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Archaea. 2005 May; 1(5): 353–363. Published online 2005 April 13. | PMCID: PMC2685549 |
Copyright © 2005 Heron Publishing—Victoria, Canada Higher-level classification of the Archaea: evolution of methanogenesis and methanogens Éric Bapteste,*1 Céline Brochier,2 and Yan Boucher3 1 Department of Biochemistry and Molecular Biology, Dalhousie University, Sir Charles Tupper Building, Halifax, NS, B3H 4H7, Canada 2 EA EGEE (Évolution, Génome, Environnement), Université Aix-Marseille I, Centre Saint-Charles, 13331 Marseille Cedex 3, France 3 Department of Biological Sciences, Macquarie University, North Ryde, NSW 2109, Australia Received January 20, 2005; Accepted March 29, 2005.
We used a phylogenetic approach to analyze the evolution of
methanogenesis and methanogens. We show that 23 vertically transmitted
ribosomal proteins do not support the monophyly of methanogens, and
propose instead that there are two distantly related groups of extant
archaea that produce methane, which we have named Class I and Class
II. Based on this finding, we subsequently investigated the uniqueness
of the origin of methanogenesis by studying both the enzymes of
methanogenesis and the proteins that synthesize its specific
coenzymes. We conclude that hydrogenotrophic methanogenesis appeared
only once during evolution. Genes involved in the seven central steps
of the methanogenic reduction of carbon dioxide (CO2) are
ubiquitous in methanogens and share a common history. This suggests
that, although extant methanogens produce methane from various
substrates (CO2, formate, acetate, methylated C-1
compounds), these archaea have a core of conserved enzymes that have
undergone little evolutionary change. Furthermore, this core of
methanogenesis enzymes seems to originate (as a whole) from the last
ancestor of all methanogens and does not appear to have been
horizontally transmitted to other organisms or between members of
Class I and Class II. The observation of a unique and ancestral form
of methanogenesis suggests that it was preserved in two independent
lineages, with some instances of specialization or added metabolic
flexibility. It was likely lost in the Halobacteriales,
Thermoplasmatales and Archaeoglobales. Given that fossil evidence for
methanogenesis dates back 2.8 billion years, a unique origin of this
process makes the methanogenic archaea a very ancient taxon.
Keywords: phylogeny, taxonomy
Methane of biological origin can be found in a wide variety of anaerobic
environments, from peat bogs to the digestive tracts of animals and
deep-sea hydrothermal vents ( McDonald et
al. 1999, Takai and Horikoshi
1999, Florin et al. 2000).
In all these locations, large quantities of methane originate from only
one type of biological methane producer, archaeal methanogens. There are
five phylogenetically divergent orders of the domain Archaea (phylum
euryarchaeota) that fall under the appellation “methanogens”
( Garrity 2001):
Methanobacteriales, Methanopyrales, Methanococcales, Methanomicrobiales
and Methanosarcinales. All of these orders contain a wide diversity of
taxa that vary greatly in their morphological and physiological
characteristics. However, they all have in common an anaerobic lifestyle
and the ability to produce methane metabolically.
Soon after it was suggested that the Archaea are a distinct taxonomic
group, microbiologists assumed that the domain would be divided along
phenotypic lines and that the methanogenic archaea would be
monophyletic. Woese and coworkers, using the 16S rRNA gene
( Woese and Olsen 1986),
demonstrated that this was not the case; the Methanomicrobiales (which
at the time included both the current Methanomicrobiales and the
Methanosarcinales) were more closely related to extremely halophilic
archaea (Halobacteriales) than to other methanogens. Moreover, shortly
after the discovery of Methanopyrus kandleri, the
sequencing of its 16S rRNA gene suggested that it was unrelated to any
other methanogens since it emerged at the base of the euryotes, leading
the authors to propose that the ancestor of euryotes could have been a
methanogen ( Burggraf et al. 1991).
Recent phylogenies of the archaeal domain based on concatenated
ribosomal proteins and on concatenated proteins involved in
transcription confirmed the absence of monophyly of methanogens
( Matte-Tailliez et al. 2002,
Brochier et al. 2004). Such
analyses also strongly suggested a close phylogenetic relationship
between Methanococcales and Methanobacteriales, but cannot resolve the
affiliation of the Methanopyrales
( Brochier et al. 2004). Genome
trees based on shared gene pairs reconstructed by
Slesarev et al. (2002) display a
strong monophyletic clustering of Methanopyrus,
Methanococcus and Methanothermobacter.
