Resolving the anatomy of mature sclerified conifer seed cones: complementarity among three methods

Phylogenetic studies of conifers that involve morphology are hindered by gaps in the anatomical characterization of seed cones, a direct result of difficulties encountered in sectioning cones in mature stages, which are often hardened due to sclerification. Here, we compare the resolving power of three methods—paraffin sectioning, petrographic thin-sectioning, and X-ray microcomputer tomography (micro-CT)—in documenting the morphology and anatomy of mature seed cones at different scales of detail. We use Taxodium as a case study, based on which we make recommendations on the complementarity of these methods, and we present a paraffin sectioning protocol for softening sclerified tissues. Paraffin sectioning, while providing high anatomical resolution, can only be used for small specimens, is labor-intensive, and hampered by hard tissues. Petrographic sectioning is fast and effective on larger specimens, but has low anatomical resolution and is limited to dry non-fleshy material. Micro-CT, if available, is fast, produces high resolution with no size limitations, and allows virtual sectioning and accurate 3D rendering; however, understanding of histology requires comparisons of CT images with results of the other methods. Although they overlap, each of the three methods provides unique insights on anatomy at different scales of detail. Thus, combining all three methods is ideal for producing high-quality data at all scales of anatomical and morphological detail.