BACKGROUND
the evolutionary history of prokaryotes includes both horizontal and vertical inheritance of genes  <cit> .
horizontal gene transfer  events are of great interest in themselves, for their roles in creating functionally new combinations of genes  <cit> , but they pose problems for investigating the phylogenetic history and divergence times of organisms.
the existence of a core of genes that has not been transferred is still under debate as hgts have been detected in genes previously considered to be immune to these events  <cit> .
although a complete absence of hgt appears to be unlikely, genes belonging to different functional categories seem to be horizontally transferred with different frequencies  <cit> .
genes forming complex interactions with other cellular components  have a lower frequency of hgt and are generally more conserved among organisms.
recent studies based on analyses of these genes have obtained similar phylogenies suggesting an underlying phylogenetic signal  <cit> .
if we accept the use of core genes for phylogeny reconstruction then they should also be of use for time estimation with molecular clocks.
moreover the increasing number of prokaryotic genomes available has facilitated the detection of hgt through more accurate detection of orthology, paralogy, and monophyletic groups, and the concatenation of gene and protein sequences has helped increase the confidence of nodes and decrease the variance of time estimates  <cit> .
temporal information concerning prokaryote evolution has come from diverse sources.
for eukaryotes, the fossil record provides an abundant source of such data, but this has not been true for prokaryotes, which are difficult to identify as fossils  <cit> .
limited information on specific groups or metabolites has been obtained from analyses of isotopic concentrations  <cit>  and detection of biomarkers  <cit> .
by making some simple assumptions – e.g., that aerobic organisms evolved after oxygen became available  <cit> - it is possible to constrain some nodes in the prokaryote timescale, but only in a coarse sense.
however, most information on the timescale of prokaryote evolution has come from analysis of dna and amino acid sequence data with molecular clocks  <cit> .
the detection of evolutionary patterns in metabolic innovations, as a consequence of a phylogeny not dominated by hgt events, allows more detailed constraints on a prokaryote timescale.
in contrast to conventional interpretations of cyanobacteria as being among the most ancient of life forms on earth  <cit> , these studies have consistently found a late origin of cyanobacteria  <cit> , nearly contemporaneous with the major proterozoic rise in oxygen at  <dig>  billion years ago , termed the great oxidation event   <cit> .
in this study we have assembled a data set of amino acid sequences from  <dig> proteins common to  <dig> species of prokaryotes and eukaryotes and estimated phylogenetic relationships and divergence times with a local clock method.
these results in turn have been used to investigate the origin of metabolic pathways of importance in evolution of the biosphere.
RESULTS
data set
the majority  of the  <dig> proteins that were used are classified in the "information storage and processes" functional category of the cog.
the other categories represented are "cellular processes" , "metabolism" , and "information storage and processing" + "metabolism" .
other studies that have analyzed prokaryote genome sequence data for phylogeny have found a similar high proportion of proteins in the "information storage and processes" functional category, presumably because hgt is more difficult with such genes that are vital for the survival of the cell  <cit> .
the concatenated and aligned data set of  <dig> proteins contained  <dig>  amino acid sites .
with alignment gaps removed, the two data sets analyzed were  <dig>  amino acid sites  and  <dig>  amino acid sites .
the data sets were complete in the sense that sequences of all taxa were present for all proteins.
phylogeny
the phylogeny of eubacteria  shows significant bootstrap support for most of the major groups and subgroups.
all proteobacteria form a monophyletic group  with the following relationships of the subgroups: )).
there has been debate about the effect of base composition and substitution rate on the phylogenetic position of the endosymbiont buchnera among γ-proteobacteria  <cit> .
its position here  differs slightly from both studies; accordingly, any conclusions concerning its divergence time should be treated with caution.
spirochaetes cluster with chlamydiae, actinobacteria with cyanobacteria and deinococcus  and the hyperthermophiles  branch basally in the tree.
these groups and relationships are similar to those found previously with analyses of prokaryote genome sequences  <cit> .
the phylogeny of archaebacteria  agrees with some but not all aspects of previous phylogenetic analyses of prokaryote genomes using sequence data  <cit>  and the presence and absence of genes  <cit> .
for example, each of the two major clades of archaebacteria  is monophyletic.
this is consistent with some analyses  <cit>  but not others  <cit> .
also, the position of crenarchaeota as closest relatives of eukaryotes , instead of euryarchaeota, has been debated  <cit> .
the faster rate of evolution in eukaryotes , as noted elsewhere  <cit> , requires some caution in drawing conclusions regarding their phylogenetic position.