The clade that includes these three methanogens is also supported by the
RNA polymerase subunit B (rpoB) tree, reconstructed by taking into
account the variation of evolutionary rate among sites, and consistent
with a shared split event of rpoB into rpoB′ and rpoB″
( Brochier et al. 2004). However,
the lack of complete genome sequence data for a member of the
Methanomicrobiales has prevented the inclusion of this order in
concatenated phylogenetic analyses or genome tree reconstructions. The
16S rRNA gene sequences, available from representatives of the
Methanomicrobiales, however, support the close relationship of this
phylum to the Methanosarcinales ( Castro
et al. 2004).
Methane can be produced by three different pathways, which vary in the
carbon compound used as the substrate, as well as the source of the
reducing potential (). The
hydrogenotrophic pathway is the most widespread, being found in all
methanogenic orders. It involves the reduction of CO 2 with
H 2 as an electron donor, and is composed of seven central
steps ()( Reeve et al. 1997).
Formate can also be converted to methane through this pathway, acting as
a source for CO 2 and reducing potential. Two other pathways
are found in the order Methanosarcinales: the aceticlastic pathway and
the methylotrophic pathway. In the aceticlastic pathway, acetate is
split into a methyl group and CO, the latter being subsequently oxidized
to provide electrons ( Meuer et al.
2002). The methyl group from the splitting of acetate is linked
to methanopterin (or sarcinapterin, for Methanosarcina)
before being reduced to methane in two enzymatic reactions, homologous
to the last two steps of the hydrogenotrophic pathway. The
methylotrophic pathway, also present in one Methanobacteriales genus
Methanosphaera ( van de
Wijngaard et al. 1991), has several possible variants
( Meuer et al. 2002). The
best-studied version is that where C-1 compounds such as methyl-amines
or methanol can be used as both an electron donor and acceptor. One
molecule of C-1 compound is oxidized (running the hydrogenotrophic
pathway in the reverse direction from methyl-CoM to CO 2) to
provide electrons for reducing three additional molecules to methane.
However, in the presence of methanol and H 2/CO 2,
some Methanosarcinales can reduce this C-1 compound using only the last
step of hydrogenotrophic methanogenesis (methyl-CoM to CH 4),
drawing electrons from H 2.
Methanosphaera presents yet another variation of the
methylotrophic pathway. This Methanobacteriale, unlike
Methanosarcinales, cannot reduce CO 2 to produce methane
( Schwörer and Thauer 1991).
Methanosphaera requires methanol and H 2 for
growth, reducing the former to methane in a process not yet fully
understood, but which has been shown to overlap with hydrogenotrophic
methanogenesis only in its last step
( Schwörer and Thauer 1991).
Because methanogenesis is found solely in the euryarchaeal branch of the
archaeal domain, it most likely originated in that phylum. Biological
production of methane requires at least 25 genes (in addition to more
than 20 biochemically characterized proteins involved in the synthesis
of the coenzymes). Genes encoding different subunits of an enzyme tend
to be clustered together in the genome, but these clusters and genes
encoding for monomers or homopolymers are scattered around the genome
( Reeve et al. 1997). Some
Methanosarcinales are estimated to have over 250 genes involved in
different aspects of methanogenesis
( Galagan et al. 2002). The number
of genes involved, as well as their scattered genomic arrangement, makes
it unlikely that methanogenesis could be acquired by lateral gene
transfers (LGT). However, portions of the pathways involved in this
process, along with single genes, very likely have been transferred
across vast phylogenetic distances
( Galagan et al. 2002). For
example, homologs of enzymes catalyzing the first three steps of the
methanogenic reduction of CO 2 are used for formaldehyde
oxidation in methylotrophic Proteobacteria and Planctomycetes
( Vorholt et al. 1999,
Chistoserdova et al. 2004). It is
extremely unlikely that these enzymes were present in the common
ancestor of Archaea and Bacteria and lost in all but a few lineages of
prokaryotes. Interdomain transfer(s) is a much more parsimonious
possibility ( Chistoserdova et al.
1998).
