BACKGROUND
elongation factor- <dig> alpha  is a core element of the translation apparatus and member of the gtpase protein family.
the gene has been widely used as a phylogenetic marker in eukaryotes; either to resolve their early evolution  or more recent phylogenetic patterns .
the evolutionary history of genes used for such inferences should closely match that of the organisms and not be affected by ancient paralogy or lateral gene transfer  <cit> .
a gene related to but clearly distinguishable from ef-1α, called elongation factor-like , appears to substitute ef-1α in a scattered pattern: several unrelated eukaryote lineages have representatives that encode efl and others that possess ef-1α.
the efl and ef-1α genes are mutually exclusive in all but two organisms: the zygomycete fungus basidiobolus and the diatom thalassiosira  <cit> .
although efl is found in several eukaryotic lineages, ef-1α is thought to be the most abundant of both  <cit> .
so far, efl has been reported in chromalveolates , the plant lineage , rhizarians , unikonts  and centrohelids  <cit> ].
the mutually exclusive distribution of ef-1α and efl suggests similar functionality.
the main function of ef-1α is translation initiation and termination, by delivering aminoacyl trnas to the ribosomes  <cit> .
other functions include interactions with cytoskeletal proteins: transfer, immobilization and translation of mrna and involvement in the ubiquitine-dependent proteolytic system, as such forming an intriguing link between protein synthesis and degradation  <cit> .
in contrast, the function of efl is barely known.
it is assumed to have a translational function because the putative ef-1β, aa-trna, and gtp/gdp binding sites do not differ between ef-1α and efl  <cit> .
based on a reverse transcriptase quantitative pcr assay in the diatom thalassiosira, which possesses both genes, it was proposed that efl had a translation function while ef-1α performed the auxiliary functions  <cit> .
the apparently scattered distribution of efl across eukaryotes raises questions about the gain-loss patterns of genes with an important role in the cell.
this mutually exclusive and seemingly scattered distribution can be explained by two different mechanisms: ancient paralogy and lateral gene transfer.
ancient paralogy was considered unlikely because this would imply that both genes were present in ancestral eukaryotic genomes during extended periods of evolutionary history while the genes rarely coexist in extant species  <cit> .
furthermore, a prolonged coexistence of both genes in early eukaryotes would have likely resulted in either functional divergence or pseudogene formation of one or the other copy  <cit> , as is suggested for efl and ef-1α coexisting in the diatom thalassiosira  <cit> .
keeling and inagaki  <cit>  proposed lateral gene transfer of the efl gene between eukaryotic lineages as the most likely explanation for the scattered distribution of both genes.
in the green plants , ef-1α and efl seem to show a mutually exclusive distribution.
of the two major green plant lineages, the chlorophyta were shown to have efl with the exception of acetabularia where ef-1α is found, and the streptophyta were shown to possess ef-1α with the exception of mesostigma, which has efl  <cit> .
noble et al.
<cit>  proposed the hypothesis that efl was introduced once in the ancestor of the green lineage, followed by differential loss of ef-1α or efl in the principal clades of the viridiplantae .
the goals of the present study are to extend our knowledge of the distribution pattern of ef-1α and efl in the green algae and investigate patterns of gain and loss of these key genes of the translational apparatus.
we applied a rt-pcr and sequencing-based screening approach across a broad spectrum of green algae, with emphasis on the ulvophycean relatives of acetabularia.
to test the hypothesis of noble et al.
<cit> , we modeled patterns of gene gain and loss.
to this goal, a reference phylogeny based on three commonly used loci was inferred, and gain-loss dynamics of efl and ef-1α were optimized along this phylogeny using continuous-time markov models.
RESULTS
distribution of elongation factors in the green algae
ef-1α sequences were retrieved from streptophytes entransia  and chlorokybus , confirming previous observations that all streptophyta except mesostigma have ef-1α.
we found efl sequences in chlorella , acrochaete and bolbocoleon , nephroselmis and tetraselmis striata , further confirming the formerly established distribution pattern within the chlorophyta.
we reaffirmed the presence of efl in chlamydomonas and scenedesmus , ulva intestinalis and u. fenestra  and ostreococcus , previously shown by noble et al.
