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Horizontal transfer of a eukaryotic plastid-targeted protein gene to cyanobacteria Rogers, Matthew B; Patron, Nicola J; Keeling, Patrick J Jun 20, 2007

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ralssBioMed CentBMC BiologyOpen AcceResearch articleHorizontal transfer of a eukaryotic plastid-targeted protein gene to cyanobacteriaMatthew B Rogers, Nicola J Patron and Patrick J Keeling*Address: Canadian Institute for Advanced Research, Department of Botany, University of British Columbia, 3529-6270 University Boulevard, Vancouver, BC V6T 1Z4, CanadaEmail: Matthew B Rogers - mbrogers@interchange.ubc.ca; Nicola J Patron - nicolapatron@mac.com; Patrick J Keeling* - pkeeling@interchange.ubc.ca* Corresponding author    AbstractBackground: Horizontal or lateral transfer of genetic material between distantly relatedprokaryotes has been shown to play a major role in the evolution of bacterial and archaealgenomes, but exchange of genes between prokaryotes and eukaryotes is not as well understood.In particular, gene flow from eukaryotes to prokaryotes is rarely documented with strong support,which is unusual since prokaryotic genomes appear to readily accept foreign genes.Results: Here, we show that abundant marine cyanobacteria in the related genera Synechococcusand Prochlorococcus acquired a key Calvin cycle/glycolytic enzyme from a eukaryote. Two non-homologous forms of fructose bisphosphate aldolase (FBA) are characteristic of eukaryotes andprokaryotes respectively. However, a eukaryotic gene has been inserted immediately upstream ofthe ancestral prokaryotic gene in several strains (ecotypes) of Synechococcus and Prochlorococcus. Inone lineage this new gene has replaced the ancestral gene altogether. The eukaryotic gene is mostclosely related to the plastid-targeted FBA from red algae. This eukaryotic-type FBA once replacedthe plastid/cyanobacterial type in photosynthetic eukaryotes, hinting at a possible functionaladvantage in Calvin cycle reactions. The strains that now possess this eukaryotic FBA are scatteredacross the tree of Synechococcus and Prochlorococcus, perhaps because the gene has been transferredmultiple times among cyanobacteria, or more likely because it has been selectively retained only incertain lineages.Conclusion: A gene for plastid-targeted FBA has been transferred from red algae tocyanobacteria, where it has inserted itself beside its non-homologous, functional analogue. Itscurrent distribution in Prochlorococcus and Synechococcus is punctate, suggesting a complex historysince its introduction to this group.BackgroundComparative genomics has generated a large pool ofmolecular data, and one of the major debates to emergeprokaryote and prokaryote to eukaryote transfers are man-ifold, and their importance has dominated this debate [2-4]. However, relatively few cases of eukaryote to prokary-Published: 20 June 2007BMC Biology 2007, 5:26 doi:10.1186/1741-7007-5-26Received: 21 December 2006Accepted: 20 June 2007This article is available from: http://www.biomedcentral.com/1741-7007/5/26© 2007 Rogers et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 8(page number not for citation purposes)from this data is the importance of horizontal gene trans-fer in genomic evolution [1]. Cases of prokaryote toote transfers have been reported, and many are not sup-ported by broad representation or strong phylogeneticBMC Biology 2007, 5:26 http://www.biomedcentral.com/1741-7007/5/26results [4]. The apparent paucity of such events in relationto transfers between prokaryotes could reflect biologicallimitations on gene transfer from eukaryotes to prokaryo-tes. Such limitations might include the presence of spli-ceosomal introns, or differences in promoter structuresand ribosomal binding sites. Another important consider-ation, however, is that prokaryotes are far more abundantthan eukaryotes in most microbial ecosystems, so theremight be limited opportunities for such transfers to occur.Nevertheless, a small number of well-supported examplesof eukaryote to prokaryote gene transfer are known. Oneof the most intriguing