rRNA Phylogeny of Arcellinida (Amoebozoa) Reveals that the Largest Arcellinid Genus, <i>Difflugia</i> Leclerc 1815, is not Monophyletic

The systematics of lobose testate amoebae (Arcellinida), a diverse group of shelled free-living unicellular eukaryotes, is still mostly based on morphological criteria such as shell shape and composition. Few molecular phylogenetic studies have been performed on these organisms to date, and their phylogeny suffers from typical under-sampling artefacts, resulting in a still mostly unresolved tree. In order to clarify the phylogenetic relationships among arcellinid testate amoebae at the inter-generic and inter-specific level, and to evaluate the validity of the criteria used for taxonomy, we amplified and sequenced the SSU rRNA gene of nine taxa - <i>Difflugia bacillariarum, D. hiraethogii, D. acuminata, D. lanceolata, D. achlora, Bullinularia gracilis, Netzelia oviformis, Physochila griseola</i> and <i>Cryptodifflugia oviformis</i>. Our results, combined with existing data demonstrate the following: 1) Most arcellinids are divided into two major clades, 2) the genus <i>Difflugia</i> is not monophyletic, and the genera <i>Netzelia</i> and <i>Arcella</i> are closely related, and 3) <i>Cryptodifflugia</i> branches at the base of the Arcellinida clade. These results contradict the traditional taxonomy based on shell composition, and emphasize the importance of general shell shape in the taxonomy of arcellinid testate amoebae.