<cit> .
in addition to acetabularia, ef-1α was discovered in representatives of the ulvophycean orders dasycladales , bryopsidales , siphonocladales  and in ignatius .
the rt-pcr approach did not reveal the presence of both genes in any of the screened species despite the fact that our primers could amplify the target genes across the viridiplantae.
our rt-pcr experiments on two species whose genomes have been sequenced  yielded a single gene for each species, a result in compliance with the knowledge derived from their genome sequences  <cit> .
the reference phylogeny, inferred from a dna matrix consisting of  <dig> taxa representing all major plant lineages and three loci , is in accordance with recent phylogenetic studies, including the position of mesostigma within the streptophyta  <cit> .
figure  <dig> shows the phylogenetic relationships among the taxa for which we have information on elongation factors; the full 72-taxon phylogeny can be found as an online supplement .
even though the tree shows improved resolution from previous studies, large parts of the backbone remained poorly resolved.
in order to obtain a solid hypothesis of green algal evolution, much additional sequence data may have to be gathered.
the occurrence of ef-1α and efl in terminal taxa was plotted on the reference phylogeny in figure  <dig>  mesostigma is the only streptophyte which encodes efl.
within the chlorophytan class ulvophyceae, the order ulvales possesses efl whereas the other orders encode ef-1α .
phylogenies of ef-1α and efl
all green plant ef-1α sequences form a monophyletic group clearly differentiated from ef-1α sequences of a variety of other eukaryotes .
even though the viridiplantae form a strongly supported group, resolution among and within streptophyta and chlorophyta is generally low, which could in part be due to some short ef-1α sequences included in the analysis.
in contrast, green plant efl genes do not form a monophyletic lineage .
although the backbone of the phylogeny is moderately resolved, monophyly of green plant efl genes is unlikely because it is not observed in the mcmc output .
efl sequences of the viridiplantae can be found in several clades.
the chlorophytes, trebouxiophytes, ulvophytes and prasinophyte tetraselmis form a single monophyletic group.
the other prasinophyte efl sequences form two separate groups.
the last clade consists of the streptophyte mesostigma.
to obtain an accurate root position for our efl tree, we included related subfamilies of the gtpase translation factor superfamily: ef-1α, eukaryotic release factor  <dig> , heat shock protein  <dig> subfamily b suppressor  and archaebacterial ef-1α sequences in our analyses.
in accordance with keeling and inagaki  <cit> , the tree is rooted with archaebacterial ef-1α sequences.
all analyses  resulted in a phylogeny very similar to the one shown in figure 2b, the complete phylogeny with all related subfamilies can be found as an online supplement .
this phylogeny shows seven efl clades, with the following branching order: bigelowiella, the diatoms, planoglabratella, the cryptophyte goniomonas, red algae, choanozoans, and a large clade containing the green plant lineage, chromalveolates , fungi and rhaphidiophrys .
deep branches generally received low statistical support, preventing strong conclusions about the relationship between the seven clades.
gain-loss dynamics
the scattered distribution of ef-1α and efl in the green plant lineage is a remarkable phenomenon that raises questions about evolutionary patterns of gain and loss of both genes.
noble et al.
<cit>  proposed the hypothesis that ef-1α was present in the common ancestor of the plant lineage, followed by a single gain of efl early in evolution of the green lineage and subsequent differential loss of one or the other gene in the various lineages.
our aim was to test this hypothesis by modeling gain-loss dynamics and inferring ancestral presence-absence patterns of both genes in a maximum likelihood framework.
gene gain and loss rates were estimated by maximum likelihood  optimization, using a dataset of presence-absence patterns of ef-1α and efl and a reference phylogeny derived from the bayesian analysis of three commonly used loci .
a first analysis, based on the reference tree, shows uncertain character state probabilities along the backbone of the viridiplantae and suggests a loss of ef-1α in early chlorophyta evolution and regain in some ulvophyceae .
because branch lengths play a crucial role in model optimization, the analysis was repeated on an alternative version of the reference tree in which branch lengths were transformed using a rate smoothing approach.
since our tree deviates from the molecular clock, we performed rate smoothing to obtain branch lengths roughly proportional to time.
rate smoothing techniques relax the assumption of constant rates of evolution throughout the tree: differences in rates of molecular evolution are smoothed out by assuming that evolutionary rates change gradually throughout the phylogeny.
the result is an ultrametric tree in which branch lengths are roughly proportional to evolutionary time instead of amounts of molecular evolution.
modeling gain-loss dynamics of elongation factor genes along the rate-smoothed tree yields results that strongly deviate from those obtained with the original reference tree: probabilities of the character states along the major part of backbone are now around 50% for efl and around 50% for the presence of both genes .
subsequently, an additional level of realism was introduced by taking phylogenetic uncertainty into account because several nodes in the reference tree are poorly supported.
to this goal, all post-burnin mcmc trees were rate-smoothed and analyzed individually.
the results were summarized on the rate-smoothed reference tree.
taking phylogenetic uncertainty into consideration had a minor influence on the probabilities of the characters states .
