BACKGROUND
elongation factor 1α  proteins in eukaryotes and archaebacteria, and their orthologues in bacteria , are gtpases required for the central process of translation  <cit> .
the primary sequence of ef-1α is highly conserved across the tree of life, suggesting that this protein was established in the last universal common ancestor, and inherited by extant organisms  <cit> .
however, genomic and transcriptomic data from diverse organisms have shown that some eukaryotic lineages lack ef-1α, and these lineages instead were found to possess a putative ef-1α-related gtpase  <cit> .
these elongation factor-like  proteins are believed to perform the same function in translation as ef-1α, as there is no significant functional divergence in the regions that are critical for ef-1α function  <cit> .
the functional equivalence of efl and ef-1α would explain the mutually exclusive distributions of efl and ef-1α genes amongst eukaryotes since ef-1α would be functionally redundant in eukaryotes with efl-mediated translation elongation, and vice versa.
intensive surveys for efl genes in phylogenetically diverse eukaryotes revealed a number of groups that have both ‘ef-1α-containing’ and ‘efl-containing’ species  <cit> .
the co-existence of ef-1α-containing and efl-containing species in a monophyletic group can be explained by the ancestral co-occurrence of ef-1α and efl, and subsequent losses of either of the two elongation factors in the descendants.
henceforth, we designate the above scenario simply as the ‘differential loss’ hypothesis  <cit> .
many aspects of this hypothesis are difficult to test experimentally.
nonetheless, dual expression of ef-1α and efl proteins in trypanosoma brucei cells, which corresponds to the ancestral state assumed in the differential loss hypothesis, had no apparent impact on cell viability  <cit> .
it was previously found that examined diatom species were either ef-1α-containing or efl-containing, except for a single species, thalassiosira pseudonana, whose genome encodes both ef-1α and efl genes  <cit> .
according to the differential loss hypothesis described above, the ef-1α/efl gene data from diatoms can be explained as follows:  the ancestral diatom genome was ‘dual-ef-containing,’  the t. pseudonana genome retains the ancestral state, and  the ef-1α  gene was lost in the extant efl-containing  descendants  <cit> .
a similar situation has been proposed for fungi; although the vast majority of fungal species are either ef-1α-containing or efl-containing, a single species, basidiobolus ranarum, was found to be dual-ef-containing  <cit> .
under the differential loss hypothesis, t. pseudonana and b. ranarum retain the ancestral state of diatom and fungal genomes, respectively.
the differential loss hypothesis is an increasingly popular explanation of the current ef-1α/efl gene distribution in the tree of eukaryotes.
nevertheless, dual-ef-containing species, which are believed to reflect the ancestral state of their phylogenetic relatives containing either ef-1α or efl, have, to date, only been described in diatoms and fungi.
in this study, by experimental surveys and data mining in publicly available genome and/or transcriptomic data, four independent lineages—stramenopiles, apusomonadida, goniomonadida, and fungi—were found to contain at least one dual ef-containing species .
all ef-1α genes in the dual ef-containing species examined here appear to be divergent, and are transcribed at a much lower level than the co-occurring efl genes, suggesting that ef-1α has functionally diverged in these species.
we propose that the re-modeling of the original ef-1α functions seemingly occurred in several independent branches of the tree of eukaryotes.
RESULTS
we successfully isolated/identified  <dig> and  <dig> previously unidentified ef-1α and efl sequences, respectively, by a pcr survey or mining publicly available and in-house genomic/transcriptomic databases .
five diatoms, three oomycetes, one goniomonad, one apusomonad, and a chytridiomycete fungus were found to be dual-ef-containing in this study, in addition to the two previously reported dual-ef-containing species, the diatom t. pseudonana <cit>  and a fungus of uncertain taxonomic affiliation, b. ranarum <cit> .
we updated ef-1α and efl alignments by adding the new sequences listed in table  <dig>  and both alignments were analyzed with maximum-likelihood  and bayesian phylogenetic methods .