of these is the transfer of eukaryoticalpha- and beta-tubulin subunits to the bacterium Pros-thecobacter [5,6]. Other examples are described in therecent analysis of eukaryotic shikimate pathway enzymes[7]. This study has revealed a possible eukaryote toprokaryote transfer in the gene encoding a class II 3-deoxy-D-arabino-heptulosonate 7-phosphate(DAHP)synthase. Another proposed eukaryote toprokaryote transfer might have occurred in the spirocha-ete Treponema pallidum, which contains a eukaryotic-typeGAPDH that is phylogenetically related to euglenozoanhomologues [8]. Here, we report another example ofeukaryote to prokaryote gene transfer with a number ofunique functional and evolutionary implications.Fructose bisphosphate aldolase (FBA, or aldolase) is acore carbon metabolic enzyme responsible for the aldolcleavage of fructose-bisphosphate into glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate(DHAP) sugars in glycolytic reactions, and the reversealdol condensation of these triose sugars to fructose-bisphosphate in Calvin cycle and gluconeogenic reac-tions. FBA exists in two non-homologous but functionallyequivalent forms, referred to as class I and class II [9].Eukaryotes typically use class I FBA, although there are afew noteworthy exceptions [10-13]. In contrast, prokaryo-tes typically use the class II form of the enzyme, althoughonce again a handful of species also encode a class I formof unknown function. One other interesting exception isfound in the plastid (chloroplast) of plants and manyalgae. A large number of cyanobacterial genomes havebeen shown to encode a canonical, bacterial class II FBA,indicating that this FBA is ancestral to the group, as onewould expect for prokaryotes. As the descendants of endo-symbiotic cyanobacteria, plastids would also be expectedto contain this cyanobacterial class II FBA, but thisenzyme has been replaced by a duplicate of the host cyto-plasmic class I enzyme in the ancestors of plants, red andgreen algae, resulting in two class I enzymes in thesegroups [14]. Hence, glaucophyte plastids are unique inhaving retained their ancestral protein [12].marine cyanobacteria. The complete genomes of severalstrains of both Prochlorococcus and Synechococcus encode aclass I FBA gene. We show that these are evolutionarilyrelated to the gene for the plastid-targeted protein of redalgae. In some strains, the ancestral class II gene has alsobeen lost, suggesting that the eukaryotic gene has com-pletely taken over its function. The position of the trans-ferred gene is also of interest, as it is immediatelyupstream of the class II FBA in genomes where both arepresent. In genomes where the class II FBA has been lost,the new class I gene therefore occupies the position previ-ously occupied by class II FBA.Results and DiscussionClass I and class II FBA in Prochlorococcus and SynechococcusCyanobacterial genomes typically encode a class II FBA foruse in both glycolysis and the Calvin cycle. However, intwo closely related genera, Synechococcus and Prochlorococ-cus, we have found the situation is more complex. Theorder of genes surrounding the FBA locus is highly con-served among completely sequenced representativegenomes from both genera. While many strains encodeonly the ancestral cyanobacterial class II FBA, in three Syn-echococcus strains (BL107, CC9902, and 9311) and threeProchlorococcus strains (SS120, AS9601, and MIT9515), aeukaryote-derived class I FBA is found immediatelyupstream of the ancestral cyanobacterial gene (Figure 1).Moreover, in two Prochlorococcus strains (NATL1A andNATL2A) the cyanobacterial class II FBA has been lost,effectively leaving the eukaryotic class I FBA in its place(Figure 1). Prochlorococcus MIT9303 also has a homologueof this gene elsewhere in the genome, but both it and thecanonical class II FBA contain several stop codons, indi-cating either they are pseudogenes or sequencing errors.To compare the distribution of FBA genes to the phylog-eny of the organisms, a phylogeny of Prochlorococcus andSynechococcus strains was constructed using a locus inde-pendent of FBA, the 16S-23S internal transcribed spacer(ITS). This