although the exact numbers differ between analyses, gene gain rates were always lower than gene loss rates, reinforcing the notion that gene transfers are rare events in comparison to losses of redundant genes  <cit> .
whereas the analysis based on the original reference tree returned faster gain and loss rates for efl than for ef-1α, analyses based on rate-smoothed trees  suggested the inverse, marking the sensitivity of markov models to the unit of operational time.
from these results, it seems fair to conclude that there is major uncertainty about the ancestral states for a variety of reasons, including sensitivity to branch lengths and lack of prior knowledge about ancestral states or rates of gene gain and loss.
considering that the ancestors must have had either ef-1α, efl or both genes opens perspectives for hypothesis comparison in a likelihood framework.
additionally, information about rates of gene gain and loss could be gleaned from the ef-1α and efl phylogenies.
analyses constrained with various hypotheses about ancestral gene content resulted in a confidence set of  <dig> trees that differ extensively .
the fact that strongly different hypotheses are also present in the confidence set denotes that the likelihood surface is too flat to draw firm conclusions in favor of one or another hypothesis.
the last option to reduce uncertainty is to inform the markov models with information on gains and losses gleaned from the ef-1α and efl trees .
because green plant ef-1α sequences form a monophyletic and strongly supported lineage, it seems fair to assume vertical descent of ef-1α throughout the viridiplantae.
this knowledge can be introduced in our markov model by setting a very low gain rate of ef-1α.
if the analysis is constrained in this way, both efl and ef-1α were inferred to be present along the backbone of the viridiplantae in the original reference tree  and a 50/ <dig> probability for the presence of ef-1α or both genes was obtained in the rate-smoothed trees .
comparison of hypotheses about ancestral gene content constrained with a very low ef-1α gain rate reduced the confidence set to  <dig> trees in which either ef-1α or both genes are present along the backbone .
the ml solution  assumes that only ef-1α was present along the backbone of the tree and consequently shows independent gains of efl in mesostigma, prasinophytes, chlorophyceae, trebouxiophyceae and ulvales.
an alternative scenario  in the confidence set has ef-1α at the base of the viridiplantae, a gain of efl in the ancestor of the chlorophyta, and subsequent differential loss of one or the other gene in the various lineages.
information from the efl phylogeny may provide clues for further distinction between either multiple transfers or ancient paralogy with subsequent losses.
the green efl sequences form a highly supported clade together with dinoflagellates, cryptophytes, haptophytes, fungi and rhaphidiophrys, suggesting lateral gene transfer of the efl gene between these distant eukaryotic lineages  <cit> .
considering the ability of chromalveolates  and raphidiophrys to feed through phagocytosis  <cit>  and the absence of this behavior in green algae, it would be tempting to assume that lateral gene transfer occurred from green algae to the dinoflagellates, cryptophytes, haptophytes and raphidiophrys instead of the other way around.
phagotrophic eukaryotes have been shown to have elevated rates of lateral gene transfer  <cit>  because this feeding mechanism enables them to continually recruit genes from engulfed prey  <cit> .
lateral gene transfers to fungi, although known to exist  <cit> , would require a different explanation because neither phagotrophy nor endosymbiosis occur in fungi.
however, in the light of this peripheral information, it would be tempting to conclude that both ef-1α and efl essentially show vertical descent in green plants and that the observed mutually exclusive pattern of efl and ef-1α sequences results from differential loss.
in this scenario, lateral gene transfer must have occurred from green algal cells to other eukaryotic lineages.
in previous studies of functionally similar eukaryotic genes with mutually exclusive distributions, distinction between ancient paralogy with subsequent losses and multiple transfers was made based on two main criteria  <cit> .
the first criterion states that if one gene dominates the tree and the other occurs in only a few lineages, multiple independent transfers should be regarded as the most probable explanation whereas equal representation would suggest common ancestry with subsequent differential loss.
the second criterion is about the age of the taxa involved.
if the mutually exclusive pattern occurs between closely related species, one can conclude common ancestry with subsequent losses.
if the pattern is more ancient, multiple lateral transfers are a more probable explanation.
it is obvious that such criteria are very difficult to apply in real situations.
these difficulties can be overcome by taking a probabilistic angle on the problem and modeling gain-loss dynamics with continuous-time markov models.
this approach brings statistical rigor to the analysis of gene presence-absence patterns and has the potential to discriminate between the alternative scenarios of ancient paralogy with differential losses and multiple independent lateral transfers.
application of this technique to our dataset of green algal elongation factors revealed the difficulty of arriving at firm conclusions about ancient gene transfer events because of a relatively flat likelihood surface and, consequently, ambiguous probabilities for gene content at ancestral nodes.
when informed with external information, the analyses allow somewhat more definitive conclusions.
the broader eukaryotic picture
in addition to the information gained about elongation factor evolution in green algae, our results also highlight misinterpretations in recent literature on efl evolution across the eukaryotes.
previous studies have not been explicit about whether or how their phylogenetic trees were rooted, but have drawn conclusions that require directionality in the tree.
kamikawa et al.