In the present work, we focus on the enzymes catalyzing the seven steps
of the hydrogenotrophic methanogenesis pathway that are ubiquitous to
methanogens, with the possible exception of
Methanosphaera, of which its specialized lifestyle
might lead to the loss of several methanogenesis enzymes as well as the
cofactor methanofuran ( van de Wijngaard
et al. 1991). This ubiquity strongly suggests that
hydrogenotrophic methanogenesis is the ancestral form of methane
production, other pathways being subsequent innovations of individual
lineages of methanogens. By combining phylogenies of methanogenesis
genes and concatenated ribosomal proteins that include data from the
partially sequenced genomes of the methanosarcinale
Methanococcoides burtonii as well as the
methanomicrobiale Methanogenium frigidum, we set out to
determine if the phylogenetic relationships of methanogen orders
suggested (i) by genome trees (Methanopyrales + Methanococcales +
Methanobacteriales) and (ii) by 16S rRNA phylogenies (Methanomicrobiales
+ Methanosarcinales) could be considered as monophyletic. This
information seems essential because the recently revised Bergey’s
Manual of Systematic Bacteriology presents a taxonomic grouping (the
inclusion of Methanomicrobiales and Methanosarcinales along with
Methanococcales in the class Methanococci) that is clearly polyphyletic
and places Methanopyrus kandleri in a separate class
because of its unresolved phylogenetic position. We also show that,
contrary to expectation, most of the enzymes involved in methanogenesis
have evolved by vertical descent. This finding is important because it
is not obvious from previous work if these operational enzymes have
evolved by vertical descent in methanogens, or if they have been
subjected to extensive LGT and gene loss. Finally, we discuss briefly
why the identification of vertically inherited pathways could serve a
purpose in nomenclature, notwithstanding the discovery of an
ever-increasing number of laterally transferred operational genes in
prokaryotes.
Constitution of the three data sets and preliminary phylogenetic analyses
The data sets of individual ribosomal proteins were obtained from
Brochier et al. (2004). These 53
ribosomal proteins (rpl1, rpl2, rpl3, rpl4, rpl5, rpl6, rpl10, rpl10e,
rpl11, rpl13, rpl14, rpl15, rpl16, rpl18, rpl18e, rpl19e, rpl20a,
rpl21e, rpl22, rpl23, rpl24, rpl24e, rpl29, rpl30, rpl31e, rpl32e,
rpl34e, rpl35ae, rpl37e, rpl39e, rps2, rps3, rps3ae, rps4, rps4e,
rps5, rps6e, rps7, rps8, rps8e, rps9, rps10, rps11, rps13, rps15,
rps17, rps17e, rps19, rps19e, rps24e, rps27ae, rps27e and rps28e) were
obtained from BLASTP and TBLASTN at the NCBI server
( http://www.ncbi.nlm.nih.gov/)
and aligned using CLUSTALW ( Thompson
et al. 1994) and the program ED of the MUST package
( Philippe 1993). Sequences from
the two Methanosarcinales Methanosarcina mazei and
Methanosarcina acetivorans and the Nanoarchaeon
Nanoarchaeum equitans, for which complete genome
sequences are now available, were added to these ribosomal data sets.
In addition, we included two methanogens for which the genome has been
partially sequenced: Methanogenium frigidum and
Methanococcoides burtonii
( Saunders et al.
2003). The first is a psychrophilic euryarchaeon belonging to
the order Methanomicrobiales, based on 16S rRNA phylogenetic analysis
( Franzmann et al. 1997),
whereas the second is a mesophilic euryarchaeon of the
Methanosarcinales order ( Franzmann et
al. 1992). Sequences were retrieved by TBLASTN from genome
sequencing web sites for M. burtonii and M.
frigidum, or by using BLASTP in NCBI for N.
equitans, M. acetivorans and M.
mazei ( Altschul et al.
1990). New sequences were manually edited and added to the data
sets. Regions where the alignment was ambiguous were removed from each
data set. Data sets for proteins of methanogenesis and coenzyme
synthesis were obtained at the NCBI. Biochemically characterized
enzymes were used as queries to retrieve orthologs using BLASTP. We
performed TBLASTN to look for M. burtonii and
M. frigidum ( Altschul
et al. 1990). Amino acid sequences were aligned with CLUSTALW
(default settings). Ambiguously aligned regions were deleted from the
alignments.
Maximum likelihood (ML) phylogenetic analyses were performed with
PROML with the JTT amino acid substitution matrix
( Jones et al. 1992), a rate
heterogeneity model with gamma-distributed rates over four categories,
with the α parameter estimated using TREE-PUZZLE, global
rearrangements and randomized input order of sequences (10 jumbles).
Bootstrap support values represent a consensus (obtained using
CONSENSE) of 100 Fitch-Margoliash distance trees (obtained using
PUZZLEBOOT and FITCH) from pseudo-replicates (obtained using SEQBOOT)
of the original alignment. The settings of PUZZLEBOOT were the same as
those used for PROML, except that global rearrangements and randomized
input order of sequences are unavailable in this program. PROML,
CONSENSE, FITCH and SEQBOOT are from the PHYLIP package Version 3.6a
( http://evolution.genetics.washington.edu/phylip.html).
We obtained TREE-PUZZLE and PUZZLEBOOT from
http://www.tree-puzzle.de.
