The evolution of upright limb posture in mammals may have enabled modifications of the forelimb for diverse locomotor ecologies. A rich fossil record of non-mammalian synapsids holds the key to unraveling the transition from "sprawling" to "erect" limb function in the precursors to mammals, but a detailed understanding of muscle functional anatomy is a necessary prerequisite to reconstructing postural evolution in fossils. Here we characterize the gross morphology and internal architecture of muscles crossing the shoulder joint in two morphologically-conservative extant amniotes that form a phylogenetic and morpho-functional bracket for non-mammalian synapsids: the Argentine black and white tegu Salvator merianae and the Virginia opossum Didelphis virginiana. By combining traditional physical dissection of cadavers with nondestructive three-dimensional digital dissection, we find striking similarities in muscle organization and architectural parameters. Despite the wide phylogenetic gap between our study species, distal muscle attachments are notably similar, while differences in proximal muscle attachments are driven by modifications to the skeletal anatomy of the pectoral girdle that are well-documented in transitional synapsid fossils. Further, correlates for force production, physiological cross-sectional area (PCSA), muscle gearing (pennation), and working range (fascicle length) are statistically indistinguishable for an unexpected number of muscles. Functional tradeoffs between force production and working range reveal muscle specializations that may facilitate increased girdle mobility, weight support, and active stabilization of the shoulder in the opossum-a possible signal of postural transformation. Together, these results create a foundation for reconstructing the musculoskeletal anatomy of the non-mammalian synapsid pectoral girdle with greater confidence, as we demonstrate by inferring shoulder muscle PCSAs in the fossil non-mammalian cynodont Massetognathus pascuali.How to cite this article Fahn-Lai P, Biewener AA, Pierce SE. 2020. Broad similarities in shoulder muscle architecture and organization across two amniotes: implications for reconstructing non-mammalian synapsids. PeerJ 8:e8556

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The sprawling-parasagittal postural shift was a major transition during synapsid evolution, underpinned by reorganization of the forelimb, and considered key to mammalian ecological diversity. Determining when and how this transition occurred in the fossil record is challenging owing to limited comparative data on extant species. Here, we built forelimb musculoskeletal models of three extant taxa that bracket sprawling-parasagittal postures-tegu lizard, echidna, and opossumand tested the relationship between three-dimensional joint mobility, muscle action, and posture. Results demonstrate clear functional variation between postural grades, with the parasagittal opossum occupying a distinct region of pose space characterized by a highly retracted and depressed shoulder joint that emphasizes versatility and humeral elevation. Applying our data to the fossil record support trends of an increasingly retracted humerus and greater elevation muscle moment arms indicative of more parasagittal postures throughout synapsid evolution.

In evolutionary biomechanics, musculoskeletal computer models of extant and extinct taxa are often used to estimate joint range of motion (ROM) and muscle moment arms (MMAs), two parameters which form the basis of functional inferences. However, relatively few experimental studies have been performed to validate model outputs. Previously, we built a model of the short-beaked echidna (Tachyglossus aculeatus) forelimb using a traditional modelling workflow, and in this study we evaluate its behaviour and outputs using experimental data. The echidna is an unusual animal representing an edge-case for model validation: it uses a unique form of sprawling locomotion, and possesses a suite of derived anatomical features, in addition to other features reminiscent of extinct early relatives of mammals. Here we use diffusible iodine-based contrast-enhanced computed tomography (diceCT) alongside digital and traditional dissection to evaluate muscle attachments, modelled muscle paths, and the effects of model alterations on the MMA outputs. We use X-ray Reconstruction of Moving Morphology (XROMM) to compare ex vivo joint ROM to model estimates based on osteological limits predicted via single-axis rotation, and to calculate experimental MMAs from implanted muscles using a novel geometric method. We also add additional levels of model detail, in the form of muscle architecture, to evaluate how muscle torque might alter the inferences made from MMAs alone, as is typical in evolutionary studies. Our study identifies several key findings that can be applied to future models. 1) A light-touch approach to model building can generate reasonably accurate muscle paths, and small alterations in attachment site seem to have minimal effects on model output. 2) Simultaneous movement through multiple degrees of freedom, including rotations and translation at joints, are necessary to ensure full joint ROM is captured; however, single-axis ROM can provide a reasonable approximation of mobility depending on the modelling objectives. 3) Our geometric method of calculating MMAs is consistent with model-predicted MMAs calculated via partial velocity, and is a potentially useful tool for others to create and validate musculoskeletal models. 4) Inclusion of muscle architecture data can change some functional inferences, but in many cases reinforced conclusions based on MMA alone.

The sprawling-parasagittal postural shift was a major transition during synapsid evolution and is considered key to mammalian ecological diversity. Despite a good fossil record, debate remains over when the shift to parasagittal posture occurred, primarily due to limited comparative biomechanical data on extant species. Here, we built forelimb musculoskeletal models of three extant taxa that bracket the sprawling-parasagittal transition: a tegu lizard, an echidna, and an opossum. We measured shoulder joint range of motion (ROM) about all three degrees of rotational freedom and characterized shoulder muscle moment arms (MMAs) across the entire pose space. Our results show that both the opossum and the tegu had high shoulder joint ROM, and both were substantially higher than the echidna. However, the opossum occupied a distinct region of pose space characterized by high humeral retraction angles. There are clear interspecific differences in MMAs related to posture, with the sprawling tegu and echidna emphasizing humeral depression, and the parasagittal opossum emphasizing humeral elevation. There are also notable differences between our sprawling taxa, with the echidna possessing much greater moment arms for humeral pronation than the tegu. We demonstrate clear functional variation between locomotor grades and use these data to hypothesize major shifts in forelimb function and posture along the mammalian stem.