<cit>  concluded that lateral gene transfer from a foraminifer  to the ancestor of the diatoms must have occurred because the diatom sequences were nested within the rhizaria .
in case their tree was unrooted, this conclusion is flawed due to a lack of directionality in the tree.
in their presentation of the tree, choanozoans are used as one of the basal clades, probably because they were the earliest-branching lineage in the tree presented by keeling and inagaki  <cit> .
our efl tree, which includes ef-1α, erf <dig> and hbs <dig> sequences and is rooted with archaebacterial ef-1α sequences, indicates that the directionality inferred by kamikawa et al.
<cit>  is likely to be wrong.
our phylogram  suggest that the root position of efl lies on the branch leading towards the cercozoan bigelowiella, but support is lacking for the basal relationships.
a plot of the posterior distribution of root placements  illustrates the uncertainty about the root placement more clearly.
it is evident from this plot that the choanozoans are not the oldest diverging lineage.
this finding overturns the conclusion from kamikawa et al.
<cit>  because the nested position of the diatom efl genes within the rhizaria sequences can no longer be maintained.
our efl phylogeny supports the presence of lateral gene transfer between eukaryotic lineages, however, the direction of lateral gene transfer is difficult to evaluate.
CONCLUSIONS
the mutually exclusive nature of ef-1α and efl is confirmed in a large sample of green algae.
the streptophyta possess ef-1α with the exception of mesostigma, which has efl.
the chlorophyta encode efl with the exception of dasycladales, bryopsidales, siphonocladales and ignatius, where ef-1α is found.
this result establishes ef-1α as a widespread gene among green algae.
gain-loss models revealed that the probabilities of the presence of ef-1α, efl or both genes along the backbone of the plant phylogeny are highly uncertain, and that a previously published hypothesis  <cit>  is as likely as several other hypotheses.
model refinements based on insights gained from the ef-1α phylogeny were unable to distinguish between three possibilities:  multiple, independent gains of efl throughout the plant lineage,  a single gain of efl early in evolution of the plant lineage followed by differential loss, or  independent gains of efl in mesostigma and the ancestor of the chlorophyta followed by differential loss of one or the other gene in the various lineages .
further research into the gain-loss dynamics of elongation factors of green plants and eukaryotes in general is needed to come to more definitive conclusions about their evolution.
first, the efl phylogeny should be refined by obtaining full-length sequences for a set of relevant taxa to confirm or reject the presence of multiple independent green lineages in this tree.
the use of codon models may help to achieve this  <cit> .
an alternative approach would be to learn about the processes responsible for lateral transfer of elongation factors by studying their flanking regions for signature sequences of mobile elements  <cit> .
finally, studying gain-loss dynamics across a wider spectrum of eukaryotic supergroups should lead to more stable conclusions.
in addition to yielding more precise parameter estimates for gene gain and loss rates, a eukaryote-wide study would allow the use of more specific models for lateral gene transfer because both donor and recipient lineages would be present in the analysis  <cit> .
it remains an enigma that the evolution of elongation factors, genes crucial for cell functioning, is marked by such complex gain-loss patterns.