*co-occurred with efl, ¶co-occurred with ef-1α.
accession numbers for the sequences obtained by public database search are not described, but their protein sequences were shown in additional file  <dig>
dual-ef-containing species in diatoms
the majority of diatom species, in which ef-1α/efl sequences have been characterized to date, appear to possess efl genes, except for the genomes of phaeodactylum tricornutum <cit> , which encodes only an ef-1α gene, and t. pseudonana, which encodes both ef-1α and efl genes  <cit> .
in this study, we surveyed ef-1α/efl genes in diatoms further, and identified five more dual-ef-containing species, indicating that dual-ef-containing species are quite prevalent amongst diatoms.
ef-1α transcripts were detected in detonula confervacea, achnanthes kuwaitensis, fragilariopsis cylindrus, thalassionema nitzschioides, and asterionella glacialis, all of which were previously considered to be ‘efl-containing’.
in the ef-1α ml tree, all diatom homologues grouped together with an ml bootstrap value  of 57% , and this group branches with the ef-1α homologues of the bolidophyte bolidomonas pacifica.
although the statistical support for the diatom-bolidomonas affiliation was moderate , this particular affiliation found in the ef-1α phylogeny is consistent with their close  relationships  <cit> .
thus we concluded that there had been vertical descent of ef-1α genes in the diatom-bolidomonas clade.
as shown in previous studies e.g.,  <cit> , the updated efl phylogeny also includes a diatom clade, indicating the vertical descent of efl genes in this lineage .
quantitative reverse transcriptase pcr  assays revealed that the expression level of the efl gene is much greater than that of the ef-1α gene in each of the dual ef-containing diatom species identified in this study , except for f. cylindrus, for which these assays were not performed.
however, ef-1α transcripts are likely much less abundant than efl transcripts in f. cylindrus as well, since only efl transcripts were detected in the f. cylindrus transcriptomic data publicly available from the joint genome institute .
notes—normalized by the copy number of α-tubulin transcripts.
dual-ef-containing species in oomycetes
only ef-1α homologues were identified in well-studied members of the oomycetes , but some of us have recently reported efl genes in pythium oligandrum and pythium ultimum <cit> .
in this study, we resurveyed ef-1α/efl sequences in  <dig> members of the genus pythium, and identified pythium intermedium, py.
ultimum, and py.
apleroticum as dual-ef-containing.
the ef-1α phylogenetic analysis successfully recovered the monophyly of all of oomycetes, suggesting that py.
intermedium, py.
ultimum, and py.
apleroticum vertically inherited their ef-1α genes from a common oomycete ancestor.
we suspect that the ml bootstrap support for the oomycete clade in the ef-1α analysis was lowered due to the divergent nature of the py.
intermedium, py.
ultimum, and py.
apleroticum homologues .
the efl phylogeny also robustly unites all oomycete efl sequences, including those of the three dual-ef-containing pythium spp.
.
the ef-1α gene of py.
ultimum is seemingly much less transcribed than its efl gene.
in illumina transcriptomic data, the k-mer frequency for efl contig was significantly higher than that for a cytoskeletal protein, α-tubulin .
in sharp contrast, no contig for ef-1α was obtained in the transcriptomic data , even though our rt-pcr successfully detected ef-1α transcripts in py.
ultimum .
dual-ef-containing species in goniomonads
prior to this study, ef-1α/efl data were available for only two goniomonad species: ef-1α transcripts were detected in goniomonas pacifica <cit> , while an efl gene was isolated from goniomonas amphinema <cit> .
in this study, we experimentally surveyed ef-1α/efl sequences in five goniomonas strains .
of these, strain atcc  <dig> appeared to be dual-ef-containing .
a qrt-pcr assay revealed that efl transcripts were more abundant than ef-1α transcripts in strain atcc  <dig> .
the ef-1α sequences amplified from strains atcc  <dig>  atcc pra <dig>  nies- <dig>  and ccap 980_ <dig>  together with that of g. pacifica, formed a clade in the ef-1α phylogeny .
the new efl homologues from strains nies  <dig> and atcc  <dig> showed a close relationship to the g. amphinema homologue .
both ef-1α and efl phylogenies suggest vertical inheritance of the genes encoding the two elongation factors in this lineage.
other dual-ef-containing species in apusomonadida and fungi
we detected both efl and ef-1α sequences in both whole-genome shotgun and transcriptomic data from the apusomonad thecamonas trahens .
the ef-1α sequences of two apusomonads, t. trahens and apusomonas proboscidea, grouped together in the ml tree topology , consistent with their organismal relationship.
the large discrepancy in branch length between the two apusomonad sequences is likely responsible for the low ml bootstrap support.
in the efl phylogeny, the t. trahens sequence branched at the base of the diatom-oomycete clade .
