Inferring the root of Isoetes:: Exploring alternatives in the absence of an acceptable outgroup

The ancient divergence between the lycopsid genus Isoetes and its closest living relative (Selaginella) has resulted in considerable morphological and genetic disparity, yet within Isoetes there is remarkable morphological and genetic uniformity. This has made it difficult to identify the phylogenetic root of the genus. In this study, we addressed this problem and characterized the early branching patterns within Isoetes using an expanded set of taxa and three molecular markers. We assessed the saturation in the molecular data sets, tested for differences in evolutionary rate, determined the stability of the ingroup topology, and evaluated the applicability of the molecular clock. We then explored three alternative rooting approaches: outgroup, midpoint, and maximum likelihood under the assumption of a molecular clock. Attempts to infer the root of Isoetes using the outgroup approach were severely hindered by the effects of saturation, but the results from midpoint rooting and the enforcement of the molecular clock were highly consistent among the data sets. We identify the root of Isoetes to be located among three major, highly supported clades.