phylogeny (Figure 1) largely agrees with previ-ously published analyses [15], with a distinction betweenthe two genera and a further subdivision between high-light and low-light adapted strains of Prochlorococcus [15].However, the phylogeny is incompatible with any simpleexplanation for the distribution of FBA genes. Mostimportantly, the organisms that possess the eukaryoticclass I FBA do not form a unique clade in the ITS phylog-eny, but are instead scattered across the tree. This is exem-plified by two pairs of Prochlorococcus strains (MIT9301/AS9601 and MED4/MIT9515) that share virtually identi-cal ITS sequences, and are thus inferred to be closelyrelated, but in both cases the former strain encodes onlyPage 2 of 8(page number not for citation purposes)Here we show that this eukaryotic plastid-targeted class IFBA has itself been transferred to a specific group ofthe ancestral class II FBA whereas the latter encodes bothclasses. The two Prochlorococcus strains that encode onlyBMC Biology 2007, 5:26 http://www.biomedcentral.com/1741-7007/5/26Page 3 of 8(page number not for citation purposes)ITS Phylogeny of Prochlorococcus and Synechococcus showing gene order surrounding FBA I locusFigure 1ITS Phylogeny of Prochlorococcus and Synechococcus showing gene order surrounding FBA I locus. Phylogeny of Prochlorococcus and Synechococcus strains showing the distribution of class I and class II FBA. Maximum likelihood tree based on rRNA ITS sequences with bootstrap support shown for major nodes with greater than 70% support. The branch leading to one clade of Prochlorococcus (indicated by double hatch marks) has been truncated to fit. The genomic context of FBA genes is shown for completely sequenced genomes. Class I FBA (eukaryotic) is shown in red and class II (prokaryotic) is shown in green. Black arrows correspond to up and downstream genes that are conserved in order and direction in most genomes, whereas grey arrows are the few exceptions to this conservation.SynechococcusProchlorococcusSBMIT9301 FBAIIAS9601 FBAI FBAIIMIT9201RS810MIT9311MIT9312 FBAIIMIT9314MIT9123MIT9107MIT9116MIT9302GP2MIT9322MIT9401MIT9321MIT9202MIT9215MIT9515MED4 FBAIIFBAI FBAIINATL1APAC1NATL2AFBAIFBAIMIT9211SS51SS2SS120SS35LGFBAIIFBAI FBAIIMIT9313MIT9303FBAII CC9605 WH6501 WH8005 WH8109 WH8002 WH8012 CC9902 RS8015WH8102WH8103WH8113WH8406WH8112FBAI FBAIIFBAII MITS9220WH8101RS9705WH7803WH9908WH8020WH8015WH8016FBAIIBL107RS9917CC9311WH5701WH7805 FBAIIFBAI FBAIIFBAIIFBAII0.05FBAIIRS9916906685-100989910010095725554-91981001009810084-10010010010064-695061-77100FBAI FBAIIFBAIIFBAIBMC Biology 2007, 5:26 http://www.biomedcentral.com/1741-7007/5/26the eukaryotic class I gene (NATL1A and NATL2A) areclosely related in ITS phylogeny, suggesting the class IIFBA was lost once in their common ancestor.Origin of class I FBA in Synechococcus and ProchlorococcusThe relatively restricted distribution of this eukaryoticgene in a few strains of Synechococcus and Prochlorococcussuggests either a very recent origin in these genera, or hor-izontal gene transfer between strains. To determine if thegenes originated once in cyanobacteria and from whatkind of eukaryote they might be derived, phylogeneticanalyses were performed to determine the position of thecyanobacterial genes relative to eukaryotic class I FBAs.Class I FBA phylogeny has been shown previously to lacksufficient resolution between many major subgroups[14,16], and the present analysis was no exception to this(Figure 2). However, the cyanobacterial genes formed aunique clade with 100% support, indicating a single ori-gin of the gene in Synechococcus and Prochlorococcus. Mostimportantly, the cyanobacterial genes formed a specificand strongly-supported group with the nuclear-encoded,plastid-targeted FBAs from red algae (100% support).These genes are very distantly related to the discrete cladeof class I FBAs already known from some prokaryotes,which is also present in three cyanobacteria Trichodesmiumerythraeum, Crocosphaera watsonii, and SynechococystisPCC6803. These genera are not closely related to Prochlo-rococcus and Synechococcus. The Prochlorococcus and Syne-chococcus