methanogens were found to be monophyletic in some previous analyses  <cit>  but were paraphyletic in other analyses  <cit>  and in our analysis .
the phylogenetic position of one species of methanogen in particular, methanopyrus kandleri, has differed among previous studies  <cit> .
however, it is difficult to make direct comparisons among various studies because they have included different sets of taxa.
time estimation
times of divergence were estimated for all nodes in the phylogenies of eubacteria  and archaebacteria  using the alternative constraints  described in the methods.
the eubacteria time estimates show an average 7% increase from the molecular to the geologic  calibration point.
two other additional geologic calibration points were used in the analyses ,  <dig>  ga fixed and  <dig>  ga minimum, which showed respectively 10% younger and 11% older time estimates compared with the  <dig>  ga minimum calibration point.
the times estimated with the fossil calibration point in the archaebacteria data set were on average only 10% younger than the ones estimated with the molecular calibration.
moreover there was even a smaller effect on the time estimates of the deepest nodes, which were the ones of interest in this study .
this variation is due not only to the different calibration times but also to the type of constraints used .
a single timetree  was constructed from the phylogenetic and divergence time data.
the time estimates summarized in that tree derive only from the best-justified calibrations.
for eubacteria, the  <dig>  ga minimum calibration , from the geologic record, was chosen because it encompasses all of the hypothesized time estimates for the origin of cyanobacteria.
for archaebacteria, the  <dig>  ga calibration , from the red algae fossil record, was selected because it provides a conservative constraint on the divergence of plants and animals.
time estimates and 95% credibility intervals for all nodes under all calibrations are presented elsewhere , and those data are summarized for selected nodes and calibrations for eubacteria and archaebacteria .
although some undetected hgt could be a source of bias in the time estimates, the direction of the bias  would depend on the specific node and groups involved, and it is unlikely to have had a major affect on the results, even if present.
a averages of the divergence times estimated using the  <dig>  ga minimum constraint and the five ingroup root constraints  and using the  <dig>  ±  <dig>  ga constraint and the five ingroup root constraints .
b credibility interval
divergence times within eubacteria  show a pattern seen previously  <cit>  whereby most major groups diverge from one another  in a relatively limited time interval, approximately between  <dig> – <dig>  ga. the position of the hyperthermophiles has been debated, with some studies showing them in a basal position whereas others place them more derived.
the high g-c composition of these taxa is believed to be responsible for this difficulty in phylogenetic placement.
here, they branch basally , but this should be interpreted with caution for this reason.
the divergence of escherichia coli from salmonella typhimurium  is consistent with the time estimated previously from consideration of mammalian host evolution   <cit> .
on the other hand an inconsistency with the fossil record is represented by the divergence of unicellular  and heterocyst-forming  cyanobacteria.
our time estimate for this divergence is  <dig> – <dig>  ga  while microfossils of both groups have been identified in mesoproterozoic  and paleoproterozoic  rocks  <cit> .
however the identification of these latter fossils has been debated  <cit> .
branch lengths of cyanobacteria in our protein tree and in 16s ribosomal rna trees  <cit>  do not suggest obvious substitutional biases or rate changes as they are neither unusually long nor unusually short.
the reason for the discrepancy between the molecular and fossil times remains unclear but a possible misinterpretation of the fossil record cannot be dismissed.
divergence times of most internal nodes among archaebacteria  are closely spaced in time and relatively ancient, approximately between  <dig> – <dig>  ga, regardless of the initial setting  for the ingroup root.
node p is the earliest divergence, separating euryarchaeota from crenarchaeota+eukaryotes.
node o represents the common ancestor of the methanogens in our analysis .
therefore, methanogenesis presumably arose between nodes p and o, or between  <dig>  ga  and  <dig>  ga  .
if the position of methanopyrus kandleri is not considered, in lieu of the current debate concerning its relationships , node n , the minimum time for the origin of methanogenesis drops only slightly, from  <dig>  ga  to  <dig>  ga .
discussion
origin of life on earth
neither the time for the origin of life, nor the divergence of archaebacteria and eubacteria, was estimated directly in this study.
nonetheless, one divergence within archaebacteria was estimated to be as old as  <dig>  ga , suggesting even earlier dates for the last common ancestor of living organisms and the origin of life.
this is in agreement with previous molecular clock analyses using mostly different data sets and methodology  <cit> .
a hadean  origin for life on earth is also consistent with the early establishment of a hydrosphere  <cit> .
