(A) Best maximum likelihood (ML) ribosomal tree obtained from fusion analyses of 53 markers and separate analyses of 23 markers. Two classes of methanogens are proposed. (B) Best ML tree based on the fusion analysis of seven orthologous proteins involved in the synthesis of cofactors (1432 positions). A non-methanogen sequence supposed to be a priori outside these two groups was also retained for the coenzyme data set: either Archaeoglobus fulgidus or Ralstonia solanacearum, if the former was not present. (C) Best ML tree, based on the fusion of nine orthologous proteins of the hydrogenotrophic methanogenesis (2382 positions). For A, B and C, only bootstrap values > 50% are indicated.

Pathways of methanogenesis: hydrogenotrophic (double-lined arrows), aceticlastic (solid arrows) and methylotrophic (broken gray arrows). The hydrogenotrophic pathway can operate from formate or H₂/CO₂, obtaining reducing potential from formate or H₂, respectively. The aceticlastic pathway obtains electrons from the oxidation of CO produced by the splitting of acetate. The methylotrophic pathway can operate in two ways: (1) the methylated C-1 compound is a source for a methyl group as well as electrons (one molecule oxidized to CO₂ provides electrons for three molecules to be reduced to methane, dotted green arrows); and (2) the methylated C-1 compound is reduced to methane using electrons from H₂. The names of the proteins studied here are indicated on the pathway. For the synthesis of the cofactors, mptH plays a role in the biosynthesis of tetrahydrofolate of M. jannaschii, and mptN is involved in the biosynthesis of methanopterin. Both tetrahydrofolate and methanopterin are intermediate C1 carriers in methanogenesis. The biosynthesis of the coenzyme F420 rests on the cof enzymes (cofC, cofD, cofE, cofG and cofH). The aks enzymes (aksA, aksD, aksE and aksF) participate in 2-oxoacid elongation steps of coenzyme B9 biosynthesis, which partners with coenzyme M in the final step of methanogenesis. This lastcoenzyme is the smallest known organic cofactor, but an essential terminal methyl carrier during methanogenesis and is produced by the com enzymes (comA, comB, comC, comD and comE) (Graham and White 2002).

Enzymes are listed along the hydrogenotrophic pathway. Enzymes supporting the monophyly of methanogens are in italics (BV >75%). Enzymes supporting the monophyly of both Class I and Class II are in bold. Enzymes present in methanogens only are underlined. Genes present in Class I only are indicated by an asterisk. Phylogeny supporting the monophyly of Class II only are indicated by a +. Genes without lateral gene transfers between Class I/ Class II are in quotes. Genes with duplication in methanogenes, leading to incongruent position of a species in the tree are denoted by a superscript 2. Sixteen genes out of 20 coding for the seven core enzymes of hydrogenotrophic methanogenesis (cofD, fwdA, hmdI, mtrA, mtrB, mtrC, mtrD, mtrE, mtrF, mtrG, mtrH, mcrA, mcrB, mcrC, mcrD and mcrG) are consistent with the monophyly of methanogens. In other words, proteins of methanogenesis themselves seem in contradiction with the reference tree based on ribosomal proteins. In fact, 13 of the 15 proteins consistent with the monophyly of methanogens are exclusively present in methanogens (an additional protein is exclusively found in Class I, see Table 1). Such a restricted taxonomical sampling obviously increases the number of markers with monophyletic methanogens, but does not really help to test this monophyly. A crude interpretation could be that methanogenesis is a plesiomorphic trait, ancestral to most of the euryarchaea. It would have been lost several times independently in the Thermoplasmatales, Archaeoglobales and Halobacteriales lineages.