 elongation factor- alpha is a core element of the translation apparatus and member of the gtpase protein family. the gene has been widely used as a phylogenetic marker in eukaryotes; either to resolve their early evolution or more recent phylogenetic patterns. the evolutionary history of genes used for such inferences should closely match that of the organisms and not be affected by ancient paralogy or lateral gene transfer. a gene related to but clearly distinguishable from ef-1α, called elongation factor-like, appears to substitute ef-1α in a scattered pattern: several unrelated eukaryote lineages have representatives that encode efl and others that possess ef-1α. the efl and ef-1α genes are mutually exclusive in all but two organisms: the zygomycete fungus basidiobolus and the diatom thalassiosira. although efl is found in several eukaryotic lineages, ef-1α is thought to be the most abundant of both. so far, efl has been reported in chromalveolates, the plant lineage, rhizarians, unikonts and centrohelids ]. the mutually exclusive distribution of ef-1α and efl suggests similar functionality. the main function of ef-1α is translation initiation and termination, by delivering aminoacyl trnas to the ribosomes. other functions include interactions with cytoskeletal proteins: transfer, immobilization and translation of mrna and involvement in the ubiquitine-dependent proteolytic system, as such forming an intriguing link between protein synthesis and degradation. in contrast, the function of efl is barely known. it is assumed to have a translational function because the putative ef-1β, aa-trna, and gtp/gdp binding sites do not differ between ef-1α and efl. based on a reverse transcriptase quantitative pcr assay in the diatom thalassiosira, which possesses both genes, it was proposed that efl had a translation function while ef-1α performed the auxiliary functions. the apparently scattered distribution of efl across eukaryotes raises questions about the gain-loss patterns of genes with an important role in the cell. this mutually exclusive and seemingly scattered distribution can be explained by two different mechanisms: ancient paralogy and lateral gene transfer. ancient paralogy was considered unlikely because this would imply that both genes were present in ancestral eukaryotic genomes during extended periods of evolutionary history while the genes rarely coexist in extant species. furthermore, a prolonged coexistence of both genes in early eukaryotes would have likely resulted in either functional divergence or pseudogene formation of one or the other copy, as is suggested for efl and ef-1α coexisting in the diatom thalassiosira. keeling and inagaki proposed lateral gene transfer of the efl gene between eukaryotic lineages as the most likely explanation for the scattered distribution of both genes. in the green plants, ef-1α and efl seem to show a mutually exclusive distribution. of the two major green plant lineages, the chlorophyta were shown to have efl with the exception of acetabularia where ef-1α is found, and the streptophyta were shown to possess ef-1α with the exception of mesostigma, which has efl. noble et al. proposed the hypothesis that efl was introduced once in the ancestor of the green lineage, followed by differential loss of ef-1α or efl in the principal clades of the viridiplantae. the goals of the present study are to extend our knowledge of the distribution pattern of ef-1α and efl in the green algae and investigate patterns of gain and loss of these key genes of the translational apparatus. we applied a rt-pcr and sequencing-based screening approach across a broad spectrum of green algae, with emphasis on the ulvophycean relatives of acetabularia. to test the hypothesis of noble et al., we modeled patterns of gene gain and loss. to this goal, a reference phylogeny based on three commonly used loci was inferred, and gain-loss dynamics of efl and ef-1α were optimized along this phylogeny using continuous-time markov models.  distribution of elongation factors in the green algae ef-1α sequences were retrieved from streptophytes entransia and chlorokybus, confirming previous observations that all streptophyta except mesostigma have ef-1α. we found efl sequences in chlorella, acrochaete and bolbocoleon, nephroselmis and tetraselmis striata, further confirming the formerly established distribution pattern within the chlorophyta. we reaffirmed the presence of efl in chlamydomonas and scenedesmus, ulva intestinalis and u. fenestra and ostreococcus, previously shown by noble et al.. in addition to acetabularia, ef-1α was discovered in representatives of the ulvophycean orders dasycladales, bryopsidales, siphonocladales and in ignatius. the rt-pcr approach did not reveal the presence of both genes in any of the screened species despite the fact that our primers could amplify the target genes across the viridiplantae. our rt-pcr experiments on two species whose genomes have been sequenced yielded a single gene for each species, a result in compliance with the knowledge derived from their genome sequences. the reference phylogeny, inferred from a dna matrix consisting of taxa representing all major plant lineages and three loci, is in accordance with recent phylogenetic studies, including the position of mesostigma within the streptophyta. figure shows the phylogenetic relationships among the taxa for which we have information on elongation factors; the full 72-taxon phylogeny can be found as an online supplement. even though the tree shows improved resolution from previous studies, large parts of the backbone remained poorly resolved. in order to obtain a solid hypothesis of green algal evolution, much additional sequence data may have to be gathered. the occurrence of ef-1α and efl in terminal taxa was plotted on the reference phylogeny in figure mesostigma is the only streptophyte which encodes efl. within the chlorophytan class ulvophyceae, the order ulvales possesses efl whereas the other orders encode ef-1α. phylogenies of ef-1α and efl all green plant ef-1α sequences form a monophyletic group clearly differentiated from ef-1α sequences of a variety of other eukaryotes. even though the viridiplantae form a strongly supported group, resolution among and within streptophyta and chlorophyta is generally low, which could in part be due to some short ef-1α sequences included in the analysis. in contrast, green plant efl genes do not form a monophyletic lineage. although the backbone of the phylogeny is moderately resolved, monophyly of green plant efl genes is unlikely because it is not observed in the mcmc output. efl sequences of the viridiplantae can be found in several