Concatenations, separate analyses of multiple markers and test for lateral gene transfers in the data sets
Phylogenetic analyses of the ribosomal data set and selection
of the best tree Selection of the best tree was based on the
concatenation of the ribosomal proteins (called fusion), followed by
the separate analyses of 23 of these proteins (rpl2p, rpl15p, rpl18p,
rpl22p, rpl23p, rpl30p, rpl37ae, rpl3p, rpl44e, rpl4p, rps10p, rps13,
rps15p, rps17e, rps19e, rps19p, rps2p, rps3p, rps4p, rps5p, rps6e,
rps7p and rps8e). The concatenation of the 53 markers (6384
positions), representing 20 to 23 species of archaea, was used to
calculate the most likely tree by PROML, JTT, and eight categories
estimated from TREE-PUZZLE. To further test the relationships between
methanogens, as well as the robustness of the best ML tree based on
the fusion of the ribosomal proteins, 14 additional alternative
topologies were constructed. These topologies were created to test
specific hypotheses of relationships. Four topologies were created by
local rearrangements of the fusion tree and 10 others were designed
independently of it. Briefly, they explore some combinations of
relationships between the following euryarchaeal taxa or groups: the
( Ferroplasmatales/ Thermoplasmatales),
Archaeoglobus, the Halobacteriales,
the Pyrococcales and the methanogens. For instance,
in one tree, all these taxa emerge simultaneously; in others, the taxa
M. kandleri, M. thermoautotrophicus,
M. jannaschii and M. maripaludis are
associated and considered as either a late or early lineage. Similar
test trees explored the late/early emergence of the group of
M. frigidum, M. burtonii and the
Methanosarcinales. The monophyly of all the
methanogens was also investigated (Appendix 1). Likelihoods of these
different topologies were compared by an Approximately Unbiased (AU)
test ( Shimodaira 2002), using
CONSEL ( Shimodaira and Hasegawa
2001) to identify the best tree for the concatenated data set.
This statistical test estimates if the likelihoods of trees harboring
the same species, but with different relationships, differ
significantly or not. When the AU test associates a P
value that is < 0.05 to one of the topologies under study,
then this tree can be trended as significantly different and worse
than the other topologies for a given data set, at a threshold of 5%.
However, concatenation is not the most accurate approach for choosing
among topologies when dealing simultaneously with multiple markers
( Bapteste et al. 2002). A
concatenation enforces a mean rate of evolution for species, and a
mean alpha parameter for all sequences; however, not all the markers
evolve at the same rate, nor have the same alpha parameter (data not
shown). More appropriate than a simple concatenation is a separate
analysis of the markers present in all the species. Here the separate
analysis consists in testing the support/rejection for the 15
topologies by 23 individual markers, retaining as the best organismal
tree the topology that receives the largest number of individual
supports, the smallest number of individual rejections and the highest
average P value in the AU test.
We also determined if these ribosomal proteins were free of LGT
events. To evaluate their phylogenetic signal and to identify
potential LGTs, we manually designed a set of 197 topologies, many of
which could be explained by LGT. The species part of the groups
mentioned above, i.e., the members of the euryotes, of the crenotes,
of the
Ferroplasmatales/Thermoplasmatales,
of the Halobacteriales, of the
Pyrococcales, and of each of two groups of
methanogens and Archaeoglobus were mixed in our
test-trees in non-conventional groups to break the accepted
relationships, as LGTs between species of these groups would do. Some
of these trees were fully dichotomic (entirely resolved), but most of
them presented soft polytomies, allowing alternative orders of
emergence inside and between the sets of species. These topologies are
available from the authors. The AU test was applied to this set of
topologies at the level of 5%. If some phylogenetic signal is present
in the markers, we expect the trees with deep polytomy (i.e., star
phylogenies) to be rejected, and unless recent LGT occurred in our
markers, no topology describing an LGT event should be supported.
Phylogenetic analyses of the enzymes of methanogenesis and of the proteins involved in the synthesis of its coenzymes
We reconstructed individual ML phylogenies of 20 proteins involved
in hydrogenotrophic methanogenesis and of 15 proteins involved in
the synthesis of the coenzymes of this pathway. The names and
functions of these operational enzymes are listed in
. Bootstrap values for
these individual phylogenies were calculated with PUZZLEBOOT. These
analyses allowed the identification of two categories of markers:
likely orthologs (presenting a single copy for each species of
methanogens) and non-orthologs (presenting more than one copy for
some species of methanogens) (Appendix 1). Only unambigous orthologs
were retained for further analyses to test the hypothetical
monophyly of the two groups of methanogens on the basis of these two
data sets. First, the two largest cores of genes of methanogens were
defined. Two data sets were analyzed: (i) seven proteins of coenzyme
synthesis (aksD, aksE,
aksF, cofD, cofG,
cofH, comD) and (ii) nine
proteins/subunits of hydrogenotrophic methanogenesis
(ftr, fwdA, mcrA,
mer, mtd, mtrB,
mtrC, mtrD, mtrE)
(used for the trees in and ,
respectively). The proteins of each of these data sets were
concatenated, not to study the monophyly of methanogens, but to
investigate the relationships between these archaea. To test that no
alternative tree was more robustly supported than the fusion tree,
nor preferred by any individual marker, we rearranged these best
fusion trees to allow single species to be located at any
alternative position in each of them, generating 65 and 44
rearranged trees, respectively. The rejection/acceptance of these
topologies was evaluated by an AU test at the 5% level.
