BACKGROUND
a cyanobacterium-like ancestor gave rise via primary endosymbiosis to a distinctive endosymbiotic organelle, the plastid , in eukaryotic cells  <cit> .
some eukaryotic lineages retained the plastid through successive generations, and its photosynthetic ability enabled them to grow autotrophically.
some may have lost the plastid, and returned to their previous heterotrophic state, whereas others may have never experienced such an endosymbiotic event.
green plants , glaucophyta and red algae are primary plastid-containing photosynthetic eukaryotes.
they are classified into a single super-group, archaeplastida, among the six 'super-groups' proposed by adl et al.
<cit> .
it is generally believed that the majority of the cyanobacterial genes  found in the nuclear genomes of extant archaeplastida were recruited from cyanobacterium-like endosymbionts via endosymbiotic gene transfer   <cit> .
other algae in several independent lineages are thought to have secondarily acquired plastids by engulfing primary photosynthetic eukaryotes.
these have evolved into secondary plastid-containing photosynthetic eukaryotes   <cit> .
most secondary plastids in the super-group chromalveolata, which consists of stramenopiles, alveolata, haptophyta and cryptophyta, are derived from red algae.
chlorarachniophyta in the rhizaria group and euglenida in the excavata group possess secondary plastids derived from green algal ancestors  <cit> .
a large number of plastid-related cyanobacterial genes were further introduced into nuclear genomes of secondary phototrophs via secondary egt  <cit> .
although several studies have reported cyanobacterial genes in plastid-lacking eukaryotes  <cit> , gnd genes are remarkable in their broad distribution among primary and secondary plastid-containing photosynthetic eukaryotes as well as among plastid-lacking protists  <cit> .
the gnd gene encodes an oxidative pentose phosphate pathway enzyme, 6-phosphogluconate dehydrogenase, which is important in regulating sugar metabolism and intracellular redox state.
previous studies reported that the gnd gene is widely conserved among eukaryotes and eubacteria  <cit> , and showed that there are two types of gnd genes; one is phylogenetically close to cyanobacterial gnd genes , and the other resembles cytosol-localized gnd genes in opisthokonta .
cyanobacterial gnd genes are present not only in primary and secondary phototrophs, but also in plastid-lacking protists.
these include the plant pathogen phytophthora that is classified into the super-group chromalveolata, and the heterolobosean amoebo-flagellates that are classified into the super-group excavata  <cit> .
these pioneering studies suggested a possible scenario that cyanobacterial gnd genes were introduced via primary or secondary endosymbiosis  <cit> .
nevertheless, the origin and evolutionary relationships of these genes in photosynthetic and plastid-lacking eukaryotes remains inconclusive.
we present here an extended analysis of the phylogeny of gnd genes with emphasis on the plastid-lacking excavate protists.
we also discuss the origin and evolutionary history of the cyanobacterial genes in plastid-lacking protists, within the scope of previously proposed hypotheses on ancient lateral gene transfer  and egt events.
methods
culture material
diplonema papillatum  was axenically cultured at 25°c in artificial seawater supplemented with 1% horse serum ,  <dig> × daigo imk medium  and  <dig> % tryptone.
peranema trichophorum cells, co-cultured with chlorogonium sp., were provided by dr. toshinobu suzaki .
euglena gracilis z  was cultured as described previously  <cit> .
cdna library construction and pcr-based gene isolation
d. papillatum genomic dna was extracted using the dneasy plant mini kit .
p. trichophorum full-length cdna sequences were synthesized using the sv total rna isolation system  and the capfishing full-length cdna kit .
glaucophyte cdnas  were prepared as described in the previous study  <cit> , and used as templates for gene isolation.
fragments of gnd genes were amplified using nested-degenerated primers based on the conserved amino acid motif glavmgqn for forward primers  and qaqrdffg for reverse primers .
pcr products and cdna clones were sequenced directly or after ta-cloning, using an abi prism  <dig> genetic analyzer  with a bigdye terminator cycle sequencing ready reaction kit v.  <dig>  .
expressed sequence tags  of euglena gracilis  were generated by sequencing cdna clones selected at random from a cdna library  constructed using a cdna synthesis kit .
the est sequencing was performed at the dragon genomics center, takara bio inc. .
a clone harboring the full-length gnd gene sequence was identified by blast search.
phylogenetic analysis
the data matrix of gnd genes was based on the amino acid alignment in andersson and roger  <cit> .
we excluded amitochondrial and/or parasitic eukaryotes, which might cause long branch attraction due to unusual nucleotide substitutions  <cit> .
we included the novel sequences determined in this study , and sequences identified by the blast program from the galdieria sulphuraria genome database  <cit> , the joint genome institute  <cit>  and the acanthamoeba castellanii est database in tbestdb  <cit> .
the sequences were aligned using clustal x  <cit>  and manually refined using seaview  <cit> .
the data matrix was made with  <dig> taxa and  <dig> amino acid sites .
data matrices excluding heterolobosea  and including amitochondrial and/or parasitic eukaryotes  were also prepared to construct additional trees .
ew signature residues conserved among cyanobacterial gnd genes  <cit>  are indicated in bold.
bayesian inference was performed with the program mrbayes version  <dig> .
<dig>  <cit>  using the wag matrix of amino acid replacements assuming a proportion of invariant positions and four gamma-distributed rates .
for the mrbayes consensus trees,  <dig> , <dig> generations were completed with trees collected every  <dig> generations.
one thousand replicates of bootstrap analyses by maximum likelihood  method were performed using phyml version  <dig> .
