Muscle architecture is a major determinant of muscle performance and, in mammalian lineages, has been correlated with both feeding ecology and locomotor behaviors. Over the past decade, contrast‐enhanced micro‐CT (DiceCT) has emerged as an alternative to traditional dissection‐based measurement. DiceCT allows the collection of myological data without damaging the specimen, and while preserving 3D relationships inside muscular tissues. However, manual segmentation of DiceCT datasets involves a major time investment and requires subjective judgments that can introduce bias. To address these shortcomings, several algorithmic approaches to tracing muscle fascicles have been described; however, these have not yet been rigorously tested in complex vertebrate muscle. Here, we present a standardized protocol for algorithmic fiber tracking using the commercial software extension XFiber within the Amira suite and compared its performance to manual segmentation, an open source algorithm ( GoodFibes ), and dissection values from the literature. Fascicle length and tortuosity (curvature) were measured in the jaw muscles of eight mammalian species spanning a wide range of cranial morphologies, diets, and body sizes. XFiber produced fascicle lengths that were generally similar to both gross dissection and manual segmentation, regardless of muscle identity or taxonomic group. All three digital methods tended to overestimate fascicle lengths relative to dissection, with XFiber and manual segmentation performing similarly (~15% overestimation). GoodFibes yielded substantially longer fascicle lengths, but its tortuosity values were closer to manual segmentation than those from XFiber . Given its major advantages in terms of time investment and inter‐operator reliability, we suggest this workflow may represent a promising method for large‐scale comparative studies.

Muscle loading is known to influence skeletal morphology. Therefore, modification of the biomechanical environment is expected to cause coordinated morphological changes to the bony and cartilaginous tissues. Understanding how this musculoskeletal coordination contributes to morphological variation has relevance to health sciences, developmental biology, and evolutionary biology. To investigate how muscle loading influences skeletal morphology, we replicate a classic in ovo embryology experiment in the domestic chick (Gallus gallus domesticus) while harnessing modern methodologies that allow us to quantify skeletal anatomy more precisely and in situ. We induced rigid muscle paralysis in developing chicks mid-incubation, then compared the morphology of the cranium and mandible between immobilized and untreated embryos using microcomputed tomography and landmark-based geometric morphometric methods. Like earlier studies, we found predictable differences in the size and shape of the cranium and mandible in paralyzed chicks. These differences were concentrated in areas known to experience high strains during feeding, including the jaw joint and jaw muscle attachment sites. These results highlight specific areas of the skull that appear to be mechanosensitive and suggest muscles that could produce the biomechanical stimuli necessary for normal hatchling morphology. Interestingly, these same areas correspond to areas that show the greatest disparity and fastest evolutionary rates across the avian diversity, which suggests that the musculoskeletal integration observed during development extends to macroevolutionary scales. Thus, selection and evolutionary changes to muscle physiology and architecture could generate large and predictable changes to skull morphology. Building upon previous work, the adoption of modern imaging and morphometric techniques allows richer characterization of musculoskeletal integration that empowers researchers to understand how tissue-to-tissue interactions contribute to overall phenotypic variation.

Understanding the locomotor characteristics of early diverging ground-walking chameleons (members of the genera Brookesia, Rhampholeon, Palleon, and Rieppeleon) can help to explain how their unique morphology is adapted to fit their environment and mode of life. However, nearly all quantitative studies of chameleon locomotion thus far have focused on the larger "true arboreal" chameleons. We investigated kinematics and spatiotemporal gait characteristics of the Brown Leaf Chameleon (Brookesia superciliaris) on different substrates and compared them with true arboreal chameleons, nonchameleon lizards, and other small arboreal animals. Brookesia exhibits a combination of locomotor traits, some of which are traditionally arboreal, others more terrestrial, and a few that are very unusual. Like other chameleons, Brookesia moved more slowly on narrow dowels than on broad planks (simulating arboreal and terrestrial substrates, respectively), and its speed was primarily regulated by stride frequency rather than stride length. While Brookesia exhibits the traditionally arboreal trait of a high degree of humeral protraction at the beginning of stance, unlike most arboreal tetrapods, it uses smaller shoulder and hip excursions on narrower substrates, possibly reflecting its more terrestrial habits. When moving at very slow speeds, Brookesia often adopts an unusual footfall pattern, lateral-sequence lateral-couplets. Because Brookesia is a member of one of the earliest-diverging groups of chameleons, its locomotion may provide a good model for an intermediate stage in the evolution of arboreal chameleons. Thus, the transition to a fully arboreal way of life in "true arboreal" chameleons may have involved changes in spatiotemporal and kinematic characteristics as well as morphology.