Methanogens are not monophyletic, but can be separated into two classes
Methanogens can be defined functionally and ecologically as methane
producers. Here we questioned their monophyly, which a priori is
unexpected. If their monophyly is supported, we could infer that the
production of methane evolved once in their last common ancestor. In
contrast, the question of unique versus multiple (and independent)
origins of methanogenesis needs to be answered if we conclude that
methanogens are paraphyletic or polyphyletic. As in previous studies,
our reference organismal tree resulting from the separate analysis of
23 ribosomal proteins rejected the monophyly of methanogens
().
The fusion of the 53 ribosomal proteins significantly favored a tree
(P value of the AU test = 0.874) in which the
monophyly of all methanogens was rejected. This tree supported two
monophyletic groups of methanogens: (1) the Methanobacteriales and
Methanococcales; and (2) the Methanomicrobiales and Methanosarcinales.
Methanopyrus kandleri emerged on its own after the
Pyrococcales divergence. All alternative topologies under study,
except the one retained for (P value of the AU test = 0.126) were
rejected. Moreover, a separate analysis favored this second topology,
which proposes two monophyletic groups of methanogens over the basic
fusion-tree (16/23 genes preferred it). The difference between the
fusion-tree and this tree concerns the positioning of
Methanopyrus kandleri. In the second tree, it groups
with Methanococcales and Methanobacteriales (BV = 50%;
), and
Methanomicrobiales strongly grouped with Methanosarcinales (BV = 100%,
).
The tree shown in may
be treated as reasonable reference, because we tested that most of the
genes used to build it reject most of the trees with simulated LGT.
There is an average of 5% of non-rejected trees by gene, suggesting
that our 23 ribosomal genes contain a significant phylogenetic signal,
free of the LGT effect. Only one gene coding for the 50S ribosomal
protein L37Ae showed strong support for a unique topology with LGT
(between Haloarcula and the Methanococcales, as
previously described by Matte-Tailliez
et al. (2002)), and showed no support for any other tree. Nine
other genes (rpl18, rpl22, rps10, rps8e, rps19, rps19e, rps6e, rps17e
and rpl23) did not reject several different topologies (≥ 5%)
with LGT, and notably rps6e, rps17e and rpl23 did not reject several
topologies involving LGT of methanogenic species. This indicates that
the hypothesis of LGT involving methanogens cannot be discarded
entirely nor proved for these nine markers, although this observation
could also be the result of a weakness of the individual phylogenetic
signals of these markers. Therefore, we assumed that, apart from
rpl37ae, these 23 ribosomal proteins were vertically inherited (i.e.,
that their histories were free of LGT events), or that, if they
contained LGT, these events would have an insignificant impact on our
phylogenetic reconstruction. Thus, our analyses strongly rejected the
existence of a large clade regrouping all the methanogens (BV = 99%).
Similarly, individual phylogenies of proteins involved in coenzyme
biosynthesis have never supported the monophyly of a large group of
methanogens.
Although the monophyly of methanogens is rejected by our reference
tree, taxa producing methane are grouped into two clades: (1)
Methanopyrales + Methanobacteriales + Methanococcales, which we call
Class I (BV < 50%, but statistically favored by the AU test); and
(2) Methanomicrobiales + Methanosarcinales, which we call Class II
(strongly supported, BV = 100%), in agreement with the most recent
version of the Bergey’s taxonomy
( Garrity 2001) (see details at
http://141.150.157.80/bergeysoutline/main.htm).
