From Serial Histological Sections to Three-Dimensional Anatomy: Reconstructing Internal Morphology for Systematic Studies

Natural history collections are increasingly being digitised to improve accessibility, preserve valuable specimens, and expand opportunities for biological research. While three-dimensional (3D) imaging techniques such as micro-computed tomography have transformed morphological studies, methods for generating comparable 3D datasets from serial histological sections remain underutilised. Here, we evaluate the potential of reconstructing digitised histological series as a taxonomic tool using two polyclad flatworms, Cestoplana rubrocincta (Cotylea) and Notoplana australis (Acotylea), as proof-of-concept case studies. Both species lack reliable external diagnostic characters and are traditionally distinguished using internal reproductive anatomy. Whole-slide images of serial sagittal histological sections were digitally aligned and reconstructed into interactive 3D volumes using an open-source workflow. The resulting reconstructions closely matched conventional anatomical interpretations while providing enhanced visualisation of the pharyngeal and reproductive systems from any orientation. Virtual specimens enabled non-destructive extraction of quantitative anatomical data, including measurements of internal organs from multiple viewing planes, illustrating the potential to derive morphometric characters unavailable through conventional histological examination alone. Although measurements were obtained from only one individual of each species and are therefore not representative of species-level variation, they demonstrate the capacity of reconstructed histological datasets to generate quantitative anatomical information that may prove useful for future taxonomic investigations. We stress, however, that this study is a methodological representation with taxonomic potential that needs validation. With validation, this approach could transform traditional histological collections into interactive, reusable digital resources that preserve anatomical fidelity while expanding analytical capability. We propose that 3D reconstruction of archived histological material provides a valuable complement to molecular and morphological approaches in systematics, with broad applications for taxonomy, biodiversity research, museum digitisation, and education.