Layer-by-layer soft-tissue effects on flexion-extension-dominant passive ex vivo limb joint ROM in quadrupedal...

Functional reconstructions of extinct mammals often infer joint mobility from osteological geometry, yet the mobility envelope in life emerged from bones embedded within a layered soft-tissue system. More broadly, passive joint mobility provides an anatomical bridge between osteological form, soft-tissue constraints, and biological movement. Ex vivo range-of-motion (ROM) datasets that explicitly partition soft-tissue contributions across multiple joints and postural types remain scarce for mammals. Here, we quantify layer-by-layer effects on flexion-extension-dominant passive ex vivo limb joint ROM across six major limb joints (shoulder, elbow, wrist [carpal joint], hip, knee [stifle] and ankle [hock]) in four quadrupedal mammals spanning differing postures: rabbit (Oryctolagus cuniculus), chilla fox (Lycalopex griseus), pig (Sus scrofa domestica) and pudu deer (Pudu puda) (one specimen per taxon). ROM was measured sequentially under four anatomical conditions that progressively isolate tissue contributions: intact (S+M+CL+O), myofascial (M+CL+O), capsulo-ligamentous (CL+O) and osteology-only (O). Three trained evaluators passively moved each joint to maximal flexion and extension endpoints. Endpoints were recorded using calibrated photographs, and joint angles were quantified via vector-based analysis by a single experienced assessor. Between-evaluator dispersion was summarised using SD and coefficient of variation (CV%). Passive ROM did not increase monotonically with tissue removal. Instead, trajectories were strongly joint- and taxon-specific, with frequent intermediate-condition maxima and, in several distal joints, marked reductions in the osteology-only condition after capsulo-ligamentous removal. The wrist showed the most pronounced non-monotonicity, commonly peaking at CL+O and decreasing sharply in O, in some cases, to values below the intact state. By contrast, proximal joints more often exhibited large net expansions across the dissection sequence, although the condition producing maximal ROM varied among taxa and joints. Measurement dispersion also varied by joint and condition, tending to be higher in intact states and in joints that were more complex to manipulate consistently, especially distal joints, consistent with less sharply defined passive endpoints when multiple layers contribute distributed resistance. These results indicate that osteology-only ROM is not a reliable upper bound on biologically feasible flexion-extension-dominant passive motion, because periarticular tissues can both constrain excursion and stabilise alignment within multi-element joint complexes. Accordingly, layer-resolved ROM series provide an empirical anatomical line of evidence that can guide sensitivity analyses in musculoskeletal reconstructions and support cautious calibration of morphofunctional spaces for movement interpretation, including future palaeobiological applications.