unfortunately, the current analysis does not allow us to determine if efl genes were the result of descent through vertical inheritance in apusomonads, because:  only one efl sequence is known for apusomonads, and  t. trahens and opisthokonts were distant from each other in the efl phylogeny, in contrast to the close organismal relationship between apusomonads and opisthokonts e.g.,  <cit> .
our ef-1α/efl gene survey also identified the genome of the chytridiomycote fungus spizellomyces punctatus as encoding both kinds of elongation factors.
the ef-1α sequences of s. punctatus and b. ranarum bore the well-known opisthokonta-specific insertion , and formed a clade with other fungal sequences in the phylogenetic analyses , suggesting that the ef-1α genes of s. punctatus and b. ranarum and those of other fungal species share an exclusive ancestry.
again, the grouping of the two long-branched sequences of s. punctatus and b. ranarum with other fungal sequences did not receive high ml bootstrap support.
we are currently unsure whether the extant efl genes in fungi are the descendents of a single gene in the ancestral fungal species: the monophyly of fungi was not recovered in the ml tree inferred from the efl alignment , but the approximately unbiased test  <cit>  failed to reject the alternative hypothesis, in which all fungal efl sequences were enforced to be monophyletic, at the 5% level .
in both t. trahens and s. punctatus there is a large difference in transcriptional levels between ef-1α and efl genes.
in the transcriptomic data of the two species, the k-mer frequency for efl was much greater than that for ef-1α , as seen in other dual-ef-containing species .
new ef-1α/efl data from other eukaryotes
our ef-1α/efl survey successfully revealed that the taxa katablepharida, amoebozoa , and ancyromonadida contain both efl-containing and ef-1α-containing species.
for amoebozoans and ancyromonads, only ef-1α-containing species were known prior to this study , however, we detected efl sequences in the amoebozoan copromyxa protea and the ancyromonad fabomonas tropica .
likewise, the first-surveyed katablepharid leucocryptos marina was efl-containing  <cit> , but a rt-pcr survey of a secondly-surveyed katablepharid, roombia sp., identified ef-1α transcripts .
discussion
several eukaryote lineages include multiple dual-ef-containing species
ancestral co-occurrence of ef-1α and efl followed by differential loss of one of the two elongation factors most likely shaped the current ef-1α/efl distribution within eukaryotes.
in this scenario, the extant dual-ef-containing species retain the ancestral state and thus are analogous to the inferred intermediates that led to descendant lineages that contain either ef-1α or efl .
in this study, we found  <dig> new dual-ef-containing species in four distantly related lineages:  goniomonadida,  apusomonadida,  stramenopiles , and  fungi .
in light of the differential loss process proposed for ef-1α/efl evolution, we speculate that more dual-ef-containing species remain undetected in other lineages that contain both ef-1α-containing and efl-containing species, including: viridiplantae  <cit> , euglenozoa  <cit> , choanoflagellata  <cit> , endomyxa  <cit> , filosa  <cit> , rhodophyta  <cit> , katablepharida , amoebozoa , and ancyromonadida  .
considering the revised distribution of ef-1α/efl genes, we cannot exclude the possibility that the last eukaryotic common ancestor was dual-ef-containing.
finally, it will be of interest to continue surveying dual-ef-containing species, especially within stramenopiles and fungi.
kamikawa et al.
<cit>  postulated that the dual-ef status can be traced back to the ancestral stramenopile species, based on the monophyly of stramenopiles in ef-1α phylogenies , and of diatoms and oomycetes in efl phylogenies .
thus, we predict that dual-ef-containing species should be found in so-far unsampled stramenopiles.
similarly, s. punctatus and b. ranarum are unlikely to be the sole fungal species with a dual-ef status, given that the most recent common ancestral fungus was proposed to be dual-ef-containing  <cit> .
parallel re-modeling of ef-1α function in eukaryotic evolution
in the dual ef-containing diatom t. pseudonana, some of us  <cit>  proposed that the ef-1α homolog performs only a subset of its original functions, and does not participate in protein synthesis as an elongation factor, for the following reasons.
firstly, in an ef-1α phylogeny, the t. pseudonana homologue was much more divergent than that of a closely related ef-1α-containing species, p. tricornutum, suggesting that the former is under fewer functional constraints than the latter.
secondly, ef-1α transcripts were much less abundant in t. pseudonana than the transcripts of efl or of an α-tubulin gene.
as observed in t. pseudonana, the five dual ef-containing diatoms identified in this study  appeared to possess divergent ef-1α genes .