Regionalization of the vertebral column can help animals adapt to different kinds of locomotion, including arboreal locomotion. Although functional axial regionalization has been described in both chameleons and arboreal mammals, no morphological basis for this functional regionalization in chameleons has been proposed. However, recent studies have described regionalization in the presacral vertebral column of other extant squamates. To investigate possible morphological regionalization in the vertebral column of chameleons, we took morphometric measurements from the presacral vertebrae of 28 chameleon species representing all extant chameleon genera, both fully arboreal and ground-dwelling, and performed comparative analyses. Our results support chameleons exhibiting three or four presacral morphological regions that correspond closely to those in other sauropsids, but we detected evolutionary shifts in vertebral traits occurring in only arboreal chameleons. Specifically, the anterior dorsal region in arboreal chameleons has more vertically oriented zygapophyseal joints, predicting decreased mediolateral flexibility. This shift is functionally significant because stiffening of the anterior thoracic vertebral column has been proposed to help bridge gaps between supports in primates. Thus, specialization of existing morphological regions in the vertebral column of chameleons may have played an important role in the evolution of extreme arboreal locomotion, paralleling the adaptations of arboreal primates.

EDITORIAL article Front. Ecol. Evol., 19 July 2022Sec. Paleontology https://doi.org/10.3389/fevo.2022.968979

BACKGROUND:Squamate reptiles (lizards, snakes, and amphisbaenians) exhibit incredible diversity in their locomotion, behavior, morphology, and ecological breadth. Although they often are used as models of locomotor diversity, surprisingly little attention has been given to muscle development in squamate reptiles. In fact, the most detailed examination was conducted almost 80 years ago and solely focused on the proximal limb regions. Herein, we present forelimb and hindlimb muscle morphogenesis data for three lizard species with different locomotion and feeding strategies: the desert grassland whiptail lizard, the central bearded dragon, and the veiled chameleon. This study fills critical gaps in our understanding of muscle morphogenesis in squamate reptiles and presents a comparative and temporospatial analysis of muscle development.RESULTS:Our results reveal a conserved pattern of early muscle development among lizards with different adult morphologies and ecologies. The variations that exist are concentrated in distal regions, particularly the specialized autopodia of chameleons, where differentiation of muscles associated with the digits is delayed.CONCLUSIONS:The chameleon autopod provides an example of major evolutionary modifications to the skeleton with only minor disruption of the conserved order and pattern of limb muscle development. This robustness of muscle patterning facilitates the evolution of extreme yet functional phenotypes.