<dig>  <cit>  with the wag+i+Γ <dig> model on two sunfire 15k machines, each of which has  <dig> cpus.
bootstrap values  based on maximum parsimony  analysis were calculated with paup  <dig>  b <dig> with tbr heuristic search  <cit> .
for exhaustive ml analysis, topology-dependent sitewise likelihood values were calculated using tree-puzzle version  <dig>  under a wag+f+Γ <dig> model  <cit> .
alternative tree topologies were analyzed with the approximately unbiased   <cit>  and kishino-hasegawa   <cit>  tests, and the resampling estimated log-likelihood  bootstrap support values  <cit> , using the consel package  <cit> .
RESULTS
phylogenetic and statistical analysis of gnd genes
fig.
<dig> shows a bayesian consensus tree from a matrix with  <dig> taxa, with bayesian posterior probabilities  of 70% or more, and ml and mp bootstrap support values of 50% or more.
as reported previously  <cit> , all the red algae examined have both cyanobacterial and eukaryotic ancestral gnd genes.
although several excavate gnd genes  were cyanobacterial in agreement with the previous studies  <cit> , the gnd gene from another excavate species, d. papillatum, was found to group with opisthokonta and red algal eukaryotic ancestral genes .
several proteobacterial species  showed a weak affinity to eukaryotic genes , and amoebozoa was located outermost in the eukaryotic ancestral clade .
notably, red algae and excavate genes shared basal positions within each of the cyanobacterial and eukaryotic ancestral clades.
as shown in trypanosoma, giardia and trichomonas  <cit> , the ew sequence signature, which is unique to the cyanobacterial gnd genes, was absent in the d. papillatum gnd gene , confirming its non-cyanobacterial origin.
however, the parasitic excavates were positioned outside of the eukaryotic ancestral clade with weak support values in the tree of  <dig> taxa , possibly due to long branch attraction.
whether the genes from parasitic excavata truly shared the same origin as known free-living excavata genes, or were independently acquired via prokaryote-to-eukaryote lgt is open to further investigation of evolutionary signals and functional characterization.
our results and currently available genome information suggest that, while each red algal species possesses both cyanobacterial and eukaryotic ancestral genes and supposedly use them in different cellular compartments, free-living excavata examined to date have just one or the other.
cyanobacterial genes from bikonts  <cit>   were robustly monophyletic  and showed a strong affiliation with the genes from cyanobacteria  .
in the cyanobacterial gene clade, each of the three divisions of archaeplastida  was monophyletic but separately located .
glaucophyte gnd genes formed a monophyletic group with green plants, euglenida, and stramenopiles with moderate support values .
secondary phototrophs and the plastid-lacking heterotrophic relatives from euglenida and stramenopiles were robustly monophyletic .
plastid-lacking heterolobosean protists and red algae were located at the basal position in the cyanobacterial clade, weakly forming a monophyletic group .
to test the possibility that the plastid-lacking excavate protists acquired gnd genes via secondary endosymbiosis of a green alga  <cit> , we carried out an exhaustive ml analysis for calculating the likelihood values of alternative tree topologies.
first, based on the topology in fig.
<dig>  we defined six groups in which monophyly was confirmed by all three methods : green plants , glaucophyta , stramenopiles + euglenida , heterolobosea , red algae  and others .
then, we constructed all possible  <dig> trees, fixing the intra-group topologies of the six monophyletic groups as in fig.
<dig>  and calculated probabilities of each tree for au and kh tests .
all possible  <dig> trees supporting the monophyly of green + htrl were rejected by both au and kh tests at the 5% confidence level.
all possible nine trees supporting monophyly of green + eust + htrl groups were also rejected by both tests at the 5% confidence level .
abest tree  and trees supporting the monophyly of green plants + heterolobosea among all the possible  <dig> trees retaining six monophyletic groups in fig.
<dig>
bgreen, green plants; htrl, heterolobosea; glauco, glaucophyta; eust, euglenida and stramenopiles; red, red algae; out, eukaryotic ancestral clade and cyanobacteria.
intra-group topologies of six groups are fixed as shown in fig.
<dig>
cdifference in the log-likelihood value of alternative tree versus the 'best' tree.
dprobability values of the approximately unbiased  and kishino-hasegawa  tests.
ebootstrap support value of resampling estimated log-likelihood with  <dig>  replicates.
asterisks indicate the topologies supporting the monophyly of green + htrl + eust.
although our tree topology in fig.
<dig> suggests that cyanobacterial genes from bikonts were originally acquired via a single gene transfer event from cyanobacteria, there are two possible explanations of their origin as discussed in the previous study  <cit> ; early primary egt from the ancestral plastid genome, or prokaryote-to-eukaryote lgt from a close relative of extant cyanobacteria independently of egt.
we favor the former scenario for the following reasons: 1) the gnd gene product is functionally plastid-related, and is enzymatically localized to the plastid in green plants  <cit> ; and 2) the overall tree topology in fig.