in each of the five diatoms, the transcriptional level of the ef-1α gene was heavily suppressed compared to that of the co-occurring efl gene .
thus, the five dual-ef-containing diatoms most likely use efl as the principal elongation factor, while a sub-set of the original ef-1α functions is assigned to the divergent ef-1α.
these dual-ef-containing diatoms have most likely re-modeled their ef-1α functions, such that they carry out only the auxiliary roles that the proteins originally performed, such as interactions with cytoskeletal proteins and ubiquitin-dependent protein degradation  <cit> .
it is likely that similar re-modeling of ef-1α function has also occurred in other dual-ef-containing lineages.
in the non-diatom dual-ef-containing species, the ef-1α sequences were also divergent , and were transcribed at a low level compared to the co-occurring efl genes .
these results strongly suggest that dual-ef-containing species in general utilize ef-1α for subsets of the original functions, while efl participates in translation as a core factor.
significantly, the re-modeling of ef-1α function probably took place separately in stramenopiles , goniomonadida, apusomonadida, and fungi, as these lineages are distantly related to one another in the organismal phylogeny.
moreover, diatoms  and oomycetes  may have also re-modeled their ef-1α functions in parallel as they are relatively distantly related within stramenopiles.
we also suspect that parallel re-modeling of ef-1α function occurred within fungi, as s. punctatus and b. ranarum are not particularly close relatives  <cit> .
we are currently unsure about the precise functions of the divergent ef-1α in the dual-ef-containing species.
under the parallel re-modeling scenario proposed above, the suite of retained ef-1α functions could vary between any of two dual-ef-containing lineages.
however, the overall substitution patterns in divergent ef-1α sequences in distantly related dual-ef-containing species are found to be similar to each other .
this observation hints at parallel loss of the same aspects of ef-1α function and retention of a subset of original functions in multiple dual-ef-containing lineages scattered over the tree of eukaryotes.
these speculations could be tested more directly by biochemical studies of ef-1α function in selected representatives of these lineages.
CONCLUSIONS
according to the differential loss hypothesis for ef-1α/efl evolution, a dual-ef-containing ancestor likely gave rise to two types of descendants—one containing only efl and the other containing only ef-1α.
nevertheless, ef-1α/efl surveys, including this study, have identified an additional type of descendent retaining the ancestral arrangement  in multiple branches of the tree of eukaryotes.
if ef-1α/efl sequences are surveyed in a broader spectrum of eukaryotes, it is highly likely that the number and diversity of known dual-ef-containing species will grow further.
curiously, all dual-ef-containing species identified so far appear to retain divergent, low-expressed ef-1α genes , which are analogous to the hypothetical intermediate leading to efl-containing descendants .
we suspect that the multiple functions of the canonical ef-1α may have prevented the dual-ef-containing cells from losing this protein immediately after efl took over from ef-1α as the core translation factor.
the presence of dual-ef-containing species indicates that the adoption of efl as the dominant core factor in translation does not necessarily lead to the elimination of ef-1α from the entire cellular system.
curiously, we found little evidence for living analogues of the hypothetical intermediate that led to ef-1α-containing descendants, which would possess a divergent, low-transcribed efl gene.
the presence or absence of dual-ef-containing species, in which a divergent efl gene is transcribed at lower levels than the co-occurring ef-1α gene, would be crucial to understanding the evolutionary processes that shaped the current ef-1α/efl gene distribution across the tree of eukaryotes.
we need to re-examine efl sequences in the species currently recognized as ‘ef-1α-containing’ since low-expressed efl genes might be overlooked in these taxa, especially if genomic or high-coverage transcriptomic data is lacking.