nevertheless, the earliest geologic and fossil evidence for life has been debated  <cit>  leaving no direct support for such old time estimates.
methanogenesis
the lower luminosity of the sun during the hadean and archean predicts that surface water would have been frozen during that time.
instead there is evidence of liquid water and moderate to high surface temperatures  <cit> .
the long term carbon cycle , which acts as a temperature buffer, combined with greenhouse gases, probably explain this "faint young sun paradox"  <cit> .
arguments have been made in support of either methane  <cit>  or carbon dioxide  <cit>  as the major greenhouse gas involved.
if methane was important, it would have necessarily come from organisms , given the volume required.
archaebacteria are the only prokaryotes known to produce methane.
our time estimate of between  <dig>  ga  and  <dig>  ga  for the origin of methanogenesis suggests that methanogens were present on earth during the archean, consistent with the methane greenhouse theory  <cit> .
nonetheless, this does not rule out the alternative  explanation  <cit> .
anaerobic methanotrophy
anaerobic methanotrophy, or anaerobic oxidation of methane , is a metabolism associated with anoxic marine sediments rich in methane.
this metabolism is characterized by the coupling of two reactions, oxidation of methane and sulfate reduction.
the methane oxidizers are represented by archaebacteria phylogenetically related to the methanosarcinales, while the sulfate reducers, when present, are eubacterial members of the δ-proteobacteria division  <cit> .
these two groups of prokaryotes have been found associated in syntrophies, thus suggesting the coupling of these two pathways  <cit> .
archaebacteria have been found also isolated in monospecific clusters, oxidizing methane through an unknown reaction.
it has been suggested that they may use elements of both the methanogenesis and sulfate-reducing pathways  <cit> .
an example of coexistence of genes from both of these pathways is archaeoglobus fulgidus.
the particular condition of this archaebacterium has been explained with an ancient horizontal gene transfer from an eubacterial lineage, most likely a δ-proteobacterium  <cit> .
the phylogenetic position of the anaerobic methanotrophs with the methanosarcinales places the maximum date for the origin of this metabolism at  <dig>   ga .
the minimum time estimate of  <dig>  ga  , probably a substantial underestimate of the true time, results from the limited phylogenetic sampling available for this group.
aerobic methanotrophy
aerobic methanotrophs are represented in the α and γ divisions of the proteobacteria.
this suggests an origin for this metabolism between node c  and node b  .
shared genes from this pathway and from methanogenesis also have been found in the planctomycetales  <cit> .
this has suggested a revision of the direction of the hgt, usually considered from archaebacteria to eubacteria  <cit> , that presumably has spread these genes in the two domains.
however the absence of planctomycetales from our dataset and its controversial phylogenetic position  <cit>  does not allow us to discriminate among these possibilities.
both anaerobic and aerobic methanotrophy have been used to explain the highly depleted carbon isotopic values found in  <dig> – <dig>  ga geologic formations  <cit> .
our time estimates for these two metabolisms are both compatible with the isotopic record.
molecular clock methods have estimated the origin of cyanobacteria at  <dig>  ga   <cit> .
because oxygenic photosynthesis would have been necessary for aerobic methanotrophy  <cit> , an anaerobic metabolism seems more likely to explain the isotopic record.
phototrophy
the ability to utilize light as an energy source  is restricted to eubacteria among prokaryotes.
phototrophic eubacteria are found in five major phyla , including proteobacteria, green sulfur bacteria, green filamentous bacteria, gram positive heliobacteria, and cyanobacteria  <cit> .
only cyanobacteria produce oxygen.
there are three explanations for this broad taxonomic distribution of phototrophic metabolism; it evolved in one lineage of eubacteria and spread at a later time to other lineages by horizontal transfer, the common ancestor of these groups possessed this metabolism and genetic machinery, or there was a combination of horizontal transfer and vertical inheritance  <cit> .
because two of the three explanations require a phototrophic common ancestor, and because some features of the archean geologic record require this metabolism if biologically produced  <cit> , we have assumed here that the common ancestor  was phototrophic.
therefore, we estimate that phototrophy evolved prior to  <dig>   ga .
because the hyperthermophiles aquifex and thermotoga are not phototrophic and branch more basally,  <dig>   ga  can be considered a maximum date for phototrophy.
however, if those hyperthermophiles instead occupy a more derived position on the tree, as some analyses have indicated  <cit> , then the maximum date is no longer constrained in this analysis.