<dig> is consistent with a recent multigene phylogeny of eukaryotes based on slowly evolving nuclear genes  <cit> .
origins of plastid-lacking excavate gnd genes
heterolobosean gnd genes occupied the basal positions in the cyanobacterial clade and weakly formed a monophyletic group with red algae.
although our tree topology suggests that euglenid and heterolobosean gnd genes are distantly related, previous studies have not clearly excluded the single secondary-plastid origin of these genes  <cit> .
to test whether the heterolobosean gnd genes could originate with secondary egt as suggested by the 'plastids-early' hypothesis for secondary plastids in euglenida  <cit> , we verified the possibility that the cyanobacterial gnd genes in plastid-lacking heterolobosean protists and green plants could be potentially monophyletic, using confidence tests based on exhaustive ml analyses .
according to the plastids-early hypothesis for secondary plastids in euglenida  <cit> , the secondary endosymbiosis of green alga occurred in the common ancestor of euglenida and heterolobosea, and extant plastid-lacking protists within these taxa have secondarily lost their plastids and photosynthesis-related genes.
although this hypothesis is contentious  <cit> , it is worth verifying because this is the leading explanation for the acquisition of cyanobacterial genes through secondary endosymbionts in heterolobosea.
considering that the orientation of lgt between the ancestors of stramenopiles and euglenida is unknown, we examined two possibilities on the origin of the euglenid and heterolobosean gnd genes.
first, we examined the possibility that ancient euglenid gnd was transferred into the common ancestor of stramenopiles, which postulates the monophyly of stramenopiles, euglenida, heterolobosea and green plants.
then we examined the second possibility that an ancient stramenopile gnd was acquired by the euglenid ancestor, which assumes that heterolobosea and green plants are exclusively monophyletic.
all the trees supporting first or second possibilities were rejected by au and kh tests at the 5% confidence level .
these results suggested that heterolobosean gnd genes were not secondary green plastid-derived, and that the gnd gene phylogeny did not support the plastids-early hypothesis  <cit> .
taken together, our data disallowed the plastids-early hypothesis, and showed that a secondary endosymbiotic origin of the gnd genes from green alga into plastid-lacking excavate protists is unlikely.
it is striking that euglenida is monophyletic with stramenopiles in the cyanobacterial clade .
recent phylogenetic analyses of the plastid-encoded and nuclear-encoded plastid-targeted genes suggest that the ancestor of euglenid secondary plastids branches within green algae, inconsistent with our gnd tree topology  <cit> .
the monophyly of cyanobacterial gnd genes from e. gracilis and plastid-lacking p. trichophorum further suggests that euglenid gnd genes have not been recruited via secondary egt of a green alga, because the 'plastids-recent' hypothesis argues that eukaryovorous euglenid species such as p. trichophorum diverged before the secondary endosymbiotic event in the euglenida lineage  <cit> .
meanwhile, the presence of the cyanobacterial genes in stramenopiles, including photosynthetic algae and the plastid-lacking oomycete phytophthora, is apparently consistent with the 'chromalveolate hypothesis'  <cit> , which suggests that secondary plastids of chromalveolata have been acquired through a single secondary endosymbiotic event.
the most likely explanation is that the ancestor of the euglenida host cells acquired a gnd gene via ancient lgt from the stramenopile lineage before their divergence.
this also explains well why euglenida and heterolobosea are robustly separated in the gnd phylogeny  despite the close relatedness of these two lineages based on ssu rrna gene phylogeny  <cit>  and multiple nuclear-encoded protein phylogenies  <cit> .
evolutionary history of gnd genes and plastid-lacking excavate genomes
although our gnd tree topology appears unexpected compared with the prevailing view of plastid evolution  <cit> , several gene phylogenies that suggested imprints of gene transfer between euglenida and stramenopiles have been reported.
in the plastid-targeted phosphoribulokinase  gene phylogeny  <cit> , red algal genes were basal in the eukaryotic clade and were separated from chromalveolate and green plant genes.
furthermore, euglenid and chromalveolate prk genes were monophyletic and sister to green plants, and the authors reasoned that these secondary phototrophs might acquire prk genes via independent lgt events.
as discussed above, it is likely that euglenida has acquired a cyanobacterial gnd gene from the ancestor of stramenopiles via lgt.
although prk genes are found only in photosynthetic organisms  and the origin of euglenid prk genes was phylogenetically unresolved, one can argue that prk and cyanobacterial gnd genes might have gone through similar evolutionary histories.
a phylogenetic analysis of plastid-targeted fructose- <dig> -bisphosphatase  genes illustrated another case of lgt between euglenida and chromalveolata  <cit> .
thus these genes might have been transferred from the stramenopile lineage to the euglenid lineage via multiple lgt events, perhaps phagocytosis of secondary phototrophs by a phagotrophic ancestor as suggested in the chlorarachniophyte bigelowiella natans  <cit> .
in the cyanobacterial gnd gene subtree, the red algal clade was at the basal position and was moderately separated from green plants and glaucophyta.
an additional phylogenetic analysis excluding heterolobosea recovered the basal position of red algae in this subtree, suggesting that long branch attraction or artificial misplacement of red algae by heterolobosean sequences was unlikely .
additionally, provided that the cyanobacterial genes from bikonts were robustly monophyletic , in contrast to well-characterized examples of prokaryote-to-eukaryote lgts  <cit> , it is unlikely that the cyanobacterial gnd genes from bikonts had been acquired via multiple lgts from cyanobacteria to eukaryotes.
recently, two competing hypotheses on archaeplastida phylogeny were proposed   <cit> .
the phylogenetic position of red algae in fig.
<dig> is inconsistent with the monophyletic hypothesis of the archaeplastida  <cit>  unless multiple eukaryote-to-eukaryote lgts are hypothesized .
although red algal and glaucophyte ancestries of the heterolobosean genes were not significantly dismissed, au tests rejected the possible secondary egt from green alga to heterolobosea .
hence, the eukaryote-to-eukaryote lgts shown in fig.
