The over-designed limb: Skeletal robustness and locomotor adaptation in terrestrial and freshwater turtles

The morphology and internal structure of tetrapod limb bones reflect complex interactions between functional demands, mechanical loading, and environmental conditions. In most tetrapods, limb bone microanatomy shows a clear ecological signal, with aquatic taxa typically displaying increased bone compactness and terrestrial taxa exhibiting more open medullary cavities. Testudines represent a notable exception to this pattern, as both terrestrial and aquatic turtles possess unusually compact long bones, suggesting that factors beyond habitat alone shape their skeletal design. Here, we present the first comprehensive whole-bone, longitudinal analysis of cortical bone mass, geometry, tissue quality, and mechanostat-related distribution/quality (d/q) relationships in turtle long bones, aiming to evaluate how locomotor ecology modulates bone design within the constraints of the turtle bauplan. We analyzed femora and humeri of four turtle species spanning a terrestrial-aquatic gradient (Chelonoidis carbonarius, Chelonoidis chilensis, Trachemys dorbignii, and Phrynops hilarii) using peripheral quantitative computed tomography (pQCT). Ten serial cross-sections along each bone were assessed to quantify cortical bone mineral content, cortical area, volumetric bone mineral density, cross-sectional moments of inertia, bone strength indices, and d/q relationships between tissue stiffness and cross-sectional design. Statistical comparisons focused on mid-diaphyseal regions, with interspecific differences evaluated using ANOVA, and mechanostat-related patterns examined through correlation analyses and ANCOVA. All species exhibited the characteristic turtle "over-designed" condition, with abundant cortical bone and peak mass and density at midshaft. However, marked interspecific differences emerged in bone geometry and in the organization of cortical tissue. The terrestrial species, particularly Chelonoidis carbonarius, showed higher cortical mass and larger moments of inertia, reflecting enhanced resistance to bending under weight-bearing locomotion. In contrast, the more aquatic Phrynops hilarii displayed lower moments of inertia and flatter or near-zero d/q slopes, indicating a biomechanical strategy favoring compressive stiffness via endosteal infilling rather than structural optimization for bending. Intermediate species occupied transitional positions along this gradient. These results demonstrate that, despite strong phylogenetic constraints associated with the turtle bauplan, the spatial distribution of cortical bone is finely tuned to locomotor ecology. The bone mechanostat in turtles operates within an "over-designed" framework to produce distinct structural solutions for terrestrial versus aquatic loading regimes. Our findings provide a quantitative link between habitat, locomotor mode, and long-bone design, revealing that turtle limbs are not simply overbuilt, but precisely engineered for their mechanical environments.