FIB-SEM enhances the potential taxonomic significance of internal pollen wall structure at the generic level

•A new technique (FIB-SEM) allowing 3D views of internal pollen grain walls.•Identification of distinct, internal pollen wall features.•Potential application of internal pollen wall structures for taxonomy and phylogeny.

The external pollen morphology of Justicia is diverse and offers a wealth of characters currently used for classifying species. The pollen morphology of 12 species of Justicia and six species from five closely related genera was investigated using a focused ion beam-scanning electron microscope (FIB-SEM). This method allows for three dimensional examination of the internal pollen wall structure. The internal pollen wall structure can be characterised by the structure of the ectexine in the mesocolpium region. This internal wall layer contains elongate, arched, walled cavities or polygonal, walled chambers, each with or without a central columella. A morphometric analysis using a combination of documented external characters and the internal wall structure characters obtained in this study was conducted. The resulting tree clustered the taxa (the 18 selected species) into separate groups broadly in line with existing classifications. This verifies that the characters identified by means of FIB-SEM have the potential to be applicable in taxonomic studies at genus level.