Longamoebia is not monophyletic: Phylogenomic and cytoskeleton analyses provide novel and well-resolved relationships of amoebozoan subclades.

Longamoebia is one of the most morphologically diverse member of Amoebozoa. It includes the human pathogen Acanthamoeba, which causes minor skin and serious eye infections as well as fatal central nervous system complications. The taxonomy and phylogeny of Longamoebia is poorly understood partly due to the growing number of molecular studies that report unsuspected affiliations of lineages with extremely different morphotypes in the group. A recent molecular study questioned the monophyly of Longamoebia. In this study, we conducted a more comprehensive phylogenomic analysis including all of putative members of Longamoebia to assess its monophyly. We conducted extensive analyses to see effects of outgroup choice, missing data, and gene and taxon sampling on resulting phylogenies. We also collected morphological characters derived from the cytoskeleton using immunocytochemistry to assess homologies of pseudopodia at a finer scale. Our phylogenomic analysis yielded a well-resolved tree of Amoebozoa and highly supported novel relationships. Discosea is recovered as a monophyletic group with all of its known taxonomic orders. However, its within-group relationships dramatically differed from those originally proposed. Our study strongly demonstrates that Longamoebia sensu Smirnov et al. (2011) is not monophyletic and an invalid taxon. Thecamoebida forms a strongly supported sister group relationship with clade Flabellinea (Dactylopodida and Vannellida), while Dermamoebida (Mayorella+Dermamoeba) form an independent branch basal to other members of Discosea. The remaining groups including members of Centramoebida form a consistently well-supported clade that was shown to form a sister group relationship with Himatismenida. This robust clade shares the unique cytoskeletal features of coiled cytoplasmic microtubule network and F-actin characters. Our analyses demonstrated that placement of unstable taxa in large-scale analysis with varying levels of missing data might be compromised by some confounding factors such as outgroup choice and gene and taxon sampling.