The monophylies of Class I and Class II are supported by 11 individual
ML phylogenies of the hydrogenotrophic methanogenesis enzymes and one
coenzyme biosynthesis protein (Appendix 1). The fusion of nine
orthologs of the hydrogenotrophic pathway
() and of seven genes of
coenzyme synthesis ()
also support these two monophylies. The presence of Class II as a
group is strongly supported by phylogenetic analyses of ribosomal
proteins, methanogenesis enzymes and methanogenesis coenzyme
biosynthesis proteins. This result is particularly important because
it strengthens the existence of Class II methanogens, which was
originally proposed based solely on 16S rRNA phylogenies. In contrast,
ribosomal proteins only weakly support the presence of Class I, and
individual phylogenies of the genes involved in the synthesis of
coenzymes indicate that the paraphyly of Class I is often not rejected
(three to six genes can accept a topology with paraphyletic Class I),
making this group more suggested than supported by phylogenetic
analyses of the coenzyme data set. Yet, methanogenesis enzymes display
this group as strongly supported. Furthermore, the presence of five
proteins ( hmdI in methanogenesis +
cofC, comA, comB
and comC in coenzyme synthesis) solely in
representatives of Class I suggests that these species may be members
of a clade. The presence of these genes in all members of Class I
could be viewed as Class I innovations or Class II losses. In
addition, genome trees (based on either gene content or conserved gene
pairs) also support the monophyly of Class I
( Slesarev et al. 2002).
Slesarev et al. (2002) observed
that three representatives of Class I methanogens share 59 COGs
(Cluster of Ortholog Genes) that are not represented in any other
archaea or bacteria and therefore appear to comprise a genomic
signature of this group. Two of the three orders found in Class I,
Methanopyrales and Methanobacteriales, also share a distinct
feature—both have pseudomurein as a major component of their
cellular envelope ( Konig et al.
1989). Pseudomurein is not found in other organisms and is
therefore likely to have originated in the common ancestor of the
Methanopyrales and Methanobacteriales, making it a strong shared
characteristic. Furthermore, all orders of Class I display
hyperthermophiles, whereas none are found in Class II.
We propose restructuring the classification of methanogens at the
class level, leaving all other taxonomic categories intact. The
monophyletic groups observed in our analyses, Class I and Class II,
would represent the only two classes of methanogens. The orders
Methanobacteriales, Methanococcales and Methanopyrales would be
grouped under Class I, and the orders Methanomicrobiales and
Methanosarcinales would stay in the Methanomicrobia or Class II. This
division of methanogens into two distinct classes improves on the old
classification by being consistent with phylogeny (the old class
Methanococci described in Bergey’s Manual of Systematic
Bacteriology 2001 is polyphyletic and the classes Methanococci and
Methanobacteria are unrelated in the 2004 taxonomic update of this
manual). Further phylogenomic investigations are needed to validate
these groups.
Evolution of the hydrogenotrophic methanogenesis
It is not obvious from previous work if the enzymes of the
hydrogenotrophic methanogenesis and the synthesis of its cofactor have
evolved by vertical descent in methanogens, or if they have been
subjected to extensive LGT and gene loss among methanogens, as
reported for other “operational” proteins that can be a
priori exchanged quite easily ( Boucher
et al. 2003). In fact, the history of this pathway is unlikely
to be simple. First, it is obvious that hydrogenotrophic
methanogenesis and the synthesis of coenzymes evolved mostly
independently (Appendix 1). Enzymes involved in the synthesis of
coenzymes are more broadly distributed than those of hydrogenotrophic
methanogenesis (they are not restricted to the Archaea). In addition,
these proteins seem to have undergone intricate evolutionary processes
(duplications/losses and transfers although not between methanogens).
The taxonomic distribution of the enzymes of hydrogenotrophic
methanogenesis is more restricted, but is not limited to extant
methanogens.
At the time that we performed this analysis, five genomes of
methanogens had been completely sequenced: M.
jannaschii, M. kandleri, M.
acetivorans, M. mazei and M.
thermautotrophicus ( Methanococcus
maripaludis was completed afterwards). All these species
harbor the vast majority of the proteins involved in hydrogenotrophic
methanogenesis and the synthesis of its specific coenzymes
( Table 1). The only exceptions
are hmdI, lacking in Class II, and
mtrF, absent in M. kandleri for
methanogenesis, as well as cofC, absent in Class
II and M. kandleri, and
comABC, absent in Class II for the synthesis
of coenzymes. This shows that, although additional pathways are
present for methanogenesis in some of these species, their existence
was unaccompanied by multiple specific losses of steps of the
hydrogenotrophic pathway studied here. The most parsimonious scenario
would thus be that proteins for methanogenesis appeared in the context
of a unique pathway (the hydrogenotrophic pathway)
( Ferry 1999) and were conserved
in species producing methane (with the possible exception of the
specialized Methanosphaera)
( van de Wijngaard et al. 1991).
| Table 1.