2a are likely sources of gnd genes in plastid-lacking protists, in terms of the monophyletic hypothesis of the archaeplastida  <cit> .
however, monophyly of red algae plus stramenopiles  was not rejected in our statistical tests , suggesting that the stramenopile genes might be attributed to secondary egt of red alga.
on the other hand, an increasing number of multigene phylogenies showed that monophyly of archaeplastida had limited or no support  <cit> .
therefore it is advisable to discuss the evolutionary history of gnd genes, taking a different point of view on the plastid evolution into consideration .
in terms of the non-monophyly hypothesis of the archaeplastida, it is reasonable to suggest that the gnd gene phylogeny may reflect the host cell phylogeny as recently resolved by a multiple slowly evolving nuclear gene phylogeny  <cit> , which demonstrated the non-monophyly of archaeplastida and the most basal positioning of red algae plus excavata within the bikonts .
possible evolutionary scenarios of plastid and host nuclear genomes
we propose evolutionary scenarios in which the common ancestor of eukaryotes possessed a eubacteria-derived eukaryotic ancestral gnd gene, and the bikonts lineage additionally acquired the cyanobacterial gnd gene via a single primary endosymbiosis  <cit>  , and then diversified into archaeplastida, chromalveolata, excavata  .
given that recent large-scale molecular phylogenies demonstrated the monophyly of bikonts  <cit>  based on the rooting of eukaryotes  <cit> , and no data providing evidence on primary and secondary plastids in the unikonts has been shown, we illustrated two likely scenarios in fig.
<dig>  in scenario a, we assumed monophyly of archaeplastida , and accordingly, at least two gains of cyanobacterial gnd genes via lgt and multiple losses of eukaryotic ancestral genes in separate lineages of bikonts.
in scenario b, we presumed that all the bikonts including secondary phototrophs and plastid-lacking bikonts had at one time acquired the primary plastid  <cit> .
green plants, glaucophyta and chromalveolata then lost the eukaryotic ancestral gnd gene, red algae retained both, and excavata lost either one.
in the ancestors of excavata, loss of primary photosynthetic plastids might have triggered concurrent gene loss of either cyanobacterial or eukaryotic ancestral gnd.
only a single lgt event from stramenopiles into euglenida is considered in scenario b. although both scenarios are compatible with our phylogenetic analysis and statistical tests, we reason that scenario b is parsimonious and more likely to explain the evolutionary history of the gnd genes in that less lgt events need to be presupposed.
broader sampling from various eukaryotic groups  will be critical to devise a more reliable evolutionary history of eukaryotic gnd genes, and host lineages  <cit> .
it is also important to note that concatenated nuclear gene phylogeny of eukaryotic  lineages and data mining for cyanobacterial genes in plastid-lacking protists are supposed to be independent approaches for exploring the origin of plants.
future research will be focused on how deeply primary endosymbiosis is rooted within the bikonts, and which lineage could experience primary endosymbiosis early in the evolution of bikonts.
CONCLUSIONS
our present study demonstrates that  free-living excavata possess either cyanobacterial or eukaryotic ancestral gnd genes,  it is statistically unlikely that heterolobosean gnd genes were acquired via ancient secondary egt of green alga, and  euglenida and stramenopiles are robustly monophyletic.
although the sister relationship of this monophyletic group to any archaeplastida lineage is not rejected by the statistical tests , it is moderately separated from red algae , suggesting that the gnd genes in stramenopiles are not of secondary endosymbiont origin.
one explanation is that a unique primary egt of cyanobacterial gnd genes into archaeplastida was followed by independent eukaryote-to-eukaryote lgts into stramenopiles and heterolobosea, and then by an additional lgt from stramenopiles into euglenida .
alternatively, our results favor an evolutionary scenario that the gnd gene phylogeny reflects host cell phylogeny, and that the common ancestor of bikonts has acquired cyanobacterial gnd genes via primary endosymbiotic gene transfer early in eukaryotic evolution .
authors' contributions
sm participated in the design of the study and coordination, carried out the molecular phylogenetic and statistical studies, and drafted the manuscript.
km participated in the phylogenetic and statistical studies.
mi and mw participated in the cdna library construction and sequence analysis.
hn conceived of the study, and participated in its design and helped to draft the manuscript.
all authors read and approved the final manuscript.
supplementary material
additional file 1
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additional file 2
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additional file 3
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additional file 4
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acknowledgements
we thank dr. toshinobu suzaki  for a generous gift of the p. trichophorum culture, and dr. takashi nakada  for help with phylogenetic analysis.
computation time was provided by the super computer system, human genome center, institute of medical science, university of tokyo.
this work was supported by grants-in-aid for creative scientific research  and for scientific research  from the ministry of education, culture, sports, science, and technology, japan.