Understanding the locomotor characteristics of early diverging “pygmy” or “leaf” chameleons can help to explain how their unique morphology is adapted to fit their environment and mode of life. However, nearly all quantitative studies of chameleon locomotion thus far have focused on the larger, highly arboreal “true” chameleons. Unlike “true chameleons, “pygmy” chameleons spend most of their time on the ground and are considered terrestrial. We investigated forearm kinematics in the Brown Leaf Chameleon Brookesia superciliaris to quantify and compare the locomotor characteristics of B. superciliaris relative to “true” chameleons such as Trioceros and Bradypodion. We predicted that elbow joint angles would be smaller in B. superciliaris, corresponding to a less upright posture, and that wrist range of motion would be more limited because of their simplified wrist structure. Kinematic data were collected from B. superciliaris and Trioceros jacksonii, a three horned chameleon native to east Africa. Markers were painted on the skin, and locomotion was recorded using four high speed cameras at 120 Hz while the chameleons traversed a horizontal dowel. Five events (recordings) for B. superciliaris and one event for T. jacksonii were analyzed. Markers on the elbow, forearm, wrist, and manual digits were tracked throughout one stride in each event. Outcome measures were range of motion at the wrist, elbow angle, and footfall patterns. Elbow joint angles in B. superciliaris at mid-stance and touch-down were similar to values reported in the literature forBradypodion pumilim, a larger arboreal chameleon found in parts of south Africa. The elbow joint angle of B. superciliaris at lift off was smaller when compared to B. pumilim. T. jacksonii had a much smaller elbow range of motion than B. superciliaris and B. pumilim. Wrist range of motion was similar between B. superciliaris and T. jacksonii. B. superciliaris used a lateral sequence walk with a lower step frequency and higher duty factor than T. jacksonii, which used a walking trot. Our results do not support the hypothesis that “pygmy” chameleons have more restricted ranges of motion at the wrist than “true” chameleons, but they indicate that the elbow is slightly more flexed and has a smaller range of motion. However, the greatest difference between the two groups was that B. superciliaris moved much more slowly and kept at least three feet in contact with the substrate, trading speed for increased stability. Thus, the transition to a fully arboreal way of life in “true” chameleons may have involved changes in limb kinematics as well as morphology.

The size and shape of articular cartilage in the limbs of extant vertebrates are highly variable, yet they are critical for understanding joint and limb function in an evolutionary context. For example, inferences about unpreserved articular cartilage in early tetrapods have implications for how limb length, joint range of motion, and muscle leverage changed over the tetrapod water-land transition. Extant salamanders, which are often used as functional models for early limbed vertebrates, have much thicker articular cartilage than most vertebrate groups, but the exact proportion of cartilage and how it varies across salamander species is unknown. I aimed to quantify this variation in a sample of 13 salamanders representing a broad range of sizes, modes of life, and genera. Using contrast-enhanced micro-CT, cartilage dimensions and bone length were measured non-destructively in the humerus, radius, ulna, femur, tibia, and fibula of each specimen. Cartilage correction factors were calculated as the combined thickness of the proximal and distal cartilages divided by the length of the bony shaft. Articular cartilage added about 30% to the length of the long bones on average. Cartilage was significantly thicker in aquatic salamanders (42 ± 14% in the humerus and 35 ± 8 in the femur) than in terrestrial salamanders (21 ± 7% in both humerus and femur). There was no consistent relationship between relative cartilage thickness and body size or phylogenetic relatedness. In addition to contributing to limb length, cartilage caps increased the width and breadth of the epiphyses by amounts that varied widely across taxa. To predict the effect of salamander-like cartilage correction factors on muscle leverage, a simplified model of the hindlimb of the Devonian stem tetrapod Acanthostega was built. In this model, the lever arms of muscles that cross the hip at an oblique angle to the femur was increased by up to six centimeters. Future reconstructions of osteological range of motion and muscle leverage in stem tetrapods and stem amphibians can be made more rigorous by explicitly considering the possible effects of unpreserved cartilage and justifying assumptions based on available data from extant taxa, including aquatic and terrestrial salamanders.