genes show elevated rates of evolution, but withthe exception of the possible pseudogene in Prochlorococ-cus MIT9303, none show any signs of being non-func-tional or otherwise unusual.An affinity between genes for plastid-targeted proteinsand cyanobacterial homologues is generally not surpris-ing. However, this result is unusual in that the red andgreen algal plastid FBAs are not derived from the cyano-bacterial endosymbiont. Instead, these genes have beeninferred to result from a duplication of and replacementby the non-homologous, cytosolic class I FBA [14]. Alto-gether, this eukaryotic class I FBA has invaded a prokaryo-tic, photosynthetic environment twice: originallyinvading the endosymbiotic plastid in the ancestor of redand green algae (glaucophytes being the only primaryalgae that retain the ancestral class II FBA in their plastid[11,12]), and subsequently invading the cyanobacteriallineage itself in the genera Synechococcus and Prochlorococ-cus.FBA distribution within Synechococcus and ProchlorococcusThe phylogeny of class I FBA shows the Prochlorococcus/cifically involving the gene for its plastid-targeted FBA.However, such an event would lead to a simple distribu-tion of class I FBA in cyanobacteria where all taxa possess-ing the new gene were closely related to one another. Thiscontrasts with the observed distribution shown in Figure1, and is best exemplified by Prochlorococcus strainsAS9601/MIT9301 and MIT9515/MED4, where apparentlyclose relatives differ in the presence or absence of class IFBA. The complexity of this distribution suggests the evo-lution of class I FBA within Prochlorococcus and Synechococ-cus has been characterised either by further gene transferevents, or selective loss and retention of this eukaryoticgene across different strains. These two alternatives aredescribed in greater detail below.On the one hand, it is possible that the class I FBA wastransferred relatively recently from a red alga to one mem-ber of the Prochlorococcus/Synechococcus clade, and subse-quently moved to other strains to achieve its presentdistribution. While multiple transfer events appears com-plex, there are well-known precedents for gene transferbetween Prochlorococcus strains, facilitated by cross-infect-ing phage [17-19]. This leads to the possibility that thephylogeny of class I FBA might not match that of thestrains in which it is found. However, the topology of theProchlorococcus/Synechococcus clade in the class I FBA tree(Figure 2) does not differ in any strongly-supported nodefrom the phylogeny inferred from ITS (Figure 1). Moreo-ver, the fact that class I FBA is always found in the samegenomic context suggests that such inter-strain transferwould most likely have involved insertion by homolo-gous recombination. This would lead to the additionalexpectation that the flanking genes might share a greaterdegree of similarity to homologues in other strains withclass I FBA than they do to homologues from strains thatlack it. To test this, we inferred phylogenies of the ances-tral class II FBA (Figure 3) and genes upstream and down-stream of both FBA genes (mviM upstream and purQdownstream: see Additional file 1). In all three cases thephylogenies mirror the ITS phylogeny for strongly-sup-ported nodes relative to the distribution of FBA genes, andin no case do they reveal a clade consisting of the class IFBA-containing strains. These gene sequences were alsomanually examined for evidence of small-scale recombi-nation at the ends proximal to FBA, and none wasobserved. There is therefore no evidence that the evolu-tionary history of either class I or class II FBA has recentlydeviated from that of the organisms in which they arefound (inferred from ITS phylogeny).As there is no direct evidence for recombination in thisregion between strains containing class I FBA, and noindication that the evolutionary history of ProchlorococcusPage 4 of 8(page number not for citation purposes)Synechococcus genes originated once, and strongly suggeststhis was by horizontal gene transfer from a red alga, spe-and Synechococcus class I FBA genes differs from the organ-isms in which