In accord with my original intention that organisms demonstrated to have lost both characters should also be included in Chromista, I now place Alveolata, Rhizaria (phyla Cercozoa and Retaria) and centrohelid Heliozoa within Chromista (figure 1; electronic supplementary material, table S1), as multigene trees show that they belong to Chromista phylogenetically (Burki et al. 2007, 2008, 2009; Hackett et al. 2007). Like chromists, photosynthetic dinoflagellates have chlorophyll c, but were originally excluded from Chromista because their ciliary hairs and membrane topology are simpler; their closest evolutionary affinities are with Ciliophora and Apicomplexa, grouped with them as protozoan infrakingdom Alveolata. Later, extending parsimonious protein-targeting evolution arguments to embrace chromists and alveolates, I postulated that both (collectively ‘chromalveolates’) got their chloroplasts by the same secondary intracellular enslavement of a red alga (Cavalier-Smith 1999), which is now firmly established (Keeling 2009).

Cytochromes c and c1 are key components of mitochondrial respiratory chains. Encoded in the nucleus and imported into mitochondria, they originated from the α-proteobacterium enslaved to make mitochondria, not its eukaryotic host. Their haem electron carrier is covalently attached via two cysteines (one only in Euglenozoa, uniquely in nature). Attachment is catalysed within the periplasm (intermembrane space) of mitochondria or bacteria by specific enzymes—in α-proteobacteria and excavate protozoa by the Ccm system of about eight inner membrane proteins named CcmA-H (Allen et al. 2008). Unikont eukaryotes have instead a simpler system: the enzyme haem lyase (sometimes one, sometimes two specialized for cytochrome c or c1, respectively), which evolved before their last common ancestor (figure 1). Bacteria lack homologous unimolecular haem lyases, a derived character unique to neozoa; the eukaryote root cannot be within neozoa (thus not between unikonts and bikonts) unless Eozoa secondarily lost haem lyase. Some plants and chromists have Ccms instead of haem lyase; this mosaic distribution of two mechanisms within Chromista and Plantae is most simply interpreted if haem lyase originated in the ancestor of neozoa, Ccms were lost quickly by the ancestral unikont, but coexisted with lyase relatively briefly in early corticates, one or the other being repeatedly lost during their early internal diversification; this is more parsimonious than hypothetical lateral gene transfer of haem lyase (Allen et al. 2008). Mosaic distribution of lyase and Ccms within corticates cannot be explained by placing the root within Plantae or Chromista, because the symbiogeneses creating each kingdom necessarily followed the origin of eukaryotes; a position between Plantae and Chromista would not explain it or why excavates have only the ancestral Ccms. Euglenozoa have neither Ccms nor haem lyase, but an unknown unique mechanism—probably derived, thus excluding the root from within Euglenozoa (if even the most divergent Euglenozoa possess it).

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, Trimastix) 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).