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 By contrast to these examples, the stem at the base of the clade comprising opisthokonts, Apusozoa, and Amoebozoa is consistently well supported by both ML and CAT trees, showing the robustness of all Varisulca being the deepest branching of all podiates. This difference is perhaps attributable to whole genome sequences being available for many opisthokonts and Amoebozoa and one apusozoan, whereas none is for Varisulca. The basal branching of Sulcozoa and therefore podiates as a whole will probably only be more robustly resolved when at least one complete genome or near-complete transcriptome is available for each of the three varisulcan lineages as well as for a breviate. The same problem applies to the still more uncertain position of Mantamonas. The stem at the base of the clade containing it and Collodictyon on Fig. 4 is just as short. Indeed this grouping is supported (weakly) only in the full alignment and for CAT in the <40% alignment (Fig. 3) and disappears for both ML and CAT with proportionally less missing data (Fig. 3); though ML always had a glissodiscean clade, the CAT tree with <50% missing data placed Mantamonas on its own as sister neither to planomonads nor to Collodictyon, but as sister to Collodictyon plus Amoebozoa, Apusozoa, and opisthokonts (low 0.51 support). The topology of the holophyletic Varisulca in the <30% missing gene tree (Fig. 2) ought technically to be the most reliable, and is in harmony with ML trees that exclude Malawimonas, and with all the cell evolutionary arguments underlying the establishment of that subphylum (Cavalier-Smith, 2013a). Though that makes it overall the most likely topology, more nearly complete transcriptomes for taxa branching in this region are essential to test it more thoroughly. That diphylleids and Mantamonas are represented by only single species, unlike all other sulcozoan clades, makes accurate tree construction even more difﬁcult, so it will be desirable to break up these long branches by adding other members of both groups (and a rigiﬁlid, which rDNA suggests is related to diphylleids: Yabuki et al., 2013).

5. Conclusions

We conclude that Varisulca (diphylleids plus Glissodiscea) are the most divergent podiates, and probably holophyletic, with important implications for the origin and earliest evolution of podiates. In harmony with 18S rDNA trees, we conclusively show, contrary to 28S rDNA trees (Glücksman et al., 2011), that Mantamonas is more closely related to planomonads and diphylleids than to apusomonads. We show for the ﬁrst time that within breviates Subulatomonas is more closely related to Breviata than to Pygsuia and that the apusomonad genus Thecamonas as currently constituted is paraphyletic or polyphyletic. Our analyses clarify the reasons for and largely resolve previous contradictions between site-homogeneous ML and site-heterogeneous Bayesian trees with respect to the holophyly or paraphyly of Apusozoa (and similar contradictions seen here for Varisulca as well as two previously inconsistent branching patterns within opisthokonts) and conﬁrm the conclusion that Apusozoa are paraphyletic (Brown et al., 2013); as breviates and apusomonads are probably not sisters, their shared apusozoan phenotype is ancestral to that of opisthokonts.

Our trees are consistent with the subdivision of Sulcozoa into two subphyla, conﬁrm that breviates belong in Apusozoa (not Amoebozoa) and that Apusozoa are sister to opisthokonts, and show that Sulcozoa are paraphyletic and thus ancestral to all other podiate eukaryotes, with subphylum Varisulca being sister to opisthokonts plus Apusozoa and Amoebozoa. That means that the simple microtubular skeletons of animal and other opisthokont cells arose by radically simplifying a much more complex cell body plan that ﬁrst evolved in association with a ventral feeding groove in the ancestor of biciliate excavates, from which all eukaryotes other than Euglenozoa are argued to have descended (CavalierSmith, 2010a, 2013a). Overall, when initial conﬂicts between CAT and ML trees are resolved by reducing the proportion of missing data, our trees provide substantial support for the thesis that the sulcozoan dorsal pellicle, ventral pseudopodia, and posterior ciliary gliding all evolved simultaneously and coadaptively during the origin of ancestral podiates from a swimming, non-gliding, non-pseudopodial Malawimonas-like excavate (Cavalier-Smith, 2010a, 2013a). Moreover, CAT trees place Malawimonas and anaeromonad metamonads as sisters to podiates as in Brown et al. (2013), further strengthening that idea. Our analyses clarify hitherto contradictory interpretations of the phylogenetic position of malawimonads and make it likely that Loukozoa and excavates as a whole are paraphyletic.

Acknowledgments

This work was supported by the Natural Environment Research Council (Grant No. NE/E004156/1) and by the Leverhulme Trust (Grant No. R1008101). We thank Research Councils UK (15842) for open access funding.

RL thanks NERC for a research studentship. We thank Oxford University Advanced Research Computing (ARC) for access to their clusters and efﬁcient advice, Matthew Brown, Fabien Burki, Jessica Grant, Vladimir Hampl, Timothy James, Naira Rodríguez-Espeleta, Ken-Ichiro Ishida (for Paulinella), and Kamran Shalchian-Tabrizi for protein alignments or sequences.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ympev.2014.08.012.

References