they are found, there is no reason to believeBMC Biology 2007, 5:26 http://www.biomedcentral.com/1741-7007/5/26the gene has moved between strains. A more likely expla- tion is due to differential gene loss and retention events.Protein maximum likelihood tree of class I FBAFigure 2Protein maximum likelihood tree of class I FBA. Protein maximum likelihood phylogeny of class I FBA. The cyanobacte-rial and plastid-targeted red algal class I FBA genes are indicated by boxes, and all other groups are bracketed and labelled to the right. Numbers at node correspond to bootstrap support over 50% for major nodes from ML (above) and distance (below). Methods and parameters used are detailed in the Methods section.Nocardiodes sp.Thiobacillus denitrificansChlorobium limnicolaProsthecoides aestuariiRubrobacter xylanophilusBartonella quintanaBradyrhizobium japonicumLegionella pneumophilaSinorhizobium melilotiTrichodesmium erythraeumCrocosphaera watsoniiXylella fastidiosaXanthomonas axonopodisXanthomonas oryzaeBurkholderia fungorumRalstonia eutrophaPolaromonas sp.0.1Trypanosoma bruceiChlamydomonas reinhardtii Bigelowiella natans PLASTID-TARGETEDXenopus laevisSphoeroides nephelusHomo sapiensBiomphalaria glabrataCaenorhabditis elegansGlobodera rostochiensisDrosophila melanogasterToxoptera citricadaCryptosporidium parvumToxoplasma gondiiPlasmodium bergheiPlasmodium falciparumArabidopsis thalianaPandanus amaryllifoliusPisum sativumFragaria X ananassaArabidopsis thalianaPisum sativumCicer arietinumPersea americanaZea maysOryza sativaSpinacia oleraceaMesembryanthemum crystallinumDunaliella salinaChlamydomonas reinhardtiiScherffelia dubiaArabidopsis thalianaOryza sativaPisum sativumTrifolium repensOryza sativaNicotiana tabacumSpinacia oleraceaSolanum tuberosumTetrahymena thermophilaParamecium aureliaGaldieria sulphurariaCyanidioschyzon merolaePorphyra yezoensisSynechococcus CC9902Prochlorococcus MIT9515Prochlorococcus AS9601Prochlorococcus HF1088Prochlorococcus NATL2AProchlorococcus NATL1ABigelowiella natans CYTOSOLICDictyostelium discoideumEuglena gracilis Galdieria sulphuraria CYTOSOLICCyanidioschyzon merolae CYTOSOLICSynechococcus BL107Synechococcus CC9311Prochlorococcus 9303ψProchlorococcus SS120EubacteriaMetazoaPlantsCytosolicGreen algae & plantsplastid-targetedAlveolatesRed algaeplastid-targetedProchlorococcus &Synechococcus10010010010010010084/6991871001009294971001001009998100100100100100100951006796100/100859051-6869100100Page 5 of 8(page number not for citation purposes)nation is that class I FBA was acquired in the ancestor ofProchlorococcus and Synechococcus and its current distribu-As with the horizontal transfer explanation, there is also aprecedent for aldolase loss events in these genera, as theBMC Biology 2007, 5:26 http://www.biomedcentral.com/1741-7007/5/26ancestral class II FBA has clearly been lost in Prochlorococ-cus NATL1A and NATL2A. This explanation is consistentwith all phylogenetic data, but without more samplingfrom additional strains, it would be premature to defini-tively distinguish between these two alternatives, or somecombination of both.Implications for FBA evolution and functionThe existence and the punctate distribution of the eukary-otic class I FBA in Prochlorococcus and Synechococcus bothhave interesting implications for the evolution and func-tion of this enzyme in cyanobacteria.A single early origin of the eukaryotic class I FBA inProchlorococcus and Synechococcus implies that both geneswere present in their common ancestor and that redun-dancy was common throughout their evolution. Theeukaryotic enzyme would not be retained without havingacquired some function, which is most clearly demon-strated in the NATL1A and NATL2A strains where theancestral class II gene has been lost. The maintenance ofboth types of FBA in several strains today, particularly inthe minimal genome of Prochlorococcus SS120 [20], fur-ther supports the conclusion that the class I FBA has somefunction. It is possible that the two classes of FBA are func-(as might also be the case with the other discrete clade ofbacterial class I FBA genes in Figure 2). Any such differen-tiation must be