Combined LM and EM investigations of pollen from compressed fossil flowers aid in their taxonomic assignment

Flowers are delicate structures that are relatively rare in the fossil record compared to other plant organs such as dispersed pollen, leaves, fruits, or seeds. Also, due to their fragile state, fossil flowers are seldom complete, mostly with parts or complete stamens, petals, sepals, or stigma missing. Besides amber inclusions, most Cenozoic flowers discovered so far are preserved as compression fossils with diagnostic features often obscured or deformed. This makes in situ pollen grains, still present and extractable from anthers of these flowers, essential to determine their taxonomic placement. Palynology and the study of fossil pollen are primarily focused on morphology observed with light microscopy (LM) and, in some cases, scanning electron microscopy (SEM). This is despite the fact that taxonomic assignment of pollen to a particular family, genus or species is sometimes only possible using additional information related to the ultrastructure of the pollen wall. We have adjusted previous preparation methods so that a transmission electron microscope (TEM) can easily be applied to single fossil pollen grains, which have previously been investigated with both LM and SEM. The combined LM- and SEM-based morphological characteristics and the ultrastructural features documented for single pollen grains provide a higher taxonomic resolution and a more reliable affiliation to extant and/or extinct taxa. For example, we present several Paleogene pollen-bearing buds/flowers/inflorescences which were either noted as unidentified prior to our study, or the pollen suggested they should be placed in a different taxon. In some cases, the combined morphological and ultrastructural features allow for direct comparison with pollen of extant species. In this way, applying our method on previously unidentified fossils, we were able to assign a flower bud to Ludwigia (Onagraceae), flowers to Alangium (Cornaceae) and Euphorbiaceae, and an inflorescence to Ascarina (Chloranthaceae).