (lion, n = 1), Urocyon cinereoargenteus (gray fox, n = 1), Ursus arctos arctos (Eurasian brown bear, n = 1), and Viverricula indica (small Indian civet, n = 5). Due to the relatively later stage of collection for the available specimens, few heterochronic shifts were identified. Ossification sequences of feliform species showed complete agreement with the domestic cat. In caniforms, the bear and fox ossification sequences largely matched the dog, but numerous heterochronic shifts were identified in the sea lion.Conclusions We use museum specimens to generate cranial and postcranial micro-CT data on six species split between the two major carnivoran clades: Caniformia and Feliformia. Our data suggest that the ossification sequence of domestic dogs and cats are likely good models for terrestrial caniforms and feliforms, respectively, but not pinnipeds.

Turbinals are key bony elements of the mammalian nasal cavity, involved in heat and moisture conservation as well as olfaction. While turbinals are well known in some groups, their diversity is poorly understood at the scale of placental mammals, which span 21 orders. Here, we investigated the turbinal bones and associated lamellae for one representative of each extant order of placental mammals. We segmented and isolated each independent turbinal and lamella and found an important diversity of variation in the number of turbinals, as well as their size, and shape. We found that the turbinal count varies widely, from zero in the La Plata dolphin, (Pontoporia blainvillei) to about 110 in the African bush elephant (Loxodonta africana). Multiple turbinal losses and additional gains took place along the phylogeny of placental mammals. Some changes are clearly attributed to ecological adaptation, while others are probably related to phylogenetic inertia. In addition, this work highlights the problem of turbinal nomenclature in some placental orders with numerous and highly complex turbinals, for which homologies are extremely difficult to resolve. Therefore, this work underscores the importance of developmental studies to better clarify turbinal homology and nomenclature and provides a standardized comparative framework for further research.

The length of the snout in mammals has important evolutionary consequences for the functional systems housed within the rostrum. However, whether increased snout lengths lead to expanded olfactory performance has rarely been examined. Here, we investigate inner rostral function among 10 species of myrmecophagous (ant- and/or termite-eating) placental mammals and 10 closely related species. We use nondestructive computed tomography scanning methods to characterize inner rostral function based on the underlying morphology of the turbinal bones in the nasal cavity. Three approaches were chosen to address this question, including the quantification of functional turbinal surface area, the quantification of functional turbinal three-dimensional complexity, and geometric morphometrics. By including non-model species from several different mammalian orders, we were able to extend the discussion surrounding turbinal homologies to comparisons across mammals. Our results show no increased olfactory function in all myrmecophagous species relative to their sister taxa, which suggests that there is no trade-off for increased olfactory capabilities in myrmecophagous species with elongated snouts. We found no evidence of convergence in turbinal morphology among all five myrmecophagous lineages. However, we found evidence of morphological convergence in the turbinals between the giant armadillo and the aardvark, suggesting a more complex interplay between the evolution of turbinal morphology and ecological correlates. While myrmecophagy alone may not be a strong enough ecological signal to overcome phylogenetic and developmental constraints, we suggest that this might be the case at the intersection of this dietary specialization with a primarily underground lifestyle where odorants may be difficult to detect.

The evolution of endothermy in vertebrates is a major research topic in recent decades that has been tackled by a myriad of research disciplines including paleontology, anatomy, physiology, evolutionary and developmental biology. The ability of most mammals to maintain a relatively constant and high body temperature is considered a key adaptation, enabling them to successfully colonize new habitats and harsh environments. It has been proposed that in mammals the anterior nasal cavity, which houses the maxilloturbinal, plays a pivotal role in body temperature maintenance, via a bony system supporting an epithelium involved in heat and moisture conservation. The presence and the relative size of the maxilloturbinal has been proposed to reflect the endothermic conditions and basal metabolic rate in extinct vertebrates. We show that there is no evidence to relate the origin of endothermy and the development of some turbinal bones by using a comprehensive dataset of µCT-derived maxilloturbinals spanning most mammalian orders. Indeed, we demonstrate that neither corrected basal metabolic rate nor body temperature significantly correlate with the relative surface area of the maxilloturbinal. Instead, we identify important variations in the relative surface area, morpho-anatomy, and complexity of the maxilloturbinal across the mammalian phylogeny and species ecology.

For the first 100+ million years of their evolutionary history, the majority of mammals were very small, and many exhibited relatively generalized locomotor ecologies. Among extant mammals, small-bodied, generalist species share similar hindlimb bone morphology and locomotor mechanics, but details of their musculature have not been investigated. To examine whether hindlimb muscle architecture properties are also similar, we dissected hindlimb muscles of the gray short-tailed opossum ( Monodelphis domestica ) and aggregated muscle properties from the literature for three other small-bodied mammals ( Mus musculus , Rattus norvegicus , Cavia porcellus ). We then studied hindlimb musculature from a whole-limb perspective and by separating the limb into nine anatomical regions. The region analysis explained substantially more variance in the data (r 2 : 0.601 > 0.074) but only detected six statistically significant pairwise species differences in muscle architecture properties. This finding suggests either deep conservation of therian hindlimb muscle properties or, more likely, a biomechanical constraint imposed by small body size. In addition, we find specialization for either large force production (i.e., PCSA) or longer active working ranges (i.e. long muscle fascicles) in proximal limb regions but neither specialization in more distal limb regions. This functional pattern may be key for small mammals to traverse across uneven and shifting substrates, regardless of environment. These findings are particularly relevant for researchers seeking to reconstruct and model soft tissue properties of extinct mammals during the early evolutionary history of the clade.

Mammals are characterized by rich diversity in hindlimb morphology, locomotory specialization, and ecological habit. As mammalian species spread throughout the globe occupying novel environments, some modified their hindlimbs to reflect the challenges of these new environments. However, it is unclear whether disparate limb morphology was the driver or the result of this ecological diversification. Exploring the relationship between ecology and morphology during the transition from the earliest synapsids (mammalian ancestors) to basal mammals requires a comparative framework connecting morphology to locomotory function. This transition included a postural shift in hindlimb orientation. The earliest synapsids had limbs abducted to their sides that operated with the same sprawling movement common in many extant lizards and salamanders. However, the first mammals had limbs rotated underneath the body, upright, that operated primarily in a parasagittal plane of movement. Here, two species (the extinct sphenacodontid, Dimetrodon milleri, and the extant opossum, Monodelphis domestica) are used to represent an early and late character state, respectively, of hindlimb function during this transition. The opossum was stained using phosphomolybdic acid (PMA) for soft tissue contrast, and both specimens were scanned using microcomputed tomography (μCT). With three-dimensional modeling software, musculoskeletal models were created for each specimen to interpret functional morphology of the hindlimb at two stages during this evolutionary transition. The results here show that basal synapsids had increased mechanical advantage during sprawling movements (higher abduction-adduction moment arms) while the proximal hindlimb of ancestral therian mammals functioned as joint-stabilizers during parasagittal stances (negative sloping flexion-extension moment arms that cross through zero). Sensitivity analyses of two model- building parameters (neutral pose orientation and muscle attachment site) confirm that differences in functional interpretations are not caused by limitations during model construction.