reversible, however, as both classes havebeen lost at least once. If we consider only nodes withgreater than 90% support from at least one method in Fig-ure 1, then the eukaryotic class I FBA would have to havebeen lost at least once in Synechococcus and at least threetimes in Prochlorococcus. In reality these figures are likelylarger, because many nodes in the phylogeny that are nottaken into account are also consistently recovered andwell supported in the other phylogenies. The ancestralclass II gene would also have to have been lost at leastonce in the ancestor of Prochlorococcus NATL1A andNATL2A, which has remarkable implications for the func-tion of this enzyme in the Calvin cycle. Indeed, if any ofthe gene losses implied by the distribution of FBA genesled to its function being assumed or re-assumed by itsanalogue, then a closer examination of the biochemicalactivities of class I and class II FBA in strains of Prochloro-coccus and Synechococcus where both types of FBA arepresent will be interesting. Further examination mightreveal conditions under which each class of FBA isfavoured, addressing the question of why the eukaryoticprotein was retained at all.These considerations also need to take into account theunusual position of the eukaryotic gene in the genome. Itssituation immediately upstream of its analogue might bechance, but this would be extremely fortuitous. We sug-gested above that this position might have favoured theretention of the new gene because the regulation of thisposition might have been favourable for a new FBA genedue to the presence of the existing one. However, if thesetwo genes are part of an operon, functional differentiationmight have taken place without expression-level differen-tiation. For example, temporal differentiation (such aslight/dark cycles) could easily lead to the two enzymesadapting to different roles, but if they are co-expressedsuch differentiation becomes more difficult. This onceagain points to the importance of re-evaluating the func-tional differences between these two classes of FBA to seeif there is some mechanistic property that might convey aselective advantage of class I FBA over class II, perhapsonly manifested in one direction.ConclusionRecent and well-defined cases of horizontal gene transferfrom eukaryotes to prokaryotes remain rare, despite theabundance of data from prokaryotic genomes. This exam-ple stands out not only as one of the more strongly-sup-ported cases, but also has a number of other interestingimplications. The location of the gene immediatelyupstream of its non-homologous, functional analoguePHYML tree of class II FBAFigure 3PHYML tree of class II FBA. Maximum likelihood phylog-eny of Prochlorococcus and Synechococcus class II FBA using nucleic acids. Numbers at node correspond to bootstrap support over 50% for major nodes from ML (above) and dis-tance (below). Methods and parameters used are detailed in the Methods section.0.1Synechococcus sp. CC9605Synechococcus sp. WH8102Synechococcus sp. CC9902Prochlorococcus marinus MIT9313Prochlorococcus marinus MIT9211Prochlorococcus marinus SS120Prochlorococcus marinus MED4Prochlorococcus marinus MIT9312Prochlorococcus marinus MIT9515Prochlorococcus marinus MIT9303Prochlorococcus marinus MIT9301Prochlorococcus marinus AS9601Synechococcus sp. WH7805Synechococcus sp. CC9311Synechococcus sp. RS9916Synechococcus sp. RS9917Synechococcus sp. WH5701Synechococcus sp. BL10710010010010052-1001009899991009310010010092929510077-98100Page 6 of 8(page number not for citation purposes)tionally differentiated, perhaps between glycolysis and theCalvin cycle, or perhaps one recognised a new substratesuggests the genomic context of this insertion was decid-edly non-random and points to the importance of contextBMC Biology 2007, 5:26 http://www.biomedcentral.com/1741-7007/5/26in the adaptation of a newly transferred gene. The punc-tate distribution of the eukaryotic class I FBA amongstrains of Prochlorococcus and Synechococcus also suggeststhe history of this enzyme was subject to further compli-cations that might shed light on the fate of newly acquiredgenes in prokaryotic