The Backbone of Regionalisation: Refining Axial Regionalisation Through 3D Morphometric Sampling Strategies on Elapid...

Axial elongation has evolved repeatedly in vertebrates, and previous research has revealed insights into the developmental and evolutionary mechanisms that shape this body plan. Snakes, with their elongated, functionally limbless bodies, offer an exceptional system to investigate how vertebral regions are organised or modified throughout elongation. Recent work suggests that snakes retain ancestral regionalisation and that heart position coincides with the boundary between two regions, but how methodological choices such as vertebral sampling density and landmark dimensionality affect the detectability of these regions remain untested. Here, we used three-dimensional geometric morphometrics (3D GM) and segmented linear regressions on complete vertebral columns from 12 elapid snakes to investigate regionalisation and interspecific differences. We compared multiple subsampling strategies (every vertebra, or sampling every 2%, 2.5%, 4% or 5%) to assess how resolution can influence the detection of regional boundaries. We also tested various landmarking schemes, examining if previously utilised landmarking schemes differ greatly in region detection. Our analyses reveal a 4- or 5-region model to be most suitable among elapids, including a short cervical region spanning 2%-4% of the column, a robust morphological shift at ~20% of the column aligning strongly with heart position and a distinct lumbar boundary in the final 5%. Interspecific shape differences were detected but lacked sufficient distinction to identify reliably. Fine-scale sampling improves the detection of small regions, decaying with coarser subsampling. Our results determined that the coarsest reliable sampling method was found at the 2.5% mark. These findings show that despite axial elongation, snakes retained a short cervical and lumbar region, with most of the elongation happening in the thoracic region. This, in turn, has resulted in the thoracic region undergoing axial repatterning, with three modules detected within the thoracic region. This study refines our understanding of vertebral modularity and highlights how regionalisation evolves in elongated vertebrates.