Distribution of genes coding for proteins involved in the
methanogenesis pathway in various prokaryotes.
|
We obtained no evidence that the steps of this pathway were elaborated
progressively. We can only report that markers consistent with a
unique origin of all methanogens (13 enzymes) are involved in the last
two steps of the pathway (Appendix 1). This contrasts with the absence
of statistical support for this monophyly for enzymes involved in the
first steps of methanogenesis. However, this does not indicate that
the first steps would be more ancient than the last steps; it only
indicates that all methanogenesis pathways overlap at the last two
steps of methane production. A strong evolutionary pressure for the
conservation of these final enzymatic steps, while the beginning of
the pathway could be tinkered with for metabolic flexibility, likely
explains the differences in the phylogenies of genes of
methanogenesis.
It thus appears that the methanogen clade exists as a much broader
taxon than that usually distinguished as methanogens and includes, in
addition to methanogens of Classes I and II, non-methanogenic archaea
such as Thermoplasmatales, Archaeoglobales and Halobacteriales. These
last three phyla would then represent degenerated methanogens that
have lost most of the proteins involved in methanogenesis, especially
those of the last two steps. The case of A. fulgidus
is particularly interesting because it possesses five enzymes
involved in methanogenesis, with the exception of the last two steps.
It seems that these five enzymes are more likely involved with lactate
oxidation ( Vorholt et al. 1995)
than with methanogenesis (although A. fulgidus is
capable of reverse methanogenesis, resulting in CO 2
production). The absence of methyl-CoM reductase in this archaeon
eliminates the possibility of methane production by conventional
pathways ( Klenk et al. 1997)
and illustrates the idea that genes of methanogenesis are ancient in
euryarchaea and were lost independently in various lineages. This
vertical descent with differential loss may provide a good explanation
for the orthology of hydrogenotrophic methanogenesis enzymes, but
needs to be tested further.
Our AU tests indicated that six genes (out of nine) involved in
methanogenesis are highly discriminatory and reject all trees other
than the fusion-tree in which Class I and Class II are separated. Only
three orthologs ( mtrE, mtrB and
mtrC) fail to reject 3/4/5 alternative trees (in
addition to the best tree), where the Class I M.
jannaschii branches within Class II. In contrast, paralogs a
priori excluded from the fusion can accept several
rearranged trees mixing Class I and Class II, depending on which copy
was retained, to represent a given lineage. This confirms both our
choice of removing the paralogs from fusion to avoid introducing
biases, as well as the efficiency of the concatenation analysis. It
also strongly suggests that no recent transfer of genes involved in
hydrogenotrophic methanogenesis has occurred between Class I and Class
II for at least six genes located along the pathway. The analysis of
the coenzyme-synthesizing proteins led to a similar conclusion. The
monophylies of Class I and Class II are always supported, their
polyphyly always rejected, and no recent transfers seem to have
occurred between Class I and Class II for at least the seven
non-paralogous coenzyme biosynthesis proteins. The paraphyly of Class
II (two Methanosarcina plus
Methanococcoides and/or
Methanomicrobium, depending on
available genes) is never supported (except by some combination of
paralogs). Lateral gene transfer simply cannot be rejected for the
enzymes of the two last steps that underwent duplications, but if we
assume that the right paralogs among the duplicated copies can be
identified, the number of possible LGT cases decreases to three.
Transfers or recruitments sometimes occurred, but only outside the
methanogens: ftr (enzyme of step 2) is present in
four distantly related bacteria, and mch (enzyme of
step 3) is present in seven distantly related bacteria (four of which
are the same as for ftr)
( Table 1). These bacteria have
likely acquired these genes and use them to perform a different
function such as formaldehyde oxidation
( Vorholt et al. 1999).
In summary, (1) we observed homologous proteins, without evidence of
LGT, between Class I and Class II, along the whole hydrogenotrophic
pathway (and for the synthesis of some of its specific coenzymes). (2)
The non-methanogenic A. fulgidus
harbors enzymes involved in all but the last two steps of
hydrogenotrophic methanogenesis, and Halobacteriales contain some of
the proteins involved in methanogenesis. (3) Duplications were
observed in seven genes; remarkably, six of them are in the last two
steps of hydrogenotrophic methanogenesis and one in the first step.
This, as well as a gene specific to Class I in step 4, suggests that
some local specialization at crucial points of hydrogenotrophic
methanogenesis is possible, but not by LGT, and that the global
pathway is highly conserved. The hypothesis of an ancestral
methanogenesis is more likely than its transfer between two distantly
related and ecologically distinct archaeal groups, especially given
that the genes of the hydrogenotrophic pathway are not found in an
operon (only genes encoding for subunits of a single enzyme are
usually linked).
On the general interest of testing if pathways have single or multiple origins: toward a new nomenclature?