the colonization of land
the evolution of phototrophy was most likely linked to the evolution of other features essential to survival in stressful environments.
considerable biological damage can occur from exposure to ultraviolet radiation, especially prior to the goe and later formation of the protective ozone layer  <cit> .
the synthesis of pigments such as carotenoids, which function as photoprotective compounds against the reactive oxygen species created by uv radiation  <cit> , is an ability present in all the photosynthetic eubacteria and in groups that are partly or mostly associated with terrestrial habitats such as the actinobacteria, cyanobacteria, and deinococcus-thermus.
pigmentation was probably a fundamental step in the colonization of surface environments  <cit> .
besides the sharing of photoprotective compounds, these three groups  also share a high resistance to dehydration  <cit> , which further suggests that their common ancestor was adapted to land environments.
therefore we propose the name terrabacteria  for the group that includes the bacterial phyla actinobacteria, cyanobacteria, and deinococcus-thermus.
an early colonization of land is inferred to have occurred after the divergence of this terrestrial lineage with firmicutes ,  <dig>   ga, and prior to the divergence of actinobacteria with cyanobacteria + deinococcus ,  <dig>   ga. these molecular time estimates are compatible with time estimates  based on geological evidence for the earliest colonization of land by organisms   <cit> .
many groups of prokaryotes currently inhabit terrestrial environments, indicating that land has been colonized multiple times in different lineages.
oxygenic photosynthesis
from the above analyses and discussion, some of the early steps leading to oxygenic photosynthesis apparently were acquisition of protective pigments, phototrophy, and the colonization of land.
currently, hundreds of terrestrial species of cyanobacteria are known, broadly distributed among the orders, with species occurring in some of the driest environments on earth.
it is possible that a terrestrial ancestry of cyanobacteria, where stresses resulting from desiccation and solar radiation were severe, may have played a part in the evolution of oxygenic photosynthesis.
nonetheless, there is ample evidence that horizontal gene transfer also has played an important role in the assembly of photosynthetic machinery  <cit> .
although we have used the origin of cyanobacteria as a calibration , such minimum constraints permit the estimated time to be much older in a bayesian analysis.
however, in this case, the time estimated for node e  was not much older than the constraint itself.
it also agrees with an earlier molecular time estimate  based on a largely different data set and methods  <cit> .
when we used the older minimum constraint of  <dig>  ga, corresponding to 2α-methyl-hopane evidence considered to represent a biomarker of cyanobacteria  <cit> , the estimated time was likewise only slightly older .
the oldest time estimates for oxygenic photosynthesis that we obtained are still considerable younger than has been assumed – generally – in the geologic literature  <cit> .
this suggests that carbon isotope excursions, microfossils, microbial mats, stromatolites, and other pre- <dig> ga evidence ascribed to cyanobacteria should be re-evaluated.
CONCLUSIONS
the analyses presented here are based on the assumption, still under debate, that historical information  can be retrieved from genes in the prokaryote genome that have not been affected by horizontal gene transfer.
our prokaryotic timeline shows deep divergences within both the eubacterial and archaebacterial domains indicating a long evolutionary history.
the early evolution of life  and early origin of several important metabolic pathways  suggests that organisms have influenced the earth's environment since early in the history of the planet .
an inferred early presence of methanogens  is consistent with models suggesting that methane was important in keeping the earth's surface warm in the archean but does not rule out the possibility that carbon dioxide may have been equally  important.
in contrast to many classical interpretations of the early evolution of life, we find no compelling evidence for a pre- <dig> ga evolution of cyanobacteria and oxygenic photosynthesis.
this unique metabolism apparently evolved relatively late in the radiation of eubacterial clades, shortly before the great oxidation event .
the evolution of oxygenic photosynthesis may have involved a combination of adaptations to stressful terrestrial environments as well as acquisition of genes through horizontal transfer.