 a cyanobacterium-like ancestor gave rise via primary endosymbiosis to a distinctive endosymbiotic organelle, the plastid, in eukaryotic cells. some eukaryotic lineages retained the plastid through successive generations, and its photosynthetic ability enabled them to grow autotrophically. some may have lost the plastid, and returned to their previous heterotrophic state, whereas others may have never experienced such an endosymbiotic event. green plants, glaucophyta and red algae are primary plastid-containing photosynthetic eukaryotes. they are classified into a single super-group, archaeplastida, among the six'super-groups' proposed by adl et al.. it is generally believed that the majority of the cyanobacterial genes found in the nuclear genomes of extant archaeplastida were recruited from cyanobacterium-like endosymbionts via endosymbiotic gene transfer. other algae in several independent lineages are thought to have secondarily acquired plastids by engulfing primary photosynthetic eukaryotes. these have evolved into secondary plastid-containing photosynthetic eukaryotes. most secondary plastids in the super-group chromalveolata, which consists of stramenopiles, alveolata, haptophyta and cryptophyta, are derived from red algae. chlorarachniophyta in the rhizaria group and euglenida in the excavata group possess secondary plastids derived from green algal ancestors. a large number of plastid-related cyanobacterial genes were further introduced into nuclear genomes of secondary phototrophs via secondary egt. although several studies have reported cyanobacterial genes in plastid-lacking eukaryotes, gnd genes are remarkable in their broad distribution among primary and secondary plastid-containing photosynthetic eukaryotes as well as among plastid-lacking protists. the gnd gene encodes an oxidative pentose phosphate pathway enzyme, 6-phosphogluconate dehydrogenase, which is important in regulating sugar metabolism and intracellular redox state. previous studies reported that the gnd gene is widely conserved among eukaryotes and eubacteria, and showed that there are two types of gnd genes; one is phylogenetically close to cyanobacterial gnd genes, and the other resembles cytosol-localized gnd genes in opisthokonta. cyanobacterial gnd genes are present not only in primary and secondary phototrophs, but also in plastid-lacking protists. these include the plant pathogen phytophthora that is classified into the super-group chromalveolata, and the heterolobosean amoebo-flagellates that are classified into the super-group excavata. these pioneering studies suggested a possible scenario that cyanobacterial gnd genes were introduced via primary or secondary endosymbiosis. nevertheless, the origin and evolutionary relationships of these genes in photosynthetic and plastid-lacking eukaryotes remains inconclusive. we present here an extended analysis of the phylogeny of gnd genes with emphasis on the plastid-lacking excavate protists. we also discuss the origin and evolutionary history of the cyanobacterial genes in plastid-lacking protists, within the scope of previously proposed hypotheses on ancient lateral gene transfer and egt events. methods culture material diplonema papillatum was axenically cultured at 25°c in artificial seawater supplemented with 1% horse serum, × daigo imk medium and % tryptone. peranema trichophorum cells, co-cultured with chlorogonium sp., were provided by dr. toshinobu suzaki. euglena gracilis z was cultured as described previously. cdna library construction and pcr-based gene isolation d. papillatum genomic dna was extracted using the dneasy plant mini kit. p. trichophorum full-length cdna sequences were synthesized using the sv total rna isolation system and the capfishing full-length cdna kit. glaucophyte cdnas were prepared as described in the previous study, and used as templates for gene isolation. fragments of gnd genes were amplified using nested-degenerated primers based on the conserved amino acid motif glavmgqn for forward primers and qaqrdffg for reverse primers. pcr products and cdna clones were sequenced directly or after ta-cloning, using an abi prism genetic analyzer with a bigdye terminator cycle sequencing ready reaction kit v.. expressed sequence tags of euglena gracilis were generated by sequencing cdna clones selected at random from a cdna library constructed using a cdna synthesis kit. the est sequencing was performed at the dragon genomics center, takara bio inc.. a clone harboring the full-length gnd gene sequence was identified by blast search. phylogenetic analysis the data matrix of gnd genes was based on the amino acid alignment in andersson and roger. we excluded amitochondrial and/or parasitic eukaryotes, which might cause long branch attraction due to unusual nucleotide substitutions. we included the novel sequences determined in this study, and sequences identified by the blast program from the galdieria sulphuraria genome database, the joint genome institute and the acanthamoeba castellanii est database in tbestdb. the sequences were aligned using clustal x and manually refined using seaview. the data matrix was made with taxa and amino acid sites. data matrices excluding heterolobosea and including amitochondrial and/or parasitic eukaryotes were also prepared to construct additional trees. ew signature residues conserved among cyanobacterial gnd genes are indicated in bold. bayesian inference was performed with the program mrbayes version. using the wag matrix of amino acid replacements assuming a proportion of invariant positions and four gamma-distributed rates. for the mrbayes consensus trees,, generations were completed with trees collected every generations. one thousand replicates of bootstrap analyses by maximum likelihood method were performed using phyml version. with the wag+i+Γ model on two sunfire 15k machines, each of which has cpus. bootstrap values based on maximum parsimony analysis were calculated with paup b with tbr heuristic search. for exhaustive ml analysis, topology-dependent sitewise likelihood values were calculated using tree-puzzle version under a wag+f+Γ model. alternative tree topologies were analyzed with the approximately unbiased and kishino-hasegawa tests, and the resampling estimated log-likelihood bootstrap support values, using the consel package.  