Philip G. Cox

Micro-computed tomography with iodine staining resolves the arrangement of muscle fibres

We illustrate here microCT images in which contrast between muscle and connective tissue has been achieved by means of staining with iodine. Enhancement is shown to be dependent on the concentration of iodine solution (I(2)KI), time in solution and specimen size. Histological examination confirms that the arrangement of individual muscle fibres can be visualised on the enhanced microCT images, and that the iodine accumulates in the muscle fibres in preference to the surrounding connective tissues. We explore the application of this technique to describe the fibrous structure of skeletal muscle, and conclude that it has the potential to become a non-destructive and cost-effective method for investigating muscle fascicle architecture, particularly in comparative morphological studies.

Functional Evolution of the Feeding System in Rodents

The masticatory musculature of rodents has evolved to enable both gnawing at the incisors and chewing at the molars. In particular, the masseter muscle is highly specialised, having extended anteriorly to originate from the rostrum. All living rodents have achieved this masseteric expansion in one of three ways, known as the sciuromorph, hystricomorph and myomorph conditions. Here, we used finite element analysis (FEA) to investigate the biomechanical implications of these three morphologies, in a squirrel, guinea pig and rat. In particular, we wished to determine whether each of the three morphologies is better adapted for either gnawing or chewing. Results show that squirrels are more efficient at muscle-bite force transmission during incisor gnawing than guinea pigs, and that guinea pigs are more efficient at molar chewing than squirrels. This matches the known diet of nuts and seeds that squirrels gnaw, and of grasses that guinea pigs grind down with their molars. Surprisingly, results also indicate that rats are more efficient as well as more versatile feeders than both the squirrel and guinea pig. There seems to be no compromise in biting efficiency to accommodate the wider range of foodstuffs and the more general feeding behaviour adopted by rats. Our results show that the morphology of the skull and masticatory muscles have allowed squirrels to specialise as gnawers and guinea pigs as chewers, but that rats are high-performance generalists, which helps explain their overwhelming success as a group.

Diffusible iodine-based contrast-enhanced computed tomography (diceCT) : an emerging tool for rapid, high-resolution, 3-D imaging of metazoan soft tissues.

Morphologists have historically had to rely on destructive procedures to visualize the three‐dimensional (3‐D) anatomy of animals. More recently, however, non‐destructive techniques have come to the forefront. These include X‐ray computed tomography (CT), which has been used most commonly to examine the mineralized, hard‐tissue anatomy of living and fossil metazoans. One relatively new and potentially transformative aspect of current CT‐based research is the use of chemical agents to render visible, and differentiate between, soft‐tissue structures in X‐ray images. Specifically, iodine has emerged as one of the most widely used of these contrast agents among animal morphologists due to its ease of handling, cost effectiveness, and differential affinities for major types of soft tissues. The rapid adoption of iodine‐based contrast agents has resulted in a proliferation of distinct specimen preparations and scanning parameter choices, as well as an increasing variety of imaging hardware and software preferences. Here we provide a critical review of the recent contributions to iodine‐based, contrast‐enhanced CT research to enable researchers just beginning to employ contrast enhancement to make sense of this complex new landscape of methodologies. We provide a detailed summary of recent case studies, assess factors that govern success at each step of the specimen storage, preparation, and imaging processes, and make recommendations for standardizing both techniques and reporting practices. Finally, we discuss potential cutting‐edge applications of diffusible iodine‐based contrast‐enhanced computed tomography (diceCT) and the issues that must still be overcome to facilitate the broader adoption of diceCT going forward.

Reviewing the Morphology of the Jaw‐Closing Musculature in Squirrels, Rats, and Guinea Pigs with Contrast‐Enhanced MicroCt

Rodents are defined by their unique masticatory apparatus and are frequently separated into three nonmonophyletic groups—sciuromorphs, hystricomorphs, and myomorphs—based on the morphology of their masticatory muscles. Despite several comprehensive dissections in previous work, inconsistencies persist as to the exact morphology of the rodent jaw‐closing musculature, particularly, the masseter. Here, we review the literature and document for the first time the muscle architecture noninvasively and in 3D by using iodine‐enhanced microCT. Observations and measurements were recorded with reference to images of three individuals, each belonging to one of the three muscle morphotypes (squirrel, guinea pig, and rat). Results revealed an enlarged superficial masseter muscle in the guinea pig compared with the rat and squirrel, but a reduced deep masseter (possibly indicating reduced efficiency at the incisors). The deep masseter had expanded forward to take an origin on the rostrum and was also separated into anterior and posterior parts in the rat and squirrel. The zygomaticomandibularis muscle was split into anterior and posterior parts in all the three specimens by the masseteric nerve, and in the rat and guinea pig had an additional rostral expansion through the infraorbital foramen. The temporalis muscle was found to be considerably larger in the rat, and its separation into anterior and posterior parts was only evident in the rat and squirrel. The pterygoid muscles were broadly similar in all three specimens, although the internal pterygoid was somewhat enlarged in the guinea pig implying greater lateral movement of the mandible during chewing in this species. Anat Rec,, 2011.