 the evolutionary history of prokaryotes includes both horizontal and vertical inheritance of genes. horizontal gene transfer events are of great interest in themselves, for their roles in creating functionally new combinations of genes, but they pose problems for investigating the phylogenetic history and divergence times of organisms. the existence of a core of genes that has not been transferred is still under debate as hgts have been detected in genes previously considered to be immune to these events. although a complete absence of hgt appears to be unlikely, genes belonging to different functional categories seem to be horizontally transferred with different frequencies. genes forming complex interactions with other cellular components have a lower frequency of hgt and are generally more conserved among organisms. recent studies based on analyses of these genes have obtained similar phylogenies suggesting an underlying phylogenetic signal. if we accept the use of core genes for phylogeny reconstruction then they should also be of use for time estimation with molecular clocks. moreover the increasing number of prokaryotic genomes available has facilitated the detection of hgt through more accurate detection of orthology, paralogy, and monophyletic groups, and the concatenation of gene and protein sequences has helped increase the confidence of nodes and decrease the variance of time estimates. temporal information concerning prokaryote evolution has come from diverse sources. for eukaryotes, the fossil record provides an abundant source of such data, but this has not been true for prokaryotes, which are difficult to identify as fossils. limited information on specific groups or metabolites has been obtained from analyses of isotopic concentrations and detection of biomarkers. by making some simple assumptions – e.g., that aerobic organisms evolved after oxygen became available - it is possible to constrain some nodes in the prokaryote timescale, but only in a coarse sense. however, most information on the timescale of prokaryote evolution has come from analysis of dna and amino acid sequence data with molecular clocks. the detection of evolutionary patterns in metabolic innovations, as a consequence of a phylogeny not dominated by hgt events, allows more detailed constraints on a prokaryote timescale. in contrast to conventional interpretations of cyanobacteria as being among the most ancient of life forms on earth, these studies have consistently found a late origin of cyanobacteria, nearly contemporaneous with the major proterozoic rise in oxygen at billion years ago, termed the great oxidation event. in this study we have assembled a data set of amino acid sequences from proteins common to species of prokaryotes and eukaryotes and estimated phylogenetic relationships and divergence times with a local clock method. these results in turn have been used to investigate the origin of metabolic pathways of importance in evolution of the biosphere.  data set the majority of the proteins that were used are classified in the "information storage and processes" functional category of the cog. the other categories represented are "cellular processes", "metabolism", and "information storage and processing" + "metabolism". other studies that have analyzed prokaryote genome sequence data for phylogeny have found a similar high proportion of proteins in the "information storage and processes" functional category, presumably because hgt is more difficult with such genes that are vital for the survival of the cell. the concatenated and aligned data set of proteins contained amino acid sites. with alignment gaps removed, the two data sets analyzed were amino acid sites and amino acid sites. the data sets were complete in the sense that sequences of all taxa were present for all proteins. phylogeny the phylogeny of eubacteria shows significant bootstrap support for most of the major groups and subgroups. all proteobacteria form a monophyletic group with the following relationships of the subgroups: )). there has been debate about the effect of base composition and substitution rate on the phylogenetic position of the endosymbiont buchnera among γ-proteobacteria. its position here differs slightly from both studies; accordingly, any conclusions concerning its divergence time should be treated with caution. spirochaetes cluster with chlamydiae, actinobacteria with cyanobacteria and deinococcus and the hyperthermophiles branch basally in the tree. these groups and relationships are similar to those found previously with analyses of prokaryote genome sequences. the phylogeny of archaebacteria agrees with some but not all aspects of previous phylogenetic analyses of prokaryote genomes using sequence data and the presence and absence of genes. for example, each of the two major clades of archaebacteria is monophyletic. this is consistent with some analyses but not others. also, the position of crenarchaeota as closest relatives of eukaryotes, instead of euryarchaeota, has been debated. the faster rate of evolution in eukaryotes, as noted elsewhere, requires some caution in drawing conclusions regarding their phylogenetic position. methanogens were found to be monophyletic in some previous analyses but were paraphyletic in other analyses and in our analysis. the phylogenetic position of one species of methanogen in particular, methanopyrus kandleri, has differed among previous studies. however, it is difficult to make direct comparisons among various studies because they have included different sets of taxa. time estimation times of divergence were estimated for all nodes in the phylogenies of eubacteria and archaebacteria using the alternative constraints described in the methods. the eubacteria time estimates show an average 7% increase from the molecular to the geologic calibration point. two other additional geologic calibration points were used in the analyses, ga fixed and ga minimum, which showed respectively 10% younger and 11% older time estimates compared with the ga minimum calibration point. the times estimated with the fossil calibration point in the archaebacteria data set were on average only 10% younger than the ones estimated with the molecular calibration. moreover there was even a smaller effect on the time estimates of the deepest nodes, which were the ones of interest in this study. this variation is due not only to the different calibration times but also to the type of constraints used. a single timetree was constructed from the phylogenetic and divergence