 elongation factor 1α proteins in eukaryotes and archaebacteria, and their orthologues in bacteria, are gtpases required for the central process of translation. the primary sequence of ef-1α is highly conserved across the tree of life, suggesting that this protein was established in the last universal common ancestor, and inherited by extant organisms. however, genomic and transcriptomic data from diverse organisms have shown that some eukaryotic lineages lack ef-1α, and these lineages instead were found to possess a putative ef-1α-related gtpase. these elongation factor-like proteins are believed to perform the same function in translation as ef-1α, as there is no significant functional divergence in the regions that are critical for ef-1α function. the functional equivalence of efl and ef-1α would explain the mutually exclusive distributions of efl and ef-1α genes amongst eukaryotes since ef-1α would be functionally redundant in eukaryotes with efl-mediated translation elongation, and vice versa. intensive surveys for efl genes in phylogenetically diverse eukaryotes revealed a number of groups that have both ‘ef-1α-containing’ and ‘efl-containing’ species. the co-existence of ef-1α-containing and efl-containing species in a monophyletic group can be explained by the ancestral co-occurrence of ef-1α and efl, and subsequent losses of either of the two elongation factors in the descendants. henceforth, we designate the above scenario simply as the ‘differential loss’ hypothesis. many aspects of this hypothesis are difficult to test experimentally. nonetheless, dual expression of ef-1α and efl proteins in trypanosoma brucei cells, which corresponds to the ancestral state assumed in the differential loss hypothesis, had no apparent impact on cell viability. it was previously found that examined diatom species were either ef-1α-containing or efl-containing, except for a single species, thalassiosira pseudonana, whose genome encodes both ef-1α and efl genes. according to the differential loss hypothesis described above, the ef-1α/efl gene data from diatoms can be explained as follows: the ancestral diatom genome was ‘dual-ef-containing,’ the t. pseudonana genome retains the ancestral state, and the ef-1α gene was lost in the extant efl-containing descendants. a similar situation has been proposed for fungi; although the vast majority of fungal species are either ef-1α-containing or efl-containing, a single species, basidiobolus ranarum, was found to be dual-ef-containing. under the differential loss hypothesis, t. pseudonana and b. ranarum retain the ancestral state of diatom and fungal genomes, respectively. the differential loss hypothesis is an increasingly popular explanation of the current ef-1α/efl gene distribution in the tree of eukaryotes. nevertheless, dual-ef-containing species, which are believed to reflect the ancestral state of their phylogenetic relatives containing either ef-1α or efl, have, to date, only been described in diatoms and fungi. in this study, by experimental surveys and data mining in publicly available genome and/or transcriptomic data, four independent lineages—stramenopiles, apusomonadida, goniomonadida, and fungi—were found to contain at least one dual ef-containing species. all ef-1α genes in the dual ef-containing species examined here appear to be divergent, and are transcribed at a much lower level than the co-occurring efl genes, suggesting that ef-1α has functionally diverged in these species. we propose that the re-modeling of the original ef-1α functions seemingly occurred in several independent branches of the tree of eukaryotes.  we successfully isolated/identified and previously unidentified ef-1α and efl sequences, respectively, by a pcr survey or mining publicly available and in-house genomic/transcriptomic databases. five diatoms, three oomycetes, one goniomonad, one apusomonad, and a chytridiomycete fungus were found to be dual-ef-containing in this study, in addition to the two previously reported dual-ef-containing species, the diatom t. pseudonana and a fungus of uncertain taxonomic affiliation, b. ranarum. we updated ef-1α and efl alignments by adding the new sequences listed in table  and both alignments were analyzed with maximum-likelihood and bayesian phylogenetic methods. *co-occurred with efl, ¶co-occurred with ef-1α. accession numbers for the sequences obtained by public database search are not described, but their protein sequences were shown in additional file dual-ef-containing species in diatoms the majority of diatom species, in which ef-1α/efl sequences have been characterized to date, appear to possess efl genes, except for the genomes of phaeodactylum tricornutum, which encodes only an ef-1α gene, and t. pseudonana, which encodes both ef-1α and efl genes. in this study, we surveyed ef-1α/efl genes in diatoms further, and identified five more dual-ef-containing species, indicating that dual-ef-containing species are quite prevalent amongst diatoms. ef-1α transcripts were detected in detonula confervacea, achnanthes kuwaitensis, fragilariopsis cylindrus, thalassionema nitzschioides, and asterionella glacialis, all of which were previously considered to be ‘efl-containing’. in the ef-1α ml tree, all diatom homologues grouped together with an ml bootstrap value of 57%, and this