genomes.One of the major outstanding questions of this study iswhether the eukaryotic class I FBA confers any advantageover its analogue, as opposed to simply replacing the ana-logue to no great consequence. In either case, the func-tional differences between class I and class II FBA proteinsshould be re-investigated in greater detail, as the possibil-ity that class I FBAs might be more effective in Calvin cyclereactions has broad implications for the evolution of pho-tosynthesis in prokaryotes and eukaryotes, and for the ori-gin and evolution of plastid organelles.MethodsIdentification of FBA genes and genomic contextAll complete cyanobacterial genomes (and all prokaryoticgenomes in general) were searched for both class I andclass II FBA gene sequences using Blastp and Blastx. Thered alga-derived class I FBA was found in several strains ofProchlorococcus and Synechococcus but in no other cyano-bacterium and no other prokaryotic genome. The anno-tated identity of the flanking genes was compared andconfirmed by database comparisons. Genomic data fromunpublished genomes of Prochlorococcus AS9601,MIT9211, MIT9301, MIT9303, MIT9312, MIT9315,NATL1A were kindly provided by ML Coleman and SWChisholm (Massachusetts Institute of Technology,Department of Civil and Environmental Engineering, 15Vassar Street Bldg., Cambridge, MA).Phylogenetic methodsProtein phylogeny of class I FBA was generated from analignment of 75 taxa and 312 characters using PhyML2.4.4 [21] with the WAG substitution matrix, four gammacategories and one category of invariable sites. All substi-tutions matrices were determined using Modelgenerator0.83 [22]. The gamma shape parameter alpha and the pro-portion of invariable sites i were estimated by PhyML tobe 1.14 and 0.07, respectively. One hundred bootstrapreplicates were preformed in the same way, using alphaand i parameters estimated from the original data. Dis-tances were calculated using TREE-PUZZLE v. 5.2 with theWAG substitution matrix, four gamma categories and onecategory of invariable sites using the same alpha and i esti-mates. Trees were constructed using WEIGHBOR 1.0.1aand 500 bootstraps carried out using puzzleboot [23].Nucleotide based phylogenies of ITS, FBA II, purQ andmviM from Prochlorococcus and Synechococcus were carriedmodel of substitution with four gamma categories andone category of invariable sites was used, with the alphaparameter, proportion of invariable sites, and ts:tv ratioestimated from the data (0.40, 0.12, and 3.05, respec-tively). For class II FBA (18 taxa and 1021 positions), purQ(20 taxa and 639 positions) and mviM (20 taxa and 1003positions) the GTR model of substitution with fourgamma categories and one category of invariable sites wasused. The alpha parameters were estimated to be 2.96,1.57, and 1.55, and the proportions of invariable siteswere estimated to be 0.51, 0.31 and 0.20, for class II FBA,purQ and mviM, respectively.Authors' contributionsMBR, NJP and PJK contributed to the concept of this man-uscript. Phylogenetic analyses were performed by MBRand PJK. The original alignment of class I FBAs was pro-vided by NJP. The manuscript was written by MBR andPJK.Additional materialAcknowledgementsWe wish to thank ML Coleman and SW Chisholm for data from unpub-lished genomes of Prochlorococcus AS9601, MIT9211, MIT9301, MIT9303, MIT9312, MIT9315 and NATL1A. This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada. PJK is a Fellow of the Canadian Institute for Advanced Research and a senior inves-tigator of the Michael Smith Foundation for Health Research.References1. Doolittle WF: Phylogenetic classification and the universaltree.  Science 1999, 284:2124-2129.2. 