phylogenetic and statistical analysis of gnd genes fig. shows a bayesian consensus tree from a matrix with taxa, with bayesian posterior probabilities of 70% or more, and ml and mp bootstrap support values of 50% or more. as reported previously, all the red algae examined have both cyanobacterial and eukaryotic ancestral gnd genes. although several excavate gnd genes were cyanobacterial in agreement with the previous studies, the gnd gene from another excavate species, d. papillatum, was found to group with opisthokonta and red algal eukaryotic ancestral genes. several proteobacterial species showed a weak affinity to eukaryotic genes, and amoebozoa was located outermost in the eukaryotic ancestral clade. notably, red algae and excavate genes shared basal positions within each of the cyanobacterial and eukaryotic ancestral clades. as shown in trypanosoma, giardia and trichomonas, the ew sequence signature, which is unique to the cyanobacterial gnd genes, was absent in the d. papillatum gnd gene, confirming its non-cyanobacterial origin. however, the parasitic excavates were positioned outside of the eukaryotic ancestral clade with weak support values in the tree of taxa, possibly due to long branch attraction. whether the genes from parasitic excavata truly shared the same origin as known free-living excavata genes, or were independently acquired via prokaryote-to-eukaryote lgt is open to further investigation of evolutionary signals and functional characterization. our results and currently available genome information suggest that, while each red algal species possesses both cyanobacterial and eukaryotic ancestral genes and supposedly use them in different cellular compartments, free-living excavata examined to date have just one or the other. cyanobacterial genes from bikonts were robustly monophyletic and showed a strong affiliation with the genes from cyanobacteria. in the cyanobacterial gene clade, each of the three divisions of archaeplastida was monophyletic but separately located. glaucophyte gnd genes formed a monophyletic group with green plants, euglenida, and stramenopiles with moderate support values. secondary phototrophs and the plastid-lacking heterotrophic relatives from euglenida and stramenopiles were robustly monophyletic. plastid-lacking heterolobosean protists and red algae were located at the basal position in the cyanobacterial clade, weakly forming a monophyletic group. to test the possibility that the plastid-lacking excavate protists acquired gnd genes via secondary endosymbiosis of a green alga, we carried out an exhaustive ml analysis for calculating the likelihood values of alternative tree topologies. first, based on the topology in fig. we defined six groups in which monophyly was confirmed by all three methods: green plants, glaucophyta, stramenopiles + euglenida, heterolobosea, red algae and others. then, we constructed all possible trees, fixing the intra-group topologies of the six monophyletic groups as in fig. and calculated probabilities of each tree for au and kh tests. all possible trees supporting the monophyly of green + htrl were rejected by both au and kh tests at the 5% confidence level. all possible nine trees supporting monophyly of green + eust + htrl groups were also rejected by both tests at the 5% confidence level. abest tree and trees supporting the monophyly of green plants + heterolobosea among all the possible trees retaining six monophyletic groups in fig. bgreen, green plants; htrl, heterolobosea; glauco, glaucophyta; eust, euglenida and stramenopiles; red, red algae; out, eukaryotic ancestral clade and cyanobacteria. intra-group topologies of six groups are fixed as shown in fig. cdifference in the log-likelihood value of alternative tree versus the'best' tree. dprobability values of the approximately unbiased and kishino-hasegawa tests. ebootstrap support value of resampling estimated log-likelihood with replicates. asterisks indicate the topologies supporting the monophyly of green + htrl + eust. although our tree topology in fig. suggests that cyanobacterial genes from bikonts were originally acquired via a single gene transfer event from cyanobacteria, there are two possible explanations of their origin as discussed in the previous study; early primary egt from the ancestral plastid genome, or prokaryote-to-eukaryote lgt from a close relative of extant cyanobacteria independently of egt. we favor the former scenario for the following reasons: 1) the gnd gene product is functionally plastid-related, and is enzymatically localized to the plastid in green plants; and 2) the overall tree topology in fig. is consistent with a recent multigene phylogeny of eukaryotes based on slowly evolving nuclear genes. origins of plastid-lacking excavate gnd genes heterolobosean gnd genes occupied the basal positions in the cyanobacterial clade and weakly formed a monophyletic group with red algae. although our tree topology suggests that euglenid and heterolobosean gnd genes are distantly related, previous studies have not clearly excluded the single secondary-plastid origin of these genes. to test whether the heterolobosean gnd genes could originate with secondary egt as suggested by the'plastids-early' hypothesis for secondary plastids in euglenida, we verified the possibility that the cyanobacterial gnd genes in plastid-lacking heterolobosean protists and green plants could be potentially monophyletic, using confidence tests based on exhaustive ml analyses. according to the plastids-early hypothesis for secondary plastids in euglenida, the secondary endosymbiosis of green alga occurred in the common ancestor of euglenida and heterolobosea, and extant plastid-lacking protists within these taxa have secondarily lost their plastids and photosynthesis-related genes. although this hypothesis is contentious, it is worth verifying because this is the leading explanation for the acquisition of cyanobacterial genes through secondary endosymbionts in heterolobosea. considering that the orientation of lgt between the ancestors of stramenopiles and euglenida is unknown, we examined two possibilities on the origin of the euglenid and heterolobosean gnd genes. first, we examined the possibility that ancient euglenid gnd was transferred into the common ancestor of stramenopiles, which postulates the monophyly of stramenopiles, euglenida, heterolobosea and green plants. then we examined the second possibility that an ancient stramenopile gnd was acquired by the euglenid ancestor, which assumes that heterolobosea and green plants are exclusively monophyletic. all the trees supporting first or second possibilities were rejected by au and kh tests at the 5% confidence level. these results suggested that heterolobosean gnd genes were not secondary green plastid-derived, and that the gnd gene phylogeny did not support the plastids-early hypothesis. taken together, our data disallowed the plastids-early hypothesis, and