time data. the time estimates summarized in that tree derive only from the best-justified calibrations. for eubacteria, the ga minimum calibration, from the geologic record, was chosen because it encompasses all of the hypothesized time estimates for the origin of cyanobacteria. for archaebacteria, the ga calibration, from the red algae fossil record, was selected because it provides a conservative constraint on the divergence of plants and animals. time estimates and 95% credibility intervals for all nodes under all calibrations are presented elsewhere, and those data are summarized for selected nodes and calibrations for eubacteria and archaebacteria. although some undetected hgt could be a source of bias in the time estimates, the direction of the bias would depend on the specific node and groups involved, and it is unlikely to have had a major affect on the results, even if present. a averages of the divergence times estimated using the ga minimum constraint and the five ingroup root constraints and using the ± ga constraint and the five ingroup root constraints. b credibility interval divergence times within eubacteria show a pattern seen previously whereby most major groups diverge from one another in a relatively limited time interval, approximately between – ga. the position of the hyperthermophiles has been debated, with some studies showing them in a basal position whereas others place them more derived. the high g-c composition of these taxa is believed to be responsible for this difficulty in phylogenetic placement. here, they branch basally, but this should be interpreted with caution for this reason. the divergence of escherichia coli from salmonella typhimurium is consistent with the time estimated previously from consideration of mammalian host evolution. on the other hand an inconsistency with the fossil record is represented by the divergence of unicellular and heterocyst-forming cyanobacteria. our time estimate for this divergence is – ga while microfossils of both groups have been identified in mesoproterozoic and paleoproterozoic rocks. however the identification of these latter fossils has been debated. branch lengths of cyanobacteria in our protein tree and in 16s ribosomal rna trees do not suggest obvious substitutional biases or rate changes as they are neither unusually long nor unusually short. the reason for the discrepancy between the molecular and fossil times remains unclear but a possible misinterpretation of the fossil record cannot be dismissed. divergence times of most internal nodes among archaebacteria are closely spaced in time and relatively ancient, approximately between – ga, regardless of the initial setting for the ingroup root. node p is the earliest divergence, separating euryarchaeota from crenarchaeota+eukaryotes. node o represents the common ancestor of the methanogens in our analysis. therefore, methanogenesis presumably arose between nodes p and o, or between ga and ga. if the position of methanopyrus kandleri is not considered, in lieu of the current debate concerning its relationships, node n, the minimum time for the origin of methanogenesis drops only slightly, from ga to ga. discussion origin of life on earth neither the time for the origin of life, nor the divergence of archaebacteria and eubacteria, was estimated directly in this study. nonetheless, one divergence within archaebacteria was estimated to be as old as ga, suggesting even earlier dates for the last common ancestor of living organisms and the origin of life. this is in agreement with previous molecular clock analyses using mostly different data sets and methodology. a hadean origin for life on earth is also consistent with the early establishment of a hydrosphere. nevertheless, the earliest geologic and fossil evidence for life has been debated leaving no direct support for such old time estimates. methanogenesis the lower luminosity of the sun during the hadean and archean predicts that surface water would have been frozen during that time. instead there is evidence of liquid water and moderate to high surface temperatures. the long term carbon cycle, which acts as a temperature buffer, combined with greenhouse gases, probably explain this "faint young sun paradox". arguments have been made in support of either methane or carbon dioxide as the major greenhouse gas involved. if methane was important, it would have necessarily come from organisms, given the volume required. archaebacteria are the only prokaryotes known to produce methane. our time estimate of between ga and ga for the origin of methanogenesis suggests that methanogens were present on earth during the archean, consistent with the methane greenhouse theory. nonetheless, this does not rule out the alternative explanation. anaerobic methanotrophy anaerobic methanotrophy, or anaerobic oxidation of methane, is a metabolism associated with anoxic marine sediments rich in methane. this metabolism is characterized by the coupling of two reactions, oxidation of methane and sulfate reduction. the methane oxidizers are represented by archaebacteria phylogenetically related to the methanosarcinales, while the sulfate reducers, when present, are eubacterial members of the δ-proteobacteria division. these two groups of prokaryotes have been found associated in syntrophies, thus suggesting the coupling of these two pathways. archaebacteria have been found also isolated in monospecific clusters, oxidizing methane through an unknown reaction. it has been suggested that they may use elements of both the methanogenesis and sulfate-reducing pathways. an example of coexistence of genes from both of these pathways is archaeoglobus fulgidus. the particular condition of this archaebacterium has been explained with an ancient horizontal gene transfer from an eubacterial lineage, most likely a δ-proteobacterium. the phylogenetic position of the anaerobic methanotrophs with the methanosarcinales places the maximum date for the origin of this metabolism at ga. the minimum time estimate of ga, probably a substantial underestimate of the true time, results from the limited phylogenetic sampling available for this group. aerobic methanotrophy aerobic methanotrophs are represented in the α and γ divisions of the proteobacteria. this suggests an origin for this metabolism between node c and node b. shared genes from this pathway and from methanogenesis also have been found in the planctomycetales. this has suggested a revision of the direction of the hgt, usually considered from archaebacteria to eubacteria, that