3D pollination biology using micro-computed tomography and geometric morphometrics in Theobroma cacao

Imaging technologies that capture three-dimensional (3D) variation in floral morphology at micro- and nano-resolutions are increasingly accessible. In herkogamous flowers, such as those of , structural barriers between anthers and stigmas represent bottlenecks that restrict pollinator size and access to reproductive organs. To study the unresolved pollination biology of cacao, we present a novel application of micro-computed tomography (micro-CT) using floral dimensions to quantify pollinator functional size limits. We generated micro-CT data sets from field-collected flowers and museum specimens of potential pollinators. To compare floral variation, we used 3D Slicer to place landmarks on the surface models and performed a geometric morphometric (GMM) analysis using geomorph R. We identified the petal side door (an opening between the petal hoods and filament) as the main bottleneck for pollinator access. We compared its mean dimensions with proposed pollinators to identify viable candidates. We identified three levels of likelihood for putative pollinators based on the number of morphological (body) dimensions that fit through the petal side door. We also found floral reward microstructures whose presence and location were previously unclear. Using micro-CT and GMM to study the 3D pollination biology of cacao provides new evidence for predicting unknown pollinators. Incorporating geometry and floral rewards will strengthen plant-pollinator trait matching models for cacao and other species.