Finite element modelling of squirrel, guinea pig and rat skulls: using geometric morphometrics to assess sensitivity

Rodents are defined by a uniquely specialized dentition and a highly complex arrangement of jaw‐closing muscles. Finite element analysis (FEA) is an ideal technique to investigate the biomechanical implications of these specializations, but it is essential to understand fully the degree of influence of the different input parameters of the FE model to have confidence in the model’s predictions. This study evaluates the sensitivity of FE models of rodent crania to elastic properties of the materials, loading direction, and the location and orientation of the models’ constraints. Three FE models were constructed of squirrel, guinea pig and rat skulls. Each was loaded to simulate biting on the incisors, and the first and the third molars, with the angle of the incisal bite varied over a range of 45°. The Young’s moduli of the bone and teeth components were varied between limits defined by findings from our own and previously published tests of material properties. Geometric morphometrics (GMM) was used to analyse the resulting skull deformations. Bone stiffness was found to have the strongest influence on the results in all three rodents, followed by bite position, and then bite angle and muscle orientation. Tooth material properties were shown to have little effect on the deformation of the skull. The effect of bite position varied between species, with the mesiodistal position of the biting tooth being most important in squirrels and guinea pigs, whereas bilateral vs. unilateral biting had the greatest influence in rats. A GMM analysis of isolated incisor deformations showed that, for all rodents, bite angle is the most important parameter, followed by elastic properties of the tooth. The results here elucidate which input parameters are most important when defining the FE models, but also provide interesting glimpses of the biomechanical differences between the three skulls, which will be fully explored in future publications.

Patterns of covariation in the masticatory apparatus of hystricognathous rodents: implications for evolution and diversification.

The mammalian masticatory apparatus is a highly plastic region of the skull. In this study, a quantification of shape variation, the separation of phylogeny from ecology in the genesis of shape brings new insights on the relationships between morphological changes in the cranium, mandible, and muscle architecture. Our study focuses on the Ctenohystrica, a clade that is remarkably diverse and exemplifies a rich evolutionary history in the Old and New World. Current and past rodent diversity brings out the limitations of the qualitative descriptive approach and highlights the need for using integrative quantitative methods. We present here the first descriptive comparison of the whole masticatory apparatus within the Ctenohystrica, by combining geometric morphometric approaches with a noninvasive method of dissection in 3D, iodine‐enhanced microcomputed tomography. We used these methods to explore the patterns of covariation between the cranium and the mandible, and the interspecific morphological variation of the skull with regard to several factors such as phylogeny, activity period, type of habitat, and diet. Our study revealed strong phylogenetic and ecological imprints on the morphological traits associated with masticatory mechanics. We showed that, despite a high diversification of lineages, the evolutionary history of Ctenohystrica comprises only a small number of morphotypes for the skull and mandible. The position of the eye was suggested as a key factor determining morphological evolution of the masticatory apparatus by limiting the number of possible pathways and promoting convergent evolution toward new habitats and diets between different clades. J. Morphol. 2012.

Semicircular canals and agility: the influence of size and shape measures

The semicircular canals of the inner ear sense angular accelerations and decelerations of the head and enable co‐ordination of posture and body movement, as well as visual stability. Differences of agility and spatial sensitivity among species have been linked to interspecific differences in the relative size of the canals, particularly the radius of curvature (R) and the ratio of the canal plane area to streamline length (P/L). Here we investigate the scaling relationships of these two size variables and also out‐of‐plane torsion in the three semicircular canals (anterior, posterior and lateral), in order to assess which is more closely correlated with body size and locomotor agility. Measurements were computed from 3D landmarks taken from magnetic resonance images of a diverse sample of placental mammals encompassing 16 eutherian orders. Body masses were collected from the literature and an agility score was assigned to each species. The R and P/L of all three semicircular canals were found to have highly significant positive correlations with each other and no statistical difference was found between the slope of 2P/L against R and 1. This indicated that, contrary to initial hypotheses, there is little difference between 2P/L and R as measures of semicircular canal size. A measure of the in‐plane circularity of the canal was obtained by dividing 2P/L by R and out‐of‐plane torsion was measured as angular deviation from a plane of best fit. It was predicted that deviations from in‐plane and out‐of‐plane circularity would increase at small body size due to the constraints of fitting a proportionately larger canal into a smaller petrous bone. However, neither measurement was found to have a significant correlation with body mass, indicating that deviations from circularity (both in‐plane and out‐of‐plane) are not sufficient to alter P/L to an extent that would impact the sensitivity of the canals. 2P/L and R were both shown to be significantly correlated with locomotor agility. The posterior canal was the least correlated with agility, suggesting that it may be generally less closely aligned to the direction of movement than the anterior canal. Of the three canals, the lateral canal was the most highly correlated with agility. In particular, it could be used to distinguish between species that move in a largely 2D environment and those that locomote in 3D space (aerial, arboreal and aquatic species). This complements previous work suggesting that the lateral canal primarily commands navigation, whereas the vertical canals control reflex adjustments. It was also found that 2P/L is substantially better correlated with agility than is R in the lateral canal. This result is intriguing given the above finding that there is no statistical difference between 2P/L and R, and requires further investigation.