group branches with the ef-1α homologues of the bolidophyte bolidomonas pacifica. although the statistical support for the diatom-bolidomonas affiliation was moderate, this particular affiliation found in the ef-1α phylogeny is consistent with their close relationships. thus we concluded that there had been vertical descent of ef-1α genes in the diatom-bolidomonas clade. as shown in previous studies e.g.,, the updated efl phylogeny also includes a diatom clade, indicating the vertical descent of efl genes in this lineage. quantitative reverse transcriptase pcr assays revealed that the expression level of the efl gene is much greater than that of the ef-1α gene in each of the dual ef-containing diatom species identified in this study, except for f. cylindrus, for which these assays were not performed. however, ef-1α transcripts are likely much less abundant than efl transcripts in f. cylindrus as well, since only efl transcripts were detected in the f. cylindrus transcriptomic data publicly available from the joint genome institute. notes—normalized by the copy number of α-tubulin transcripts. dual-ef-containing species in oomycetes only ef-1α homologues were identified in well-studied members of the oomycetes, but some of us have recently reported efl genes in pythium oligandrum and pythium ultimum. in this study, we resurveyed ef-1α/efl sequences in members of the genus pythium, and identified pythium intermedium, py. ultimum, and py. apleroticum as dual-ef-containing. the ef-1α phylogenetic analysis successfully recovered the monophyly of all of oomycetes, suggesting that py. intermedium, py. ultimum, and py. apleroticum vertically inherited their ef-1α genes from a common oomycete ancestor. we suspect that the ml bootstrap support for the oomycete clade in the ef-1α analysis was lowered due to the divergent nature of the py. intermedium, py. ultimum, and py. apleroticum homologues. the efl phylogeny also robustly unites all oomycete efl sequences, including those of the three dual-ef-containing pythium spp.. the ef-1α gene of py. ultimum is seemingly much less transcribed than its efl gene. in illumina transcriptomic data, the k-mer frequency for efl contig was significantly higher than that for a cytoskeletal protein, α-tubulin. in sharp contrast, no contig for ef-1α was obtained in the transcriptomic data, even though our rt-pcr successfully detected ef-1α transcripts in py. ultimum. dual-ef-containing species in goniomonads prior to this study, ef-1α/efl data were available for only two goniomonad species: ef-1α transcripts were detected in goniomonas pacifica, while an efl gene was isolated from goniomonas amphinema. in this study, we experimentally surveyed ef-1α/efl sequences in five goniomonas strains. of these, strain atcc appeared to be dual-ef-containing. a qrt-pcr assay revealed that efl transcripts were more abundant than ef-1α transcripts in strain atcc. the ef-1α sequences amplified from strains atcc atcc pra nies- and ccap 980_ together with that of g. pacifica, formed a clade in the ef-1α phylogeny. the new efl homologues from strains nies and atcc showed a close relationship to the g. amphinema homologue. both ef-1α and efl phylogenies suggest vertical inheritance of the genes encoding the two elongation factors in this lineage. other dual-ef-containing species in apusomonadida and fungi we detected both efl and ef-1α sequences in both whole-genome shotgun and transcriptomic data from the apusomonad thecamonas trahens. the ef-1α sequences of two apusomonads, t. trahens and apusomonas proboscidea, grouped together in the ml tree topology, consistent with their organismal relationship. the large discrepancy in branch length between the two apusomonad sequences is likely responsible for the low ml bootstrap support. in the efl phylogeny, the t. trahens sequence branched at the base of the diatom-oomycete clade. unfortunately, the current analysis does not allow us to determine if efl genes were the result of descent through vertical inheritance in apusomonads, because: only one efl sequence is known for apusomonads, and t. trahens and opisthokonts were distant from each other in the efl phylogeny, in contrast to the close organismal relationship between apusomonads and opisthokonts e.g.,. our ef-1α/efl gene survey also identified the genome of the chytridiomycote fungus spizellomyces punctatus as encoding both kinds of elongation factors. the ef-1α sequences of s. punctatus and b. ranarum bore the well-known opisthokonta-specific insertion, and formed a clade with other fungal sequences in the phylogenetic analyses, suggesting that the ef-1α genes of s. punctatus and b. ranarum and those of other fungal species share an exclusive ancestry. again, the grouping of the two long-branched sequences of s. punctatus and b. ranarum with other fungal sequences did not receive high ml bootstrap support. we are currently unsure whether the extant efl genes in fungi are the descendents of a single gene in the ancestral fungal species: the monophyly of fungi was not recovered in the ml tree inferred from the efl alignment, but the approximately unbiased test failed to reject the alternative hypothesis, in which all fungal efl sequences were enforced to be monophyletic, at the 5% level. in both t. trahens and s. punctatus there