Since the early 1900s, researchers have attempted to unravel the origin and evolution of tetrapod limb muscles using a combination of comparative anatomy, phylogeny, and development. The methods for reconstructing soft tissues in extinct animals have been refined over time as our ability to determine muscle homology and phylogenetic relationships between tetrapods has improved. Since many muscles do not leave osteological correlates, muscle reconstruction in extinct animals is largely based on anatomy and development in extant animals. While muscle anatomy in extant tetrapods is quite conservative, the homologies of certain muscles between taxonomic groups are still uncertain. Comparative developmental studies can help to resolve these controversies, as well as revealing general patterns of muscle morphogenesis across tetrapod groups. We review the methods, results, and controversies in the muscle reconstructions of early members of the amniote, mammalian, and lissamphibian lineages, including recent attempts to reconstruct limb muscles in members of the tetrapod stem group. We also review the contribution of recent comparative developmental studies toward understanding the evolution of tetrapod limb muscles, including morphogenic gradients, the origin of paired fins, and the evolution of morphological complexity. Finally, we discuss the role of broad, comparative myological studies as part of an integrative research program on vertebrate evolutionary biology.

Investigating structure‐function relationships in the locomotor system requires an understanding of the complex interactions between bones, cartilage and muscles. This is especially true when examining how these musculoskeletal structures evolved over time and across different environments in both extant and extinct species. We aimed to quantify the effect of different assumptions about cartilage thickness on reconstructions of femur length, hip range of motion (ROM), and muscle leverage in Acanthostega gunnari, one of the earliest known tetrapods and an important species in the vertebrate water‐land transition. We began by examining the hindlimbs of two extant salamanders, Dicamptodon ensatus and Ambystoma mexicanum, using contrast‐enhanced micro‐CT in order to extrapolate the degree to which cartilage thickness might affect femur length in a fossil taxon with salamander‐like morphology. Data visualization software was used to measure the distal and proximal cartilage caps of the femora in each of the extant species. Three‐dimensional modeling software was then used to estimate the center of rotation of the hip joint of Acanthostega. Then, cartilage correction factors were applied based on the measurements from the extant taxa. The limit of ROM was defined as the point at which either the femur contacted the pelvis, or less than 50% of the femoral head remained within the acetabulum. Finally, we used biomechanical modeling software to map muscles onto the bones of Acanthostega and plotted their leverage to examine how assumptions about cartilage affect reconstructions of muscle leverage. Cartilage caps on the proximal end of the femur in Dicamptodon and Ambystoma measured 36% and 14% of femur length, respectively. Taking the most conservative estimate, we modeled Acanthostega with cartilage caps of 0, 7.5 and 13% femur length. The greatest variation in muscle leverage occurred in muscles whose axes of movement were nearly perpendicular to the femur (70–120% of the mean moment arm), as opposed to those muscles whose axes were nearly parallel (less than 10%). We also found that assuming the highest percentage of cartilage increased ROM in protraction and retraction by 15–20° and in elevation and depression by 15–30° but had little to no effect on long‐axis rotation. These results will help to predict the effects of unpreserved soft tissues in future studies that reconstruct ROM and muscle leverage in extinct animals.Effect of different cartilage correction factors on reconstructed hip muscle leverage in the early tetrapod Acanthostega gunnari.The y‐axis shows coefficient of variation of muscle leverage (standard deviation/mean moment arms) across cartilage correction factors of 0, 7.5 and 13% femur length. Moment arms were measured in 3 axes of movement for muscles that cross the hip joint. The greatest effects occurred in muscles with lines of action nearly perpendicular to the femur.Figure 1

Lobe-fins transformed into limbs during the Devonian period, facilitating the water-to-land transition in tetrapods. We traced the evolution of well-articulated skeletons across the fins-to-limbs transition, using a network-based approach to quantify and compare topological features of fins and limbs. We show that the topological arrangement of bones in pectoral and pelvic appendages evolved in parallel during the fins-to-limbs transition, occupying overlapping regions of the morphospace, following a directional trend, and decreasing their disparity over time. We identify the presence of digits as the morphological novelty triggering topological changes that discriminated limbs from fins. The origin of digits caused an evolutionary shift toward appendages that were less densely and heterogeneously connected, but more assortative and modular. Disparity likewise decreased for both appendages, more markedly until a time concomitant with the earliest-known tetrapod tracks. Last, we rejected the presence of a pectoral-pelvic similarity bottleneck at the origin of tetrapods.