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ProcNatl Acad Sci USA 2005, 102:9170-9175.Additional file 1Figure showing phylogenies of purQ, mviM generated from nucleic acids Numbers at node correspond to bootstrap support over 50% for major nodes from ML (above) and distance (below). Methods and parameters used are detailed in the Methods section.Click here for file[http://www.biomedcentral.com/content/supplementary/1741-7007-5-26-S1.eps]Page 7 of 8(page number not for citation purposes)out using the same methods and the same numbers ofbootstraps. For ITS (58 taxa and 212 positions), the HKY7. Richards TA, Dacks JB, Campbell SA, Blanchard JL, Foster PG,McLeod R, Roberts CW: Evolutionary origins of the eukaryoticshikimate pathway: gene fusions, horizontal gene transfer,Publish with BioMed Central   and  every scientist can read your work free of charge"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime."Sir Paul Nurse, Cancer Research UKYour research papers will be:available free of charge to the entire biomedical communitypeer reviewed and published immediately upon acceptancecited in PubMed and archived on PubMed Central BMC Biology 2007, 5:26 http://www.biomedcentral.com/1741-7007/5/26and endosymbiotic replacements.  Eukaryot Cell 2006,5:1517-1531.8. Figge RM, Schubert M, Brinkmann H, Cerff R: Glyceraldehyde-3-phosphate dehydrogenase gene diversity in eubacteria andeukaryotes: evidence for intra- and inter-kingdom genetransfer.  Mol Biol Evol 1999, 16:429-440.9. Marsh JJ, Lebherz HG: Fructose-bisphosphate aldolases: an evo-lutionary history.  Trends Biochem Sci 1992, 17:110-113.10. Sanchez L, Horner D, Moore D, Henze K, Embley T, Müller M: Fruc-tose-1,6-bisphosphate aldolases in amitochondriate protistsconstitute a single protein subfamily with eubacterial rela-tionships.  Gene 2002, 295:51-59.11. Rogers MB, Keeling PJ: Lateral gene transfer and re-compart-mentalisation of Calvin cycle enzymes in plants and algae.  JMol Evol 2003, 58:367-375.12. Nickol AA, Muller NE, Bausenwein U, Bayer MG, Maier TL, SchenkHE: Cyanophora paradoxa : nucleotide sequence and phylog-eny of the nucleus encoded muroplast fructose-1,6-bisphos-phate aldolase.  Z Naturforsch 2000, 55(11-12):991-1003.13. Patron NJ, Rogers MB, Keeling PJ: Gene replacement of fructose-1,6-bisphosphate aldolase (FBA) supports a single photosyn-thetic ancestor of chromalveolates.  Eukaryot Cell 2004,3:1169-1175.14. Gross W, Lenze D, Nowitzki U, Weiske J, Schnarrenberger C: Char-acterization, cloning, and evolutionary history of the chloro-plast and cytosolic class I aldolases of the red alga Galdieriasulphuraria.  Gene 1999, 230:7-14.15. Rocap G, Distel DL, Waterbury JB, Chisholm SW: Resolution ofProchlorococcus and Synechococcus ecotypes by using 16S-23S ribosomal DNA internal transcribed spacer sequences.Appl Environ Microbiol 2002, 68:1180-1191.16. Plaumann M, Pelzer-Reith B, Martin WF, Schnarrenberger C: Multi-ple recruitment of class-I aldolase to chloroplasts and eubac-terial origin of eukaryotic class-II aldolases revealed bycDNAs from Euglena gracilis.  Curr Genet 1997, 31:430-438.17. Coleman ML, Sullivan MB, Martiny AC, Steglich C, Barry K, Delong EF,Chisholm SW: Genomic islands and the ecology and evolutionof Prochlorococcus.  Science 2006, 311:1768-1770.18. Sullivan MB, Coleman ML, Weigele P, Rohwer F, Chisholm SW:Three Prochlorococcus cyanophage genomes: signature fea-tures and ecological interpretations.  PLoS Biol 2005, 3:e144.19. Lindell D, Sullivan MB, Johnson ZI, Tolonen AC, Rohwer F, ChisholmSW: Transfer of photosynthesis genes to and from Prochloro-coccus viruses.  Proc Natl Acad Sci USA 2004, 101:11013-8.20. Dufresne A, Salanoubat M, Partensky F, Artiguenave F, Axmann IM,Barbe V, Duprat S, Galperin MY, Koonin EV, Le Gall F, et al.:Genome sequence of the cyanobacterium Prochlorococcusmarinus SS120, a nearly minimal oxyphototrophic genome.Proc Natl Acad Sci USA 2003, 100:10020-10025.21. Guindon S, Gascuel O: A simple, fast, and accurate algorithmto estimate large phylogenies by maximum likelihood.  SystBiol 2003, 52:696-704.22. Keane TM, Creevey CJ, Pentony MM, Naughton TJ, McLnerney JO:Assessment of methods for amino acid matrix selection andtheir use on empirical data shows that ad hoc assumptionsfor choice of matrix are not justified.  BMC Evol Biol 2006, 6:29.23. Roger A, Holder M: Puzzleboot.   [http://www.tree-puzzle.de].yours — you keep the copyrightSubmit your manuscript here:http://www.biomedcentral.com/info/publishing_adv.aspBioMedcentralPage 8 of 8(page number not for citation purposes)


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