clades. the chlorophytes, trebouxiophytes, ulvophytes and prasinophyte tetraselmis form a single monophyletic group. the other prasinophyte efl sequences form two separate groups. the last clade consists of the streptophyte mesostigma. to obtain an accurate root position for our efl tree, we included related subfamilies of the gtpase translation factor superfamily: ef-1α, eukaryotic release factor, heat shock protein subfamily b suppressor and archaebacterial ef-1α sequences in our analyses. in accordance with keeling and inagaki, the tree is rooted with archaebacterial ef-1α sequences. all analyses resulted in a phylogeny very similar to the one shown in figure 2b, the complete phylogeny with all related subfamilies can be found as an online supplement. this phylogeny shows seven efl clades, with the following branching order: bigelowiella, the diatoms, planoglabratella, the cryptophyte goniomonas, red algae, choanozoans, and a large clade containing the green plant lineage, chromalveolates, fungi and rhaphidiophrys. deep branches generally received low statistical support, preventing strong conclusions about the relationship between the seven clades. gain-loss dynamics the scattered distribution of ef-1α and efl in the green plant lineage is a remarkable phenomenon that raises questions about evolutionary patterns of gain and loss of both genes. noble et al. proposed the hypothesis that ef-1α was present in the common ancestor of the plant lineage, followed by a single gain of efl early in evolution of the green lineage and subsequent differential loss of one or the other gene in the various lineages. our aim was to test this hypothesis by modeling gain-loss dynamics and inferring ancestral presence-absence patterns of both genes in a maximum likelihood framework. gene gain and loss rates were estimated by maximum likelihood optimization, using a dataset of presence-absence patterns of ef-1α and efl and a reference phylogeny derived from the bayesian analysis of three commonly used loci. a first analysis, based on the reference tree, shows uncertain character state probabilities along the backbone of the viridiplantae and suggests a loss of ef-1α in early chlorophyta evolution and regain in some ulvophyceae. because branch lengths play a crucial role in model optimization, the analysis was repeated on an alternative version of the reference tree in which branch lengths were transformed using a rate smoothing approach. since our tree deviates from the molecular clock, we performed rate smoothing to obtain branch lengths roughly proportional to time. rate smoothing techniques relax the assumption of constant rates of evolution throughout the tree: differences in rates of molecular evolution are smoothed out by assuming that evolutionary rates change gradually throughout the phylogeny. the result is an ultrametric tree in which branch lengths are roughly proportional to evolutionary time instead of amounts of molecular evolution. modeling gain-loss dynamics of elongation factor genes along the rate-smoothed tree yields results that strongly deviate from those obtained with the original reference tree: probabilities of the character states along the major part of backbone are now around 50% for efl and around 50% for the presence of both genes. subsequently, an additional level of realism was introduced by taking phylogenetic uncertainty into account because several nodes in the reference tree are poorly supported. to this goal, all post-burnin mcmc trees were rate-smoothed and analyzed individually. the results were summarized on the rate-smoothed reference tree. taking phylogenetic uncertainty into consideration had a minor influence on the probabilities of the characters states. although the exact numbers differ between analyses, gene gain rates were always lower than gene loss rates, reinforcing the notion that gene transfers are rare events in comparison to losses of redundant genes. whereas the analysis based on the original reference tree returned faster gain and loss rates for efl than for ef-1α, analyses based on rate-smoothed trees suggested the inverse, marking the sensitivity of markov models to the unit of operational time. from these results, it seems fair to conclude that there is major uncertainty about the ancestral states for a variety of reasons, including sensitivity to branch lengths and lack of prior knowledge about ancestral states or rates of gene gain and loss. considering that the ancestors must have had either ef-1α, efl or both genes opens perspectives for hypothesis comparison in a likelihood framework. additionally, information about rates of gene gain and loss could be gleaned from the ef-1α and efl phylogenies. analyses constrained with various hypotheses about ancestral gene content resulted in a confidence set of trees that differ extensively. the fact that strongly different hypotheses are also present in the confidence set denotes that the likelihood surface is too flat to draw firm conclusions in favor of one or another hypothesis. the last option to reduce uncertainty is to inform the markov models with information on gains and losses gleaned from the ef-1α and efl trees. because green plant ef-1α sequences form a monophyletic and strongly supported lineage, it seems fair to assume vertical descent of ef-1α throughout the viridiplantae. this knowledge can be introduced in our markov model by setting a very low gain rate of ef-1α. if the analysis is constrained in this way, both efl and ef-1α were inferred to be present along the backbone of the viridiplantae in the original reference tree and a 50/ probability for the presence of ef-1α or both genes was obtained in the rate-smoothed trees. comparison of hypotheses about ancestral gene content constrained with a very low ef-1α gain rate reduced the confidence set to trees in which either ef-1α or both genes are present along the backbone. the ml solution assumes that only ef-1α was present along the backbone of the tree and consequently shows independent gains of efl in mesostigma, prasinophytes, chlorophyceae, trebouxiophyceae and ulvales. an alternative scenario in the confidence set has ef-1α at the base of the viridiplantae, a gain of efl in the ancestor of the chlorophyta, and subsequent differential loss of one or the other gene in the various lineages. information from the efl phylogeny may provide clues for further distinction between either multiple transfers or ancient paralogy with subsequent losses. the green efl sequences form a highly supported clade together with dinoflagellates, cryptophytes, haptophytes, fungi and rhaphidiophrys, suggesting lateral gene transfer of the efl gene between these