presumably has spread these genes in the two domains. however the absence of planctomycetales from our dataset and its controversial phylogenetic position does not allow us to discriminate among these possibilities. both anaerobic and aerobic methanotrophy have been used to explain the highly depleted carbon isotopic values found in – ga geologic formations. our time estimates for these two metabolisms are both compatible with the isotopic record. molecular clock methods have estimated the origin of cyanobacteria at ga. because oxygenic photosynthesis would have been necessary for aerobic methanotrophy, an anaerobic metabolism seems more likely to explain the isotopic record. phototrophy the ability to utilize light as an energy source is restricted to eubacteria among prokaryotes. phototrophic eubacteria are found in five major phyla, including proteobacteria, green sulfur bacteria, green filamentous bacteria, gram positive heliobacteria, and cyanobacteria. only cyanobacteria produce oxygen. there are three explanations for this broad taxonomic distribution of phototrophic metabolism; it evolved in one lineage of eubacteria and spread at a later time to other lineages by horizontal transfer, the common ancestor of these groups possessed this metabolism and genetic machinery, or there was a combination of horizontal transfer and vertical inheritance. because two of the three explanations require a phototrophic common ancestor, and because some features of the archean geologic record require this metabolism if biologically produced, we have assumed here that the common ancestor was phototrophic. therefore, we estimate that phototrophy evolved prior to ga. because the hyperthermophiles aquifex and thermotoga are not phototrophic and branch more basally, ga can be considered a maximum date for phototrophy. however, if those hyperthermophiles instead occupy a more derived position on the tree, as some analyses have indicated, then the maximum date is no longer constrained in this analysis. the colonization of land the evolution of phototrophy was most likely linked to the evolution of other features essential to survival in stressful environments. considerable biological damage can occur from exposure to ultraviolet radiation, especially prior to the goe and later formation of the protective ozone layer. the synthesis of pigments such as carotenoids, which function as photoprotective compounds against the reactive oxygen species created by uv radiation, is an ability present in all the photosynthetic eubacteria and in groups that are partly or mostly associated with terrestrial habitats such as the actinobacteria, cyanobacteria, and deinococcus-thermus. pigmentation was probably a fundamental step in the colonization of surface environments. besides the sharing of photoprotective compounds, these three groups also share a high resistance to dehydration, which further suggests that their common ancestor was adapted to land environments. therefore we propose the name terrabacteria for the group that includes the bacterial phyla actinobacteria, cyanobacteria, and deinococcus-thermus. an early colonization of land is inferred to have occurred after the divergence of this terrestrial lineage with firmicutes, ga, and prior to the divergence of actinobacteria with cyanobacteria + deinococcus, ga. these molecular time estimates are compatible with time estimates based on geological evidence for the earliest colonization of land by organisms. many groups of prokaryotes currently inhabit terrestrial environments, indicating that land has been colonized multiple times in different lineages. oxygenic photosynthesis from the above analyses and discussion, some of the early steps leading to oxygenic photosynthesis apparently were acquisition of protective pigments, phototrophy, and the colonization of land. currently, hundreds of terrestrial species of cyanobacteria are known, broadly distributed among the orders, with species occurring in some of the driest environments on earth. it is possible that a terrestrial ancestry of cyanobacteria, where stresses resulting from desiccation and solar radiation were severe, may have played a part in the evolution of oxygenic photosynthesis. nonetheless, there is ample evidence that horizontal gene transfer also has played an important role in the assembly of photosynthetic machinery. although we have used the origin of cyanobacteria as a calibration, such minimum constraints permit the estimated time to be much older in a bayesian analysis. however, in this case, the time estimated for node e was not much older than the constraint itself. it also agrees with an earlier molecular time estimate based on a largely different data set and methods. when we used the older minimum constraint of ga, corresponding to 2α-methyl-hopane evidence considered to represent a biomarker of cyanobacteria, the estimated time was likewise only slightly older. the oldest time estimates for oxygenic photosynthesis that we obtained are still considerable younger than has been assumed – generally – in the geologic literature. this suggests that carbon isotope excursions, microfossils, microbial mats, stromatolites, and other pre- ga evidence ascribed to cyanobacteria should be re-evaluated.  the analyses presented here are based on the assumption, still under debate, that historical information can be retrieved from genes in the prokaryote genome that have not been affected by horizontal gene transfer. our prokaryotic timeline shows deep divergences within both the eubacterial and archaebacterial domains indicating a long evolutionary history. the early evolution of life and early origin of several important metabolic pathways suggests that organisms have influenced the earth's environment since early in the history of the planet. an inferred early presence of methanogens is consistent with models suggesting that methane was important in keeping the earth's surface warm in the archean but does not rule out the possibility that carbon dioxide may have been equally important. in contrast to many classical interpretations of the early evolution of life, we find no compelling evidence for a pre- ga evolution of cyanobacteria and oxygenic photosynthesis. this unique metabolism apparently evolved relatively late in the radiation of eubacterial clades, shortly before the great oxidation event. the evolution of oxygenic photosynthesis may have involved a combination of adaptations to stressful terrestrial environments as well as acquisition of genes through horizontal transfer. 