is a large difference in transcriptional levels between ef-1α and efl genes. in the transcriptomic data of the two species, the k-mer frequency for efl was much greater than that for ef-1α, as seen in other dual-ef-containing species. new ef-1α/efl data from other eukaryotes our ef-1α/efl survey successfully revealed that the taxa katablepharida, amoebozoa, and ancyromonadida contain both efl-containing and ef-1α-containing species. for amoebozoans and ancyromonads, only ef-1α-containing species were known prior to this study, however, we detected efl sequences in the amoebozoan copromyxa protea and the ancyromonad fabomonas tropica. likewise, the first-surveyed katablepharid leucocryptos marina was efl-containing, but a rt-pcr survey of a secondly-surveyed katablepharid, roombia sp., identified ef-1α transcripts. discussion several eukaryote lineages include multiple dual-ef-containing species ancestral co-occurrence of ef-1α and efl followed by differential loss of one of the two elongation factors most likely shaped the current ef-1α/efl distribution within eukaryotes. in this scenario, the extant dual-ef-containing species retain the ancestral state and thus are analogous to the inferred intermediates that led to descendant lineages that contain either ef-1α or efl. in this study, we found new dual-ef-containing species in four distantly related lineages: goniomonadida, apusomonadida, stramenopiles, and fungi. in light of the differential loss process proposed for ef-1α/efl evolution, we speculate that more dual-ef-containing species remain undetected in other lineages that contain both ef-1α-containing and efl-containing species, including: viridiplantae, euglenozoa, choanoflagellata, endomyxa, filosa, rhodophyta, katablepharida, amoebozoa, and ancyromonadida. considering the revised distribution of ef-1α/efl genes, we cannot exclude the possibility that the last eukaryotic common ancestor was dual-ef-containing. finally, it will be of interest to continue surveying dual-ef-containing species, especially within stramenopiles and fungi. kamikawa et al. postulated that the dual-ef status can be traced back to the ancestral stramenopile species, based on the monophyly of stramenopiles in ef-1α phylogenies, and of diatoms and oomycetes in efl phylogenies. thus, we predict that dual-ef-containing species should be found in so-far unsampled stramenopiles. similarly, s. punctatus and b. ranarum are unlikely to be the sole fungal species with a dual-ef status, given that the most recent common ancestral fungus was proposed to be dual-ef-containing. parallel re-modeling of ef-1α function in eukaryotic evolution in the dual ef-containing diatom t. pseudonana, some of us proposed that the ef-1α homolog performs only a subset of its original functions, and does not participate in protein synthesis as an elongation factor, for the following reasons. firstly, in an ef-1α phylogeny, the t. pseudonana homologue was much more divergent than that of a closely related ef-1α-containing species, p. tricornutum, suggesting that the former is under fewer functional constraints than the latter. secondly, ef-1α transcripts were much less abundant in t. pseudonana than the transcripts of efl or of an α-tubulin gene. as observed in t. pseudonana, the five dual ef-containing diatoms identified in this study appeared to possess divergent ef-1α genes. in each of the five diatoms, the transcriptional level of the ef-1α gene was heavily suppressed compared to that of the co-occurring efl gene. thus, the five dual-ef-containing diatoms most likely use efl as the principal elongation factor, while a sub-set of the original ef-1α functions is assigned to the divergent ef-1α. these dual-ef-containing diatoms have most likely re-modeled their ef-1α functions, such that they carry out only the auxiliary roles that the proteins originally performed, such as interactions with cytoskeletal proteins and ubiquitin-dependent protein degradation. it is likely that similar re-modeling of ef-1α function has also occurred in other dual-ef-containing lineages. in the non-diatom dual-ef-containing species, the ef-1α sequences were also divergent, and were transcribed at a low level compared to the co-occurring efl genes. these results strongly suggest that dual-ef-containing species in general utilize ef-1α for subsets of the original functions, while efl participates in translation as a core factor. significantly, the re-modeling of ef-1α function probably took place separately in stramenopiles, goniomonadida, apusomonadida, and fungi, as these lineages are distantly related to one another in the organismal phylogeny. moreover, diatoms and oomycetes may have also re-modeled their ef-1α functions in parallel as they are relatively distantly related within stramenopiles. we also suspect that parallel re-modeling of ef-1α function occurred within fungi, as s. punctatus and b. ranarum are not particularly close relatives. we are currently unsure about the precise functions of the divergent ef-1α in the dual-ef-containing species. under the parallel re-modeling scenario proposed above, the suite of retained ef-1α functions could vary between any of two dual-ef-containing lineages. however, the overall substitution patterns in divergent ef-1α sequences in distantly related dual-ef-containing species are found to be similar to each other. this observation hints at parallel loss of the same aspects of ef-1α function and retention of a subset of original functions in multiple dual-ef-containing lineages scattered over the tree of eukaryotes. these speculations could be tested more directly by biochemical studies of ef-1α function in selected representatives of these lineages.  