distant eukaryotic lineages. considering the ability of chromalveolates and raphidiophrys to feed through phagocytosis and the absence of this behavior in green algae, it would be tempting to assume that lateral gene transfer occurred from green algae to the dinoflagellates, cryptophytes, haptophytes and raphidiophrys instead of the other way around. phagotrophic eukaryotes have been shown to have elevated rates of lateral gene transfer because this feeding mechanism enables them to continually recruit genes from engulfed prey. lateral gene transfers to fungi, although known to exist, would require a different explanation because neither phagotrophy nor endosymbiosis occur in fungi. however, in the light of this peripheral information, it would be tempting to conclude that both ef-1α and efl essentially show vertical descent in green plants and that the observed mutually exclusive pattern of efl and ef-1α sequences results from differential loss. in this scenario, lateral gene transfer must have occurred from green algal cells to other eukaryotic lineages. in previous studies of functionally similar eukaryotic genes with mutually exclusive distributions, distinction between ancient paralogy with subsequent losses and multiple transfers was made based on two main criteria. the first criterion states that if one gene dominates the tree and the other occurs in only a few lineages, multiple independent transfers should be regarded as the most probable explanation whereas equal representation would suggest common ancestry with subsequent differential loss. the second criterion is about the age of the taxa involved. if the mutually exclusive pattern occurs between closely related species, one can conclude common ancestry with subsequent losses. if the pattern is more ancient, multiple lateral transfers are a more probable explanation. it is obvious that such criteria are very difficult to apply in real situations. these difficulties can be overcome by taking a probabilistic angle on the problem and modeling gain-loss dynamics with continuous-time markov models. this approach brings statistical rigor to the analysis of gene presence-absence patterns and has the potential to discriminate between the alternative scenarios of ancient paralogy with differential losses and multiple independent lateral transfers. application of this technique to our dataset of green algal elongation factors revealed the difficulty of arriving at firm conclusions about ancient gene transfer events because of a relatively flat likelihood surface and, consequently, ambiguous probabilities for gene content at ancestral nodes. when informed with external information, the analyses allow somewhat more definitive conclusions. the broader eukaryotic picture in addition to the information gained about elongation factor evolution in green algae, our results also highlight misinterpretations in recent literature on efl evolution across the eukaryotes. previous studies have not been explicit about whether or how their phylogenetic trees were rooted, but have drawn conclusions that require directionality in the tree. kamikawa et al. concluded that lateral gene transfer from a foraminifer to the ancestor of the diatoms must have occurred because the diatom sequences were nested within the rhizaria. in case their tree was unrooted, this conclusion is flawed due to a lack of directionality in the tree. in their presentation of the tree, choanozoans are used as one of the basal clades, probably because they were the earliest-branching lineage in the tree presented by keeling and inagaki. our efl tree, which includes ef-1α, erf and hbs sequences and is rooted with archaebacterial ef-1α sequences, indicates that the directionality inferred by kamikawa et al. is likely to be wrong. our phylogram suggest that the root position of efl lies on the branch leading towards the cercozoan bigelowiella, but support is lacking for the basal relationships. a plot of the posterior distribution of root placements illustrates the uncertainty about the root placement more clearly. it is evident from this plot that the choanozoans are not the oldest diverging lineage. this finding overturns the conclusion from kamikawa et al. because the nested position of the diatom efl genes within the rhizaria sequences can no longer be maintained. our efl phylogeny supports the presence of lateral gene transfer between eukaryotic lineages, however, the direction of lateral gene transfer is difficult to evaluate.  the mutually exclusive nature of ef-1α and efl is confirmed in a large sample of green algae. the streptophyta possess ef-1α with the exception of mesostigma, which has efl. the chlorophyta encode efl with the exception of dasycladales, bryopsidales, siphonocladales and ignatius, where ef-1α is found. this result establishes ef-1α as a widespread gene among green algae. gain-loss models revealed that the probabilities of the presence of ef-1α, efl or both genes along the backbone of the plant phylogeny are highly uncertain, and that a previously published hypothesis is as likely as several other hypotheses. model refinements based on insights gained from the ef-1α phylogeny were unable to distinguish between three possibilities: multiple, independent gains of efl throughout the plant lineage, a single gain of efl early in evolution of the plant lineage followed by differential loss, or independent gains of efl in mesostigma and the ancestor of the chlorophyta followed by differential loss of one or the other gene in the various lineages. further research into the gain-loss dynamics of elongation factors of green plants and eukaryotes in general is needed to come to more definitive conclusions about their evolution. first, the efl phylogeny should be refined by obtaining full-length sequences for a set of relevant taxa to confirm or reject the presence of multiple independent green lineages in this tree. the use of codon models may help to achieve this. an alternative approach would be to learn about the processes responsible for lateral transfer of elongation factors by studying their flanking regions for signature sequences of mobile elements. finally, studying gain-loss dynamics across a wider spectrum of eukaryotic supergroups should lead to more stable conclusions. in addition to yielding more precise parameter estimates for gene gain and loss rates, a eukaryote-wide study would allow the use of more specific models for lateral gene transfer because both donor and recipient lineages would be present in the analysis. it remains an enigma that the evolution of elongation factors, genes crucial for cell functioning, is marked by such complex gain-loss patterns. 