however, the relatively large number of genome sequences currently available has provided a better opportunity to control for potential biases such as horizontal gene transfer and rate differences among lineages. our early time estimates for methanogenesis support the consideration of methane, in addition to carbon dioxide, as a greenhouse gas responsible for the early warming of the earths' surface. we assembled a data set of sequences from proteins common to species and estimated phylogenetic relationships and divergence times with a local clock method. our divergence times for the origin of anaerobic methanotrophy are compatible with highly depleted carbon isotopic values found in rocks dated – ga. an early origin of phototrophy is consistent with the earliest bacterial mats and structures identified as stromatolites, but a ga origin of cyanobacteria suggests that those archean structures, if biologically produced, were made by anoxygenic photosynthesizers. our phylogenetic results support most of the currently recognized higher-level groupings of prokaryotes. divergence time estimates for the major groups of eubacteria are between – billion years ago while those for archaebacteria are mostly between – ga. the time estimates suggest a hadean origin of life, an early origin of methanogenesis, an origin of anaerobic methanotrophy after ga, an origin of phototrophy prior to ga, an early colonization of land – ga, and an origin of aerobic methanotrophy – ga. the timescale of prokaryote evolution has been difficult to reconstruct because of a limited fossil record and complexities associated with molecular clocks and deep divergences. of particular interest is a well-supported group of three major lineages of eubacteria that we call terrabacteria and associate with an early colonization of land. the resistance to desiccation of terrabacteria and their elaboration of photoprotective compounds suggests that the common ancestor of this group inhabited land. if true, then oxygenic photosynthesis may owe its origin to terrestrial adaptations.
 the timescale of prokaryote evolution has been difficult to reconstruct because of a limited fossil record and complexities associated with molecular clocks and deep divergences. however, the relatively large number of genome sequences currently available has provided a better opportunity to control for potential biases such as horizontal gene transfer and rate differences among lineages. we assembled a data set of sequences from proteins common to species and estimated phylogenetic relationships and divergence times with a local clock method.  our phylogenetic results support most of the currently recognized higher-level groupings of prokaryotes. of particular interest is a well-supported group of three major lineages of eubacteria that we call terrabacteria and associate with an early colonization of land. divergence time estimates for the major groups of eubacteria are between – billion years ago while those for archaebacteria are mostly between – ga. the time estimates suggest a hadean origin of life, an early origin of methanogenesis, an origin of anaerobic methanotrophy after ga, an origin of phototrophy prior to ga, an early colonization of land – ga, and an origin of aerobic methanotrophy – ga.  our early time estimates for methanogenesis support the consideration of methane, in addition to carbon dioxide, as a greenhouse gas responsible for the early warming of the earths' surface. our divergence times for the origin of anaerobic methanotrophy are compatible with highly depleted carbon isotopic values found in rocks dated – ga. an early origin of phototrophy is consistent with the earliest bacterial mats and structures identified as stromatolites, but a ga origin of cyanobacteria suggests that those archean structures, if biologically produced, were made by anoxygenic photosynthesizers. the resistance to desiccation of terrabacteria and their elaboration of photoprotective compounds suggests that the common ancestor of this group inhabited land. if true, then oxygenic photosynthesis may owe its origin to terrestrial adaptations. 