Digital dissection of the masticatory muscles of the naked mole-rat, Heterocephalus glaber (Mammalia, Rodentia)

The naked mole-rat, Heterocephalus glaber, of the family Bathyergidae is a subterranean rodent that feeds on underground roots and tubers and digs extensive tunnel systems with its incisors. It is a highly unusual mammal with regard to its social structure, longevity, pain insensitivity and cancer resistance, all of which have made it the subject of a great deal of research in recent years. Yet, much of the basic anatomy of this species remains undocumented. In this paper, we describe the morphology of the jaw-closing musculature of the naked mole-rat, as revealed by contrast-enhanced micro-computed tomography. This technique uses an iodine stain to enable the imaging of soft tissues with microCT. The iodine-enhanced scans were used to create 3D reconstructions of the naked mole-rat masticatory muscles from which muscle masses were calculated. The jaw-closing musculature of Heterocephalus glaber is relatively very large compared to other rodents and is dominated by the superficial masseter, the deep masseter and the temporalis. The temporalis in particular is large for a rodent, covering the entirety of the braincase and much of the rear part of the orbit. The morphology of the masseter complex described here differs from two other published descriptions of bathyergid masticatory muscles, but is more similar to the arrangement seen in other rodent families. The zygomaticomandibularis (ZM) muscle does not protrude through the infraorbital foramen on to the rostrum and thus the naked mole-rat should be considered protrogomorphous rather than hystricomorphous, and the morphology is consistent with secondarily lost hystricomorphy as has been previously suggested for Bathyergidae. Overall, the morphology of the masticatory musculature indicates a species with a high bite force and a wide gape–both important adaptations for a life dominated by digging with the incisors.

Open data and digital morphology

Over the past two decades, the development of methods for visualizing and analysing specimens digitally, in three and even four dimensions, has transformed the study of living and fossil organisms. However, the initial promise that the widespread application of such methods would facilitate access to the underlying digital data has not been fully achieved. The underlying datasets for many published studies are not readily or freely available, introducing a barrier to verification and reproducibility, and the reuse of data. There is no current agreement or policy on the amount and type of data that should be made available alongside studies that use, and in some cases are wholly reliant on, digital morphology. Here, we propose a set of recommendations for minimum standards and additional best practice for three-dimensional digital data publication, and review the issues around data storage, management and accessibility.

A quantitative analysis of the Eutherian orbit: correlations with masticatory apparatus

The mammalian orbit, or eye‐socket, is a highly plastic region of the skull. It comprises between seven and nine bones, all of which vary widely in their contribution to this region among the different mammalian orders and families. It is hypothesised that the structure of the mammalian orbit is principally influenced by the forces generated by the jaw‐closing musculature. In order to quantify the orbit, fourteen linear, angular and area measurements were taken from 84 species of placental mammals using a Microscribe‐3D digitiser. The results were then analysed using principal components analysis. The results of the multivariate analysis on untransformed data showed a clear division of the mammalian taxa into temporalis‐dominant forms and masseter‐dominant forms. This correlation between orbital structure and masticatory musculature was reinforced by results from the size‐corrected data, which showed a separation of the taxa into the three specialised feeding types proposed by Turnbull (1970) : i.e. ‘carnivore‐shear’, ‘ungulate‐grinding’ and ‘rodent‐gnawing’. Moreover, within the rodents there was a clear distinction between species in which the masseter is highly developed and those in which the temporalis has more prominence. These results were reinforced by analysis of variance which showed significant differences in the relative orbital areas of certain bones between temporalis‐dominant and masseter‐dominant taxa. Subsequent cluster analysis suggested that most of the variables could be grouped into three assemblages: those associated with the length of the rostrum; those associated with the width of the skull; and those associated with the relative size of the orbit and the shape of the face. However, the relative area of the palatine bone showed weak correlations with the other variables and did not fit into any group. Overall the relative area of the palatine was most closely correlated with feeding type, and this measure that appeared to be most strongly associated with the arrangement of the masticatory musculature. These results give a strong indication that, although orbital structure is in part determined by the relative size and orientation of the orbits, the forces generated by the muscles of mastication also have a large effect.