according to the differential loss hypothesis for ef-1α/efl evolution, a dual-ef-containing ancestor likely gave rise to two types of descendants—one containing only efl and the other containing only ef-1α. nevertheless, ef-1α/efl surveys, including this study, have identified an additional type of descendent retaining the ancestral arrangement in multiple branches of the tree of eukaryotes. if ef-1α/efl sequences are surveyed in a broader spectrum of eukaryotes, it is highly likely that the number and diversity of known dual-ef-containing species will grow further. curiously, all dual-ef-containing species identified so far appear to retain divergent, low-expressed ef-1α genes, which are analogous to the hypothetical intermediate leading to efl-containing descendants. we suspect that the multiple functions of the canonical ef-1α may have prevented the dual-ef-containing cells from losing this protein immediately after efl took over from ef-1α as the core translation factor. the presence of dual-ef-containing species indicates that the adoption of efl as the dominant core factor in translation does not necessarily lead to the elimination of ef-1α from the entire cellular system. curiously, we found little evidence for living analogues of the hypothetical intermediate that led to ef-1α-containing descendants, which would possess a divergent, low-transcribed efl gene. the presence or absence of dual-ef-containing species, in which a divergent efl gene is transcribed at lower levels than the co-occurring ef-1α gene, would be crucial to understanding the evolutionary processes that shaped the current ef-1α/efl gene distribution across the tree of eukaryotes. we need to re-examine efl sequences in the species currently recognized as ‘ef-1α-containing’ since low-expressed efl genes might be overlooked in these taxa, especially if genomic or high-coverage transcriptomic data is lacking. 
in the dual-ef-containing species we identified, the ef-1α genes appeared to be highly divergent in sequence and suppressed at the transcriptional level compared to the co-occurring efl genes. the patchy distribution of the two elongation factor types across global eukaryotic phylogeny is suggestive of a ‘differential loss’ hypothesis that assumes that ef-1α and efl were present in the most recent common ancestor of eukaryotes followed by independent differential losses of one of the two factors in the descendant lineages. phylogenetic analyses suggested vertical inheritance of both genes in each of the dual-ef lineages. we predict that dual-ef-containing species retain the divergent ef-1α homologues only for a sub-set of the original functions. as the dual-ef-containing species are distantly related to each other, we propose that independent re-modelling of ef-1α function took place in multiple branches in the tree of eukaryotes. in this study, we characterized new ef-1α/efl sequences from phylogenetically diverse eukaryotes. according to the known ef-1α/efl distribution, the differential loss process should have occurred independently in diverse eukaryotic lineages, and more dual-ef-containing species remain unidentified.
 elongation factor-1α and elongation factor-like proteins are functionally homologous to one another, and are core components of the eukaryotic translation machinery. the patchy distribution of the two elongation factor types across global eukaryotic phylogeny is suggestive of a ‘differential loss’ hypothesis that assumes that ef-1α and efl were present in the most recent common ancestor of eukaryotes followed by independent differential losses of one of the two factors in the descendant lineages. to date, however, just one diatom and one fungus have been found to have both ef-1α and efl.  in this study, we characterized new ef-1α/efl sequences from phylogenetically diverse eukaryotes. in so doing we identified previously unreported dual-ef-containing species from diverse eukaryote groups including the stramenopiles, apusomonadida, goniomonadida, and fungi. phylogenetic analyses suggested vertical inheritance of both genes in each of the dual-ef lineages. in the dual-ef-containing species we identified, the ef-1α genes appeared to be highly divergent in sequence and suppressed at the transcriptional level compared to the co-occurring efl genes.  according to the known ef-1α/efl distribution, the differential loss process should have occurred independently in diverse eukaryotic lineages, and more dual-ef-containing species remain unidentified. we predict that dual-ef-containing species retain the divergent ef-1α homologues only for a sub-set of the original functions. as the dual-ef-containing species are distantly related to each other, we propose that independent re-modelling of ef-1α function took place in multiple branches in the tree of eukaryotes. diatomsdifferential gene lossef-1αeflfunctional remodelinggoniomonaspythiumspizellomycesthecamonas 