The phylogenetic study of the evolution of a pathway is interesting to
an evolutionist, especially when, as in the present study, it confirms
the unique origin of the pathway. Indeed, in the context of possible
LGTs ( Doolittle 2000), the
evolution of genes in a pathway would show little correlation and the
duration of their association would be limited. It is now accepted
that the genomes of organisms (especially prokaryotic organisms) are
mosaics and that the evolution of genomes cannot be accurately
described by a unique tree ( Doolittle
2000). The descriptive power of a notion such as
“monophyly,” which is typically relevant in a tree-like
context only, could then be limted and it could be misleading to rely
only on it in classification if the molecular parts of organisms have
multiple evolutionary histories. This claim has conceptually
far-reaching consequences. Monophyly is generally considered the key
to the definition of natural groups by phylogeneticists and many taxa
have been reclassified accordingly to minimize paraphyly and
polyphyly. In the context of LGT, however, natural groups of
operational genes can still exist. Sets of molecular characters with a
unique origin, which have evolved and have been transmitted as a unit
thereafter, could be identified as some of the building blocks from
which species are made. The evolution of the hydrogenotrophic
methanogenesis described in this paper is an example of such a
situation. Identifying this vertically transmitted set of genes allows
the re-introduction of some accuracy and naturality in organismal
descriptions, even in the absence of a global Tree of Life on which
classifications can be based.
We thus suggest, as a future challenge for evolutionists, to elaborate
a partial but “natural and accurate” evolutionary
description of organisms based on their long-lasting molecular units.
This description would take the form of a chemical formula, in which
identified units of genes are considered atoms. These atoms would
receive a coefficient index to summarize what we know about their
evolutionary history. For instance, coefficients like
“–i” would indicate that these
units have been lost i times in the ancestors of the
species, coefficients like “+i” would
indicate different degrees i of evolution of the unit
present in the organism, and a zero would indicate that the species
never harbored this unit. One advantage of this representation is that
it would have a high explanatory power, indicating the origins of
organismal properties or atoms (i.e., vertical descent or horizontal
transfer). If we take only the example of hydrogenotrophic
methanogenesis, denoted as “HM,” we could describe species
containing the HM atom as follows: Class I Methanopyrus
kandleri would be HM+1,Class II
Methanococcoides burtonii would be HM+2, the
secondarily amethanogenic Archaeoglobus would be
HM–1, whereas Aeropyrum pernix,
which always lacked hydrogenotrophic methanogenesis, would be
considered HM0. Obviously, the more numerous the molecular
units identified, the more complex, accurate and discriminatory the
specific formula will be for each organism.
Hydrogenotrophic methanogenesis is an evolutionary unit that can be
lost, as is probably the case for Archaeoglobales and most likely for
Halobacteriales and Thermoplasmatales. It can also be tinkered with, as
seen in the acquisition of methylotrophic and aceticlastic
methanogenesis by Methanosarcinales and the apparent specialization of
Methanosphaera in hydrogen-fueled methanol reduction.
We observed no evidence of transfer events where bacteria, eukarya or
other archaea would have recently acquired the core of the
hydrogenotrophic methanogenesis. Furthermore, even in the context of
LGT, where the exchange of metabolic enzymes between species is said to
be frequent, a pathway such as hydrogenotrophic methanogenesis seems to
have been mostly maintained by vertical descent. No transfers of enzymes
of the hydrogenotrophic methanogenesis pathway seem to have occurred
between the two classes of methanogens described here, which is
unexpected for operational genes. Here, at maximum, LGT would have a
role tinkering with end of this ubiquitous pathway, rather than in
facilitating the evolution of prokaryotes, through the exchange of a
functional package. We conclude that these archaeal genes, the ribosomal
genes and those involved in the hydrogenotrophic methanogenesis, were
vertically inherited at higher phylogenetic levels, and that a broader
taxonomical monophyletic unit embedding the two classes of methanogens
likely had a methanogenic ancestor. We also conclude that
hydrogenotrophic methanogenesis has subsequently been lost in several
lineages. For these reasons, methanogenesis is likely ancient (as
suggested by the fossil record) ( Brocks
et al. 1999), and if these data are correct, methanogenic archaea
are likely ancient as well, based on our conclusion that this process
has a unique origin. The phylogenetic study of hydrogenotrophic
methanogenesis has led us to propose an alternative
“natural” nomenclature, which we hope will stimulate studies
of the evolution and origins of biochemical pathways and of their
taxonomical distribution, allowing us to identify some minimal and
meaningful patterns of gene associations in the flux of genome
evolution.
We thank D. Walsh for critical reading of the manuscript. This work was
supported by Genome Atlantic and by a grant from CIHR (MOP4467) to W.F.
Doolittle. Additional information on the test topologies used for the AU
test, and the topologies involving casual gene transfer between
methanogens are available from the corresponding authors.
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