in the green plant lineage, the chlorophyta encode efl except acetabularia where ef-1α is found, and the streptophyta possess ef-1α except mesostigma, which has efl. these results raise questions about evolutionary patterns of gain and loss of ef-1α and efl. in order to gain more insight in the distribution of ef-1α and efl in green plants and test this hypothesis we screened the presence of the genes in a large sample of green algae and analyzed their gain-loss dynamics in a maximum likelihood framework using continuous-time markov models. ef-1α is much more common among green algae than previously thought. hypotheses about the gain-loss dynamics of elongation factor genes are hard to test analytically due to a relatively flat likelihood surface, even if prior knowledge is incorporated. a previous study launched the hypothesis that ef-1α was the primitive state and that efl was gained once in the ancestor of the green plants, followed by differential loss of ef-1α or efl in the principal clades of the viridiplantae. model refinements based on insights gained from the ef-1α phylogeny reduce uncertainty but still imply several equally likely possibilities: a primitive ef-1α state with multiple independent efl gains or coexistence of both genes in the ancestor of the viridiplantae or chlorophyta followed by differential loss of one or the other gene in the various lineages. the mutually exclusive distribution of ef-1α and efl is confirmed in a large sample of green plants. phylogenetic analysis of efl genes indicates misinterpretations in the recent literature due to uncertainty regarding the root position. two key genes of the translational apparatus, elongation factor- alpha and elongation factor-like have an almost mutually exclusive distribution in eukaryotes. models describing gene gain-loss dynamics revealed that the presence of ef-1α, efl or both genes along the backbone of the green plant phylogeny is highly uncertain due to sensitivity to branch lengths and lack of prior knowledge about ancestral states or rates of gene gain and loss.
 two key genes of the translational apparatus, elongation factor- alpha and elongation factor-like have an almost mutually exclusive distribution in eukaryotes. in the green plant lineage, the chlorophyta encode efl except acetabularia where ef-1α is found, and the streptophyta possess ef-1α except mesostigma, which has efl. these results raise questions about evolutionary patterns of gain and loss of ef-1α and efl. a previous study launched the hypothesis that ef-1α was the primitive state and that efl was gained once in the ancestor of the green plants, followed by differential loss of ef-1α or efl in the principal clades of the viridiplantae. in order to gain more insight in the distribution of ef-1α and efl in green plants and test this hypothesis we screened the presence of the genes in a large sample of green algae and analyzed their gain-loss dynamics in a maximum likelihood framework using continuous-time markov models.  within the chlorophyta, ef-1α is shown to be present in three ulvophycean orders and the genus ignatius. models describing gene gain-loss dynamics revealed that the presence of ef-1α, efl or both genes along the backbone of the green plant phylogeny is highly uncertain due to sensitivity to branch lengths and lack of prior knowledge about ancestral states or rates of gene gain and loss. model refinements based on insights gained from the ef-1α phylogeny reduce uncertainty but still imply several equally likely possibilities: a primitive ef-1α state with multiple independent efl gains or coexistence of both genes in the ancestor of the viridiplantae or chlorophyta followed by differential loss of one or the other gene in the various lineages.  ef-1α is much more common among green algae than previously thought. the mutually exclusive distribution of ef-1α and efl is confirmed in a large sample of green plants. hypotheses about the gain-loss dynamics of elongation factor genes are hard to test analytically due to a relatively flat likelihood surface, even if prior knowledge is incorporated. phylogenetic analysis of efl genes indicates misinterpretations in the recent literature due to uncertainty regarding the root position. 