Multigene phylogenetic reconstruction of the Tubulinea (Amoebozoa) corroborates four of the six major lineages, while additionally revealing that shell composition does not predict phylogeny in the Arcellinida.

Tubulinea is a phylogenetically stable higher-level taxon within Amoebozoa, morphologically characterized by monoaxially streaming and cylindrical pseudopods. Contemporary phylogenetic reconstructions have largely relied on SSU rDNA, and to a lesser extent, on actin genes to reveal the relationships among these organisms. Additionally, the test (shell) forming Arcellinida, one of the most species-rich amoebozoan groups, is nested within Tubulinea and suffers from substantial under-sampling of taxa. Here, we increase taxonomic and gene sampling within the Tubulinea, characterizing molecular data for 22 taxa and six genes (SSU rDNA, actin, α- and β-tubulin, elongation factor 2 and the 14-3-3 regulatory protein). We perform concatenated phylogenetic analyses using these genes as well as approximately unbiased tests to assess evolutionary relationships within the Tubulinea. We confirm the monophyly of Tubulinea and four of the six included lineages (Echinamoeboidea, Leptomyxida, Amoebida and Poseidonida). Arcellinida and Hartmanellidae, the remaining lineages, are not monophyletic in our reconstructions, although statistical testing does not allow rejection of either group. We further investigate more fine-grained morphological evolution of previously defined groups, concluding that relationships within Arcellinida are more consistent with general test and aperture shape than with test composition. We also discuss the implications of this phylogeny for interpretations of the Precambrian fossil record of testate amoebae.