showed that a secondary endosymbiotic origin of the gnd genes from green alga into plastid-lacking excavate protists is unlikely. it is striking that euglenida is monophyletic with stramenopiles in the cyanobacterial clade. recent phylogenetic analyses of the plastid-encoded and nuclear-encoded plastid-targeted genes suggest that the ancestor of euglenid secondary plastids branches within green algae, inconsistent with our gnd tree topology. the monophyly of cyanobacterial gnd genes from e. gracilis and plastid-lacking p. trichophorum further suggests that euglenid gnd genes have not been recruited via secondary egt of a green alga, because the'plastids-recent' hypothesis argues that eukaryovorous euglenid species such as p. trichophorum diverged before the secondary endosymbiotic event in the euglenida lineage. meanwhile, the presence of the cyanobacterial genes in stramenopiles, including photosynthetic algae and the plastid-lacking oomycete phytophthora, is apparently consistent with the'chromalveolate hypothesis', which suggests that secondary plastids of chromalveolata have been acquired through a single secondary endosymbiotic event. the most likely explanation is that the ancestor of the euglenida host cells acquired a gnd gene via ancient lgt from the stramenopile lineage before their divergence. this also explains well why euglenida and heterolobosea are robustly separated in the gnd phylogeny despite the close relatedness of these two lineages based on ssu rrna gene phylogeny and multiple nuclear-encoded protein phylogenies. evolutionary history of gnd genes and plastid-lacking excavate genomes although our gnd tree topology appears unexpected compared with the prevailing view of plastid evolution, several gene phylogenies that suggested imprints of gene transfer between euglenida and stramenopiles have been reported. in the plastid-targeted phosphoribulokinase gene phylogeny, red algal genes were basal in the eukaryotic clade and were separated from chromalveolate and green plant genes. furthermore, euglenid and chromalveolate prk genes were monophyletic and sister to green plants, and the authors reasoned that these secondary phototrophs might acquire prk genes via independent lgt events. as discussed above, it is likely that euglenida has acquired a cyanobacterial gnd gene from the ancestor of stramenopiles via lgt. although prk genes are found only in photosynthetic organisms and the origin of euglenid prk genes was phylogenetically unresolved, one can argue that prk and cyanobacterial gnd genes might have gone through similar evolutionary histories. a phylogenetic analysis of plastid-targeted fructose- -bisphosphatase genes illustrated another case of lgt between euglenida and chromalveolata. thus these genes might have been transferred from the stramenopile lineage to the euglenid lineage via multiple lgt events, perhaps phagocytosis of secondary phototrophs by a phagotrophic ancestor as suggested in the chlorarachniophyte bigelowiella natans. in the cyanobacterial gnd gene subtree, the red algal clade was at the basal position and was moderately separated from green plants and glaucophyta. an additional phylogenetic analysis excluding heterolobosea recovered the basal position of red algae in this subtree, suggesting that long branch attraction or artificial misplacement of red algae by heterolobosean sequences was unlikely. additionally, provided that the cyanobacterial genes from bikonts were robustly monophyletic, in contrast to well-characterized examples of prokaryote-to-eukaryote lgts, it is unlikely that the cyanobacterial gnd genes from bikonts had been acquired via multiple lgts from cyanobacteria to eukaryotes. recently, two competing hypotheses on archaeplastida phylogeny were proposed. the phylogenetic position of red algae in fig. is inconsistent with the monophyletic hypothesis of the archaeplastida unless multiple eukaryote-to-eukaryote lgts are hypothesized. although red algal and glaucophyte ancestries of the heterolobosean genes were not significantly dismissed, au tests rejected the possible secondary egt from green alga to heterolobosea. hence, the eukaryote-to-eukaryote lgts shown in fig. 2a are likely sources of gnd genes in plastid-lacking protists, in terms of the monophyletic hypothesis of the archaeplastida. however, monophyly of red algae plus stramenopiles was not rejected in our statistical tests, suggesting that the stramenopile genes might be attributed to secondary egt of red alga. on the other hand, an increasing number of multigene phylogenies showed that monophyly of archaeplastida had limited or no support. therefore it is advisable to discuss the evolutionary history of gnd genes, taking a different point of view on the plastid evolution into consideration. in terms of the non-monophyly hypothesis of the archaeplastida, it is reasonable to suggest that the gnd gene phylogeny may reflect the host cell phylogeny as recently resolved by a multiple slowly evolving nuclear gene phylogeny, which demonstrated the non-monophyly of archaeplastida and the most basal positioning of red algae plus excavata within the bikonts. possible evolutionary scenarios of plastid and host nuclear genomes we propose evolutionary scenarios in which the common ancestor of eukaryotes possessed a eubacteria-derived eukaryotic ancestral gnd gene, and the bikonts lineage additionally acquired the cyanobacterial gnd gene via a single primary endosymbiosis, and then diversified into archaeplastida, chromalveolata, excavata. given that recent large-scale molecular phylogenies demonstrated the monophyly of bikonts based on the rooting of eukaryotes, and no data providing evidence on primary and secondary plastids in the unikonts has been shown, we illustrated two likely scenarios in fig. in scenario a, we assumed monophyly of archaeplastida, and accordingly, at least two gains of cyanobacterial gnd genes via lgt and multiple losses of eukaryotic ancestral genes in separate lineages of bikonts. in scenario b, we presumed that all the bikonts including secondary phototrophs and plastid-lacking bikonts had at one time acquired the primary plastid. green plants, glaucophyta and chromalveolata then lost the eukaryotic ancestral gnd gene, red algae retained both, and excavata lost either one. in the ancestors of excavata, loss of primary photosynthetic plastids might have triggered concurrent gene loss of either cyanobacterial or eukaryotic ancestral gnd. only a single lgt event from stramenopiles into euglenida is considered in scenario b. although both scenarios are compatible with our phylogenetic analysis and statistical tests, we reason that scenario b is parsimonious and more likely to explain the evolutionary history of the gnd genes in that less lgt events need to be presupposed. broader sampling from various eukaryotic groups will be critical to devise a more reliable evolutionary history of eukaryotic gnd genes, and host lineages. it is also important to note that concatenated nuclear gene phylogeny of eukaryotic lineages and data mining for cyanobacterial genes in plastid-lacking protists are supposed to be independent approaches for exploring the origin of plants. future research will be focused on how deeply primary endosymbiosis is rooted within the bikonts, and which lineage could experience primary endosymbiosis early in the evolution of bikonts.  