Supplemental data for Comparative rhizotaxy of fossil and living isoetalean rhizomorphs reveals development through rootlet intercalation within a triangular lattice

Supplemental data: Background and Aims: The Isoetales are a clade of lycopsids that evolved colossal arborescent forms during their Palaeozoic prime but today are represented solely by the small herbaceous monogeneric Isoetes. Despite the differences in scale of taxa in the clade the rooting system of all members consists of two-parts, a rhizomorph from which rootlets develop in a regular pattern termed rhizotaxy. Rhizomorphs are highly diverse in different members leading to different terminology being used to describe rhizotaxy in different lineages. Here we set out to investigate how conserved rhizotaxy was between taxa and to provide a standard geometric definition, and developmental interpretation of rhizotaxy applicable to all members. Methods: We developed a pipeline to quantitatively describe rhizotaxy. This pipeline allowed rootlet arrangement to be captured in 3D, before being visualsed on a 2D lattice where Deluany Triangulation could be applied. This method offers a standard method to investigate rhizotaxy across disparate rhizomorphs. Next, to investigate the evolution and development of rhizotaxy we applied our pipeline to a 3D model we generated of the rooting system of the extinct Carboniferous lycopsids Oxroadia. Finally, we made direct observations of rootlet development in Isoetes using time course imaging. Key Results: We demonstrate that rhizotaxy can be described as an equallateral triangular lattice for all members of the Isoetales including Oxroadia. By combining evidence from direct observation of rootlet development in Isoetes with inferences of rootlet development and the early stages of sporophyte ontogeny of Oxroadia we conclude that the conserved rhizotaxy developed by the process of rootlet intercalation. Conclusions: We provide a single geometric definition, and predicted developmental mechanism for rhizotaxy that applies to all Isoetales. Our findings call into question the literal interpretation that the rhizomorph is a modified shoot.