biogenesis puts the eukaryotic root either within excavates (because of their ancestral Ccms) or between excavates and either neozoa or Euglenozoa, consistently with former reasons for placing the root outside bikonts now being invalid (Roger & Simpson 2009
; see also electronic supplementary material, note 3). As Euglenozoa differ more profoundly from excavates and all other eukaryotes in genomic, mitochondrial and cytological organization than does any other phylum (electronic supplementary material, table S2), I now argue that the root is between Euglenozoa and all other eukaryotes (or possibly deeply within Euglenozoa). The most convincing new evidence is the absence of mitochondrial protein Translocator of the Outer Membrane (TOM) complex and origin recognition complex (ORC) genes from all three completely sequenced trypanosomatid genomes (Schneider et al. 2008
; Godoy et al. 2009
Tom40, a cylindrical β-barrel channel protein in the mitochondrial outer membrane (OM), is vital to almost all eukaryotes (even secondary anaerobes: Microsporidia; metamonads, e.g. Giardia
) for importing nuclear-coded proteins. It ultimately evolved from a proteobacterial porin precursor like Usher (Cavalier-Smith 2006
). Tom40 could never be lost without replacing its vital function with another protein. As TOM must interact with and recognize hundreds of mitochondrial protein presequences, changeover to radically different machinery is mechanistically almost inconceivable, making it highly unlikely that the trypanosomatid absence of Toms is secondary. Trypanosome mitochondrial protein-import machinery is radically simpler than in other eukaryotes: as postulated for the earliest mitochondria (Cavalier-Smith 2006
), TIM translocase is one and not three proteins, and presequences are shorter (Schneider et al. 2008
); but similar states in microsporidia must be simplifications, making this less compelling evidence for primitiveness than the absence of TOM. Unlike microsporidia, the aerobic trypanosomes lack obvious reasons for simplification; their Imp complex for presequence cleavage is also simpler. I suggest that after neokaryotes and Euglenozoa diverged, Tom40 evolved in the ancestral neokaryote by gene duplication and divergence of the ancestor of the sole trypanosomatid porin channel (VDAC). Originally, VDAC might have mediated both OM metabolite exchange and protein import, providing a remarkably simple way of originating mitochondria. Characterizing protein-import machinery in phylogenetically diverse Euglenozoa would test this and whether its greater simplicity in trypanosomes (and I suggest all Euglenozoa) is primitive. On VDAC/Tom40 mitochondrial porin trees, the root of the VDAC half is precisely between Euglenozoa and neokaryotes (Pusnik et al. 2009
), as in my hypothesis.
Another clearly primitive trypanosome character is absence of the neozoan six-protein DNA replication ORC; they have only a Cdc6 single-protein replication initiator, like archaebacteria (Godoy et al. 2009
). TOM complex and multicomponent ORC are arguably synapomorphies for neokaryotes (if no Euglenozoa have them) or neokaryotes plus some Euglenozoa (if some Euglenozoa have them).
Other characters also substantially differ in trypanosomatids from neokaryotes. Some markedly simpler states in cytoskeleton and endomembranes than in neokaryotes (Berriman et al. 2005
) might be consequences of parasitism; but most could, like Tom and ORC absence, be ancestral for all Euglenozoa, representing a simpler phase of eukaryote cell evolution before neokaryotes evolved. Irrespective of where within Eozoa the root is, the ancestral eozoan was non-amoeboid, with a rigid surface pellicle with cortical microtubules, not actomyosin, being cytoskeletally dominant (amoeboid surfaces evolved secondarily within Percolozoa and Metamonada). Such a rigid microtubule-supported cortex was previously thought to be essential for evolving mitosis and cytokinesis when cell surface-based DNA segregation machinery of bacteria became ineffective once phagotrophy originated and internalized DNA/membrane links (Cavalier-Smith 1987
). Trypanosomatid emphasis on microtubules and relative deficiency in actomyosin-related machinery might be ancestral for Euglenozoa, possibly even eukaryotes, not parasitic reduction. Their mitotic kinetochore machinery is far simpler; they lack actin-severing and bundling machinery and activators of the actin-related protein complex Arp2/3, many proteins for microtubule ends and lateral decoration, and ciliary tektins (Berriman et al. 2005
), all general for neozoa. Possibly, all evolved only in neokaryotes and not in the earliest eukaryotes. The hypothesis that the eukaryotic root is between Euglenozoa and neokaryotes neatly rationalizes this simplicity. Another 25 unusual genomic or cellular properties of Euglenozoa, trypanosomatids or euglenoids can be similarly interpreted (see electronic supplementary material, which highlights nine as further evidence for the root being between Euglenozoa and neokaryotes–and indicating the likely primitive state for the earliest eukaryotes).