our present study demonstrates that free-living excavata possess either cyanobacterial or eukaryotic ancestral gnd genes, it is statistically unlikely that heterolobosean gnd genes were acquired via ancient secondary egt of green alga, and euglenida and stramenopiles are robustly monophyletic. although the sister relationship of this monophyletic group to any archaeplastida lineage is not rejected by the statistical tests, it is moderately separated from red algae, suggesting that the gnd genes in stramenopiles are not of secondary endosymbiont origin. one explanation is that a unique primary egt of cyanobacterial gnd genes into archaeplastida was followed by independent eukaryote-to-eukaryote lgts into stramenopiles and heterolobosea, and then by an additional lgt from stramenopiles into euglenida. alternatively, our results favor an evolutionary scenario that the gnd gene phylogeny reflects host cell phylogeny, and that the common ancestor of bikonts has acquired cyanobacterial gnd genes via primary endosymbiotic gene transfer early in eukaryotic evolution. authors' contributions sm participated in the design of the study and coordination, carried out the molecular phylogenetic and statistical studies, and drafted the manuscript. km participated in the phylogenetic and statistical studies. mi and mw participated in the cdna library construction and sequence analysis. hn conceived of the study, and participated in its design and helped to draft the manuscript. all authors read and approved the final manuscript. supplementary material additional file 1 click here for file additional file 2 click here for file additional file 3 click here for file additional file 4 click here for file acknowledgements we thank dr. toshinobu suzaki for a generous gift of the p. trichophorum culture, and dr. takashi nakada for help with phylogenetic analysis. computation time was provided by the super computer system, human genome center, institute of medical science, university of tokyo. this work was supported by grants-in-aid for creative scientific research and for scientific research from the ministry of education, culture, sports, science, and technology, japan.
plastids have inherited their own genomes from a single cyanobacterial ancestor, but the majority of cyanobacterial genes, once retained in the ancestral plastid genome, have been lost or transferred into the eukaryotic host nuclear genome via endosymbiotic gene transfer. we propose an evolutionary scenario in which plastid-lacking excavata acquired cyanobacterial gnd genes via eukaryote-to-eukaryote lateral gene transfer or primary endosymbiotic gene transfer early in eukaryotic evolution, and then lost either their pre-existing or cyanobacterial gene. although previous studies showed that cyanobacterial gnd genes, which encode 6-phosphogluconate dehydrogenase, are present in several plastid-lacking protists as well as primary and secondary plastid-containing phototrophic eukaryotes, the evolutionary paths of these genes remain elusive. our analysis demonstrated the patchy distribution of the excavate genes in the gnd gene phylogeny. statistical tests based on exhaustive maximum likelihood analyses strongly rejected that heterolobosean gnd genes were derived from a secondary plastid of green lineage. the diplonema gene was related to cytosol-type genes in red algae and opisthokonta, while heterolobosean genes occupied basal phylogenetic positions with plastid-type red algal genes within the monophyletic eukaryotic group that is sister to cyanobacterial genes. in addition, the cyanobacterial gnd genes from phototrophic and phagotrophic species in euglenida were robustly monophyletic with stramenopiles, and this monophyletic clade was moderately separated from those of red algae. these data suggest that these secondary phototrophic groups might have acquired the cyanobacterial genes independently of secondary endosymbioses. here we show an extended phylogenetic analysis including novel gnd gene sequences from excavata and glaucophyta.
 plastids have inherited their own genomes from a single cyanobacterial ancestor, but the majority of cyanobacterial genes, once retained in the ancestral plastid genome, have been lost or transferred into the eukaryotic host nuclear genome via endosymbiotic gene transfer. although previous studies showed that cyanobacterial gnd genes, which encode 6-phosphogluconate dehydrogenase, are present in several plastid-lacking protists as well as primary and secondary plastid-containing phototrophic eukaryotes, the evolutionary paths of these genes remain elusive.  here we show an extended phylogenetic analysis including novel gnd gene sequences from excavata and glaucophyta. our analysis demonstrated the patchy distribution of the excavate genes in the gnd gene phylogeny. the diplonema gene was related to cytosol-type genes in red algae and opisthokonta, while heterolobosean genes occupied basal phylogenetic positions with plastid-type red algal genes within the monophyletic eukaryotic group that is sister to cyanobacterial genes. statistical tests based on exhaustive maximum likelihood analyses strongly rejected that heterolobosean gnd genes were derived from a secondary plastid of green lineage. in addition, the cyanobacterial gnd genes from phototrophic and phagotrophic species in euglenida were robustly monophyletic with stramenopiles, and this monophyletic clade was moderately separated from those of red algae. these data suggest that these secondary phototrophic groups might have acquired the cyanobacterial genes independently of secondary endosymbioses.  we propose an evolutionary scenario in which plastid-lacking excavata acquired cyanobacterial gnd genes via eukaryote-to-eukaryote lateral gene transfer or primary endosymbiotic gene transfer early in eukaryotic evolution, and then lost either their pre-existing or cyanobacterial gene. 